@article {16622,
	title = {Inflammatory breast cancer biomarker identification by simultaneous TGIRT-seq profiling of coding and non-coding RNAs in tumors and blood},
	journal = {medRxiv},
	year = {2023},
	abstract = {Inflammatory breast cancer (IBC) is the most aggressive and lethal breast cancer subtype, but lags in biomarker identification. Here, we used an improved Thermostable Group II Intron Reverse Transcriptase RNA sequencing (TGIRT-seq) method to simultaneously profile coding and non-coding RNAs from tumors, PBMCs, and plasma of IBC and non-IBC patients and healthy donors. Besides RNAs from known IBC-relevant genes, we identified hundreds of other overexpressed coding and non-coding RNAs (p<=0.001) in IBC tumors and PBMCs, including higher proportions with elevated intron-exon depth ratios (IDRs), likely reflecting enhanced transcription resulting in accumulation of intronic RNAs. As a consequence, differentially represented protein-coding gene RNAs in IBC plasma were largely intron RNA fragments, whereas those in healthy donor and non-IBC plasma were largely fragmented mRNAs. Potential IBC biomarkers in plasma included T-cell receptor pre-mRNA fragments traced to IBC tumors and PBMCs; intron RNA fragments correlated with high IDR genes; and LINE-1 and other retroelement RNAs that we found globally up-regulated in IBC and preferentially enriched in plasma. Our findings provide new insights into IBC and demonstrate advantages of broadly analyzing transcriptomes for biomarker identification. The RNA-seq and data analysis methods developed for this study may be broadly applicable to other diseases.},
	url = {https://www.medrxiv.org/content/10.1101/2023.05.26.23290469v1},
	author = {Wylie, D and X Wang and Yao, J and Xu, H and Ferrick-Kiddie, EA and Iwase, T and Krishnamurthy, S and Ueno, NT and Lambowitz, A M}
}
@article {16621,
	title = {Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2},
	journal = {Nature Cell Biology},
	volume = {25},
	year = {2023},
	pages = {592{\textendash}603},
	abstract = {Cells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Aminoacyl-tRNA synthetases, enzymes that catalyse the first step of protein synthesis, can also mediate cell signalling. Here we show that depletion of arginine during inflammation decreased levels of nuclear-localized arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts and co-localizes with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal and nuclear speckle protein, and that decreased levels of nuclear ArgRS correlated with changes in condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in the synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a mechanism whereby an aminoacyl-tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.},
	url = {https://www.nature.com/articles/s41556-023-01118-8},
	author = {Cui, H and Diedrich, JK and Wu, DC and Lim, JJ and Nottingham, RM and Moresco, JJ and Yates, JR and Blencowe, BJ and Lambowitz, A M and Schimmel, P}
}
@article {15474,
	title = {Group II-like Reverse Transcriptases Function in Double Strand Break Repair},
	journal = {Cell},
	volume = {185},
	number = {20},
	year = {2022},
	pages = {3671-3688},
	abstract = {Bacteria encode free-standing reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. Here, we found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when transferred into Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase q and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) in vitro and in vivo. We also found that a group II intron RT can function similarly to G2L4 RT in DNA repair, with reciprocal substitutions at the active site showing an I residue favors MMEJ and an A residue favors primer extension in both enzymes. The DNA repair functions of these enzymes utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have an inherent ability to function in DSBR in a wide range of organisms.},
	url = {https://www.sciencedirect.com/science/article/pii/S0092867422010637?via\%3Dihub},
	author = {S.K. Park and G. Mohr and J. Yao and Russell, R. and A.M. Lambowitz}
}
@article {15472,
	title = {Abstract P5-07-03: Disease classification modeling of inflammatory breast cancer based on simultaneous profiling of coding and non-coding RNAs in tumor and blood samples by TGIRT-sequencing},
	journal = {Cancer Res},
	volume = {82},
	year = {2022},
	abstract = {Background:\&nbsp;Inflammatory breast cancer (IBC) is the most aggressive and lethal breast cancer subtype but lags in disease-specific RNA biomarkers due in part to its paucity of large discrete tumors. A strategy to overcome this challenge is to identify blood-based RNA biomarkers that are minimally invasive and reflect the state of both the diseased breast tissue and the patient{\textquoteright}s immune response. Here, we identified IBC-specific RNA biomarkers by thermostable group II intron reverse transcriptase sequencing (TGIRT-seq), a recently developed comprehensive RNA-seq technology that enables simultaneous profiling of all RNA biotypes from small amounts of starting material. We used these biomarkers to develop novel disease classification models for IBC based on coding and non-coding RNAs from FFPE tumor slices, PBMCs, and plasma.\&nbsp;Methods:\&nbsp;We obtained biological samples including FFPE, PBMC, and plasma from a cohort of ten patients with IBC and compared them to samples from six patients with non-IBC and sixteen healthy donors using TGIRT-seq technology.\&nbsp;Results:\&nbsp;TGIRT-seq of FFPE tumor slices identified differentially expressed mRNAs and miRNAs found previously to distinguish IBC from non-IBC tumors, as well as numerous additional differentially expressed mRNAs and small non-coding RNAs characteristic of IBC. Surprisingly, TGIRT-seq revealed that the differentially expressed protein-coding gene transcripts fall into two categories: mature mRNAs with reads confined to exons, and pre-mRNAs-derived transcripts with reads distributed across exons and introns, to our knowledge, a distinction not made previously for any cancer type. Differentially expressed miRNAs included both mature miRNAs and other transcripts of miRNA loci. IBC PBMCs showed a characteristic inflammatory response not seen in PBMCs from non-IBC patients, as well as differentially expressed tRNAs, snoRNAs, and other sncRNAs, while plasma samples, although of variable quality, included coding and non-coding RNAs distinctive of IBC. Classification models using panels consisting of sets of 50 selected biomarkers profiled by TGIRT-seq achieved a high degree of accuracy under cross-validation, with models based on PBMCs and plasma RNAs correlating with those based on tumor RNAs, and models using both coding and non-coding RNA biomarkers outperforming those based on either alone.\&nbsp;Conclusions:\&nbsp;Our findings are the first to define a distinct IBC profile across three different tissue types and advance TGIRT-seq as a promising method for high-resolution RNA biomarker profiling of both primary tumors and liquid biopsies with potentially broad utility for diagnosing and defining treatment response in IBC and other cancers.\&nbsp;COI:\&nbsp;Thermostable group II intron reverse transcriptase (TGIRT) enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas to InGex, LLC. A.M.L., some former and present members of the Lambowitz laboratory, and the University of Texas are minority equity holders in InGex, and receive royalty payments from the sale of TGIRT enzymes and kits and from sublicensing of intellectual property to other companies.},
	url = {https://aacrjournals.org/cancerres/article/82/4_Supplement/P5-07-03/681245/Abstract-P5-07-03-Disease-classification-modeling},
	author = {Wylie, DC and X Wang and Yao, J and Xu, H and Iwase, T and Krishnamurthy, S and Ueno, NT and Lambowitz, A M}
}
@article {15468,
	title = {Human cells contain myriad excised linear intron RNAs with links to gene regulation and potential utility as biomarkers},
	journal = {BioRxiv},
	year = {2022},
	abstract = {By using TGIRT-seq, we identified \&gt;8,500 short full-length excised linear intron (FLEXI) RNAs in human cells. Subsets of FLEXIs accumulated in a cell-type specific manner, and \~{}200 corresponded to agotrons or mirtrons or encoded snoRNAs. Analysis of CLIP-seq datasets identified potential interactions between FLEXIs and \&gt;100 different RNA-binding proteins (RBPs), 53 of which had binding sites in >=30 different FLEXIs. In addition to proteins that function in RNA splicing, these 53 RBPs included transcription factors, chromatin remodeling proteins, and cellular growth regulators that impacted FLEXI host gene alternative splicing and/or mRNA levels in knockdown datasets. We computationally identified six groups of RBPs whose binding sites were enriched in different subsets of FLEXIs: AGO1-4 and DICER associated with agotrons and mirtrons; AATF, DKC1, NOLCI, and SMNDC1 associated with snoRNA-encoding FLEXIs; two different combinations of alternative splicing factors found in stress granules; and two novel RBP-intron combinations, one including LARP4 and PABC4, which function together in the cytoplasm to regulate ribosomal protein translation. Our results suggest a model in which proteins involved in transcriptional regulation, alternative splicing, or post-splicing secondary functions bind and stabilize cell-type specific subsets of FLEXIs that perform different biological functions and have potential utility as biomarkers.},
	url = {https://www.biorxiv.org/content/10.1101/2020.09.07.285114v4},
	author = {Yao, Jun and Shelby Winans and Hengyi Xu and Elizabeth A. Ferrick-Kiddie and Manuel Ares Jr. and Alan M. Lambowitz}
}
@article {15467,
	title = {
	High-grade ovarian cancer associated H/ACA snoRNAs promote cancer cell proliferation and survival



	Laurence Faucher-Gigu{\`e}re, Audrey Roy, Gabrielle Deschamps-Francoeur, Sonia Couture, Ryan M Nottingham, Alan M Lambowitz, Michelle S Scott, Sherif},
	journal = {NAR Cancer},
	volume = {4},
	number = {1},
	year = {2022},
	abstract = {Small nucleolar RNAs (snoRNAs) are an omnipresent class of non-coding RNAs involved in the modification and processing of ribosomal RNA (rRNA). As snoRNAs are required for ribosome production, the increase of which is a hallmark of cancer development, their expression would be expected to increase in proliferating cancer cells. However, assessing the nature and extent of snoRNAs{\textquoteright} contribution to cancer biology has been largely limited by difficulties in detecting highly structured RNA. In this study, we used a dedicated midsize non-coding RNA (mncRNA) sensitive sequencing technique to accurately survey the snoRNA abundance in independently verified high-grade serous ovarian carcinoma (HGSC) and serous borderline tumour (SBT) tissues. The results identified SNORA81, SNORA19 and SNORA56 as an H/ACA snoRNA signature capable of discriminating between independent sets of HGSC, SBT and normal tissues. The expression of the signature SNORA81 correlates with the level of ribosomal RNA (rRNA) modification and its knockdown inhibits 28S rRNA pseudouridylation and accumulation leading to reduced cell proliferation and migration. Together our data indicate that specific subsets of H/ACA snoRNAs may promote tumour aggressiveness by inducing rRNA modification and synthesis.},
	url = {https://pubmed.ncbi.nlm.nih.gov/35047824/},
	author = {Laurence Faucher-Gigu{\`e}re and Audrey Roy and Gabrielle Deschamps-Francoeur and Sonia Couture and Ryan M. Nottingham and Alan M. Lambowitz and Michelle S. Scott and Sherif Abou Elela}
}
@article {15463,
	title = {Group II Intron-Like Reverse Transcriptases Function in Double-Strand Break Repair by Microhomology-Mediated End Joining},
	journal = {bioRxiv},
	volume = {484287},
	year = {2022},
	abstract = {Bacteria encode free-standing reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. Here, we found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when transferred into Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase q and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) in vitro and in vivo. We also found that a group II intron RT can function similarly to G2L4 RT in DNA repair, with reciprocal substitutions at the active site showing an I residue favors MMEJ and an A residue favors primer extension in both enzymes. The DNA repair functions of these enzymes utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have an inherent ability to function in DSBR in a wide range of organisms.},
	url = {https://www.biorxiv.org/content/10.1101/2022.03.14.484287v1},
	author = {S.K. Park and G. Mohr and J. Yao and Russell, R. and A.M. Lambowitz}
}
@article {15473,
	title = {Arg-tRNA synthetase links inflammatory metabolism to RNA splicing and nuclear trafficking via SRRM2 View ORCID Profile, , View ORCID Profile, Justin J. Lim, Ryan M. Nottingham, James J. Moresco, John R. Yates III, Benjamin J. Blencowe, Alan M. L},
	journal = {BioRxiv},
	year = {2021},
	abstract = {Cells respond to perturbations like inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Here, we found that depletion of arginine during inflammation decreased levels of a nuclear form of arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal protein and nuclear speckle component and that arginine depletion impacted both condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a novel mechanism whereby a tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.},
	url = {https://www.biorxiv.org/content/10.1101/2021.09.07.459304v2},
	author = {Haissi~Cui and Jolene K.~Diedrich and Douglas C.~Wu and Justin J.~Lim and Ryan M.~Nottingham and James J.~Moresco and John R.~Yates~III and Benjamin J.~Blencowe and Alan M.~Lambowitz and Paul~Schimmel}
}
@article {15466,
	title = {Structural basis for template switching by a group II intron\&ndash;encoded non-LTR-retroelement reverse transcriptase},
	journal = {J. Biol. Chem.},
	volume = {297},
	number = {2},
	year = {2021},
	pages = {100971},
	abstract = {Reverse transcriptases (RTs) can switch template strands during complementary DNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching (TS) plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-Seq, and may contribute to retroelement fitness by increasing evolutionary diversity and enabling continuous complementary DNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a TS complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template{\textendash} DNA primer heteroduplex with a 1-nt 30 -DNA overhang. The structure showed that the 30 end of the acceptor RNA binds in a pocket formed by an N-terminal extension present in non{\textendash}long terminal repeat{\textendash}retroelement RTs and the RT fingertips loop, with the 30 nucleotide of the acceptor base paired to the 1-nt 30 - DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structureguided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine residue near the RT active site that mediates nontemplated nucleotide addition. Mutation of the latter residue decreased multiple sequential template switches in RNA-Seq. Our results provide new insights into the mechanisms of TS and nontemplated nucleotide addition by RTs, suggest how these reactions could be improved for RNA-Seq, and reveal common structural features for TS by non{\textendash}long terminal repeat{\textendash}retroelement RTs and viral RNA{\textendash}dependent RNA polymerases.},
	url = {https://www.jbc.org/article/S0021-9258(21)00773-0/fulltext},
	author = {Alfred M. Lentzsch and Jennifer L. Stamos and Yao, Jun and Russell, Rick and Alan M. Lambowitz}
}
@article {15001,
	title = {TGIRT-seq Protocol for the Comprehensive Profiling of Coding and Non-coding RNA Biotypes in Cellular, Extracellular Vesicle, and Plasma RNAs},
	journal = {Bio-protocol},
	volume = {11},
	number = {23},
	year = {2021},
	abstract = {High-throughput RNA sequencing (RNA-seq) has extraordinarily advanced our understanding of gene expression and disease etiology, and is a powerful tool for the identification of biomarkers in a wide range of organisms. However, most RNA-seq methods rely on retroviral reverse transcriptases (RTs), enzymes that have inherently low fidelity and processivity, to convert RNAs into cDNAs for sequencing. Here, we describe an RNA-seq protocol using Thermostable Group II Intron Reverse Transcriptases (TGIRTs), which have high fidelity, processivity, and strand-displacement activity, as well as a proficient template-switching activity that enables efficient and seamless RNA-seq adapter addition. By combining these activities, TGIRT-seq enables the simultaneous profiling of all RNA biotypes from small amounts of starting material, with superior RNA-seq metrics, and unprecedented ability to sequence structured RNAs. The TGIRT-seq protocol for Illumina sequencing consists of three steps: (i) addition of a 3{\textquoteright} RNA-seq adapter, coupled to the initiation of cDNA synthesis at the 3{\textquoteright} end of a target RNA, via template switching from a synthetic adapter RNA/DNA starter duplex; (ii) addition of a 5{\textquoteright} RNA-seq adapter, by using thermostable 5{\textquoteright} App DNA/RNA ligase to ligate an adapter oligonucleotide to the 3{\textquoteright} end of the completed cDNA; (iii) minimal PCR amplification, to add capture sites and indices for Illumina sequencing. TGIRT-seq for the Illumina sequencing platform has been used for comprehensive profiling of coding and non-coding RNAs in ribodepleted, chemically fragmented cellular RNAs, and for the analysis of intact (non-chemically fragmented) cellular, extracellular vesicle (EV), and plasma RNAs, where it yields continuous full-length end-to-end sequences of structured small noncoding RNAs (sncRNAs), including tRNAs, snoRNAs, snRNAs, pre-miRNAs, and full-length excised linear intron (FLEXI) RNAs.},
	author = {Xu, H and Nottingham, Ryan M and Lambowitz, A M}
}
@article {13139,
	title = {Identification of protein-protected mRNA fragments and structured excised intron RNAs in human plasma by TGIRT-seq peak calling},
	journal = {eLife},
	year = {2020},
	abstract = {Human plasma contains \&gt;40,000 different coding and non-coding RNAs that are potential biomarkers for human diseases. Here, we used thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) combined with peak calling to simultaneously profile all RNA biotypes in apheresis-prepared human plasma pooled from healthy individuals. Extending previous TGIRT-seq analysis, we found that human plasma contains largely fragmented mRNAs from \&gt;19,000 protein-coding genes, abundant full-length, mature tRNAs and other structured small non-coding RNAs, and less abundant tRNA fragments and mature and pre-miRNAs. Many of the mRNA fragments identified by peak calling correspond to annotated protein-binding sites and/or have stable predicted secondary structures that could afford protection from plasma nucleases. Peak calling also identified novel repeat RNAs, miRNA-sized RNAs, and putatively structured intron RNAs of potential biological, evolutionary, and biomarker significance, including a family of full-length excised introns RNAs, subsets of which correspond to mirtron pre-miRNAs or agotrons.},
	url = {https://elifesciences.org/articles/60743},
	author = {Yao, Jun and Douglas C. Wu and Nottingham, Ryan M and Lambowitz, Alan M}
}
@article {11834,
	title = {Template switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq},
	journal = {Journal of Biological Chemistry},
	year = {2019},
	abstract = {
	The reverse transcriptases (RTs) encoded by mobile group II introns and other non-LTR retroelements differ from retroviral RTs in being able to template-switch efficiently from the 5 end of one template to the 3 end of another with little or no complementarity between the donor and acceptor templates. Here, to establish a complete kinetic framework for the reaction and to identify conditions that more efficiently capture acceptor RNAs or DNAs, we used a thermostable group II intron RT (TGIRT; GsI{\textendash}IIC RT) that can template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3-DNA overhang end. We found that the rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3-DNA overhang complementary to the 3 nucleotide of the acceptor RNA, suggesting a role for nontemplated nucleotide addition of a complementary nucleotide to the 3 end of cDNAs synthesized from natural templates. Longer 3-DNA overhangs progressively decreased the templateswitching rate, even when complementary to the 3 end of the acceptor template. The reliance on only a single bp with the 3 nucleotide of the acceptor together with discrimination against mismatches and the high processivity of group II intron RTs enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3 end. We discuss the possible biological functions of the template-switching activity of group II intron- and other non-LTR retroelement{\textendash} encoded RTs, as well as the optimization of this activity for adapter addition in RNAand DNA-Seq protocols.

},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/31712313},
	author = {A.M. Lentzsch and J. Yao and Russell, R. and A.M. Lambowitz}
}
@article {11370,
	title = {Distinct mechanisms of microRNA sorting into cancer cell-derived extracellular vesicle subtypes.},
	journal = {eLife},
	volume = {8},
	year = {2019},
	abstract = {Extracellular vesicles (EVs) encompass a variety of vesicles secreted into the extracellular space. EVs have been implicated in promoting tumor metastasis, but the molecular composition of tumor-derived EV sub-types and the mechanisms by which molecules are sorted into EVs remain mostly unknown. We report the separation of two small EV sub-populations from a metastatic breast cancer cell line, with biochemical features consistent with different sub-cellular origins. These EV sub-types use different mechanisms of miRNA sorting (selective and non-selective), suggesting that sorting occurs via fundamentally distinct processes, possibly dependent on EV origin. Using biochemical and genetic tools, we identified the Lupus La protein as mediating sorting of selectively packaged miRNAs. We found that two motifs embedded in miR-122 are responsible for high-affinity binding to Lupus La and sorting into vesicles formed in a cell-free reaction. Thus, tumor cells can simultaneously deploy multiple EV species using distinct sorting mechanisms that may enable diverse functions in normal and cancer biology.},
	author = {Temoche-Diaz, M. M. and M.J. Shurtleff and Nottingham, R .M. and J. Yao and Fadadu, R. P. and A.M. Lambowitz and R. Schekman}
}
@article {11369,
	title = {BCDIN3D regulates tRNAHis  3\&rsquo; fragment processing.},
	journal = {PLoS Genetics},
	volume = {15},
	number = {7},
	year = {2019},
	abstract = {5{\textquoteright} ends are important for determining the fate of RNA molecules. BCDIN3D is an RNA phospho-methyltransferase that methylates the 5{\textquoteright} monophosphate of specific RNAs. In order to gain new insights into the molecular function of BCDIN3D, we performed an unbiased analysis of its interacting RNAs by Thermostable Group II Intron Reverse Transcriptase coupled to next generation sequencing (TGIRT-seq). Our analyses showed that BCDIN3D interacts with full-length phospho-methylated tRNAHis\&nbsp;and miR-4454. Interestingly, we found that miR-4454 is not synthesized from its annotated genomic locus, which is a primer-binding site for an endogenous retrovirus, but rather by Dicer cleavage of mature tRNAHis. Sequence analysis revealed that miR-4454 is identical to the 3{\textquoteright} end of tRNAHis. Moreover, we were able to generate this {\textquoteleft}miRNA{\textquoteright}\&nbsp;in vitro\&nbsp;through incubation of mature tRNAHis\&nbsp;with Dicer. As found previously for several pre-miRNAs, a 5{\textquoteright}P-tRNAHis\&nbsp;appears to be a better substrate for Dicer cleavage than a phospho-methylated tRNAHis. Moreover, tRNAHis\&nbsp;3{\textquoteright}-fragment/{\textquoteleft}miR-4454{\textquoteright} levels increase in cells depleted for BCDIN3D. Altogether, our results show that in addition to microRNAs, BCDIN3D regulates tRNAHis\&nbsp;3{\textquoteright}-fragment processing without negatively affecting tRNAHis{\textquoteright}s canonical function of aminoacylation.},
	author = {Reinsborough, C. W. and Ipas, H. and Abell, N. S. and R.M. Nottingham and J. Yao and Devanathan, S. K. and Shelton, S. B. and A.M. Lambowitz and Xhemalce, B.}
}
@article {11190,
	title = {Group II Intron RNPs and ReverseTranscriptases: From Retroelements to Research Tools},
	journal = {Cold Spring Harbor Perspective in Biology},
	year = {2019},
	publisher = {Cold Spring Harb Perspect Biol},
	abstract = {Group II introns, self-splicing retrotransposons, serve as both targets of investigation into their structure, splicing, and retromobility and a source of tools for genome editing and RNA analysis. Here, we describe the first cryo-electron microscopy (cryo-EM) structure determination, at 3.8{\textendash}4.5 {\r A}, of a group II intron ribozyme complexed with its encoded protein, containing a reverse transcriptase (RT), required for RNA splicing and retromobility. We also describe a method called RIG-seq using a retrotransposon indicator gene for high-throughput integration profiling of group II introns and other retrotransposons. Targetrons, RNA-guided gene targeting agents widely used for bacterial genome engineering, are described next. Finally, we detail thermostable group II intron RTs, which synthesize cDNAs with high accuracy and processivity, for use in various RNA-seq applications and relate their properties to a 3.0-{\r A} crystal structure of the protein poised for reverse transcription. Biological insights from these group II intron revelations are discussed.},
	url = {https://cshperspectives.cshlp.org/content/11/4/a032375.full},
	author = {Belfort, M and Lambowitz, A M}
}
@article {11189,
	title = {Improved TGIRT-seq methods for comprehensive transcriptome profiling with decreased adapter dimer-formation and bias correction.},
	journal = {Scientific Reports},
	volume = {9},
	number = {1},
	year = {2019},
	abstract = {Thermostable group II intron reverse transcriptases (TGIRTs) with high fidelity and processivity have been used for a variety of RNA sequencing (RNA-seq) applications, including comprehensive profiling of whole-cell, exosomal, and human plasma RNAs; quantitative tRNA-seq based on the ability of TGIRT enzymes to give full-length reads of tRNAs and other structured small ncRNAs; high-throughput mapping of post-transcriptional modifications; and RNA structure mapping. Here, we improved TGIRT-seq methods for comprehensive transcriptome profiling by rationally designing RNA-seq adapters that minimize adapter dimer formation. Additionally, we developed biochemical and computational methods for remediating 5'- and 3'-end biases, the latter based on a random forest regression model that provides insight into the contribution of different factors to these biases. These improvements, some of which may be applicable to other RNA-seq methods, increase the efficiency of TGIRT-seq library construction and improve coverage of very small RNAs, such as miRNAs. Our findings provide insight into the biochemical basis of 5'- and 3'-end biases in RNA-seq and suggest general approaches for remediating biases and decreasing adapter dimer formation.},
	url = {https://www.researchgate.net/publication/333428625_Improved_TGIRT-seq_methods_for_comprehensive_transcriptome_profiling_with_decreased_adapter_dimer_formation_and_bias_correction},
	author = {Xu, H. and J. Yao and D.C. Wu and Lambowitz, A M}
}
@article {11188,
	title = {A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition.},
	journal = {Mol. Cell},
	number = {72},
	year = {2018},
	pages = {700-714},
	abstract = {Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of foreign nucleic acids (spacers) into genomic CRISPR arrays. Cas6 proteins then process CRISPR array transcripts into spacer-derived RNAs (CRISPR RNAs; crRNAs) that target Cas nucleases to matching invaders. We find that a Marinomonas mediterranea fusion protein combines three enzymatic domains (Cas6, reverse transcriptase [RT], and Cas1), which function in both crRNA biogenesis and spacer acquisition from RNA and DNA. We report a crystal structure of this divergent Cas6, identify amino acids required for Cas6 activity, show that the Cas6 domain is required for RT activity and RNA spacer acquisition, and demonstrate that CRISPR-repeat binding to Cas6 regulates RT activity. Co-evolution of putative interacting surfaces suggests a specific structural interaction between the Cas6 and RT domains, and phylogenetic analysis reveals repeated, stable association of free-standing Cas6s with CRISPR RTs in multiple microbial lineages, indicating that a functional interaction between these proteins preceded evolution of the fusion.},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/30344094},
	author = {Mohr, G and Silas, Sukrit and J. Stamos and K.S. Makarova and Markham, Laura M and J. Yao and Lucas-Elio, P. and Sanchez-Amat, Antonio and A.Z. Fire and E.V. Koonin and Lambowitz, A M}
}
@article {10021,
	title = {Simultaneous sequencing of coding and noncoding RNA reveals a human transcriptome dominated by a small number of highly expressed noncoding genes.},
	journal = {RNA},
	volume = {24},
	number = {7},
	year = {2018},
	pages = {950-965},
	abstract = {Comparing the abundance of one RNA molecule to another is crucial for understanding cellular functions but most sequencing techniques can target only specific subsets of RNA. In this study, we used a new fragmented ribodepleted TGIRT sequencing method that uses a thermostable group II intron reverse transcriptase (TGIRT) to generate a portrait of the human transcriptome depicting the quantitative relationship of all classes of nonribosomal RNA longer than 60 nt. Comparison between different sequencing methods indicated that FRT is more accurate in ranking both mRNA and noncoding RNA than viral reverse transcriptase-based sequencing methods, even those that specifically target these species. Measurements of RNA abundance in different cell lines using this method correlate with biochemical estimates, confirming tRNA as the most abundant nonribosomal RNA biotype. However, the single most abundant transcript is 7SL RNA, a component of the signal recognition particle.\&nbsp;Structured\&nbsp;noncoding\&nbsp;RNAs\&nbsp;(sncRNAs) associated with the same biological process are expressed at similar levels, with the exception of RNAs with multiple functions like U1 snRNA. In general, sncRNAs forming RNPs are hundreds to thousands of times more abundant than their mRNA counterparts. Surprisingly, only 50 sncRNA genes produce half of the non-rRNA transcripts detected in two different cell lines. Together the results indicate that the human transcriptome is dominated by a small number of highly expressed sncRNAs specializing in functions related to translation and splicing.},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/29703781},
	author = {V. Boivin and G. Deschamps-Francoeur and S. Couture and R.M. Nottingham and P. Bouchard-Bourelle and A.M. Lambowitz}
}
@article {10020,
	title = {A Highly Proliferative Group IIC Intron from Geobacillus stearothermophilus Reveals New Features of Group II Intron Mobility and Splicing.},
	journal = {Journal of Molecular Biology},
	volume = {430},
	number = {17},
	year = {2018},
	pages = {2760-2783},
	abstract = {The thermostable Geobacillus stearothermophilus GsI-IIC intron is among the few bacterial group II introns found to proliferate to high copy number in its host genome. Here, we developed a bacterial genetic assay for retrohoming and biochemical assays for protein-dependent and self-splicing of GsI-IIC. We found that GsI-IIC, like other group IIC introns, retrohomes into sites having a 5{\textquoteright}-exon DNA hairpin, typically from a bacterial transcription terminator, followed by short intron-binding sequences (IBSs) recognized by base pairing of exon-binding sequences (EBSs) in the intron RNA. Intron RNA insertion occurs preferentially but not exclusively into the parental lagging strand at DNA replication forks, using a nascent lagging strand DNA as a primer for reverse transcription. In vivo mobility assays, selections, and mutagenesis indicated that a variety of GC-rich DNA hairpins of 7-19 bp with continuous base pairs or internal elbow regions support efficient intron mobility and identified a critically recognized nucleotide (T-5) between the hairpin and IBS1, a feature not reported previously for group IIC introns. Neither the hairpin nor T-5 is required for intron excision or lariat formation during RNA splicing, but the 5{\textquoteright}-exon sequence can affect the efficiency of exon ligation. Structural modeling suggests that the 5{\textquoteright}-exon DNA hairpin and T-5 bind to the thumb and DNA-binding domains of GsI-IIC reverse transcriptase. This mode of DNA target site recognition enables the intron to proliferate to high copy number by recognizing numerous transcription terminators and then finding the best match for the EBS/IBS interactions within a short distance downstream.},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/29913158},
	author = {G. Mohr and S.Y. Kang and S.K. Park and Y. Qin and J. Grohman and J. Yao and J.L. Stamos and A.M. Lambowitz}
}
@article {10019,
	title = {Limitations of alignment-free tools in total RNA-seq quantification.},
	journal = {BMC Genomics},
	volume = {19},
	number = {1},
	year = {2018},
	pages = {510},
	abstract = {
	BACKGROUND:



	Alignment-free RNA quantification tools have significantly increased the speed of RNA-seq analysis. However, it is unclear whether these state-of-the-art RNA-seq analysis pipelines can quantify small RNAs as accurately as they do with long RNAs in the context of total RNA quantification.



	RESULT:



	We comprehensively tested and compared four RNA-seq pipelines for accuracy of gene quantification and fold-change estimation. We used a novel total RNA benchmarking dataset in which small non-coding RNAs are highly represented along with other long RNAs. The four RNA-seq pipelines consisted of two commonly-used alignment-free pipelines and two variants of alignment-based pipelines. We found that all pipelines showed high accuracy for quantifying the expression of long and highly-abundant genes. However, alignment-free pipelines showed systematically poorer performance in quantifying lowly-abundant and small RNAs.



	CONCLUSION:



	We have shown that alignment-free and traditional alignment-based quantification methods perform similarly for common gene targets, such as protein-coding genes. However, we have identified a potential pitfall in analyzing and quantifying lowly-expressed genes and small RNAs with alignment-free pipelines, especially when these small RNAs contain biological variations.

},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/29969991},
	author = {D.C. Wu and J. Yao and K.S. Ho and A.M. Lambowitz and C.O. Wilke}
}
@article {9231,
	title = {Structure of a Thermostable Group II Intron Reverse Transcriptase with Template-Primer and Its Functional and Evolutionary Implications.},
	journal = {Molecular Cell},
	volume = {68},
	year = {2017},
	pages = {926{\textendash}939},
	abstract = {Bacterial group II intron reverse transcriptases (RTs) function in both intron mobility and RNA splicing and are evolutionary predecessors of retrotransposon, telomerase, and retroviral RTs as well as the spliceosomal protein Prp8 in eukaryotes. Here we determined a crystal structure of a full-length thermostable group II intron RT in complex with an RNA template-DNA primer duplex and incoming deoxynucleotide triphosphate (dNTP) at 3.0-Ao resolution. We find that the binding of template-primer and key aspects of the RT active site are surprisingly different from retroviral RTs but remarkably similar to viral RNA-dependent RNA polymerases. The structure reveals a host of features not seen previously in RTs that may contribute to distinctive biochemical properties of group II intron RTs, and it provides a prototype for many related bacterial and eukaryotic non-LTR retroelement RTs. It also reveals how protein structural features used for reverse transcription evolved to promote the splicing of both group II and spliceosomal introns.},
	url = {http://www.cell.com/molecular-cell/fulltext/S1097-2765(17)30799-2},
	author = {J. Stamos and A.M. Lentzsch and A.M. Lambowitz}
}
@article {9230,
	title = {Detection of expanded RNA repeats using thermostable group II intron reverse transcriptase.},
	journal = {Nucleic Acids Research},
	year = {2017},
	abstract = {Cellular accumulation of repetitive RNA occurs in several dominantly-inherited genetic disorders. Expanded CUG, CCUG or GGGGCC repeats are expressed in myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), or familial amyotrophic lateral sclerosis, respectively. Expanded repeat RNAs (ER-RNAs) exert a toxic gain-of-function and are prime therapeutic targets in these diseases. However, efforts to quantify ER-RNA levels or monitor knockdown are confounded by stable structure and heterogeneity of the ER-RNA tract and background signal from non-expanded repeats. Here, we used a thermostable group II intron reverse transcriptase (TGIRT-III) to convert ER-RNA to cDNA, followed by quantification on slot blots. We found that TGIRT-III was capable of reverse transcription (RTn) on enzymatically synthesized ER-RNAs. By using conditions that limit cDNA synthesis from off-target sequences, we observed hybridization signals on cDNA slot blots from DM1 and DM2 muscle samples but not from healthy controls. In transgenic mouse models of DM1 the cDNA slot blots accurately reflected the differences of ER-RNA expression across different transgenic lines, and showed therapeutic reductions in skeletal and cardiac muscle, accompanied by improvements of the DM1-associated splicing defects. TGIRT-III was also active on CCCCGG- and GGGGCC-repeats, suggesting that ER-RNA analysis is feasible for several repeat expansion disorders.},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/29036654},
	author = {S.T. Carrell and Z. Tang and S. Mohr and A.M. Lambowitz and C.A. Thorton}
}
@article {9229,
	title = {Broad role for YBX1 in defining the small noncoding RNA composition of exosomes.},
	journal = {National Academy of Sciences},
	volume = {114},
	number = {43},
	year = {2017},
	abstract = {RNA is secreted from cells enclosed within extracellular vesicles (EVs). Defining the RNA composition of EVs is challenging due to their coisolation with contaminants, lack of knowledge of the mechanisms of RNA sorting into EVs, and limitations of conventional RNA-sequencing methods. Here we present our observations using thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) to characterize the RNA extracted from HEK293T cell EVs isolated by flotation gradient ultracentrifugation and from exosomes containing the tetraspanin CD63 further purified from the gradient fractions by immunoisolation. We found that EV-associated transcripts are dominated by full-length, mature transfer RNAs (tRNAs) and other small noncoding RNAs (ncRNAs) encapsulated within vesicles. A substantial proportion of the reads mapping to protein-coding genes, long ncRNAs, and antisense RNAs were due to DNA contamination on the surface of vesicles. Nevertheless, sequences mapping to spliced mRNAs were identified within HEK293T cell EVs and exosomes, among the most abundant being transcripts containing a 5' terminal oligopyrimidine (5' TOP) motif. Our results indicate that the RNA-binding protein YBX1, which is required for the sorting of selected miRNAs into exosomes, plays a role in the sorting of highly abundant small ncRNA species, including tRNAs, Y RNAs, and Vault RNAs. Finally, we obtained evidence for an EV-specific tRNA modification, perhaps indicating a role for posttranscriptional modification in the sorting of some RNA species into EVs. Our results suggest that EVs and exosomes could play a role in the purging and intercellular transfer of excess free RNAs, including full-length tRNAs and other small ncRNAs.},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/29073095},
	author = {M.J. Shurtleff and J. Yao and Y. Qin and R.M. Nottingham and M. Temoche-Diaz and R. Schekman and A.M. Lambowitz}
}
@article {9225,
	title = {On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.},
	journal = {American Society for Microbiology},
	volume = {8},
	number = {4},
	year = {2017},
	abstract = {Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPRassociated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium{\textemdash}Arthrospira platensis. Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1- associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown.},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/28698278},
	author = {S. Silas and K.S. Makarova and S. Shmakov and D. P{\'a}ez-Espino and G. Mohr and Y. Liu and M. Davidson and S. Roux and S.R. Krishnamurthy and B. Fu and L.L. Hansen and D. Wang and M.B. Sullivan and A. Millard and M.R. Clokie and B. Devaki and A.M. Lambowitz and N.C. Kyrpides and E.V. Koonin and A.Z. Fire}
}
@article {9224,
	title = {Facile single-stranded DNA sequencing of human plasma DNA via thermostable group II intron reverse transcriptase template switching.},
	journal = {Scientific Reports},
	volume = {7},
	number = {8421},
	year = {2017},
	abstract = {High-throughput single-stranded DNA sequencing (ssDNA-seq) of cell-free DNA from plasma and other bodily fluids is a powerful method for non-invasive prenatal testing, and diagnosis of cancers and other diseases. Here, we developed a facile ssDNA-seq method, which exploits a novel template-switching activity of thermostable group II intron reverse transcriptases (TGIRTs) for DNA-seq library construction. This activity enables TGIRT enzymes to initiate DNA synthesis directly at the 3' end of a DNA strand while simultaneously attaching a DNA-seq adapter without end repair, tailing, or ligation. Initial experiments using this method to sequence\&nbsp;E. coli\&nbsp;genomic DNA showed that the TGIRT enzyme has surprisingly robust DNA polymerase activity. Further experiments showed that TGIRT-seq of plasma DNA from a healthy individual enables analysis of nucleosome positioning, transcription factor-binding sites, DNA methylation sites, and tissues-of-origin comparably to established methods, but with a simpler workflow that captures precise DNA ends.},
	url = {https://www.nature.com/articles/s41598-017-09064-w},
	author = {Douglas C. Wu and Alan M. Lambowitz}
}
@article {7667,
	title = {DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.},
	journal = {Nat Methods},
	volume = {14},
	number = {1},
	year = {2017},
	month = {2017 Jan},
	pages = {75-82},
	abstract = {Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq{\textquoteright}s capacity to dramatically expand in vivo analysis of RNA structure.},
	issn = {1548-7105},
	doi = {10.1038/nmeth.4057},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/27819661},
	author = {Zubradt, Meghan and Gupta, Paromita and Persad, Sitara and Lambowitz, Alan M and Weissman, Jonathan S and Rouskin, Silvi}
}
@article {7666,
	title = {DUSP11 activity on triphosphorylated transcripts promotes Argonaute association with noncanonical viral microRNAs and regulates steady-state levels of cellular noncoding RNAs.},
	journal = {Genes Dev},
	volume = {30},
	number = {18},
	year = {2016},
	month = {2016 Sep 15},
	pages = {2076-2092},
	abstract = {RNA silencing is a conserved eukaryotic gene expression regulatory mechanism mediated by small RNAs. In Caenorhabditis elegans, the accumulation of a distinct class of siRNAs synthesized by an RNA-dependent RNA polymerase (RdRP) requires the PIR-1 phosphatase. However, the function of PIR-1 in RNAi has remained unclear. Since mammals lack an analogous siRNA biogenesis pathway, an RNA silencing role for the mammalian PIR-1 homolog (dual specificity phosphatase 11 [DUSP11]) was unexpected. Here, we show that the RNA triphosphatase activity of DUSP11 promotes the RNA silencing activity of viral microRNAs (miRNAs) derived from RNA polymerase III (RNAP III) transcribed precursors. Our results demonstrate that DUSP11 converts the 5{\textquoteright} triphosphate of miRNA precursors to a 5{\textquoteright} monophosphate, promoting loading of derivative 5p miRNAs into Argonaute proteins via a Dicer-coupled 5{\textquoteright} monophosphate-dependent strand selection mechanism. This mechanistic insight supports a likely shared function for PIR-1 in C. elegans Furthermore, we show that DUSP11 modulates the 5{\textquoteright} end phosphate group and/or steady-state level of several host RNAP III transcripts, including vault RNAs and Alu transcripts. This study shows that steady-state levels of select noncoding RNAs are regulated by DUSP11 and defines a previously unknown portal for small RNA-mediated silencing in mammals, revealing that DUSP11-dependent RNA silencing activities are shared among diverse metazoans.},
	keywords = {Acid Anhydride Hydrolases, Adenoviridae, Argonaute Proteins, Dual-Specificity Phosphatases, Gene Knockout Techniques, HEK293 Cells, Humans, Leukemia Virus, Bovine, MicroRNAs, Phosphorylation, RNA Polymerase III, RNA, Untranslated, RNA, Viral},
	issn = {1549-5477},
	doi = {10.1101/gad.282616.116},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/27798849},
	author = {Burke, James M and Kincaid, Rodney P and Nottingham, Ryan M and Lambowitz, Alan M and Sullivan, Christopher S}
}
@article {5932,
	title = {Structural Divergence of the Group I Intron Binding Surface in Fungal Mitochondrial Tyrosyl-tRNA Synthetases That Function in RNA Splicing.},
	journal = {J Biol Chem},
	volume = {291},
	number = {22},
	year = {2016},
	month = {2016 May 27},
	pages = {11911-27},
	abstract = {The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mitochondrial tRNA(Tyr) and act as splicing cofactors for autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon binding domains and that the catalytic domain uses a newly evolved group I intron binding surface that includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined x-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the Aspergillus fumigatus mtTyrRS showed that the overall topology of the group I intron binding surface is conserved but with variations in key intron binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions, which also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi.},
	issn = {1083-351X},
	doi = {10.1074/jbc.M116.725390},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/27036943},
	author = {Lamech, Lilian T and Saoji, Maithili and Paukstelis, Paul J and Lambowitz, Alan M}
}
@article {5930,
	title = {RNA-seq of human reference RNA samples using a thermostable group II intron reverse transcriptase.},
	journal = {RNA},
	volume = {22},
	number = {4},
	year = {2016},
	month = {2016 Apr},
	pages = {597-613},
	abstract = {Next-generation RNA sequencing (RNA-seq) has revolutionized our ability to analyze transcriptomes. Current RNA-seq methods are highly reproducible, but each has biases resulting from different modes of RNA sample preparation, reverse transcription, and adapter addition, leading to variability between methods. Moreover, the transcriptome cannot be profiled comprehensively because highly structured RNAs, such as tRNAs and snoRNAs, are refractory to conventional RNA-seq methods. Recently, we developed a new method for strand-specific RNA-seq using thermostable group II intron reverse transcriptases (TGIRTs). TGIRT enzymes have higher processivity and fidelity than conventional retroviral reverse transcriptases plus a novel template-switching activity that enables RNA-seq adapter addition during cDNA synthesis without using RNA ligase. Here, we obtained TGIRT-seq data sets for well-characterized human RNA reference samples and compared them to previous data sets obtained for these RNAs by the Illumina TruSeq v2 and v3 methods. We find that TGIRT-seq recapitulates the relative abundance of human transcripts and RNA spike-ins in ribo-depleted, fragmented RNA samples comparably to non-strand-specific TruSeq v2 and better than strand-specific TruSeq v3. Moreover, TGIRT-seq is more strand specific than TruSeq v3 and eliminates sampling biases from random hexamer priming, which are inherent to TruSeq. The TGIRT-seq data sets also show more uniform 5{\textquoteright} to 3{\textquoteright} gene coverage and identify more splice junctions, particularly near the 5{\textquoteright} ends of mRNAs, than do the TruSeq data sets. Finally, TGIRT-seq enables the simultaneous profiling of mRNAs and lncRNAs in the same RNA-seq experiment as structured small ncRNAs, including tRNAs, which are essentially absent with TruSeq.},
	issn = {1469-9001},
	doi = {10.1261/rna.055558.115},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/26826130},
	author = {Nottingham, Ryan M and Wu, Douglas C and Qin, Yidan and Yao, Jun and Hunicke-Smith, Scott and Lambowitz, Alan M}
}
@article {4077,
	title = {High-throughput sequencing of human plasma RNA by using thermostable group II intron reverse transcriptases.},
	journal = {RNA},
	volume = {22},
	number = {1},
	year = {2016},
	month = {2016 Jan},
	pages = {111-28},
	abstract = {Next-generation RNA-sequencing (RNA-seq) has revolutionized transcriptome profiling, gene expression analysis, and RNA-based diagnostics. Here, we developed a new RNA-seq method that exploits thermostable group II intron reverse transcriptases (TGIRTs) and used it to profile human plasma RNAs. TGIRTs have higher thermostability, processivity, and fidelity than conventional reverse transcriptases, plus a novel template-switching activity that can efficiently attach RNA-seq adapters to target RNA sequences without RNA ligation. The new TGIRT-seq method enabled construction of RNA-seq libraries from \&lt;1 ng of plasma RNA in \&lt;5 h. TGIRT-seq of RNA in 1-mL plasma samples from a healthy individual revealed RNA fragments mapping to a diverse population of protein-coding gene and long ncRNAs, which are enriched in intron and antisense sequences, as well as nearly all known classes of small ncRNAs, some of which have never before been seen in plasma. Surprisingly, many of the small ncRNA species were present as full-length transcripts, suggesting that they are protected from plasma RNases in ribonucleoprotein (RNP) complexes and/or exosomes. This TGIRT-seq method is readily adaptable for profiling of whole-cell, exosomal, and miRNAs, and for related procedures, such as HITS-CLIP and ribosome profiling.},
	issn = {1469-9001},
	doi = {10.1261/rna.054809.115},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/26554030},
	author = {Qin, Yidan and Yao, Jun and Wu, Douglas C and Nottingham, Ryan M and Mohr, Sabine and Hunicke-Smith, Scott and Lambowitz, Alan M}
}
@article {4076,
	title = {Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein.},
	journal = {Science},
	volume = {351},
	number = {6276},
	year = {2016},
	month = {2016 Feb 26},
	pages = {aad4234},
	abstract = {CRISPR systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in type I and II CRISPR systems by the acquisition of short segments of DNA (spacers) from invasive elements. In several type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA.},
	issn = {1095-9203},
	doi = {10.1126/science.aad4234},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/26917774},
	author = {Silas, Sukrit and Mohr, Georg and Sidote, David J and Markham, Laura M and Sanchez-Amat, Antonio and Bhaya, Devaki and Lambowitz, Alan M and Fire, Andrew Z}
}
@article {5931,
	title = {Efficient and quantitative high-throughput tRNA sequencing.},
	journal = {Nat Methods},
	volume = {12},
	number = {9},
	year = {2015},
	month = {2015 Sep},
	pages = {835-7},
	abstract = {Despite its biological importance, tRNA has not been adequately sequenced by standard methods because of its abundant post-transcriptional modifications and stable structure, which interfere with cDNA synthesis. We achieved efficient and quantitative tRNA sequencing in HEK293T cells by using engineered demethylases to remove base methylations and a highly processive thermostable group II intron reverse transcriptase to overcome these obstacles. Our method, DM-tRNA-seq, should be applicable to investigations of tRNA in all organisms.},
	keywords = {Algorithms, Base Sequence, Gene Library, HEK293 Cells, High-Throughput Nucleotide Sequencing, Humans, Molecular Sequence Data, RNA, Transfer},
	issn = {1548-7105},
	doi = {10.1038/nmeth.3478},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/26214130},
	author = {Zheng, Guanqun and Qin, Yidan and Clark, Wesley C and Dai, Qing and Yi, Chengqi and He, Chuan and Lambowitz, Alan M and Pan, Tao}
}
@article {2628,
	title = {Retrohoming of a Mobile Group II Intron in Human Cells Suggests How Eukaryotes Limit Group II Intron Proliferation.},
	journal = {PLoS Genet},
	volume = {11},
	number = {8},
	year = {2015},
	month = {2015 Aug},
	pages = {e1005422},
	abstract = {Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1\%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but \&lt;4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.},
	issn = {1553-7404},
	doi = {10.1371/journal.pgen.1005422},
	url = {https://www.ncbi.nlm.nih.gov/pubmed/26241656},
	author = {Truong, David M and Hewitt, F Curtis and Hanson, Joseph H and Cui, Xiaoxia and Lambowitz, Alan M}
}
@article {1715,
	title = {Mobile Bacterial Group II Introns at the Crux of Eukaryotic Evolution.},
	journal = {Microbiol Spectr},
	volume = {3},
	number = {1},
	year = {2015},
	month = {2015 Feb},
	abstract = {This review focuses on recent developments in our understanding of group II intron function, the relationships of these introns to retrotransposons and spliceosomes, and how their common features have informed thinking about bacterial group II introns as key elements in eukaryotic evolution. Reverse transcriptase-mediated and host factor-aided intron retrohoming pathways are considered along with retrotransposition mechanisms to novel sites in bacteria, where group II introns are thought to have originated. DNA target recognition and movement by target-primed reverse transcription infer an evolutionary relationship among group II introns, non-LTR retrotransposons, such as LINE elements, and telomerase. Additionally, group II introns are almost certainly the progenitors of spliceosomal introns. Their profound similarities include splicing chemistry extending to RNA catalysis, reaction stereochemistry, and the position of two divalent metals that perform catalysis at the RNA active site. There are also sequence and structural similarities between group II introns and the spliceosome{\textquoteright}s small nuclear RNAs (snRNAs) and between a highly conserved core spliceosomal protein Prp8 and a group II intron-like reverse transcriptase. It has been proposed that group II introns entered eukaryotes during bacterial endosymbiosis or bacterial-archaeal fusion, proliferated within the nuclear genome, necessitating evolution of the nuclear envelope, and fragmented giving rise to spliceosomal introns. Thus, these bacterial self-splicing mobile elements have fundamentally impacted the composition of extant eukaryotic genomes, including the human genome, most of which is derived from close relatives of mobile group II introns.},
	issn = {2165-0497},
	url = {http://www.asmscience.org/content/journal/microbiolspec/10.1128/microbiolspec.MDNA3-0050-2014},
	author = {Lambowitz, Alan M and Belfort, Marlene}
}
@article {309,
	title = {Protein synthesis. Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains.},
	journal = {Science},
	volume = {347},
	number = {6217},
	year = {2015},
	month = {2015 Jan 2},
	pages = {75-8},
	abstract = {In Eukarya, stalled translation induces 40S\&nbsp;dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S\&nbsp;subunit, which mediates nascent chain degradation. Here we report cryo{\textendash}electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S\&nbsp;subunit at sites exposed after 40Sdissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S\&nbsp;subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein{\textemdash}not an mRNA{\textemdash}determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions ({\textquotedblleft}CAT tails{\textquotedblright}).},
	keywords = {Cryoelectron Microscopy, Nucleic Acid Conformation, Peptide Biosynthesis, Nucleic Acid-Independent, Protein Conformation, Ribosome Subunits, Large, Eukaryotic, RNA, Messenger, RNA, Transfer, Ala, RNA, Transfer, Thr, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Ubiquitin-Protein Ligases},
	issn = {1095-9203},
	doi = {10.1126/science.1259724},
	url = {http://www.sciencemag.org/content/347/6217/75.long},
	author = {Shen, Peter S and Park, Joseph and Qin, Yidan and Li, Xueming and Parsawar, Krishna and Larson, Matthew H and Cox, James and Cheng, Yifan and Lambowitz, Alan M and Weissman, Jonathan S and Brandman, Onn and Frost, Adam}
}
@article {313,
	title = {Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis.},
	journal = {Mob DNA},
	volume = {5},
	number = {1},
	year = {2014},
	month = {2014},
	pages = {2},
	abstract = {Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into {\textquoteleft}targetrons.{\textquoteright} Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and {\textquoteleft}cut-and-pastes{\textquoteright} (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.},
	issn = {1759-8753},
	doi = {10.1186/1759-8753-5-2},
	url = {http://www.mobilednajournal.com/content/5/1/2},
	author = {Enyeart, Peter J and Mohr, Georg and Ellington, Andrew D and Lambowitz, Alan M}
}
@article {312,
	title = {Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {111},
	number = {33},
	year = {2014},
	month = {2014 Aug 19},
	pages = {12025-30},
	abstract = {Interferon (IFN) responses play key roles in cellular defense against pathogens. Highly expressed IFN-induced proteins with tetratricopeptide repeats (IFITs) are proposed to function as RNA binding proteins, but the RNA binding and discrimination specificities of IFIT proteins remain unclear. Here we show that human IFIT5 has comparable affinity for RNAs with diverse phosphate-containing 5'-ends, excluding the higher eukaryotic mRNA cap. Systematic mutagenesis revealed that sequence substitutions in IFIT5 can alternatively expand or introduce bias in protein binding to RNAs with 5' monophosphate, triphosphate, cap0 (triphosphate-bridged N7-methylguanosine), or cap1 (cap0 with RNA 2'-O-methylation). We defined the breadth of cellular ligands for IFIT5 by using a thermostable group II intron reverse transcriptase for RNA sequencing. We show that IFIT5 binds precursor and processed tRNAs, as well as other RNA polymerase III transcripts. Our findings establish the RNA recognition specificity of the human innate immune response protein IFIT5.},
	keywords = {Humans, Models, Molecular, Neoplasm Proteins, Nucleic Acid Conformation, Protein Binding, RNA},
	issn = {1091-6490},
	doi = {10.1073/pnas.1412842111},
	url = {http://www.pnas.org/content/111/33/12025.long},
	author = {Katibah, George E and Qin, Yidan and Sidote, David J and Yao, Jun and Lambowitz, Alan M and Collins, Kathleen}
}
@article {314,
	title = {The contribution of cellulosomal scaffoldins to cellulose hydrolysis by Clostridium thermocellum analyzed by using thermotargetrons.},
	journal = {Biotechnol Biofuels},
	volume = {7},
	year = {2014},
	month = {2014},
	pages = {80},
	abstract = {Clostridium thermocellum\&nbsp;is a thermophilic anaerobic bacterium that degrades cellulose by using a highly effective cellulosome, a macromolecular complex consisting of multiple cellulose degrading enzymes organized and attached to the cell surface by non-catalytic scaffoldins. However, due largely to lack of efficient methods for genetic manipulation of\&nbsp;C. thermocellum, it is still unclear how the different scaffoldins and their functional modules contribute to cellulose hydrolysis.
},
	issn = {1754-6834},
	doi = {10.1186/1754-6834-7-80},
	url = {http://www.biotechnologyforbiofuels.com/content/7/1/80},
	author = {Hong, Wei and Zhang, Jie and Feng, Yingang and Mohr, Georg and Lambowitz, Alan M and Cui, Gu-Zhen and Liu, Ya-Jun and Cui, Qiu}
}
@article {311,
	title = {Evolution of RNA-protein interactions: non-specific binding led to RNA splicing activity of fungal mitochondrial tyrosyl-tRNA synthetases.},
	journal = {PLoS Biol},
	volume = {12},
	number = {12},
	year = {2014},
	month = {2014 Dec},
	pages = {e1002028},
	abstract = {The\&nbsp;Neurospora crassa\&nbsp;mitochondrial tyrosyl-tRNA synthetase (mtTyrRS; CYT-18 protein) evolved a new function as a group I intron splicing factor by acquiring the ability to bind group I intron RNAs and stabilize their catalytically active RNA structure. Previous studies showed: (i) CYT-18 binds group I introns by using both its N-terminal catalytic domain and flexibly attached C-terminal anticodon-binding domain (CTD); and (ii) the catalytic domain binds group I introns specifically via multiple structural adaptations that occurred during or after the divergence of Peziomycotina and Saccharomycotina. However, the function of the CTD and how it contributed to the evolution of splicing activity have been unclear. Here, small angle X-ray scattering analysis of CYT-18 shows that both CTDs of the homodimeric protein extend outward from the catalytic domain, but move inward to bind opposite ends of a group I intron RNA. Biochemical assays show that the isolated CTD of CYT-18 binds RNAs non-specifically, possibly contributing to its interaction with the structurally different ends of the intron RNA. Finally, we find that the yeast mtTyrRS, which diverged from Pezizomycotina fungal mtTyrRSs prior to the evolution of splicing activity, binds group I intron and other RNAs non-specifically via its CTD, but lacks further adaptations needed for group I intron splicing. Our results suggest a scenario of constructive neutral (i.e., pre-adaptive) evolution in which an initial non-specific interaction between the CTD of an ancestral fungal mtTyrRS and a self-splicing group I intron was {\textquotedblleft}fixed{\textquotedblright} by an intron RNA mutation that resulted in protein-dependent splicing. Once fixed, this interaction could be elaborated by further adaptive mutations in both the catalytic domain and CTD that enabled specific binding of group I introns. Our results highlight a role for non-specific RNA binding in the evolution of RNA-binding proteins.},
	issn = {1545-7885},
	doi = {10.1371/journal.pbio.1002028},
	url = {http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1002028},
	author = {Lamech, Lilian T and Mallam, Anna L and Lambowitz, Alan M}
}
@article {310,
	title = {Molecular insights into RNA and DNA helicase evolution from the determinants of specificity for a DEAD-box RNA helicase.},
	journal = {Elife},
	volume = {4},
	year = {2014},
	month = {2014},
	abstract = {How different helicase families with a conserved catalytic {\textquoteleft}helicase core{\textquoteright} evolved to function on varied RNA and DNA substrates by diverse mechanisms remains unclear. In this study, we used Mss116, a yeast DEAD-box protein that utilizes ATP to locally unwind dsRNA, to investigate helicase specificity and mechanism. Our results define the molecular basis for the substrate specificity of a DEAD-box protein. Additionally, they show that Mss116 has ambiguous substrate-binding properties and interacts with all four NTPs and both RNA and DNA. The efficiency of unwinding correlates with the stability of the {\textquoteleft}closed-state{\textquoteright} helicase core, a complex with nucleotide and nucleic acid that forms as duplexes are unwound. Crystal structures reveal that core stability is modulated by family-specific interactions that favor certain substrates. This suggests how present-day helicases diversified from an ancestral core with broad specificity by retaining core closure as a common catalytic mechanism while optimizing substrate-binding interactions for different cellular functions.\&nbsp;},
	issn = {2050-084X},
	doi = {10.7554/eLife.04630},
	url = {http://elifesciences.org/content/3/e04630},
	author = {Mallam, Anna L and Sidote, David J and Lambowitz, Alan M}
}
@article {315,
	title = {Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {110},
	number = {40},
	year = {2013},
	month = {2013 Oct 1},
	pages = {E3800-9},
	abstract = {Mobile group II introns are bacterial retrotransposons thought to be evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of a catalytically active intron RNA ("ribozyme") and an intron-encoded reverse transcriptase, which function together to promote RNA splicing and intron mobility via reverse splicing of the intron RNA into new DNA sites ("retrohoming"). Although group II introns are active in bacteria, their natural hosts, they function inefficiently in eukaryotes, where lower free Mg(2+) concentrations decrease their ribozyme activity and constitute a natural barrier to group II intron proliferation within nuclear genomes. Here, we show that retrohoming of the Ll.LtrB group II intron is strongly inhibited in an Escherichia coli mutant lacking the Mg(2+) transporter MgtA, and we use this system to select mutations in catalytic core domain V (DV) that partially rescue retrohoming at low Mg(2+) concentrations. We thus identified mutations in the distal stem of DV that increase retrohoming efficiency in the MgtA mutant up to 22-fold. Biochemical assays of splicing and reverse splicing indicate that the mutations increase the fraction of intron RNA that folds into an active conformation at low Mg(2+) concentrations, and terbium-cleavage assays suggest that this increase is due to enhanced Mg(2+) binding to the distal stem of DV. Our findings indicate that DV is involved in a critical Mg(2+)-dependent RNA folding step in group II introns and demonstrate the feasibility of selecting intron variants that function more efficiently at low Mg(2+) concentrations, with implications for evolution and potential applications in gene targeting.},
	keywords = {Biotechnology, Blotting, Northern, Catalytic Domain, Directed Molecular Evolution, DNA Primers, Escherichia coli, Magnesium, Models, Molecular, Protein Conformation, Protein Engineering, Retroelements, RNA, Catalytic},
	issn = {1091-6490},
	doi = {10.1073/pnas.1315742110},
	url = {http://www.pnas.org/content/110/40/E3800.long},
	author = {Truong, David M and Sidote, David J and Russell, Rick and Lambowitz, Alan M}
}
@article {320,
	title = {Generalized bacterial genome editing using mobile group II introns and Cre-lox.},
	journal = {Mol Syst Biol},
	volume = {9},
	year = {2013},
	month = {2013},
	pages = {685},
	abstract = {Efficient bacterial genetic engineering approaches with broad-host applicability are rare. We combine two systems, mobile group II introns ({\textquoteright}targetrons{\textquoteright}) and Cre/lox, which function efficiently in many different organisms, into a versatile platform we call GETR (Genome Editing via Targetrons and Recombinases). The introns deliver lox sites to specific genomic loci, enabling genomic manipulations. Efficiency is enhanced by adding flexibility to the RNA hairpins formed by the lox sites. We use the system for insertions, deletions, inversions, and one-step cut-and-paste operations. We demonstrate insertion of a 12-kb polyketide synthase operon into the lacZ gene of Escherichia coli, multiple simultaneous and sequential deletions of up to 120 kb in E. coli and Staphylococcus aureus, inversions of up to 1.2 Mb in E. coli and Bacillus subtilis, and one-step cut-and-pastes for translocating 120 kb of genomic sequence to a site 1.5 Mb away. We also demonstrate the simultaneous delivery of lox sites into multiple loci in the Shewanella oneidensis genome. No selectable markers need to be placed in the genome, and the efficiency of Cre-mediated manipulations typically approaches 100\%.},
	keywords = {Bacillus subtilis, Base Sequence, Escherichia coli, Genetic Engineering, Genetic Loci, Genome, Bacterial, Integrases, Introns, Lac Operon, Molecular Sequence Data, Mutagenesis, Insertional, Nucleic Acid Conformation, Recombination, Genetic, Sequence Deletion, Sequence Inversion, Shewanella, Staphylococcus aureus},
	issn = {1744-4292},
	doi = {10.1038/msb.2013.41},
	url = {http://msb.embopress.org/content/9/1/685.long},
	author = {Enyeart, Peter J and Chirieleison, Steven M and Dao, Mai N and Perutka, Jiri and Quandt, Erik M and Yao, Jun and Whitt, Jacob T and Keatinge-Clay, Adrian T and Lambowitz, Alan M and Ellington, Andrew D}
}
@article {319,
	title = {Genetic and biochemical assays reveal a key role for replication restart proteins in group II intron retrohoming.},
	journal = {PLoS Genet},
	volume = {9},
	number = {4},
	year = {2013},
	month = {2013 Apr},
	pages = {e1003469},
	abstract = {Mobile group II introns retrohome by an RNP-based mechanism in which the intron RNA reverse splices into a DNA site and is reverse transcribed by the associated intron-encoded protein. The resulting intron cDNA is then integrated into the genome by cellular mechanisms that have remained unclear. Here, we used an Escherichia coli genetic screen and Taqman qPCR assay that mitigate indirect effects to identify host factors that function in retrohoming. We then analyzed mutants identified in these and previous genetic screens by using a new biochemical assay that combines group II intron RNPs with cellular extracts to reconstitute the complete retrohoming reaction in vitro. The genetic and biochemical analyses indicate a retrohoming pathway involving degradation of the intron RNA template by a host RNase H and second-strand DNA synthesis by the host replicative DNA polymerase. Our results reveal ATP-dependent steps in both cDNA and second-strand synthesis and a surprising role for replication restart proteins in initiating second-strand synthesis in the absence of DNA replication. We also find an unsuspected requirement for host factors in initiating reverse transcription and a new RNA degradation pathway that suppresses retrohoming. Key features of the retrohoming mechanism may be used by human LINEs and other non-LTR-retrotransposons, which are related evolutionarily to mobile group II introns. Our findings highlight a new role for replication restart proteins, which function not only to repair DNA damage caused by mobile element insertion, but have also been co-opted to become an integral part of the group II intron retrohoming mechanism.},
	issn = {1553-7404},
	doi = {10.1371/journal.pgen.1003469},
	url = {http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1003469},
	author = {Yao, Jun and Truong, David M and Lambowitz, Alan M}
}
@article {316,
	title = {Rapid targeted gene disruption in Bacillus anthracis.},
	journal = {BMC Biotechnol},
	volume = {13},
	year = {2013},
	month = {2013},
	pages = {72},
	abstract = {BACKGROUND: Anthrax is a zoonotic disease recognized to affect herbivores since Biblical times and has the widest range of susceptible host species of any known pathogen. The ease with which the bacterium can be weaponized and its recent deliberate use as an agent of terror, have highlighted the importance of gaining a deeper understanding and effective countermeasures for this important pathogen. High quality sequence data has opened the possibility of systematic dissection of how genes distributed on both the bacterial chromosome and associated plasmids have made it such a successful pathogen. However, low transformation efficiency and relatively few genetic tools for chromosomal manipulation have hampered full interrogation of its genome. RESULTS: Group II introns have been developed into an efficient tool for site-specific gene inactivation in several organisms. We have adapted group II intron targeting technology for application in Bacillus anthracis and generated vectors that permit gene inactivation through group II intron insertion. The vectors developed permit screening for the desired insertion through PCR or direct selection of intron insertions using a selection scheme that activates a kanamycin resistance marker upon successful intron insertion. CONCLUSIONS: The design and vector construction described here provides a useful tool for high throughput experimental interrogation of the Bacillus anthracis genome and will benefit efforts to develop improved vaccines and therapeutics.},
	keywords = {Bacillus anthracis, Chromosomes, Bacterial, Cloning, Molecular, DNA, Bacterial, Escherichia coli, Gene Targeting, Genes, Bacterial, Genetic Vectors, Introns, Mutagenesis, Insertional, Nucleic Acid Conformation, Plasmids, Selection, Genetic},
	issn = {1472-6750},
	doi = {10.1186/1472-6750-13-72},
	url = {http://www.biomedcentral.com/1472-6750/13/72},
	author = {Saldanha, Roland J and Pemberton, Adin and Shiflett, Patrick and Perutka, Jiri and Whitt, Jacob T and Ellington, Andrew and Lambowitz, Alan M and Kramer, Ryan and Taylor, Deborah and Lamkin, Thomas J}
}
@article {317,
	title = {A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum.},
	journal = {PLoS One},
	volume = {8},
	number = {7},
	year = {2013},
	month = {2013},
	pages = {e69032},
	abstract = {BACKGROUND: Targetrons are gene targeting vectors derived from mobile group II introns. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which use their combined activities to achieve highly efficient site-specific DNA integration with readily programmable DNA target specificity. METHODOLOGY/PRINCIPAL FINDINGS: Here, we used a mobile group II intron from the thermophilic cyanobacterium Thermosynechococcus elongatus to construct a thermotargetron for gene targeting in thermophiles. After determining its DNA targeting rules by intron mobility assays in Escherichia coli at elevated temperatures, we used this thermotargetron in Clostridium thermocellum, a thermophile employed in biofuels production, to disrupt six different chromosomal genes (cipA, hfat, hyd, ldh, pta, and pyrF). High integration efficiencies (67-100\% without selection) were achieved, enabling detection of disruptants by colony PCR screening of a small number of transformants. Because the thermotargetron functions at high temperatures that promote DNA melting, it can recognize DNA target sequences almost entirely by base pairing of the intron RNA with less contribution from the intron-encoded protein than for mesophilic targetrons. This feature increases the number of potential targetron-insertion sites, while only moderately decreasing DNA target specificity. Phenotypic analysis showed that thermotargetron disruption of the genes encoding lactate dehydrogenase (ldh; Clo1313_1160) and phosphotransacetylase (pta; Clo1313_1185) increased ethanol production in C. thermocellum by decreasing carbon flux toward lactate and acetate. CONCLUSIONS/SIGNIFICANCE: Thermotargetron provides a new, rapid method for gene targeting and genetic engineering of C. thermocellum, an industrially important microbe, and should be readily adaptable for gene targeting in other thermophiles.},
	keywords = {Base Pairing, Base Sequence, Binding Sites, Chromosomes, Bacterial, Clostridium thermocellum, Cyanobacteria, Escherichia coli, Gene Order, Gene Targeting, Genetic Vectors, Introns, L-Lactate Dehydrogenase, Lac Operon, Metabolome, Metabolomics, Mutagenesis, Insertional, Nucleic Acid Conformation, Phosphate Acetyltransferase, Plasmids, RNA, Bacterial, RNA, Catalytic, Temperature},
	issn = {1932-6203},
	doi = {10.1371/journal.pone.0069032},
	url = {http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069032},
	author = {Mohr, Georg and Hong, Wei and Zhang, Jie and Cui, Gu-Zhen and Yang, Yunfeng and Cui, Qiu and Liu, Ya-Jun and Lambowitz, Alan M}
}
@article {318,
	title = {Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.},
	journal = {RNA},
	volume = {19},
	number = {7},
	year = {2013},
	month = {2013 Jul},
	pages = {958-70},
	abstract = {Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81{\textdegree}C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3{\textquoteright} ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.},
	keywords = {Base Sequence, Cloning, Molecular, Conserved Sequence, DNA, Complementary, Escherichia coli, Escherichia coli Proteins, Gene Expression Profiling, Gene Library, Geobacillus stearothermophilus, HeLa Cells, Humans, Introns, MCF-7 Cells, MicroRNAs, Molecular Sequence Data, Open Reading Frames, Periplasmic Binding Proteins, Plasmids, Protein Stability, Recombinant Fusion Proteins, Reverse Transcriptase Polymerase Chain Reaction, RNA-Directed DNA Polymerase, Sequence Analysis, RNA, Temperature},
	issn = {1469-9001},
	doi = {10.1261/rna.039743.113},
	url = {http://rnajournal.cshlp.org/content/19/7/958.long},
	author = {Mohr, Sabine and Ghanem, Eman and Smith, Whitney and Sheeter, Dennis and Qin, Yidan and King, Olga and Polioudakis, Damon and Iyer, Vishwanath R and Hunicke-Smith, Scott and Swamy, Sajani and Kuersten, Scott and Lambowitz, Alan M}
}
@article {321,
	title = {Toward a molecular understanding of RNA remodeling by DEAD-box proteins.},
	journal = {RNA Biol},
	volume = {10},
	number = {1},
	year = {2013},
	month = {2013 Jan},
	pages = {44-55},
	abstract = {DEAD-box proteins are superfamily 2 helicases that function in all aspects of RNA metabolism. They employ ATP binding and hydrolysis to generate tight, yet regulated RNA binding, which is used to unwind short RNA helices non-processively and promote structural transitions of RNA and RNA-protein substrates. In the last few years, substantial progress has been made toward a detailed, quantitative understanding of the structural and biochemical properties of DEAD-box proteins. Concurrently, progress has been made toward a physical understanding of the RNA rearrangements and folding steps that are accelerated by DEAD-box proteins in model systems. Here, we review the recent progress on both of these fronts, focusing on the mitochondrial DEAD-box proteins Mss116 and CYT-19 and their mechanisms in promoting the splicing of group I and group II introns.},
	keywords = {DEAD-box RNA Helicases, RNA},
	issn = {1555-8584},
	doi = {10.4161/rna.22210},
	url = {http://www.tandfonline.com/doi/abs/10.4161/rna.22210?url_ver=Z39.88-2003\&rfr_id=ori\%3Arid\%3Acrossref.org\&rfr_dat=cr_pub\%3Dpubmed\&$\#$.VN0CjbDF990},
	author = {Russell, Rick and Jarmoskaite, Inga and Lambowitz, Alan M}
}
@article {323,
	title = {The retrohoming of linear group II intron RNAs in Drosophila melanogaster occurs by both DNA ligase 4-dependent and -independent mechanisms.},
	journal = {PLoS Genet},
	volume = {8},
	number = {2},
	year = {2012},
	month = {2012},
	pages = {e1002534},
	abstract = {Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3{\textquoteright} end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential biotechnological applications.},
	keywords = {Animals, DNA Damage, DNA End-Joining Repair, DNA Ligases, Drosophila melanogaster, Introns, Mutation, Retroelements, RNA, RNA Splicing, RNA-Directed DNA Polymerase},
	issn = {1553-7404},
	doi = {10.1371/journal.pgen.1002534},
	author = {White, Travis B and Lambowitz, Alan M}
}
@article {322,
	title = {Structural basis for RNA-duplex recognition and unwinding by the DEAD-box helicase Mss116p.},
	journal = {Nature},
	volume = {490},
	number = {7418},
	year = {2012},
	month = {2012 Oct 4},
	pages = {121-5},
	abstract = {DEAD-box proteins are the largest family of nucleic acid helicases, and are crucial to RNA metabolism throughout all domains of life. They contain a conserved {\textquoteright}helicase core{\textquoteright} of two RecA-like domains (domains (D)1 and D2), which uses ATP to catalyse the unwinding of short RNA duplexes by non-processive, local strand separation. This mode of action differs from that of translocating helicases and allows DEAD-box proteins to remodel large RNAs and RNA-protein complexes without globally disrupting RNA structure. However, the structural basis for this distinctive mode of RNA unwinding remains unclear. Here, structural, biochemical and genetic analyses of the yeast DEAD-box protein Mss116p indicate that the helicase core domains have modular functions that enable a novel mechanism for RNA-duplex recognition and unwinding. By investigating D1 and D2 individually and together, we find that D1 acts as an ATP-binding domain and D2 functions as an RNA-duplex recognition domain. D2 contains a nucleic-acid-binding pocket that is formed by conserved DEAD-box protein sequence motifs and accommodates A-form but not B-form duplexes, providing a basis for RNA substrate specificity. Upon a conformational change in which the two core domains join to form a {\textquoteright}closed state{\textquoteright} with an ATPase active site, conserved motifs in D1 promote the unwinding of duplex substrates bound to D2 by excluding one RNA strand and bending the other. Our results provide a comprehensive structural model for how DEAD-box proteins recognize and unwind RNA duplexes. This model explains key features of DEAD-box protein function and affords a new perspective on how the evolutionarily related cores of other RNA and DNA helicases diverged to use different mechanisms.},
	keywords = {Adenosine Triphosphatases, Adenosine Triphosphate, Amino Acid Motifs, Base Sequence, Catalytic Domain, Conserved Sequence, Crystallography, X-Ray, DEAD-box RNA Helicases, Evolution, Molecular, GC Rich Sequence, Models, Molecular, Nucleic Acid Conformation, Protein Structure, Tertiary, RNA, Double-Stranded, RNA-Binding Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Structure-Activity Relationship, Substrate Specificity},
	issn = {1476-4687},
	doi = {10.1038/nature11402},
	author = {Mallam, Anna L and Del Campo, Mark and Gilman, Benjamin and Sidote, David J and Lambowitz, Alan M}
}
@article {325,
	title = {ATP-dependent roles of the DEAD-box protein Mss116p in group II intron splicing in vitro and in vivo.},
	journal = {J Mol Biol},
	volume = {411},
	number = {3},
	year = {2011},
	month = {2011 Aug 19},
	pages = {661-79},
	abstract = {The yeast DEAD-box protein Mss116p functions as a general RNA chaperone in splicing mitochondrial group I and group II introns. For most of its functions, Mss116p is thought to use ATP-dependent RNA unwinding to facilitate RNA structural transitions, but it has been suggested to assist in the folding of one group II intron (aI5γ) primarily by stabilizing a folding intermediate. Here we compare three aI5γ constructs: one with long exons, one with short exons, and a ribozyme construct lacking exons. The long exons result in slower splicing, suggesting that they misfold and/or stabilize nonnative intronic structures. Nevertheless, Mss116p acceleration of all three constructs depends on ATP and is inhibited by mutations that compromise RNA unwinding, suggesting similar mechanisms. Results of splicing assays and a new two-stage assay that separates ribozyme folding and catalysis indicate that maximal folding of all three constructs by Mss116p requires ATP-dependent RNA unwinding. ATP-independent activation is appreciable for only a subpopulation of the minimal ribozyme construct and not for constructs containing exons. As expected for a general RNA chaperone, Mss116p can also disrupt the native ribozyme, which can refold after Mss116p removal. Finally, using yeast strains with mitochondrial DNA containing only the single intron aI5γ, we show that Mss116p mutants promote splicing in vivo to degrees that correlate with their residual ATP-dependent RNA-unwinding activities. Together, our results indicate that, although DEAD-box proteins play multiple roles in RNA folding, the physiological function of Mss116p in aI5γ splicing includes a requirement for ATP-dependent local unfolding, allowing the conversion of nonfunctional RNA structure into functional RNA structure.},
	keywords = {Adenosine Triphosphate, Blotting, Northern, DEAD-box RNA Helicases, Introns, Mitochondria, Molecular Chaperones, Polymerase Chain Reaction, RNA Helicases, RNA Splicing, RNA, Catalytic, RNA, Fungal, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins},
	issn = {1089-8638},
	doi = {10.1016/j.jmb.2011.05.047},
	author = {Potratz, Jeffrey P and Del Campo, Mark and Wolf, Rachel Z and Lambowitz, Alan M and Russell, Rick}
}
@article {326,
	title = {Group II introns: mobile ribozymes that invade DNA.},
	journal = {Cold Spring Harb Perspect Biol},
	volume = {3},
	number = {8},
	year = {2011},
	month = {2011 Aug},
	pages = {a003616},
	abstract = {Group II introns are mobile ribozymes that self-splice from precursor RNAs to yield excised intron lariat RNAs, which then invade new genomic DNA sites by reverse splicing. The introns encode a reverse transcriptase that stabilizes the catalytically active RNA structure for forward and reverse splicing, and afterwards converts the integrated intron RNA back into DNA. The characteristics of group II introns suggest that they or their close relatives were evolutionary ancestors of spliceosomal introns, the spliceosome, and retrotransposons in eukaryotes. Further, their ribozyme-based DNA integration mechanism enabled the development of group II introns into gene targeting vectors ("targetrons"), which have the unique feature of readily programmable DNA target specificity.},
	keywords = {Animals, DNA, Evolution, Molecular, Humans, Introns, Nucleic Acid Conformation, RNA Splicing, RNA, Catalytic, RNA-Directed DNA Polymerase},
	issn = {1943-0264},
	doi = {10.1101/cshperspect.a003616},
	author = {Lambowitz, Alan M and Zimmerly, Steven}
}
@article {324,
	title = {High-throughput genetic identification of functionally important regions of the yeast DEAD-box protein Mss116p.},
	journal = {J Mol Biol},
	volume = {413},
	number = {5},
	year = {2011},
	month = {2011 Nov 11},
	pages = {952-72},
	abstract = {The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension (CTE) induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends on ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the CTE results primarily from a steric block. Finally, we identified two conserved regions-one the previously noted post II region in the helicase core and the other in the CTE-that may help displace or sequester the opposite RNA strand during RNA unwinding.},
	keywords = {Amino Acid Motifs, Amino Acid Sequence, Binding Sites, Blotting, Northern, Conserved Sequence, Crystallography, X-Ray, DEAD-box RNA Helicases, Evolution, Molecular, Immunoblotting, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Protein Binding, Protein Conformation, RNA Splicing, RNA, Fungal, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid},
	issn = {1089-8638},
	doi = {10.1016/j.jmb.2011.09.015},
	author = {Mohr, Georg and Del Campo, Mark and Turner, Kathryn G and Gilman, Benjamin and Wolf, Rachel Z and Lambowitz, Alan M}
}
@article {328,
	title = {NMR Structure of the C-terminal domain of a tyrosyl-tRNA synthetase that functions in group I intron splicing.},
	journal = {Biochemistry},
	volume = {50},
	number = {18},
	year = {2011},
	month = {2011 May 10},
	pages = {3816-26},
	abstract = {The mitochondrial tyrosyl-tRNA synthetases (mt TyrRSs) of Pezizomycotina fungi are bifunctional proteins that aminoacylate mitochondrial tRNA(Tyr) and are structure-stabilizing splicing cofactors for group I introns. Studies with the Neurospora crassa synthetase (CYT-18 protein) showed that splicing activity is dependent upon Pezizomycotina-specific structural adaptations that form a distinct group I intron-binding site in the N-terminal catalytic domain. Although CYT-18{\textquoteright}s C-terminal domain also binds group I introns, it has been intractable to X-ray crystallography in the full-length protein. Here, we determined an NMR structure of the isolated C-terminal domain of the Aspergillus nidulans mt TyrRS, which is closely related to but smaller than CYT-18{\textquoteright}s. The structure shows an S4 fold like that of bacterial TyrRSs, but with novel features, including three Pezizomycontia-specific insertions. (15)N-(1)H two-dimensional NMR showed that C-terminal domains of the full-length A. nidulans and Geobacillus stearothermophilus synthetases do not tumble independently in solution, suggesting restricted orientations. Modeling onto a CYT-18/group I intron cocrystal structure indicates that the C-terminal domains of both subunits of the homodimeric protein bind different ends of the intron RNA, with one C-terminal domain having to undergo a large shift on its flexible linker to bind tRNA(Tyr) or the intron RNA on either side of the catalytic domain. The modeling suggests that the C-terminal domain acts together with the N-terminal domain to clamp parts of the intron{\textquoteright}s catalytic core, that at least one C-terminal domain insertion functions in group I intron binding, and that some C-terminal domain regions bind both tRNA(Tyr) and group I intron RNAs.},
	keywords = {Alternative Splicing, Aspergillus nidulans, Catalytic Domain, Crystallography, X-Ray, DNA, Geobacillus stearothermophilus, Introns, Magnetic Resonance Spectroscopy, Models, Molecular, Models, Theoretical, Molecular Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Solvents, Tyrosine-tRNA Ligase},
	issn = {1520-4995},
	doi = {10.1021/bi200189u},
	author = {Paukstelis, Paul J and Chari, Nandini and Lambowitz, Alan M and Hoffman, David}
}
@article {327,
	title = {Solution structures of DEAD-box RNA chaperones reveal conformational changes and nucleic acid tethering by a basic tail.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {108},
	number = {30},
	year = {2011},
	month = {2011 Jul 26},
	pages = {12254-9},
	abstract = {The mitochondrial DEAD-box proteins Mss116p of Saccharomyces cerevisiae and CYT-19 of Neurospora crassa are ATP-dependent helicases that function as general RNA chaperones. The helicase core of each protein precedes a C-terminal extension and a basic tail, whose structural role is unclear. Here we used small-angle X-ray scattering to obtain solution structures of the full-length proteins and a series of deletion mutants. We find that the two core domains have a preferred relative orientation in the open state without substrates, and we visualize the transition to a compact closed state upon binding RNA and adenosine nucleotide. An analysis of complexes with large chimeric oligonucleotides shows that the basic tails of both proteins are attached flexibly, enabling them to bind rigid duplex DNA segments extending from the core in different directions. Our results indicate that the basic tails of DEAD-box proteins contribute to RNA-chaperone activity by binding nonspecifically to large RNA substrates and flexibly tethering the core for the unwinding of neighboring duplexes.},
	keywords = {Binding Sites, Circular Dichroism, DEAD-box RNA Helicases, Fungal Proteins, Models, Molecular, Neurospora crassa, Nucleic Acid Conformation, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins, RNA, Fungal, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Scattering, Small Angle, Structural Homology, Protein, X-Ray Diffraction},
	issn = {1091-6490},
	doi = {10.1073/pnas.1109566108},
	author = {Mallam, Anna L and Jarmoskaite, Inga and Tijerina, Pilar and Del Campo, Mark and Seifert, Soenke and Guo, Liang and Russell, Rick and Lambowitz, Alan M}
}
@article {330,
	title = {Genetic identification of potential RNA-binding regions in a group II intron-encoded reverse transcriptase.},
	journal = {RNA},
	volume = {16},
	number = {4},
	year = {2010},
	month = {2010 Apr},
	pages = {732-47},
	abstract = {Mobile group II introns encode a reverse transcriptase that binds the intron RNA to promote RNA splicing and intron mobility, the latter via reverse splicing of the excised intron into DNA sites, followed by reverse transcription. Previous work showed that the Lactococcus lactis Ll.LtrB intron reverse transcriptase, denoted LtrA protein, binds with high affinity to DIVa, a stem-loop structure at the beginning of the LtrA open reading frame and makes additional contacts with intron core regions that stabilize the active RNA structure for forward and reverse splicing. LtrA{\textquoteright}s binding to DIVa down-regulates its translation and is critical for initiation of reverse transcription. Here, by using high-throughput unigenic evolution analysis with a genetic assay in which LtrA binding to DIVa down-regulates translation of GFP, we identified regions at LtrA{\textquoteright}s N terminus that are required for DIVa binding. Then, by similar analysis with a reciprocal genetic assay, we confirmed that residual splicing of a mutant intron lacking DIVa does not require these N-terminal regions, but does require other reverse transcriptase (RT) and X/thumb domain regions that bind the intron core. We also show that N-terminal fragments of LtrA by themselves bind specifically to DIVa in vivo and in vitro. Our results suggest a model in which the N terminus of nascent LtrA binds DIVa of the intron RNA that encoded it and nucleates further interactions with core regions that promote RNP assembly for RNA splicing and intron mobility. Features of this model may be relevant to evolutionarily related non-long-terminal-repeat (non-LTR)-retrotransposon RTs.},
	keywords = {Bacterial Proteins, Binding Sites, Down-Regulation, Escherichia coli, Introns, Lactococcus lactis, Models, Genetic, Retroelements, RNA, Bacterial, RNA-Binding Proteins, RNA-Directed DNA Polymerase},
	issn = {1469-9001},
	doi = {10.1261/rna.2007310},
	author = {Gu, Shan-Qing and Cui, Xiaoxia and Mou, Sijiong and Mohr, Sabine and Yao, Jun and Lambowitz, Alan M}
}
@article {329,
	title = {Mechanisms used for genomic proliferation by thermophilic group II introns.},
	journal = {PLoS Biol},
	volume = {8},
	number = {6},
	year = {2010},
	month = {2010},
	pages = {e1000391},
	abstract = {Mobile group II introns, which are found in bacterial and organellar genomes, are site-specific retroelements hypothesized to be evolutionary ancestors of spliceosomal introns and retrotransposons in higher organisms. Most bacteria, however, contain no more than one or a few group II introns, making it unclear how introns could have proliferated to higher copy numbers in eukaryotic genomes. An exception is the thermophilic cyanobacterium Thermosynechococcus elongatus, which contains 28 closely related copies of a group II intron, constituting approximately 1.3\% of the genome. Here, by using a combination of bioinformatics and mobility assays at different temperatures, we identified mechanisms that contribute to the proliferation of T. elongatus group II introns. These mechanisms include divergence of DNA target specificity to avoid target site saturation; adaptation of some intron-encoded reverse transcriptases to splice and mobilize multiple degenerate introns that do not encode reverse transcriptases, leading to a common splicing apparatus; and preferential insertion within other mobile introns or insertion elements, which provide new unoccupied sites in expanding non-essential DNA regions. Additionally, unlike mesophilic group II introns, the thermophilic T. elongatus introns rely on elevated temperatures to help promote DNA strand separation, enabling access to a larger number of DNA target sites by base pairing of the intron RNA, with minimal constraint from the reverse transcriptase. Our results provide insight into group II intron proliferation mechanisms and show that higher temperatures, which are thought to have prevailed on Earth during the emergence of eukaryotes, favor intron proliferation by increasing the accessibility of DNA target sites. We also identify actively mobile thermophilic introns, which may be useful for structural studies, gene targeting in thermophiles, and as a source of thermostable reverse transcriptases.},
	keywords = {Base Sequence, Cyanobacteria, DNA, Bacterial, Genome, Bacterial, Introns, Molecular Sequence Data, Open Reading Frames, Sequence Homology, Nucleic Acid},
	issn = {1545-7885},
	doi = {10.1371/journal.pbio.1000391},
	author = {Mohr, Georg and Ghanem, Eman and Lambowitz, Alan M}
}
@article {333,
	title = {Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA oligonucleotide and AMP-PNP.},
	journal = {Acta Crystallogr Sect F Struct Biol Cryst Commun},
	volume = {65},
	number = {Pt 8},
	year = {2009},
	month = {2009 Aug 1},
	pages = {832-5},
	abstract = {The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/Delta598-664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions (< or =1 mg ml(-1)), but its solubility could be increased by adding 50 mM L-arginine plus 50 mM L-glutamate and 50\% glycerol to achieve concentrations of approximately 10 mg ml(-1). Initial crystals were obtained by the microbatch method in the presence of a U(10) RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/Delta598-664 in complex with AMP-PNP and U(10) belonged to space group P2(1)2(1)2, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52 A, and diffracted X-rays to beyond 1.9 A resolution using synchrotron radiation from sector 21 at the Advanced Photon Source.},
	keywords = {Adenylyl Imidodiphosphate, Cloning, Molecular, Crystallization, Crystallography, X-Ray, DEAD-box RNA Helicases, Molecular Conformation, Recombinant Proteins, RNA, Saccharomyces cerevisiae Proteins},
	issn = {1744-3091},
	doi = {10.1107/S1744309109027225},
	author = {Del Campo, Mark and Lambowitz, Alan M}
}
@article {336,
	title = {EcI5, a group IIB intron with high retrohoming frequency: DNA target site recognition and use in gene targeting.},
	journal = {RNA},
	volume = {15},
	number = {3},
	year = {2009},
	month = {2009 Mar},
	pages = {432-49},
	abstract = {We find that group II intron EcI5, a subclass CL/IIB1 intron from an Escherichia coli virulence plasmid, is highly active in retrohoming in E. coli. Both full-length EcI5 and an EcI5-DeltaORF intron with the intron-encoded protein expressed separately from the same donor plasmid retrohome into a recipient plasmid target site at substantially higher frequencies than do similarly configured Lactococcus lactis Ll.LtrB introns. A comprehensive view of DNA target site recognition by EcI5 was obtained from selection experiments with donor and recipient plasmid libraries in which different recognition elements were randomized. These experiments suggest that EcI5, like other mobile group II introns, recognizes DNA target sequences by using both the intron-encoded protein and base-pairing of the intron RNA, with the latter involving EBS1, EBS2, and EBS3 sequences characteristic of class IIB introns. The intron-encoded protein appears to recognize a small number of bases flanking those recognized by the intron RNA, but their identity is different than in previously characterized group II introns. A computer algorithm based on the empirically determined DNA recognition rules enabled retargeting of EcI5 to integrate specifically at 10 different sites in the chromosomal lacZ gene at frequencies up to 98\% without selection. Our findings provide insight into modes of DNA target site recognition used by mobile group II introns. More generally, they show how the diversity of mobile group II introns can be exploited to provide a large variety of different target specificities and potentially other useful properties for gene targeting.},
	keywords = {Escherichia coli, Gene Targeting, Interspersed Repetitive Sequences, Introns, Plasmids},
	issn = {1469-9001},
	doi = {10.1261/rna.1378909},
	author = {Zhuang, Fanglei and Karberg, Michael and Perutka, Jiri and Lambowitz, Alan M}
}
@article {334,
	title = {Identification of proteins associated with the yeast mitochondrial RNA polymerase by tandem affinity purification.},
	journal = {Yeast},
	volume = {26},
	number = {8},
	year = {2009},
	month = {2009 Aug},
	pages = {423-40},
	abstract = {The abundance of mitochondrial (mt) transcripts varies under different conditions, and is thought to depend upon rates of transcription initiation, transcription termination/attenuation and RNA processing/degradation. The requirement to maintain the balance between RNA synthesis and processing may involve coordination between these processes; however, little is known about factors that regulate the activity of mtRNA polymerase (mtRNAP). Recent attempts to identify mtRNAP-protein interactions in yeast by means of a generalized tandem affinity purification (TAP) protocol were not successful, most likely because they involved a C-terminal mtRNAP-TAP fusion (which is incompatible with mtRNAP function) and because of the use of whole-cell solubilization protocols that did not preserve the integrity of mt protein complexes. Based upon the structure of T7 RNAP (to which mtRNAPs show high sequence similarity), we identified positions in yeast mtRNAP that allow insertion of a small affinity tag, confirmed the mature N-terminus, constructed a functional N-terminal TAP-mtRNAP fusion, pulled down associated proteins, and identified them by LC-MS-MS. Among the proteins found in the pull-down were a DEAD-box protein (Mss116p) and an RNA-binding protein (Pet127p). Previous genetic experiments suggested a role for these proteins in linking transcription and RNA degradation, in that a defect in the mt degradadosome could be suppressed by overexpression of either of these proteins or, independently, by mutations in either mtRNAP or its initiation factor Mtf1p. Further, we found that Mss116p inhibits transcription by mtRNAP in vitro in a steady-state reaction. Our results support the hypothesis that Mss116p and Pet127p are involved in modulation of mtRNAP activity.},
	keywords = {Amino Acid Sequence, Chromatography, Affinity, Chromatography, Liquid, DNA-Directed RNA Polymerases, GTP-Binding Proteins, Mitochondria, Mitochondrial Proteins, Models, Molecular, Molecular Sequence Data, Protein Structure, Tertiary, RNA, RNA, Fungal, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid, Tandem Mass Spectrometry},
	issn = {1097-0061},
	doi = {10.1002/yea.1672},
	author = {Markov, Dmitriy A and Savkina, Maria and Anikin, Michael and Del Campo, Mark and Ecker, Karen and Lambowitz, Alan M and De Gnore, Jon P and McAllister, William T}
}
@article {331,
	title = {Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {106},
	number = {43},
	year = {2009},
	month = {2009 Oct 27},
	pages = {18189-94},
	abstract = {Mobile group II introns retrohome by an RNP-based mechanism in which the excised intron lariat RNA fully reverse splices into a DNA site via 2 sequential transesterification reactions and is reverse transcribed by the associated intron-encoded protein. However, linear group II intron RNAs, which can arise by either hydrolytic splicing or debranching of lariat RNA, cannot carry out both reverse-splicing steps and were thus expected to be immobile. Here, we used facile microinjection assays in 2 eukaryotic systems, Xenopus laevis oocyte nuclei and Drosophila melanogaster embryos, to show that group II intron RNPs containing linear intron RNA can retrohome by carrying out the first step of reverse splicing into a DNA site, thereby ligating the 3{\textquoteright} end of the intron RNA to the 5{\textquoteright} end of the downstream exon DNA. The attached linear intron RNA is then reverse transcribed, yielding an intron cDNA whose free end is linked to the upstream exon DNA. Some of these retrohoming events result in the precise insertion of full-length intron. Most, however, yield aberrant 5{\textquoteright} junctions with 5{\textquoteright} exon resections, 5{\textquoteright} intron truncations, and/or extra nucleotide residues, hallmarks of nonhomologous end-joining. Our findings reveal a mobility mechanism for linear group II intron RNAs, show how group II introns can co-opt different DNA repair pathways for retrohoming, and suggest that linear group II intron RNAs might be used for site-specific DNA integration in gene targeting.},
	keywords = {Animals, Base Sequence, Cell Nucleus, DNA, Complementary, Drosophila melanogaster, Introns, Oocytes, Retroelements, RNA, Xenopus laevis},
	issn = {1091-6490},
	doi = {10.1073/pnas.0910277106},
	author = {Zhuang, Fanglei and Mastroianni, Marta and White, Travis B and Lambowitz, Alan M}
}
@article {332,
	title = {Structure of the Yeast DEAD box protein Mss116p reveals two wedges that crimp RNA.},
	journal = {Mol Cell},
	volume = {35},
	number = {5},
	year = {2009},
	month = {2009 Sep 11},
	pages = {598-609},
	abstract = {The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF(3)(-), or ADP-AlF(4)(-). The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge alpha helix bending RNA 3{\textquoteright} of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p{\textquoteright}s C-terminal extension also bends RNA 5{\textquoteright} of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which a protein uses two wedges to act as a molecular crimper.},
	keywords = {Adenosine Diphosphate, Adenosine Triphosphate, Adenylyl Imidodiphosphate, Binding Sites, Crystallography, X-Ray, DEAD-box RNA Helicases, Models, Molecular, Nucleic Acid Conformation, Organometallic Compounds, Poly U, Protein Conformation, Protein Structure, Tertiary, RNA, Saccharomyces cerevisiae Proteins, Structure-Activity Relationship},
	issn = {1097-4164},
	doi = {10.1016/j.molcel.2009.07.032},
	author = {Del Campo, Mark and Lambowitz, Alan M}
}
@article {335,
	title = {Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones.},
	journal = {J Mol Biol},
	volume = {389},
	number = {4},
	year = {2009},
	month = {2009 Jun 19},
	pages = {674-93},
	abstract = {The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and to activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements mitochondrial translation and group I and group II intron splicing defects in mss116Delta strains, stimulates the in vitro splicing of group I and group II introns, and functions indistinguishably from CYT-19 to resolve different nonnative secondary and/or tertiary structures in the Tetrahymena thermophila large subunit rRNA-DeltaP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and group II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and the vaccinia virus DExH-box protein NPH-II gave little, if any, group I or group II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and group II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. In contrast, DbpA binds group I and group II intron RNAs nonspecifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and group II intron splicing correlates primarily with their RNA-unwinding activity, which, for the protein preparations used here, was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Furthermore, this correlation holds for all group I and group II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their distinctive RNA-unwinding mechanism, which enables refolding of localized RNA regions or structures without globally disrupting RNA structure.},
	keywords = {Base Sequence, DEAD-box RNA Helicases, DNA, Mitochondrial, Genetic Complementation Test, Introns, Molecular Chaperones, Molecular Sequence Data, Neurospora crassa, Nucleic Acid Conformation, Protein Biosynthesis, RNA, RNA Splicing, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins},
	issn = {1089-8638},
	doi = {10.1016/j.jmb.2009.04.043},
	author = {Del Campo, Mark and Mohr, Sabine and Jiang, Yue and Jia, Huijue and Jankowsky, Eckhard and Lambowitz, Alan M}
}
@article {343,
	title = {Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro.},
	journal = {J Mol Biol},
	volume = {375},
	number = {5},
	year = {2008},
	month = {2008 Feb 1},
	pages = {1344-64},
	abstract = {The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended alpha-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted alpha-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the alpha-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two sub-regions of Mss116p{\textquoteright}s C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions.},
	keywords = {Adenosine Triphosphatases, Amino Acid Motifs, Amino Acid Sequence, Amino Acids, Basic, Binding Sites, Computational Biology, Conserved Sequence, Crystallography, X-Ray, DEAD-box RNA Helicases, Drosophila Proteins, Escherichia coli, Evolution, Molecular, Gene Silencing, Genetic Complementation Test, Hydrophobic and Hydrophilic Interactions, In Vitro Techniques, Introns, Isoelectric Point, Kinetics, Mitochondria, Molecular Chaperones, Molecular Sequence Data, Mutation, Missense, Plasmids, Protein Binding, Protein Biosynthesis, Protein Structure, Secondary, Protein Structure, Tertiary, RNA, RNA Splicing, RNA, Catalytic, RNA, Fungal, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Homology, Amino Acid, Temperature},
	issn = {1089-8638},
	doi = {10.1016/j.jmb.2007.11.041},
	author = {Mohr, Georg and Del Campo, Mark and Mohr, Sabine and Yang, Quansheng and Jia, Huijue and Jankowsky, Eckhard and Lambowitz, Alan M}
}
@article {340,
	title = {Group II intron protein localization and insertion sites are affected by polyphosphate.},
	journal = {PLoS Biol},
	volume = {6},
	number = {6},
	year = {2008},
	month = {2008 Jun 24},
	pages = {e150},
	abstract = {Mobile group II introns consist of a catalytic intron RNA and an intron-encoded protein with reverse transcriptase activity, which act together in a ribonucleoprotein particle to promote DNA integration during intron mobility. Previously, we found that the Lactococcus lactis Ll.LtrB intron-encoded protein (LtrA) expressed alone or with the intron RNA to form ribonucleoprotein particles localizes to bacterial cellular poles, potentially accounting for the intron{\textquoteright}s preferential insertion in the oriC and ter regions of the Escherichia coli chromosome. Here, by using cell microarrays and automated fluorescence microscopy to screen a transposon-insertion library, we identified five E. coli genes (gppA, uhpT, wcaK, ynbC, and zntR) whose disruption results in both an increased proportion of cells with more diffuse LtrA localization and a more uniform genomic distribution of Ll.LtrB-insertion sites. Surprisingly, we find that a common factor affecting LtrA localization in these and other disruptants is the accumulation of intracellular polyphosphate, which appears to bind LtrA and other basic proteins and delocalize them away from the poles. Our findings show that the intracellular localization of a group II intron-encoded protein is a major determinant of insertion-site preference. More generally, our results suggest that polyphosphate accumulation may provide a means of localizing proteins to different sites of action during cellular stress or entry into stationary phase, with potentially wide physiological consequences.},
	keywords = {Bacterial Proteins, DNA Transposable Elements, Escherichia coli, Introns, Microarray Analysis, Polyphosphates, RNA-Directed DNA Polymerase},
	issn = {1545-7885},
	doi = {10.1371/journal.pbio.0060150},
	author = {Zhao, Junhua and Niu, Wei and Yao, Jun and Mohr, Sabine and Marcotte, Edward M and Lambowitz, Alan M}
}
@article {339,
	title = {Group II intron-based gene targeting reactions in eukaryotes.},
	journal = {PLoS One},
	volume = {3},
	number = {9},
	year = {2008},
	month = {2008},
	pages = {e3121},
	abstract = {BACKGROUND: Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors ("targetrons") with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes.

METHODOLOGY/PRINCIPAL FINDINGS: By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg(2+) concentrations. By supplying additional Mg(2+), site-specific integration occurs in up to 38\% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8\% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg(2+)-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization.

CONCLUSIONS/SIGNIFICANCE: Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms.},
	keywords = {Animals, DNA, Drosophila melanogaster, Eukaryotic Cells, Gene Targeting, Genetic Vectors, Introns, Magnesium, Models, Biological, Models, Genetic, Oocytes, Plasmids, Recombination, Genetic, RNA, Xenopus laevis},
	issn = {1932-6203},
	doi = {10.1371/journal.pone.0003121},
	author = {Mastroianni, Marta and Watanabe, Kazuo and White, Travis B and Zhuang, Fanglei and Vernon, Jamie and Matsuura, Manabu and Wallingford, John and Lambowitz, Alan M}
}
@article {342,
	title = {Identification and evolution of fungal mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {105},
	number = {16},
	year = {2008},
	month = {2008 Apr 22},
	pages = {6010-5},
	abstract = {The bifunctional Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) both aminoacylates mitochondrial tRNA(Tyr) and acts as a structure-stabilizing splicing cofactor for group I introns. Previous studies showed that CYT-18 has distinct tRNA(Tyr) and group I intron-binding sites, with the latter formed by three small "insertions" in the nucleotide-binding fold and other structural adaptations compared with nonsplicing bacterial tyrosyl-tRNA synthetases. Here, analysis of genomic sequences shows that mitochondrial tyrosyl-tRNA synthetases with structural adaptations similar to CYT-18{\textquoteright}s are uniquely characteristic of fungi belonging to the subphylum Pezizomycotina, and biochemical assays confirm group I intron splicing activity for the enzymes from several of these organisms, including Aspergillus nidulans and the human pathogens Coccidioides posadasii and Histoplasma capsulatum. By combining multiple sequence alignments with a previously determined cocrystal structure of a CYT-18/group I intron RNA complex, we identify conserved features of the Pezizomycotina enzymes related to group I intron and tRNA interactions. Our results suggest that mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity evolved during or after the divergence of the fungal subphyla Pezizomycotina and Saccharomycotina by a mechanism involving the concerted differentiation of preexisting protein loop regions. The unique group I intron splicing activity of these fungal enzymes may provide a new target for antifungal drugs.},
	keywords = {Alternative Splicing, Amino Acid Sequence, Antifungal Agents, Aspergillus nidulans, Coccidioides, Conserved Sequence, Evolution, Molecular, Fungal Proteins, Histoplasma, Introns, Mitochondria, Mitochondrial Proteins, Molecular Sequence Data, Mutagenesis, Insertional, Neurospora crassa, Protein Conformation, RNA, Fungal, Sequence Alignment, Sequence Analysis, Protein, Tyrosine, Tyrosine-tRNA Ligase},
	issn = {1091-6490},
	doi = {10.1073/pnas.0801722105},
	author = {Paukstelis, Paul J and Lambowitz, Alan M}
}
@article {344,
	title = {Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA.},
	journal = {Nature},
	volume = {451},
	number = {7174},
	year = {2008},
	month = {2008 Jan 3},
	pages = {94-7},
	abstract = {The {\textquoteright}RNA world{\textquoteright} hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter{\textquoteright}s domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ({\textquoteright}RNPzymes{\textquoteright}). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world.},
	keywords = {Crystallography, X-Ray, Introns, Models, Molecular, Molecular Conformation, Neurospora crassa, Protein Binding, RNA, RNA Splicing, RNA, Catalytic, RNA-Binding Proteins, Staphylococcus Phages, Tyrosine-tRNA Ligase},
	issn = {1476-4687},
	doi = {10.1038/nature06413},
	author = {Paukstelis, Paul J and Chen, Jui-Hui and Chase, Elaine and Lambowitz, Alan M and Golden, Barbara L}
}
@article {341,
	title = {A three-dimensional model of a group II intron RNA and its interaction with the intron-encoded reverse transcriptase.},
	journal = {Mol Cell},
	volume = {30},
	number = {4},
	year = {2008},
	month = {2008 May 23},
	pages = {472-85},
	abstract = {Group II introns are self-splicing ribozymes believed to be the ancestors of spliceosomal introns. Many group II introns encode reverse transcriptases that promote both RNA splicing and intron mobility to new genomic sites. Here we used a circular permutation and crosslinking method to establish 16 intramolecular distance relationships within the mobile Lactococcus lactis Ll.LtrB-DeltaORF intron. Using these new constraints together with 13 established tertiary interactions and eight published crosslinks, we modeled a complete three-dimensional structure of the intron. We also used the circular permutation strategy to map RNA-protein interaction sites through fluorescence quenching and crosslinking assays. Our model provides a comprehensive structural framework for understanding the function of group II ribozymes, their natural structural variations, and the mechanisms by which the intron-encoded protein promotes RNA splicing and intron mobility. The model also suggests an arrangement of active site elements that may be conserved in the spliceosome.},
	keywords = {Amino Acid Sequence, Bacterial Proteins, Binding Sites, Cross-Linking Reagents, Introns, Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, Phylogeny, Protein Conformation, RNA, RNA Splicing, RNA, Catalytic, RNA-Directed DNA Polymerase},
	issn = {1097-4164},
	doi = {10.1016/j.molcel.2008.04.001},
	author = {Dai, Lixin and Chai, Dinggeng and Gu, Shan-Qing and Gabel, Jesse and Noskov, Sergei Y and Blocker, Forrest J H and Lambowitz, Alan M and Zimmerly, Steven}
}
@article {337,
	title = {DEAD-box proteins can completely separate an RNA duplex using a single ATP.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {105},
	number = {51},
	year = {2008},
	month = {2008 Dec 23},
	pages = {20203-8},
	abstract = {DEAD-box proteins are ubiquitous in RNA metabolism and use ATP to mediate RNA conformational changes. These proteins have been suggested to use a fundamentally different mechanism from the related DNA and RNA helicases, generating local strand separation while remaining tethered through additional interactions with structured RNAs and RNA-protein (RNP) complexes. Here, we provide a critical test of this model by measuring the number of ATP molecules hydrolyzed by DEAD-box proteins as they separate short RNA helices characteristic of structured RNAs (6-11 bp). We show that the DEAD-box protein CYT-19 can achieve complete strand separation using a single ATP, and that 2 related proteins, Mss116p and Ded1p, display similar behavior. Under some conditions, considerably ,
	keywords = {Adenosine Triphosphate, DEAD-box RNA Helicases, Models, Chemical, Nucleic Acid Conformation, Protein Conformation, RNA, Double-Stranded, Saccharomyces cerevisiae Proteins},
	issn = {1091-6490},
	doi = {10.1073/pnas.0811075106},
	author = {Chen, Yingfeng and Potratz, Jeffrey P and Tijerina, Pilar and Del Campo, Mark and Lambowitz, Alan M and Russell, Rick}
}
@article {338,
	title = {Toward predicting self-splicing and protein-facilitated splicing of group I introns.},
	journal = {RNA},
	volume = {14},
	number = {10},
	year = {2008},
	month = {2008 Oct},
	pages = {2013-29},
	abstract = {In the current era of massive discoveries of noncoding RNAs within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. Although studies of individual group I introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group I introns sequenced to date. Here, the self-splicing activities of 12 group I introns from various organisms were assayed under six reaction conditions that had been shown previously to promote RNA catalysis for different RNAs. Besides revealing that assessing self-splicing under only one condition can be misleading, this survey emphasizes that in vitro self-splicing efficiency is correlated with the GC content of the intron (>35\% GC was generally conductive to self-splicing), and with the ability of the introns to form particular tertiary interactions. Addition of the Neurospora crassa CYT-18 protein activated splicing of two nonself-splicing introns, but inhibited the second step of self-splicing for two others. Together, correlations between sequence, predicted structure and splicing begin to establish rules that should facilitate our ability to predict the self-splicing activity of any group I intron from its sequence.},
	keywords = {Base Composition, Base Sequence, Catalysis, Introns, Neurospora crassa, Nucleic Acid Conformation, RNA Splicing, RNA, Catalytic, RNA, Messenger, Tyrosine-tRNA Ligase},
	issn = {1469-9001},
	doi = {10.1261/rna.1027208},
	author = {Vicens, Quentin and Paukstelis, Paul J and Westhof, Eric and Lambowitz, Alan M and Cech, Thomas R}
}
@article {349,
	title = {Involvement of DEAD-box proteins in group I and group II intron splicing. Biochemical characterization of Mss116p, ATP hydrolysis-dependent and -independent mechanisms, and general RNA chaperone activity.},
	journal = {J Mol Biol},
	volume = {365},
	number = {3},
	year = {2007},
	month = {2007 Jan 19},
	pages = {835-55},
	abstract = {The RNA-catalyzed splicing of group I and group II introns is facilitated by proteins that stabilize the active RNA structure or act as RNA chaperones to disrupt stable inactive structures that are kinetic traps in RNA folding. In Neurospora crassa and Saccharomyces cerevisiae, the latter function is fulfilled by specific DEAD-box proteins, denoted CYT-19 and Mss116p, respectively. Previous studies showed that purified CYT-19 stimulates the in vitro splicing of structurally diverse group I and group II introns, and uses the energy of ATP binding or hydrolysis to resolve kinetic traps. Here, we purified Mss116p and show that it has RNA-dependent ATPase activity, unwinds RNA duplexes in a non-polar fashion, and promotes ATP-independent strand-annealing. Further, we show that Mss116p binds RNA non-specifically and promotes in vitro splicing of both group I and group II intron RNAs, as well as RNA cleavage by the aI5gamma-derived D135 ribozyme. However, Mss116p also has ATP hydrolysis-independent effects on some of these reactions, which are not shared by CYT-19 and may reflect differences in its RNA-binding properties. We also show that a non-mitochondrial DEAD-box protein, yeast Ded1p, can function almost as efficiently as CYT-19 and Mss116p in splicing the yeast aI5gamma group II intron and less efficiently in splicing the bI1 group II intron. Together, our results show that Mss116p, like CYT-19, can act broadly as an RNA chaperone to stimulate the splicing of diverse group I and group II introns, and that Ded1p also has an RNA chaperone activity that can be assayed by its effect on splicing mitochondrial introns. Nevertheless, these DEAD-box protein RNA chaperones are not completely interchangeable and appear to function in somewhat different ways, using biochemical activities that have likely been tuned by coevolution to function optimally on specific RNA substrates.},
	keywords = {Adenosine Triphosphate, Animals, DEAD-box RNA Helicases, Hydrolysis, Introns, Magnesium, Mitochondria, Molecular Chaperones, Neurospora crassa, Nucleic Acid Denaturation, Open Reading Frames, Protein Binding, RNA Splicing, RNA, Catalytic, RNA, Fungal, RNA-Binding Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Substrate Specificity, Tetrahymena thermophila},
	issn = {0022-2836},
	doi = {10.1016/j.jmb.2006.09.083},
	author = {Halls, Coralie and Mohr, Sabine and Del Campo, Mark and Yang, Quansheng and Jankowsky, Eckhard and Lambowitz, Alan M}
}
@article {345,
	title = {DEAD-box proteins unwind duplexes by local strand separation.},
	journal = {Mol Cell},
	volume = {28},
	number = {2},
	year = {2007},
	month = {2007 Oct 26},
	pages = {253-63},
	abstract = {DEAD-box proteins catalyze ATP-driven, local structural changes in RNA or RNA-protein complexes (RNP) during which only few RNA base pairs are separated. It is unclear how duplex unwinding by DEAD-box proteins differs from unwinding by canonical helicases, which can separate many base pairs by directional and processive translocation on the nucleic acid, starting from a helical end. Here, we show that two different DEAD-box proteins, Ded1p and Mss116p, can unwind RNA duplexes from internal as well as terminal helical regions and act on RNA segments as small as two nucleotides flanked by DNA. The data indicate that duplex unwinding by DEAD-box proteins is based on local destabilization of RNA helical regions. No directional movement of the enzymes through the duplex is involved. We propose a three-step mechanism in which DEAD-box proteins unwind duplexes as "local strand separators." This unwinding mode is well-suited for local structural changes in complex RNA or RNP assemblies.},
	keywords = {Adenosine Triphosphate, DEAD-box RNA Helicases, Kinetics, Models, Genetic, Nucleic Acid Conformation, RNA Stability, RNA, Double-Stranded, RNA, Fungal, RNA-Binding Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Substrate Specificity},
	issn = {1097-2765},
	doi = {10.1016/j.molcel.2007.08.016},
	author = {Yang, Quansheng and Del Campo, Mark and Lambowitz, Alan M and Jankowsky, Eckhard}
}
@article {346,
	title = {Do DEAD-box proteins promote group II intron splicing without unwinding RNA?},
	journal = {Mol Cell},
	volume = {28},
	number = {1},
	year = {2007},
	month = {2007 Oct 12},
	pages = {159-66},
	abstract = {The DEAD-box protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.},
	keywords = {DEAD-box RNA Helicases, Introns, Mutation, Nucleic Acid Conformation, Nucleic Acid Denaturation, Recombinant Fusion Proteins, RNA, RNA Splicing, Saccharomyces cerevisiae Proteins},
	issn = {1097-2765},
	doi = {10.1016/j.molcel.2007.07.028},
	author = {Del Campo, Mark and Tijerina, Pilar and Bhaskaran, Hari and Mohr, Sabine and Yang, Quansheng and Jankowsky, Eckhard and Russell, Rick and Lambowitz, Alan M}
}
@article {347,
	title = {Gene targeting in gram-negative bacteria by use of a mobile group II intron ("Targetron") expressed from a broad-host-range vector.},
	journal = {Appl Environ Microbiol},
	volume = {73},
	number = {8},
	year = {2007},
	month = {2007 Apr},
	pages = {2735-43},
	abstract = {Mobile group II introns ("targetrons") can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acid-inducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58\% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.},
	keywords = {Agrobacterium tumefaciens, Benzoates, Escherichia coli, Gene Deletion, Gene Targeting, Genetic Vectors, Gram-Negative Bacteria, Introns, Plasmids, Promoter Regions, Genetic, Pseudomonas aeruginosa},
	issn = {0099-2240},
	doi = {10.1128/AEM.02829-06},
	author = {Yao, Jun and Lambowitz, Alan M}
}
@article {348,
	title = {Probing the mechanisms of DEAD-box proteins as general RNA chaperones: the C-terminal domain of CYT-19 mediates general recognition of RNA.},
	journal = {Biochemistry},
	volume = {46},
	number = {11},
	year = {2007},
	month = {2007 Mar 20},
	pages = {3013-22},
	abstract = {The DEAD-box protein CYT-19 functions in the folding of several group I introns in vivo and a diverse set of group I and group II RNAs in vitro. Recent work using the Tetrahymena group I ribozyme demonstrated that CYT-19 possesses a second RNA-binding site, distinct from the unwinding active site, which enhances unwinding activity by binding nonspecifically to the adjacent RNA structure. Here, we probe the region of CYT-19 responsible for that binding by constructing a C-terminal truncation variant that lacks 49 amino acids and terminates at a domain boundary, as defined by limited proteolysis. This truncated protein unwinds a six-base-pair duplex, formed between the oligonucleotide substrate of the Tetrahymena ribozyme and an oligonucleotide corresponding to the internal guide sequence of the ribozyme, with near-wild-type efficiency. However, the truncated protein is activated much less than the wild-type protein when the duplex is covalently linked to the ribozyme or single-stranded or double-stranded extensions. Thus, the active site for RNA unwinding remains functional in the truncated CYT-19, but the site that binds the adjacent RNA structure has been compromised. Equilibrium binding experiments confirmed that the truncated protein binds RNA less tightly than the wild-type protein. RNA binding by the compromised site is important for chaperone activity, because the truncated protein is less active in facilitating the folding of a group I intron that requires CYT-19 in vivo. The deleted region contains arginine-rich sequences, as found in other RNA-binding proteins, and may function by tethering CYT-19 to structured RNAs, so that it can efficiently disrupt exposed, non-native structural elements, allowing them to refold. Many other DExD/H-box proteins also contain arginine-rich ancillary domains, and some of these domains may function similarly as nonspecific RNA-binding elements that enhance general RNA chaperone activity.},
	keywords = {Animals, Base Sequence, Binding Sites, DEAD-box RNA Helicases, Molecular Chaperones, Nucleic Acid Conformation, Papain, Protozoan Proteins, RNA, RNA, Catalytic, Tetrahymena thermophila},
	issn = {0006-2960},
	doi = {10.1021/bi0619472},
	author = {Grohman, Jacob K and Del Campo, Mark and Bhaskaran, Hari and Tijerina, Pilar and Lambowitz, Alan M and Russell, Rick}
}
@article {350,
	title = {Atomic force microscopy reveals DNA bending during group II intron ribonucleoprotein particle integration into double-stranded DNA.},
	journal = {Biochemistry},
	volume = {45},
	number = {41},
	year = {2006},
	month = {2006 Oct 17},
	pages = {12424-35},
	abstract = {The mobile Lactococcus lactis Ll.LtrB group II intron integrates into DNA target sites by a mechanism in which the intron RNA reverse splices into one DNA strand while the intron-encoded protein uses a C-terminal DNA endonuclease domain to cleave the opposite strand and then uses the cleaved 3{\textquoteright} end to prime reverse transcription of the inserted intron RNA. These reactions are mediated by an RNP particle that contains the intron-encoded protein and the excised intron lariat RNA, with both the protein and base pairing of the intron RNA used to recognize DNA target sequences. Here, computational analysis indicates that Escherichia coli DNA target sequences that support Ll.LtrB integration have greater predicted bendability than do random E. coli genomic sequences, and atomic force microscopy shows that target DNA is bent during the reaction with Ll.LtrB RNPs. Time course and mutational analyses show that DNA bending occurs after reverse splicing and requires subsequent interactions between the intron-encoded protein and the 3{\textquoteright} exon, which lead to two progressively larger bend angles. Our results suggest a model in which RNPs bend the target DNA by maintaining initial contacts with the 5{\textquoteright} exon while engaging in subsequent 3{\textquoteright} exon interactions that successively position the scissile phosphate for bottom-strand cleavage at the DNA endonuclease active site and then reposition the 3{\textquoteright} end of the cleaved bottom strand to the reverse transcriptase active site for initiation of cDNA synthesis. Our findings indicate that bendability of the DNA target site is a significant factor for Ll.LtrB RNP integration.},
	keywords = {Bacterial Proteins, Base Sequence, Binding Sites, DNA, Bacterial, Escherichia coli, Escherichia coli Proteins, Introns, Lactococcus lactis, Macromolecular Substances, Microscopy, Atomic Force, Models, Biological, Mutation, Nucleic Acid Conformation, Protein Binding, Ribonucleoproteins},
	issn = {0006-2960},
	doi = {10.1021/bi060612h},
	author = {Noah, James W and Park, Soyeun and Whitt, Jacob T and Perutka, Jiri and Frey, Wolfgang and Lambowitz, Alan M}
}
@article {352,
	title = {A DEAD-box protein alone promotes group II intron splicing and reverse splicing by acting as an RNA chaperone.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {103},
	number = {10},
	year = {2006},
	month = {2006 Mar 7},
	pages = {3569-74},
	abstract = {Group II intron RNAs self-splice in vitro but only at high salt and/or Mg2+ concentrations and have been thought to require proteins to stabilize their active structure for efficient splicing in vivo. Here, we show that a DEAD-box protein, CYT-19, can by itself promote the splicing and reverse splicing of the yeast aI5gamma and bI1 group II introns under near-physiological conditions by acting as an ATP-dependent RNA chaperone, whose continued presence is not required after RNA folding. Our results suggest that the folding of some group II introns may be limited by kinetic traps and that their active structures, once formed, do not require proteins or high Mg2+ concentrations for structural stabilization. Thus, during evolution, group II introns could have spliced and transposed by reverse splicing by using ubiquitous RNA chaperones before acquiring more specific protein partners to promote their splicing and mobility. More generally, our results provide additional evidence for the widespread role of RNA chaperones in folding cellular RNAs.},
	keywords = {Adenosine Triphosphate, Base Sequence, Introns, Kinetics, Magnesium, Molecular Chaperones, Nucleic Acid Conformation, Recombinant Proteins, RNA Helicases, RNA Splicing, RNA, Fungal, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Salts, Temperature},
	issn = {0027-8424},
	doi = {10.1073/pnas.0600332103},
	author = {Mohr, Sabine and Matsuura, Manabu and Perlman, Philip S and Lambowitz, Alan M}
}
@article {353,
	title = {Profile of Alan M. Lambowitz.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {103},
	number = {6},
	year = {2006},
	month = {2006 Feb 7},
	pages = {1669-71},
	keywords = {Evolution, Molecular, History, 20th Century, History, 21st Century, Humans, Microbiology, Mitochondria, Mycology, United States},
	issn = {0027-8424},
	doi = {10.1073/pnas.0508183103},
	author = {Zagorski, Nick}
}
@article {351,
	title = {Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing.},
	journal = {RNA},
	volume = {12},
	number = {7},
	year = {2006},
	month = {2006 Jul},
	pages = {1271-81},
	abstract = {We show that a targetron based on the Lactococcus lactis Ll.LtrB group II intron can be used for efficient chromosomal gene disruption in the human pathogen Staphylococcus aureus. Targetrons expressed from derivatives of vector pCN37, which uses a cadmium-inducible promoter, or pCN39, a derivative of pCN37 with a temperature-sensitive replicon, gave site-specific disruptants of the hsa and seb genes in 37\%-100\% of plated colonies without selection. To disrupt hsa, an essential gene, we used a group II intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, enabling the production of functional HSa protein. We show that because splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32 degrees C but not 43 degrees C. The temperature sensitivity of the splicing reaction suggests a general means of obtaining one-step conditional disruptions in any organism. In nature, temperature sensitivity of group II intron splicing could limit the temperature range of an organism containing a group II intron inserted in an essential gene.},
	keywords = {Base Sequence, DNA, Bacterial, Escherichia coli, Genes, Bacterial, Genes, Essential, Humans, Introns, Mutagenesis, Insertional, Operon, Reverse Transcriptase Polymerase Chain Reaction, RNA Splicing, RNA, Bacterial, Staphylococcus aureus, Thermodynamics},
	issn = {1355-8382},
	doi = {10.1261/rna.68706},
	author = {Yao, Jun and Zhong, Jin and Fang, Yuan and Geisinger, Edward and Novick, Richard P and Lambowitz, Alan M}
}
@article {362,
	title = {Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase.},
	journal = {RNA},
	volume = {11},
	number = {1},
	year = {2005},
	month = {2005 Jan},
	pages = {14-28},
	abstract = {Group II intron-encoded proteins (IEPs) have both reverse transcriptase (RT) activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active RNA structure. The LtrA protein encoded by the Lactococcus lactis Ll.LtrB group II intron contains an N-terminal RT domain, with conserved sequence motifs RT1 to 7 found in the fingers and palm of retroviral RTs; domain X, associated with maturase activity; and C-terminal DNA-binding and DNA endonuclease domains. Here, partial proteolysis of LtrA with trypsin and Arg-C shows major cleavage sites in RT1, and between the RT and X domains. Group II intron and related non-LTR retroelement RTs contain an N-terminal extension and several insertions relative to retroviral RTs, some with conserved features implying functional importance. Sequence alignments, secondary-structure predictions, and hydrophobicity profiles suggest that domain X is related structurally to the thumb of retroviral RTs. Three-dimensional models of LtrA constructed by "threading" the aligned sequence on X-ray crystal structures of HIV-1 RT (1) account for the proteolytic cleavage sites; (2) suggest a template-primer binding track analogous to that of HIV-1 RT; and (3) show that conserved regions in splicing-competent LtrA variants include regions of the RT and X (thumb) domains in and around the template-primer binding track, distal regions of the fingers, and patches on the protein{\textquoteright}s back surface. These regions potentially comprise an extended RNA-binding surface that interacts with different regions of the intron for RNA splicing and reverse transcription.},
	keywords = {Amino Acid Sequence, Bacterial Proteins, Base Sequence, Binding Sites, Genes, Bacterial, HIV Reverse Transcriptase, HIV-1, Introns, Lactococcus lactis, Models, Molecular, Molecular Sequence Data, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Retroelements, RNA, Bacterial, RNA-Directed DNA Polymerase, Sequence Homology, Amino Acid, Static Electricity},
	issn = {1355-8382},
	doi = {10.1261/rna.7181105},
	author = {Blocker, Forrest J H and Mohr, Georg and Conlan, Lori H and Qi, Li and Belfort, Marlene and Lambowitz, Alan M}
}
@article {360,
	title = {Number, position, and significance of the pseudouridines in the large subunit ribosomal RNA of Haloarcula marismortui and Deinococcus radiodurans.},
	journal = {RNA},
	volume = {11},
	number = {2},
	year = {2005},
	month = {2005 Feb},
	pages = {210-9},
	abstract = {The number and position of the pseudouridines of Haloarcula marismortui and Deinococcus radiodurans large subunit RNA have been determined by a combination of total nucleoside analysis by HPLC-mass spectrometry and pseudouridine sequencing by the reverse transcriptase method and by LC/MS/MS. Three pseudouridines were found in H. marismortui, located at positions 1956, 1958, and 2621 corresponding to Escherichia coli positions 1915, 1917, and 2586, respectively. The three pseudouridines are all in locations found in other organisms. Previous reports of a larger number of pseudouridines in this organism were incorrect. Three pseudouridines and one 3-methyl pseudouridine (m3Psi) were found in D. radiodurans 23S RNA at positions 1894, 1898 (m3Psi), 1900, and 2584, the m3Psi site being determined by a novel application of mass spectrometry. These positions correspond to E. coli positions 1911, 1915, 1917, and 2605, which are also pseudouridines in E. coli (1915 is m3Psi). The pseudouridines in the helix 69 loop, residues 1911, 1915, and 1917, are in positions highly conserved among all phyla. Pseudouridine 2584 in D. radiodurans is conserved in eubacteria and a chloroplast but is not found in archaea or eukaryotes, whereas pseudouridine 2621 in H. marismortui is more conserved in eukaryotes and is not found in eubacteria. All the pseudoridines are near, but not exactly at, nucleotides directly involved in various aspects of ribosome function. In addition, two D. radiodurans Psi synthases responsible for the four Psi were identified.},
	keywords = {Base Sequence, Deinococcus, Escherichia coli, Haloarcula marismortui, Hydro-Lyases, Nucleic Acid Conformation, Pseudouridine, RNA, Archaeal, RNA, Bacterial, RNA, Ribosomal, 23S},
	issn = {1355-8382},
	doi = {10.1261/rna.7209905},
	author = {Del Campo, Mark and Recinos, Claudia and Yanez, Giscard and Pomerantz, Steven C and Guymon, Rebecca and Crain, Pamela F and McCloskey, James A and Ofengand, James}
}
@article {361,
	title = {The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {102},
	number = {1},
	year = {2005},
	month = {2005 Jan 4},
	pages = {163-8},
	abstract = {Group I and II introns self-splice in vitro, but require proteins for efficient splicing in vivo, to stabilize the catalytically active RNA structure. Recent studies showed that the splicing of some Neurospora crassa mitochondrial group I introns additionally requires a DEAD-box protein, CYT-19, which acts as an RNA chaperone to resolve nonnative structures formed during RNA folding. Here we show that, in Saccharomyces cerevisiae mitochondria, a related DEAD-box protein, Mss116p, is required for the efficient splicing of all group I and II introns, some RNA end-processing reactions, and translation of a subset of mRNAs, and that all these defects can be partially or completely suppressed by the expression of CYT-19. Results for the aI2 group II intron indicate that Mss116p is needed after binding the intron-encoded maturase, likely for the disruption of stable but inactive RNA structures. Our results suggest that both group I and II introns are prone to kinetic traps in RNA folding in vivo and that the splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones.},
	keywords = {Amino Acid Motifs, DEAD-box RNA Helicases, Introns, Mitochondria, Molecular Chaperones, Mutation, Protein Biosynthesis, RNA, RNA Helicases, RNA Processing, Post-Transcriptional, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins},
	issn = {0027-8424},
	doi = {10.1073/pnas.0407896101},
	author = {Huang, Hon-Ren and Rowe, Claire E and Mohr, Sabine and Jiang, Yue and Lambowitz, Alan M and Perlman, Philip S}
}
@article {359,
	title = {A tyrosyl-tRNA synthetase adapted to function in group I intron splicing by acquiring a new RNA binding surface.},
	journal = {Mol Cell},
	volume = {17},
	number = {3},
	year = {2005},
	month = {2005 Feb 4},
	pages = {417-28},
	abstract = {We determined a 1.95 A X-ray crystal structure of a C-terminally truncated Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) that functions in splicing group I introns. CYT-18{\textquoteright}s nucleotide binding fold and intermediate alpha-helical domains superimpose on those of bacterial TyrRSs, except for an N-terminal extension and two small insertions not found in nonsplicing bacterial enzymes. These additions surround the cyt-18-1 mutation site and are sites of suppressor mutations that restore splicing, but not synthetase activity. Highly constrained models based on directed hydroxyl radical cleavage assays show that the group I intron binds at a site formed in part by the three additions on the nucleotide binding fold surface opposite that which binds tRNATyr. Our results show how essential proteins can progressively evolve new functions.},
	keywords = {Amino Acid Sequence, Amino Acid Substitution, Catalytic Domain, Crystallography, X-Ray, Genes, Fungal, Hydroxyl Radical, Introns, Models, Molecular, Molecular Sequence Data, Mutagenesis, Insertional, Mutagenesis, Site-Directed, Neurospora crassa, Nucleic Acid Conformation, Protein Structure, Tertiary, RNA Splicing, RNA, Fungal, RNA, Transfer, Amino Acyl, Sequence Homology, Amino Acid, Static Electricity, Tyrosine-tRNA Ligase},
	issn = {1097-2765},
	doi = {10.1016/j.molcel.2004.12.026},
	author = {Paukstelis, Paul J and Coon, Robert and Madabusi, Lakshmi and Nowakowski, Jacek and Monzingo, Arthur and Robertus, Jon and Lambowitz, Alan M}
}
@article {354,
	title = {A bacterial group II intron-encoded reverse transcriptase localizes to cellular poles.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {102},
	number = {45},
	year = {2005},
	month = {2005 Nov 8},
	pages = {16133-40},
	abstract = {The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase (LtrA protein) that binds the intron RNA to promote RNA splicing and intron mobility. Here, we used LtrA-GFP fusions and immunofluorescence microscopy to show that LtrA localizes to cellular poles in Escherichia coli and Lactococcus lactis. This polar localization occurs with or without coexpression of Ll.LtrB intron RNA, is observed over a wide range of cellular growth rates and expression levels, and is independent of replication origin function. The same localization pattern was found for three nonoverlapping LtrA subsegments, possibly reflecting dependence on common redundant signals and/or protein physical properties. When coexpressed in E. coli, LtrA interferes with the polar localization of the Shigella IcsA protein, which mediates polarized actin tail assembly, suggesting competition for a common localization determinant. The polar localization of LtrA could account for the preferential insertion of the Ll.LtrB intron in the origin and terminus regions of the E. coli chromosome, may facilitate access to exposed DNA in these regions, and could potentially link group II intron mobility to the host DNA replication and/or cell division machinery.},
	keywords = {Bacterial Proteins, DNA Replication, DNA-Binding Proteins, Escherichia coli, Green Fluorescent Proteins, Introns, Lactococcus lactis, Microscopy, Fluorescence, Recombinant Fusion Proteins, RNA-Directed DNA Polymerase, Transcription Factors},
	issn = {0027-8424},
	doi = {10.1073/pnas.0507057102},
	author = {Zhao, Junhua and Lambowitz, Alan M}
}
@article {356,
	title = {Gene targeting using randomly inserted group II introns (targetrons) recovered from an Escherichia coli gene disruption library.},
	journal = {Nucleic Acids Res},
	volume = {33},
	number = {10},
	year = {2005},
	month = {2005},
	pages = {3351-62},
	abstract = {The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse-splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it to prime reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base-pairing of the intron RNA. Consequently, group II introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, we show that an Escherichia coli gene disruption library obtained using such randomly inserting Ll.LtrB introns contains most viable E.coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. Additionally, we identified a subset of introns that insert at sites lacking T+5, a nucleotide residue critical for second-strand cleavage. All such introns tested individually gave the desired specific disruption, some by switching to an alternate retrohoming mechanism targeting single-stranded DNA and using a nascent lagging DNA strand to prime reverse transcription.},
	keywords = {Base Pairing, DNA, Escherichia coli, Gene Library, Gene Targeting, Genes, Bacterial, Introns, Lactococcus lactis, Plasmids, Polymerase Chain Reaction, Retroelements},
	issn = {1362-4962},
	doi = {10.1093/nar/gki649},
	author = {Yao, Jun and Zhong, Jin and Lambowitz, Alan M}
}
@article {355,
	title = {Recruitment of host functions suggests a repair pathway for late steps in group II intron retrohoming.},
	journal = {Genes Dev},
	volume = {19},
	number = {20},
	year = {2005},
	month = {2005 Oct 15},
	pages = {2477-87},
	abstract = {Retrohoming of group II introns occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Host repair enzymes are predicted to complete this process. Here, we screened a battery of Escherichia coli mutants defective in host functions that are potentially involved in retrohoming of the Lactococcus lactis Ll.LtrB intron. We found strong (greater than threefold) effects for several enzymes, including nucleases directed against RNA and DNA, replicative and repair polymerases, and DNA ligase. A model including the presumptive roles of these enzymes in resection of DNA, degradation of the intron RNA template, traversion of RNA-DNA junctions, and second-strand DNA synthesis is described. The completion of retrohoming is viewed as a DNA repair process, with features that may be shared by other non-LTR retroelements.},
	keywords = {Bacterial Proteins, DNA Ligases, DNA Repair, DNA Replication, DNA Transposable Elements, DNA, Bacterial, Escherichia coli, Introns, Retroelements, RNA Splicing, RNA, Bacterial},
	issn = {0890-9369},
	doi = {10.1101/gad.1345105},
	author = {Smith, Dorie and Zhong, Jin and Matsuura, Manabu and Lambowitz, Alan M and Belfort, Marlene}
}
@article {357,
	title = {Retargeting mobile group II introns to repair mutant genes.},
	journal = {Mol Ther},
	volume = {11},
	number = {5},
	year = {2005},
	month = {2005 May},
	pages = {687-94},
	abstract = {Retroposable elements such as retroviral and lentiviral vectors have been employed for many gene therapy applications. Unfortunately, such gene transfer vectors integrate genes into many different DNA sequences and unintended integration of the vector near a growth-promoting gene can engender pathological consequences. For example, retroviral vector-mediated gene transfer induced leukemia in 2 of 11 children treated for severe combined immunodeficiency, raising significant safety issues for gene transfer strategies that cannot be targeted to specific sequences. Here, we examine the use of a mobile retroposable genetic element that can be targeted to introduce therapeutic sequences site specifically into mutant genes. The data demonstrate that the mobile group II intron from Lactococcus lactis can be targeted to insert into and repair mutant lacZ (approved gene symbol GLB1) and beta-globin (approved gene symbol HBB) genes with high efficiency and fidelity in model systems in bacteria. These results suggest that these mobile genetic elements represent a novel class of agents for performing targeted genetic repair.},
	keywords = {Animals, Base Sequence, Cell Line, DNA Repair, Globins, Humans, Introns, Lac Operon, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, RNA, Catalytic, Sensitivity and Specificity, Substrate Specificity},
	issn = {1525-0016},
	doi = {10.1016/j.ymthe.2005.01.014},
	author = {Jones, John Patrick and Kierlin, Monique N and Coon, Robert G and Perutka, Jiri and Lambowitz, Alan M and Sullenger, Bruce A}
}
@article {358,
	title = {Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment.},
	journal = {Mol Microbiol},
	volume = {56},
	number = {2},
	year = {2005},
	month = {2005 Apr},
	pages = {509-24},
	abstract = {Group II introns are mobile retroelements that invade their cognate intron-minus gene in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. Previous studies of the Lactococcus lactis intron Ll.LtrB indicated that in its native host, as in Escherichia coli, retrohoming occurs by the intron RNA reverse splicing into double-stranded DNA (dsDNA) through an endonuclease-dependent pathway. However, in retrotransposition in L. lactis, the intron inserts predominantly into single-stranded DNA (ssDNA), in an endonuclease-independent manner. This work describes the retrotransposition of the Ll.LtrB intron in E. coli, using a retrotransposition indicator gene previously employed in our L. lactis studies. Unlike in L. lactis, in E. coli, Ll.LtrB retrotransposed frequently into dsDNA, and the process was dependent on the endonuclease activity of the intron-encoded protein. Further, the endonuclease-dependent insertions preferentially occurred around the origin and terminus of chromosomal DNA replication. Insertions in E. coli can also occur through an endonuclease-independent pathway, and, as in L. lactis, such events have a more random integration pattern. Together these findings show that Ll.LtrB can retrotranspose through at least two distinct mechanisms and that the host environment influences the choice of integration pathway. Additionally, growth conditions affect the insertion pattern. We propose a model in which DNA replication, compactness of the nucleoid and chromosomal localization influence target site preference.},
	keywords = {Bacterial Proteins, DNA Replication, DNA Transposable Elements, DNA, Bacterial, DNA, Single-Stranded, Escherichia coli Proteins, Introns, Lactococcus lactis, Models, Biological, Models, Genetic, Retroelements},
	issn = {0950-382X},
	doi = {10.1111/j.1365-2958.2005.04554.x},
	author = {Coros, Colin J and Landthaler, Markus and Piazza, Carol Lyn and Beauregard, Arthur and Esposito, Donna and Perutka, Jiri and Lambowitz, Alan M and Belfort, Marlene}
}
@article {364,
	title = {A group II intron-encoded maturase functions preferentially in cis and requires both the reverse transcriptase and X domains to promote RNA splicing.},
	journal = {J Mol Biol},
	volume = {340},
	number = {2},
	year = {2004},
	month = {2004 Jul 2},
	pages = {211-31},
	abstract = {Mobile group II introns encode proteins with both reverse transcriptase activity, which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active structure of the intron RNA. Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein binds first to a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of its own coding region, and then makes additional contacts with conserved catalytic core regions to stabilize the active RNA structure. Here, we developed an Escherichia coli genetic assay that links the splicing of the Ll.LtrB intron to the expression of green fluorescent protein and used it to study the in vivo splicing of wild-type and mutant introns and to delineate regions of the maturase required for splicing. Our results show that the maturase functions most efficiently when expressed in cis from the same transcript as the intron RNA. In agreement with previous in vitro assays, we find that the high-affinity binding site in DIVa is required for efficient splicing of the Ll.LtrB intron in vivo, but in the absence of DIVa, 6-10\% residual splicing occurs by the direct binding of the maturase to the catalytic core. Critical regions of the maturase were identified by statistically analyzing ratios of missense to silent mutations in functional LtrA variants isolated from a library generated by mutagenic PCR ("unigenic evolution"). This analysis shows that both the reverse transcriptase domain and domain X, which likely corresponds to the reverse transcriptase thumb, are required for RNA splicing, while the C-terminal DNA-binding and DNA endonuclease domains are not required. Within the reverse transcriptase domain, the most critical regions for maturase activity include parts of the fingers and palm that function in template and primer binding in HIV-1 reverse transcriptase, but the integrity of the reverse transcriptase active site is not required. Biochemical analysis of LtrA mutants indicates that the N terminus of the reverse transcriptase domain is required for high-affinity binding of the intron RNA, possibly via direct interaction with DIVa, while parts of domain X interact with conserved regions of the catalytic core. Our results support the hypothesis that the intron-encoded protein adapted to function in splicing by using, at least in part, interactions used initially to recognize the intron RNA as a template for reverse transcription.},
	keywords = {Base Sequence, Escherichia coli, Introns, Molecular Sequence Data, Nucleic Acid Conformation, Reverse Transcriptase Polymerase Chain Reaction, RNA Splicing, RNA, Bacterial, RNA-Directed DNA Polymerase},
	issn = {0022-2836},
	doi = {10.1016/j.jmb.2004.05.004},
	author = {Cui, Xiaoxia and Matsuura, Manabu and Wang, Qin and Ma, Hongwen and Lambowitz, Alan M}
}
@article {363,
	title = {High-affinity binding site for a group II intron-encoded reverse transcriptase/maturase within a stem-loop structure in the intron RNA.},
	journal = {RNA},
	volume = {10},
	number = {9},
	year = {2004},
	month = {2004 Sep},
	pages = {1433-43},
	abstract = {Mobile group II introns encode proteins that have reverse transcriptase and maturase activities and bind specifically to the intron RNA to promote both RNA splicing and intron mobility. Previous studies with the Lactococcus lactis Ll.LtrB intron showed that the intron-encoded protein (LtrA) has a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure containing the translation initiation region of the LtrA open reading frame, and that this binding site consists of a small stem-loop emanating from a purine-rich internal loop. The binding of LtrA to DIVa is important for translational regulation, RNA splicing, and intron mobility. Here, we show by in vitro selection that part of the purine-rich internal loop can be closed by base pairing, enabling the LtrA binding site to be represented as an extended stem-loop structure with a bulged A (A556) required for tight binding of LtrA. The deletion or pairing of A556 has relatively little effect on maturase-promoted RNA splicing, but significantly inhibits intron mobility. The wild-type DIVa structure has a second bulged A (A553), which is selected against in tightly binding variants. As expected from the selection, the deletion or pairing of A553 results in tighter binding of LtrA, but surprisingly, also inhibits intron mobility. These findings suggest that the binding of LtrA to DIVa is delicately balanced, so that either too weak or too tight binding can be deleterious. The nature of the maturase/DIVa interaction and its role in translational regulation are reminiscent of the coat protein/RNA hairpin interactions of single-stranded RNA phages.},
	keywords = {Bacterial Proteins, Base Pairing, Base Sequence, Binding Sites, Introns, Lactococcus lactis, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, Protein Biosynthesis, RNA Splicing, RNA, Bacterial, RNA-Directed DNA Polymerase, Sequence Deletion, Sequence Homology, Nucleic Acid},
	issn = {1355-8382},
	doi = {10.1261/rna.7730104},
	author = {Watanabe, Kazuo and Lambowitz, Alan M}
}
@article {367,
	title = {Mobile group II introns.},
	journal = {Annu Rev Genet},
	volume = {38},
	year = {2004},
	month = {2004},
	pages = {1-35},
	abstract = {Mobile group II introns, found in bacterial and organellar genomes, are both catalytic RNAs and retrotransposable elements. They use an extraordinary mobility mechanism in which the excised intron RNA reverse splices directly into a DNA target site and is then reverse transcribed by the intron-encoded protein. After DNA insertion, the introns remove themselves by protein-assisted, autocatalytic RNA splicing, thereby minimizing host damage. Here we discuss the experimental basis for our current understanding of group II intron mobility mechanisms, beginning with genetic observations in yeast mitochondria, and culminating with a detailed understanding of molecular mechanisms shared by organellar and bacterial group II introns. We also discuss recently discovered links between group II intron mobility and DNA replication, new insights into group II intron evolution arising from bacterial genome sequencing, and the evolutionary relationship between group II introns and both eukaryotic spliceosomal introns and non-LTR-retrotransposons. Finally, we describe the development of mobile group II introns into gene-targeting vectors, "targetrons," which have programmable target specificity.},
	keywords = {Bacteria, Base Sequence, DNA Replication, DNA, Mitochondrial, Evolution, Molecular, Gene Targeting, Introns, Models, Genetic, Models, Molecular, Molecular Sequence Data, Molecular Structure, Phylogeny, Retroelements, RNA Splicing, RNA, Catalytic, Spliceosomes, Yeasts},
	issn = {0066-4197},
	doi = {10.1146/annurev.genet.38.072902.091600},
	author = {Lambowitz, Alan M and Zimmerly, Steven}
}
@article {365,
	title = {The Neurospora crassa CYT-18 protein C-terminal RNA-binding domain helps stabilize interdomain tertiary interactions in group I introns.},
	journal = {RNA},
	volume = {10},
	number = {4},
	year = {2004},
	month = {2004 Apr},
	pages = {634-44},
	abstract = {The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by stabilizing the catalytically active RNA structure. To accomplish this, CYT-18 recognizes conserved structural features of group I intron RNAs using regions of the N-terminal nucleotide-binding fold, intermediate alpha-helical, and C-terminal RNA-binding domains that also function in binding tRNA(Tyr). Curiously, whereas the splicing of the N. crassa mitochondrial large subunit rRNA intron is completely dependent on CYT-18{\textquoteright}s C-terminal RNA-binding domain, all other group I introns tested thus far are spliced efficiently by a truncated protein lacking this domain. To investigate the function of the C-terminal domain, we used an Escherichia coli genetic assay to isolate mutants of the Saccharomyces cerevisiae mitochondrial large subunit rRNA and phage T4 td introns that can be spliced in vivo by the wild-type CYT-18 protein, but not by the C-terminally truncated protein. Mutations that result in dependence on CYT-18{\textquoteright}s C-terminal domain include those disrupting two long-range GNRA tetraloop/receptor interactions: L2-P8, which helps position the P1 helix containing the 5{\textquoteright}-splice site, and L9-P5, which helps establish the correct relative orientation of the P4-P6 and P3-P9 domains of the group I intron catalytic core. Our results indicate that different structural mutations in group I intron RNAs can result in dependence on different regions of CYT-18 for RNA splicing.},
	keywords = {Introns, Mitochondria, Mutation, Neurospora crassa, Nucleic Acid Conformation, Protein Structure, Tertiary, Saccharomyces cerevisiae, Sequence Analysis, Protein, Tyrosine-tRNA Ligase},
	issn = {1355-8382},
	author = {Chen, Xin and Mohr, Georg and Lambowitz, Alan M}
}
@article {366,
	title = {Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes.},
	journal = {J Mol Biol},
	volume = {336},
	number = {2},
	year = {2004},
	month = {2004 Feb 13},
	pages = {421-39},
	abstract = {Mobile group II introns are site-specific retroelements that use a novel mobility mechanism in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the associated intron-encoded protein. Because the DNA target site is recognized primarily by base-pairing of the intron RNA with only a small number of positions recognized by the protein, it has been possible to develop group II introns into a new type of gene targeting vector ("targetron"), which can be reprogrammed to insert into desired DNA targets simply by modifying the intron RNA. Here, we used databases of retargeted Lactococcus lactis Ll.LtrB group II introns and a compilation of nucleotide frequencies at active target sites to develop an algorithm that predicts optimal Ll.LtrB intron-insertion sites and designs primers for modifying the intron to insert into those sites. In a test of the algorithm, we designed one or two targetrons to disrupt each of 28 Escherichia coli genes encoding DExH/D-box and DNA helicase-related proteins and tested for the desired disruptants by PCR screening of 100 colonies. In 21 cases, we obtained disruptions at frequencies of 1-80\% without selection, and in six other cases, where disruptants were not identified in the initial PCR screen, we readily obtained specific disruptions by using the same targetrons with a retrotransposition-activated selectable marker. Only one DExH/D-box protein gene, secA, which was known to be essential, did not give viable disruptants. The apparent dispensability of DExH/D-box proteins in E.coli contrasts with the situation in yeast, where the majority of such proteins are essential. The methods developed here should permit the rapid and efficient disruption of any bacterial gene, the computational analysis provides new insight into group II intron target site recognition, and the set of E.coli DExH/D-box protein and DNA helicase disruptants should be useful for analyzing the function of these proteins.},
	keywords = {Algorithms, Amino Acid Motifs, Base Pairing, Base Sequence, Blotting, Southern, Computational Biology, DNA Helicases, Escherichia coli Proteins, Introns, Lactococcus lactis, Mutagenesis, Site-Directed},
	issn = {0022-2836},
	author = {Perutka, Jiri and Wang, Wenjun and Goerlitz, David and Lambowitz, Alan M}
}
@article {369,
	title = {Effects of maturase binding and Mg2+ concentration on group II intron RNA folding investigated by UV cross-linking.},
	journal = {Biochemistry},
	volume = {42},
	number = {43},
	year = {2003},
	month = {2003 Nov 4},
	pages = {12466-80},
	abstract = {The Lactococcus lactis Ll.LtrB group II intron encodes a reverse transcriptase/maturase (LtrA protein) that promotes RNA splicing by stabilizing the catalytically active RNA structure. Here, we mapped 17 UV cross-links induced in both wild-type Ll.LtrB RNA and Ll.LtrB-Delta2486 RNA, which has a branch-point deletion that prevents splicing, and we used these cross-links to follow tertiary structure formation under different conditions in the presence or absence of the LtrA protein. Twelve of the cross-links are long-range, with six near known tertiary interaction sites in the active RNA structure. In a reaction medium containing 0.5 M NH(4)Cl, eight of the 17 cross-links were detected in the absence of Mg(2+) or the presence of EDTA, and all were detected at 5 mM Mg(2+), where efficient splicing requires the LtrA protein. The frequencies of all but four cross-links increased with increasing Mg(2+) concentrations, becoming maximal between 4 and 50 mM Mg(2+), where the intron is self-splicing. These findings suggest that a high Mg(2+) concentration induces self-splicing by globally stabilizing tertiary structure, including key tertiary interactions that are required for catalytic activity. Significantly, the binding of the maturase under protein-dependent splicing conditions (0.5 M NH(4)Cl and 5 mM Mg(2+)) increased the frequency of only nine cross-links, seven of which are long-range, suggesting that, in contrast to a high Mg(2+) concentration, LtrA promotes splicing by stabilizing critical tertiary structure interactions, while leaving other regions of the intron relatively flexible. This difference may contribute to the high rate of protein-dependent splicing, relative to the rate of self-splicing. The propensity of the intron RNA to form tertiary structure even at relatively low Mg(2+) concentrations raises the possibility that the maturase functions at least in part by tertiary structure capture. Finally, an abundant central wheel cross-link, present in >50\% of the molecules at 5 mM Mg(2+), suggests models in which group II intron domains I and II are either coaxially stacked or aligned in parallel, bringing the 5{\textquoteright}-splice site together with the 3{\textquoteright}-splice site and catalytic core elements at JII/III. This and other cross-links provide new constraints for three-dimensional structural modeling of the group II intron catalytic core.},
	keywords = {Base Sequence, DNA Primers, Introns, Kinetics, Magnesium, Molecular Sequence Data, Nucleic Acid Conformation, Protein Binding, Protein Structure, Tertiary, RNA Splicing, RNA, Bacterial, RNA-Directed DNA Polymerase, Ultraviolet Rays},
	issn = {0006-2960},
	doi = {10.1021/bi035339n},
	author = {Noah, James W and Lambowitz, Alan M}
}
@article {374,
	title = {Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection.},
	journal = {Appl Environ Microbiol},
	volume = {69},
	number = {2},
	year = {2003},
	month = {2003 Feb},
	pages = {1121-8},
	abstract = {Despite their commercial importance, there are relatively few facile methods for genomic manipulation of the lactic acid bacteria. Here, the lactococcal group II intron, Ll.ltrB, was targeted to insert efficiently into genes encoding malate decarboxylase (mleS) and tetracycline resistance (tetM) within the Lactococcus lactis genome. Integrants were readily identified and maintained in the absence of a selectable marker. Since splicing of the Ll.ltrB intron depends on the intron-encoded protein, targeted invasion with an intron lacking the intron open reading frame disrupted TetM and MleS function, and MleS activity could be partially restored by expressing the intron-encoded protein in trans. Restoration of splicing from intron variants lacking the intron-encoded protein illustrates how targeted group II introns could be used for conditional expression of any gene. Furthermore, the modified Ll.ltrB intron was used to separately deliver a phage resistance gene (abiD) and a tetracycline resistance marker (tetM) into mleS, without the need for selection to drive the integration or to maintain the integrant. Our findings demonstrate the utility of targeted group II introns as a potential food-grade mechanism for delivery of industrially important traits into the genomes of lactococci.},
	keywords = {Bacterial Proteins, Base Sequence, Carboxy-Lyases, Conjugation, Genetic, DNA Transposable Elements, Genetic Engineering, Introns, Lactococcus lactis, Molecular Sequence Data, Recombination, Genetic, RNA Splicing, Tetracycline Resistance},
	issn = {0099-2240},
	author = {Frazier, Courtney L and San Filippo, Joseph and Lambowitz, Alan M and Mills, David A}
}
@article {370,
	title = {Group II intron mobility using nascent strands at DNA replication forks to prime reverse transcription.},
	journal = {EMBO J},
	volume = {22},
	number = {17},
	year = {2003},
	month = {2003 Sep 1},
	pages = {4555-65},
	abstract = {The Lactococcus lactis Ll.LtrB group II intron uses a major retrohoming mechanism in which the excised intron RNA reverse splices into one strand of a DNA target site, while the intron-encoded protein uses a C-terminal DNA endonuclease domain to cleave the opposite strand and then uses the cleaved 3{\textquoteright} end as a primer for reverse transcription of the inserted intron RNA. Here, experiments with mutant introns and target sites indicate that Ll.LtrB can retrohome without second-strand cleavage by using a nascent strand at a DNA replication fork as the primer for reverse transcription. This mechanism connecting intron mobility to target DNA replication may be used by group II intron species that encode proteins lacking the C-terminal DNA endonuclease domain and for group II intron retrotransposition to ectopic sites.},
	keywords = {Bacterial Proteins, Base Sequence, Binding Sites, DNA Replication, DNA Transposable Elements, DNA, Bacterial, Genes, Bacterial, Introns, Lactococcus lactis, Models, Biological, Mutation, RNA Splicing, RNA, Bacterial, Transcription, Genetic},
	issn = {0261-4189},
	doi = {10.1093/emboj/cdg433},
	author = {Zhong, Jin and Lambowitz, Alan M}
}
@article {371,
	title = {Mobility of the Sinorhizobium meliloti group II intron RmInt1 occurs by reverse splicing into DNA, but requires an unknown reverse transcriptase priming mechanism.},
	journal = {J Mol Biol},
	volume = {327},
	number = {5},
	year = {2003},
	month = {2003 Apr 11},
	pages = {931-43},
	abstract = {The mobile group II introns characterized to date encode ribonucleoprotein complexes that promote mobility by a major retrohoming mechanism in which the intron RNA reverse splices directly into the sense strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase/maturase cleaves the antisense strand and uses it as primer for reverse transcription of the inserted intron RNA. Here, we show that the Sinorhizobium meliloti group II intron RmInt1, which encodes a protein lacking a DNA endonuclease domain, similarly uses both the intron RNA and an intron-encoded protein with reverse transcriptase and maturase activities for mobility. However, while RmInt1 reverse splices into both single-stranded and double-stranded DNA target sites, it is unable to carry out site-specific antisense-strand cleavage due to the lack of a DNA endonuclease domain. Our results suggest that RmInt1 mobility involves reverse splicing into double-stranded or single-stranded DNA target sites, but due to the lack of DNA endonuclease function, it requires an alternate means of procuring a primer for target DNA-primed reverse transcription.},
	keywords = {Base Sequence, DNA, Bacterial, Introns, Molecular Sequence Data, RNA Splicing, RNA-Directed DNA Polymerase, Sinorhizobium meliloti},
	issn = {0022-2836},
	author = {Mu{\~n}oz-Adelantado, Estefan{\'\i}a and San Filippo, Joseph and Mart{\'\i}nez-Abarca, Francisco and Garc{\'\i}a-Rodr{\'\i}guez, Fernando M and Lambowitz, Alan M and Toro, Nicol{\'a}s}
}
@article {373,
	title = {The pathway for DNA recognition and RNA integration by a group II intron retrotransposon.},
	journal = {Mol Cell},
	volume = {11},
	number = {3},
	year = {2003},
	month = {2003 Mar},
	pages = {795-805},
	abstract = {Group II intron RNPs are mobile genetic elements that attack and invade duplex DNA. In this work, we monitor the invasion reaction in vitro and establish a quantitative kinetic framework for the steps of this complex cascade. We find that target site specificity is achieved after DNA binding, which occurs nonspecifically. RNP searches the bound DNA before undergoing a conformational change that is associated with identification of its specific binding site. The study reveals a facile equilibrium between intron invasion and splicing, indicating that RNP invasion of top strand DNA is a relatively unfavorable event. Group II mobility must therefore depend on the trapping of invasion products, potentially through interaction of the intron-encoded protein with the DNA target and/or initiation of reverse transcription.},
	keywords = {Bacterial Proteins, Base Sequence, Binding Sites, DNA, Dose-Response Relationship, Drug, Introns, Kinetics, Models, Biological, Molecular Sequence Data, Oligonucleotides, Retroelements, RNA, RNA-Directed DNA Polymerase, Time Factors},
	issn = {1097-2765},
	author = {Aizawa, Yasunori and Xiang, Qing and Lambowitz, Alan M and Pyle, Anna Marie}
}
@article {375,
	title = {Putative proteins related to group II intron reverse transcriptase/maturases are encoded by nuclear genes in higher plants.},
	journal = {Nucleic Acids Res},
	volume = {31},
	number = {2},
	year = {2003},
	month = {2003 Jan 15},
	pages = {647-52},
	abstract = {The Arabidopsis thaliana nuclear genome sequence revealed several open reading frames encoding proteins related to group II intron-encoded reverse transcriptase/maturases. Here, we show via sequence alignments that at least four such open reading frames are conserved in the nuclear genomes of A.thaliana and Oryza sativa (rice) and that they encode putative proteins belonging to two different classes (nMat-1 and nMat-2), neither of which is associated with a group II intron RNA structure. The two nMat-1 proteins have reverse transcriptase, maturase and DNA endonuclease domains characteristic of canonical group II intron-encoded proteins, while the two nMat-2 proteins have reverse transcriptase and maturase domains linked to a novel C-terminal domain. Although some nMat proteins have mutations expected to inactivate intron mobility functions, all could potentially retain the RNA splicing function. These nuclear maturase-like proteins may be imported into organelles to function in group II intron splicing and/or they may have assumed other cellular functions. Nuclear-encoded maturases could regulate organellar gene expression and may reflect a step in the evolution of mobile group II introns into spliceosomal introns.},
	keywords = {Amino Acid Sequence, Arabidopsis, Cell Nucleus, Chromosome Mapping, Chromosomes, Plant, Endoribonucleases, Genome, Plant, Introns, Molecular Sequence Data, Nucleotidyltransferases, Open Reading Frames, Oryza sativa, Phylogeny, Plant Proteins, RNA-Directed DNA Polymerase, Sequence Homology, Amino Acid},
	issn = {1362-4962},
	author = {Mohr, Georg and Lambowitz, Alan M}
}
@article {372,
	title = {Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker.},
	journal = {Nucleic Acids Res},
	volume = {31},
	number = {6},
	year = {2003},
	month = {2003 Mar 15},
	pages = {1656-64},
	abstract = {Mobile group II introns have been used to develop a novel class of gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. Here, we have developed a targetron containing a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial gene disruption at near 100\% efficiency after selection. The targetron can be generated via PCR without cloning, and after intron integration, the marker gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential disruptions. We also show that a RAM-targetron with randomized target site recognition sequences yields single insertions throughout the Escherichia coli genome, creating a gene knockout library. Analysis of the randomly selected insertion sites provides further insight into group II intron target site recognition rules. It also suggests that a subset of retrohoming events may occur by using a primer generated during DNA replication, and reveals a previously unsuspected bias for group II intron insertion near the chromosome replication origin. This insertional bias likely reflects at least in part the higher copy number of origin proximal genes, but interaction with the replication machinery or other features of DNA structure or packaging may also contribute.},
	keywords = {Base Sequence, Binding Sites, Chromosomes, Bacterial, DNA, Bacterial, Gene Targeting, Genes, Bacterial, Genetic Markers, Genetic Vectors, Genome, Bacterial, Introns, Mutagenesis, Insertional, Polymerase Chain Reaction, Recombination, Genetic, Retroelements},
	issn = {1362-4962},
	author = {Zhong, Jin and Karberg, Michael and Lambowitz, Alan M}
}
@article {368,
	title = {The DIVa maturase binding site in the yeast group II intron aI2 is essential for intron homing but not for in vivo splicing.},
	journal = {Mol Cell Biol},
	volume = {23},
	number = {23},
	year = {2003},
	month = {2003 Dec},
	pages = {8809-19},
	abstract = {Splicing of the Saccharomyces cerevisiae mitochondrial DNA group II intron aI2 depends on the intron-encoded 62-kDa reverse transcriptase-maturase protein (p62). In wild-type strains, p62 remains associated with the excised intron lariat RNA in ribonucleoprotein (RNP) particles that are essential for intron homing. Studies of a bacterial group II intron showed that the DIVa substructure of intron domain IV is a high-affinity binding site for its maturase. Here we first present in vitro evidence extending that conclusion to aI2. Then, experiments with aI2 DIVa mutant strains show that the binding of p62 to DIVa is not essential for aI2 splicing in vivo but is essential for homing. Because aI2 splicing in the DIVa mutant strains remains maturase dependent, splicing must rely on other RNA-protein contacts. The p62 that accumulates in the mutant strains has reverse transcriptase activity, but fractionation experiments at high and low salt concentrations show that it associates more weakly than the wild-type protein with endogenous mitochondrial RNAs, and that phenotype probably explains the homing defect. Replacing the DIVa of aI2 with that of the closely related intron aI1 improves in vivo splicing but not homing, indicating that DIVa contributes to the specificity of the maturase-RNA interaction needed for homing.},
	keywords = {Base Sequence, Binding Sites, DNA, Fungal, DNA, Mitochondrial, Genetic Complementation Test, Introns, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, Open Reading Frames, RNA, RNA Splicing, RNA, Fungal, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins},
	issn = {0270-7306},
	author = {Huang, Hon-Ren and Chao, Michael Y and Armstrong, Barbara and Wang, Yong and Lambowitz, Alan M and Perlman, Philip S}
}
@article {379,
	title = {tRNA-like recognition of group I introns by a tyrosyl-tRNA synthetase.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {99},
	number = {5},
	year = {2002},
	month = {2002 Mar 5},
	pages = {2630-5},
	abstract = {The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active RNA structure. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. Here, directed hydroxyl-radical cleavage assays show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNA(Tyr) cocrystal structure. Our results support the hypothesis that CYT-18 and other aminoacyl-tRNA synthetases interact with group I introns by recognizing conserved tRNA-like structural features of the intron RNAs.},
	keywords = {Cloning, Molecular, Cysteine, Hydroxyl Radical, Introns, Models, Molecular, Neurospora crassa, Nucleic Acid Conformation, Protein Structure, Quaternary, RNA, Transfer, Tyrosine-tRNA Ligase},
	issn = {0027-8424},
	doi = {10.1073/pnas.052596299},
	author = {Myers, Christopher A and Kuhla, Birte and Cusack, Stephen and Lambowitz, Alan M}
}
@article {378,
	title = {Binding of a group II intron-encoded reverse transcriptase/maturase to its high affinity intron RNA binding site involves sequence-specific recognition and autoregulates translation.},
	journal = {J Mol Biol},
	volume = {318},
	number = {2},
	year = {2002},
	month = {2002 Apr 26},
	pages = {287-303},
	abstract = {Mobile group II introns encode reverse transcriptases that bind specifically to the intron RNAs to promote both intron mobility and RNA splicing (maturase activity). Previous studies with the Lactococcus lactis Ll.LtrB intron suggested a model in which the intron-encoded protein (LtrA) binds first to a primary high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of the LtrA coding sequence, and then makes additional contacts with conserved regions of the intron to fold the RNA into the catalytically active structure. Here, we analyzed the DIVa binding site by iterative in vitro selection and in vitro mutagenesis. Our results show that LtrA binds to a small region at the distal end of DIVa that contains the ribosome-binding site and initiation codon of the LtrA open reading frame. The critical elements are in a small stem-loop structure emanating from a purine-rich internal loop, with both sequence and structure playing a role in LtrA recognition. The ribosome-binding site falls squarely within the LtrA-binding region and is sequestered directly by the binding of LtrA or by stabilization of the small stem-loop or both. Finally, by using LacZ fusions in Escherichia coli, we show that the binding of LtrA to DIVa down-regulates translation. This mode of regulation limits accumulation of the potentially deleterious intron-encoded protein and may facilitate splicing by halting ribosome entry into the intron. The recognition of the DIVa loop-stem-loop structure accounts, in part, for the intron specificity of group II intron maturases and has parallels in template-recognition mechanisms used by other reverse transcriptases.},
	keywords = {Bacterial Proteins, Base Sequence, Binding Sites, Escherichia coli, Genes, Reporter, Genetic Variation, Introns, Lac Operon, Lactococcus lactis, Models, Genetic, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, Protein Biosynthesis, RNA Splicing, RNA, Bacterial, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae Proteins},
	issn = {0022-2836},
	doi = {10.1016/S0022-2836(02)00054-2},
	author = {Singh, Ravindra N and Saldanha, Roland J and D{\textquoteright}Souza, Lisa M and Lambowitz, Alan M}
}
@article {376,
	title = {Characterization of the C-terminal DNA-binding/DNA endonuclease region of a group II intron-encoded protein.},
	journal = {J Mol Biol},
	volume = {324},
	number = {5},
	year = {2002},
	month = {2002 Dec 13},
	pages = {933-51},
	abstract = {Group II intron retrohoming occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase uses a C-terminal DNA endonuclease activity to cleave the opposite strand and then uses the cleaved 3{\textquoteright} end as a primer for reverse transcription of the inserted intron RNA. Here, we characterized the C-terminal DNA-binding/DNA endonuclease region of the LtrA protein encoded by the Lactococcus lactis Ll.LtrB intron. This C-terminal region consists of an upstream segment that contributes to DNA binding, followed by a DNA endonuclease domain that contains conserved sequence motifs characteristic of H-N-H DNA endonucleases, interspersed with two pairs of conserved cysteine residues. Atomic emission spectroscopy of wild-type and mutant LtrA proteins showed that the DNA endonuclease domain contains a single tightly bound Mg(2+) ion at the H-N-H active site. Although the conserved cysteine residue pairs could potentially bind Zn(2+), the purified LtrA protein is active despite the presence of only sub-stoichiometric amounts of Zn(2+), and the addition of exogenous Zn(2+) inhibits the DNA endonuclease activity. Multiple sequence alignments identified features of the DNA-binding region and DNA endonuclease domain that are conserved in LtrA and related group II intron proteins, and their functional importance was demonstrated by unigenic evolution analysis and biochemical assays of mutant LtrA protein with alterations in key amino acid residues. Notably, deletion of the DNA endonuclease domain or mutations in its conserved sequence motifs strongly inhibit reverse transcriptase activity, as well as bottom-strand cleavage, while retaining other activities of the LtrA protein. A UV-cross-linking assay showed that these DNA endonuclease domain mutations do not block DNA primer binding and thus likely inhibit reverse transcriptase activity either by affecting the positioning of the primer or the conformation of the reverse transcriptase domain.},
	keywords = {Amino Acid Motifs, Amino Acid Sequence, Bacterial Proteins, Binding Sites, Catalysis, Cations, Divalent, Colicins, Conserved Sequence, DNA Primers, DNA-Binding Proteins, Endodeoxyribonucleases, Introns, Lactococcus lactis, Magnesium, Models, Molecular, Molecular Sequence Data, Mutation, Protein Binding, Protein Structure, Tertiary, RNA-Directed DNA Polymerase, Sequence Alignment, Zinc},
	issn = {0022-2836},
	author = {San Filippo, Joseph and Lambowitz, Alan M}
}
@article {377,
	title = {A DEAD-box protein functions as an ATP-dependent RNA chaperone in group I intron splicing.},
	journal = {Cell},
	volume = {109},
	number = {6},
	year = {2002},
	month = {2002 Jun 14},
	pages = {769-79},
	abstract = {The Neurospora crassa CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in splicing group I introns by inducing formation of the catalytically active RNA structure. Here, we identified a DEAD-box protein (CYT-19) that functions in concert with CYT-18 to promote group I intron splicing in vivo and vitro. CYT-19 does not bind specifically to group I intron RNAs and instead functions as an ATP-dependent RNA chaperone to destabilize nonnative RNA structures that constitute kinetic traps in the CYT-18-assisted RNA-folding pathway. Our results demonstrate that a DExH/D-box protein has a specific, physiologically relevant chaperone function in the folding of a natural RNA substrate.},
	keywords = {Adenosine Triphosphatases, Adenosine Triphosphate, Alternative Splicing, Base Sequence, Blotting, Northern, Cloning, Molecular, Dose-Response Relationship, Drug, Fungal Proteins, Immunoblotting, Introns, Kinetics, Models, Genetic, Molecular Sequence Data, Mutation, Neurospora crassa, Nucleic Acid Conformation, Open Reading Frames, Phenotype, RNA, Substrate Specificity, Temperature, Time Factors},
	issn = {0092-8674},
	author = {Mohr, Sabine and Stryker, John M and Lambowitz, Alan M}
}
@article {385,
	title = {Function of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase in RNA splicing. Role of the idiosyncratic N-terminal extension and different modes of interaction with different group I introns.},
	journal = {J Mol Biol},
	volume = {307},
	number = {1},
	year = {2001},
	month = {2001 Mar 16},
	pages = {75-92},
	abstract = {The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by helping the intron RNA fold into the catalytically active structure. The regions required for splicing include an idiosyncratic N-terminal extension, the nucleotide-binding fold domain, and the C-terminal RNA-binding domain. Here, we show that the idiosyncratic N-terminal region is in fact comprised of two functionally distinct parts: an upstream region consisting predominantly of a predicted amphipathic alpha-helix (H0), which is absent from bacterial tyrosyl-tRNA synthetases (TyrRSs), and a downstream region, which contains predicted alpha-helices H1 and H2, corresponding to features in the X-ray crystal structure of the Bacillus stearothermophilus TyrRS. Bacterial genetic assays with libraries of CYT-18 mutants having random mutations in the N-terminal region identified functionally important amino acid residues and supported the predicted structures of the H0 and H1 alpha-helices. The function of N and C-terminal domains of CYT-18 was investigated by detailed biochemical analysis of deletion mutants. The results confirmed that the N-terminal extension is required only for splicing activity, but surprisingly, at least in the case of the N. crassa mitochondrial (mt) large ribosomal subunit (LSU) intron, it appears to act primarily by stabilizing the structure of another region that interacts directly with the intron RNA. The H1/H2 region is required for splicing activity and TyrRS activity with the N. crassa mt tRNA(Tyr), but not for TyrRS activity with Escherichia coli tRNA(Tyr), implying a somewhat different mode of recognition of the two tyrosyl-tRNAs. Finally, a CYT-18 mutant lacking the N-terminal H0 region is totally defective in binding or splicing the N. crassa ND1 intron, but retains substantial residual activity with the mt LSU intron, and conversely, a CYT-18 mutant lacking the C-terminal RNA-binding domain is totally defective in binding or splicing the mt LSU intron, but retains substantial residual activity with the ND1 intron. These findings lead to the surprising conclusion that CYT-18 promotes splicing via different sets of interactions with different group I introns. We suggest that these different modes of promoting splicing evolved from an initial interaction based on the recognition of conserved tRNA-like structural features of the group I intron catalytic core.},
	keywords = {Adenosine Monophosphate, Amino Acid Sequence, Gene Deletion, Introns, Kinetics, Molecular Sequence Data, Neurospora crassa, Protein Conformation, Protein Structure, Tertiary, Recombinant Proteins, RNA, RNA Splicing, Sequence Homology, Amino Acid, Tyrosine, Tyrosine-tRNA Ligase},
	issn = {0022-2836},
	doi = {10.1006/jmbi.2000.4460},
	author = {Mohr, G and Rennard, R and Cherniack, A D and Stryker, J and Lambowitz, A M}
}
@article {381,
	title = {Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria.},
	journal = {Nat Biotechnol},
	volume = {19},
	number = {12},
	year = {2001},
	month = {2001 Dec},
	pages = {1162-7},
	abstract = {Mobile group II introns can be retargeted to insert into virtually any desired DNA target. Here we show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1-22\%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria.},
	keywords = {Base Sequence, Blotting, Southern, Chromosomes, Codon, Terminator, Escherichia coli, Gene Library, Gene Transfer Techniques, Genes, Bacterial, Genetic Engineering, Genetic Vectors, Introns, Molecular Sequence Data, Plasmids, Point Mutation, Polymerase Chain Reaction, Recombination, Genetic},
	issn = {1087-0156},
	doi = {10.1038/nbt1201-1162},
	author = {Karberg, M and Guo, H and Zhong, J and Coon, R and Perutka, J and Lambowitz, A M}
}
@article {383,
	title = {Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference.},
	journal = {J Mol Biol},
	volume = {309},
	number = {2},
	year = {2001},
	month = {2001 Jun 1},
	pages = {361-86},
	abstract = {Group II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA. The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences. Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches. In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position -25 to +19 from the intron-insertion site on the top strand and from -28 to +16 on the bottom strand. Our results suggest that the protein first recognizes a small number of specific bases in the distal 5{\textquoteright}-exon region of the DNA target site via major-groove interactions. These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions -12 to +3 for reverse splicing. Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3{\textquoteright} exon. Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3{\textquoteright} exon, the most critical being T+5 in the now single-stranded downstream region of the target site. Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site.},
	keywords = {Adenine, Base Pairing, Base Sequence, Binding Sites, Deoxyribonuclease I, Deoxyuridine, DNA, DNA Footprinting, DNA Methylation, DNA-Binding Proteins, Endonucleases, Exons, Introns, Kinetics, Lactococcus lactis, Models, Molecular, Molecular Sequence Data, Mutation, Nucleic Acid Denaturation, Potassium Permanganate, Protein Structure, Tertiary, Ribonucleoproteins, RNA Splicing, Substrate Specificity},
	issn = {0022-2836},
	doi = {10.1006/jmbi.2001.4658},
	author = {Singh, N N and Lambowitz, A M}
}
@article {384,
	title = {Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) with the group I intron P4-P6 domain. Thermodynamic analysis and the role of metal ions.},
	journal = {J Mol Biol},
	volume = {308},
	number = {2},
	year = {2001},
	month = {2001 Apr 27},
	pages = {165-90},
	abstract = {The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron{\textquoteright}s catalytic core. Previous studies suggested a model in which the protein binds first to the intron{\textquoteright}s P4-P6 domain, and then makes additional contacts with the P3-P9 domain to stabilize the two domains in the correct relative orientation to form the intron{\textquoteright}s active site. Here, we analyzed the interaction of CYT-18 with a small RNA (P4-P6 RNA) corresponding to the isolated P4-P6 domain of the N. crassa mitochondrial large subunit ribosomal RNA intron. RNA footprinting and modification-interference experiments showed that CYT-18 binds to this small RNA around the junction of the P4-P6 stacked helices on the side opposite the active-site cleft, as it does to the P4-P6 domain in the intact intron. The binding is inhibited by chemical modifications that disrupt base-pairing in P4, P6, and P6a, indicating that a partially folded structure of the P4-P6 domain is required. The temperature-dependence of binding indicates that the interaction is driven by a favorable enthalpy change, but is accompanied by an unfavorable entropy change. The latter may reflect entropically unfavorable conformational changes or decreased conformational flexibility in the complex. CYT-18 binding is inhibited at > or =125 mM KCl, indicating a strong dependence on phosphodiester-backbone interactions. On the other hand, Mg(2+) is absolutely required for CYT-18 binding, with titration experiments showing approximately 1.5 magnesium ions bound per complex. Metal ion-cleavage experiments identified a divalent cation-binding site near the boundary of P6 and J6/6a, and chemical modification showed that Mg(2+) binding induces RNA conformational changes in this region, as well as elsewhere, particularly in J4/5. Together, these findings suggest a model in which the binding of Mg(2+) near J6/6a and possibly at one additional location in the P4-P6 RNA induces formation of a specific phosphodiester-backbone geometry that is required for CYT-18 binding. The binding of CYT-18 may then establish the correct structure at the junction of the P4/P6 stacked helices for assembly of the P3-P9 domain. The interaction of CYT-18 with the P4-P6 domain appears similar to the TyrRS interaction with the D-/anticodon arm stacked helices of tRNA(Tyr).},
	keywords = {Base Sequence, Cations, Diethyl Pyrocarbonate, Entropy, Ethylnitrosourea, Introns, Iodine, Magnesium, Metals, Mitochondria, Molecular Sequence Data, Neurospora crassa, Nucleic Acid Conformation, Pliability, Potassium Chloride, Protein Binding, RNA, Fungal, RNA, Transfer, Tyr, RNA-Binding Proteins, Sulfuric Acid Esters, Temperature, Tyrosine-tRNA Ligase},
	issn = {0022-2836},
	doi = {10.1006/jmbi.2001.4581},
	author = {Caprara, M G and Myers, C A and Lambowitz, A M}
}
@article {380,
	title = {Mechanism of maturase-promoted group II intron splicing.},
	journal = {EMBO J},
	volume = {20},
	number = {24},
	year = {2001},
	month = {2001 Dec 17},
	pages = {7259-70},
	abstract = {Mobile group II introns encode reverse transcriptases that also function as intron-specific splicing factors (maturases). We showed previously that the reverse transcriptase/maturase encoded by the Lactococcus lactis Ll.LtrB intron has a high affinity binding site at the beginning of its own coding region in an idiosyncratic structure, DIVa. Here, we identify potential secondary binding sites in conserved regions of the catalytic core and show via chemical modification experiments that binding of the maturase induces the formation of key tertiary interactions required for RNA splicing. The interaction with conserved as well as idiosyncratic regions explains how maturases in some organisms could evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns.},
	keywords = {Aldehydes, Base Sequence, Introns, Iodine, Molecular Sequence Data, Nucleic Acid Conformation, RNA, RNA Splicing, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae Proteins},
	issn = {0261-4189},
	doi = {10.1093/emboj/20.24.7259},
	author = {Matsuura, M and Noah, J W and Lambowitz, A M}
}
@article {382,
	title = {Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {98},
	number = {23},
	year = {2001},
	month = {2001 Nov 6},
	pages = {13207-12},
	abstract = {Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addition to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.},
	keywords = {Base Sequence, DNA Primers, DNA, Fungal, Introns, Mutation, Polymerase Chain Reaction, Retroelements, RNA, Fungal, Saccharomyces cerevisiae},
	issn = {0027-8424},
	doi = {10.1073/pnas.231494498},
	author = {Dickson, L and Huang, H R and Liu, L and Matsuura, M and Lambowitz, A M and Perlman, P S}
}
@article {389,
	title = {Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid.},
	journal = {Nucleic Acids Res},
	volume = {28},
	number = {13},
	year = {2000},
	month = {2000 Jul 1},
	pages = {1514-24},
	abstract = {We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64)and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich pallindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5{\textquoteright}- and 3{\textquoteright}-end processing and 3{\textquoteright} nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like tmRNA. Our results suggest that mtRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.},
	keywords = {Base Sequence, Blotting, Southern, Cloning, Molecular, Deoxyribonuclease EcoRI, DNA, Fungal, DNA, Mitochondrial, Genetic Variation, Introns, Mitochondria, Molecular Sequence Data, Mutagenesis, Insertional, Neurospora, Neurospora crassa, Nucleic Acid Conformation, Physical Chromosome Mapping, Plasmids, Reverse Transcriptase Polymerase Chain Reaction, RNA Processing, Post-Transcriptional, RNA, Fungal, RNA, Transfer, Trp, Transcription, Genetic},
	issn = {1362-4962},
	author = {Mohr, S and Wanner, L A and Bertrand, H and Lambowitz, A M}
}
@article {390,
	title = {Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid.},
	journal = {Nucleic Acids Res},
	volume = {28},
	number = {7},
	year = {2000},
	month = {2000 Apr 1},
	pages = {1514-24},
	abstract = {We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64) and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74 A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich palindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5{\textquoteright}- and 3{\textquoteright}-end processing and 3{\textquoteright} nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like mtRNA. Our results suggest that mt tRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.},
	keywords = {Base Sequence, Blotting, Southern, Cloning, Molecular, Deoxyribonuclease EcoRI, DNA Primers, DNA, Fungal, DNA, Mitochondrial, Genetic Variation, Introns, Mitochondria, Molecular Sequence Data, Mutagenesis, Insertional, Neurospora, Neurospora crassa, Nucleic Acid Conformation, Physical Chromosome Mapping, Plasmids, Reverse Transcriptase Polymerase Chain Reaction, RNA, Fungal, RNA, Transfer, Trp, Transcription, Genetic},
	issn = {1362-4962},
	author = {Mohr, S and Wanner, L A and Bertrand, H and Lambowitz, A M}
}
@article {391,
	title = {Rules for DNA target-site recognition by a lactococcal group II intron enable retargeting of the intron to specific DNA sequences.},
	journal = {Genes Dev},
	volume = {14},
	number = {5},
	year = {2000},
	month = {2000 Mar 1},
	pages = {559-73},
	abstract = {Group II intron homing occurs primarily by a mechanism in which the intron RNA reverse splices into a DNA target site and is then reverse transcribed by the intron-encoded protein. The DNA target site is recognized by an RNP complex containing the intron-encoded protein and the excised intron RNA. Here, we analyzed DNA target-site requirements for the Lactococcus lactis Ll.LtrB group II intron in vitro and in vivo. Our results suggest a model similar to yeast mtDNA introns, in which the intron-encoded protein first recognizes a small number of nucleotide residues in double-stranded DNA and causes DNA unwinding, enabling the intron RNA to base-pair with the DNA for reverse splicing. Antisense-strand cleavage requires additional interactions between the protein and 3{\textquoteright} exon. Key nucleotide residues are recognized directly by the intron-encoded protein independent of sequence context, and there is a stringent requirement for fixed spacing between target site elements recognized by the protein and RNA components of the endonuclease. Experiments with DNA substrates containing GC-clamps or "bubbles" indicate a requirement for DNA unwinding in the 3{\textquoteright} exon but not the distal 5{\textquoteright} exon region. Finally, by applying the target-site recognition rules, we show that the L1.LtrB intron can be modified to insert at new sites in a plasmid-borne thyA gene in Escherichia coli. This strategy should be generally applicable to retargeting group II introns and to delivering foreign sequences to specific sites in heterologous genomes.},
	keywords = {Base Pairing, Base Sequence, DNA, Antisense, DNA, Bacterial, Escherichia coli, Exons, Introns, Lactococcus lactis, Models, Genetic, Molecular Sequence Data, Nucleic Acid Conformation, Polymerase Chain Reaction, Saccharomyces cerevisiae, Sequence Analysis, DNA, Templates, Genetic},
	issn = {0890-9369},
	author = {Mohr, G and Smith, D and Belfort, M and Lambowitz, A M}
}
@article {387,
	title = {Function of tyrosyl-tRNA synthetase in splicing group I introns: an induced-fit model for binding to the P4-P6 domain based on analysis of mutations at the junction of the P4-P6 stacked helices.},
	journal = {J Mol Biol},
	volume = {301},
	number = {2},
	year = {2000},
	month = {2000 Aug 11},
	pages = {265-83},
	abstract = {We used an Escherichia coli genetic assay based on the phage T4 td intron to test the ability of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) to suppress mutations that cause structural defects around its binding site in the P4-P6 domain of the group I intron catalytic core. We analyzed all possible combinations of nucleotides at either P4 bp-1 or P6 bp-1, which together form the junction of the P4-P6 stacked helices, and looked for synergistic effects in double mutants. Most mutations at either position inhibit self-splicing, but can be suppressed by CYT-18. CYT-18 can compensate efficiently for mutations that disrupt base-pairing at either P4 bp-1 or P6 bp-1, for mutations at P6 bp-1 that disrupt the base-triple interaction with J3/4-3, and for nucleotide substitutions at either position that are predicted to be suboptimal for base stacking, based on the analysis of DNA four-way junctions. However, CYT-18 has difficulty suppressing combinations of mutations at P4 bp-1 and P6 bp-1 that simultaneously disrupt base-pairing and base stacking. Thermal denaturation and Fe(II)-EDTA analysis showed that mutations at the junction of the P4-P6 stacked helices lead to grossly impaired tertiary-structure formation centered in the P4-P6 domain. CYT-18-suppressible mutants bind the protein with K(d) values up to 79-fold higher than that for the wild-type intron, but in all cases tested, the k(off) value for the complex remains within twofold of the wild-type value, suggesting that the binding site can be formed properly and that the increased K(d) value reflects primarily an increased k(on) value for the binding of CYT-18 to the misfolded intron. Our results indicate that the P4/P6 junction is a linchpin region, where even small nucleotide substitutions grossly disrupt the catalytically-active group I intron tertiary structure, and that CYT-18 binding induces the formation of the correct structure in this region, leading to folding of the group I intron catalytic core.},
	keywords = {Bacteriophage T4, Catalytic Domain, Edetic Acid, Escherichia coli, Introns, Models, Biological, Mutation, Neurospora crassa, Nucleic Acid Conformation, Protein Denaturation, Protein Structure, Secondary, Protein Structure, Tertiary, RNA Splicing, Suppression, Genetic, Tyrosine-tRNA Ligase},
	issn = {0022-2836},
	doi = {10.1006/jmbi.2000.3963},
	author = {Chen, X and Gutell, R R and Lambowitz, A M}
}
@article {388,
	title = {Group II introns designed to insert into therapeutically relevant DNA target sites in human cells.},
	journal = {Science},
	volume = {289},
	number = {5478},
	year = {2000},
	month = {2000 Jul 21},
	pages = {452-7},
	abstract = {Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy.},
	keywords = {Base Pairing, Base Sequence, Cell Line, DNA, DNA, Viral, Escherichia coli, Gene Targeting, Genes, pol, Genetic Therapy, HIV-1, Humans, Introns, Lactococcus lactis, Molecular Sequence Data, Proviruses, Receptors, CCR5, Recombination, Genetic, RNA, Catalytic, Transfection},
	issn = {0036-8075},
	author = {Guo, H and Karberg, M and Long, M and Jones, J P and Sullenger, B and Lambowitz, A M}
}
@article {386,
	title = {Multiple homing pathways used by yeast mitochondrial group II introns.},
	journal = {Mol Cell Biol},
	volume = {20},
	number = {22},
	year = {2000},
	month = {2000 Nov},
	pages = {8432-46},
	abstract = {The yeast mitochondrial DNA group II introns aI1 and aI2 are retroelements that insert site specifically into intronless alleles by a process called homing. Here, we used patterns of flanking marker coconversion in crosses with wild-type and mutant aI2 introns to distinguish three coexisting homing pathways: two that were reverse transcriptase (RT) dependent (retrohoming) and one that was RT independent. All three pathways are initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, with the sense strand cleaved by partial or complete reverse splicing, and the antisense strand cleaved by the intron-encoded protein. The major retrohoming pathway in standard crosses leads to insertion of the intron with unidirectional coconversion of upstream exon sequences. This pattern of coconversion suggests that the major retrohoming pathway is initiated by target DNA-primed reverse transcription of the reverse-spliced intron RNA and completed by double-strand break repair (DSBR) recombination with the donor allele. The RT-independent pathway leads to insertion of the intron with bidirectional coconversion and presumably occurs by a conventional DSBR recombination mechanism initiated by cleavage of the recipient DNA target site by the intron-encoded endonuclease, as for group I intron homing. Finally, some mutant DNA target sites shift up to 43\% of retrohoming to another pathway not previously detected for aI2 in which there is no coconversion of flanking exon sequences. This new pathway presumably involves synthesis of a full-length cDNA copy of the inserted intron RNA, with completion by a repair process independent of homologous recombination, as found for the Lactococcus lactis Ll.LtrB intron. Our results show that group II intron mobility can occur by multiple pathways, the ratios of which depend on the characteristics of both the intron and the DNA target site. This remarkable flexibility enables group II introns to use different recombination and repair enzymes in different host cells.},
	keywords = {Base Sequence, Crosses, Genetic, DNA Repair, DNA, Complementary, Endonucleases, Exons, Introns, Mitochondria, Molecular Sequence Data, Mutation, Recombination, Genetic, Retroelements, RNA-Directed DNA Polymerase, Yeasts},
	issn = {0270-7306},
	author = {Eskes, R and Liu, L and Ma, H and Chao, M Y and Dickson, L and Lambowitz, A M and Perlman, P S}
}
@article {394,
	title = {Group II intron reverse transcriptase in yeast mitochondria. Stabilization and regulation of reverse transcriptase activity by the intron RNA.},
	journal = {J Mol Biol},
	volume = {289},
	number = {3},
	year = {1999},
	month = {1999 Jun 11},
	pages = {473-90},
	abstract = {Group II introns encode reverse transcriptases that function in both intron mobility and RNA splicing. The proteins bind specifically to unspliced precursor RNA to promote splicing, and then remain associated with the excised intron to form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription. Here, immunoblotting and UV cross-linking experiments show that the reverse transcriptase activity encoded by the yeast mtDNA group II intron aI2 is associated with an intron-encoded protein of 62 kDa (p62). p62 is bound tightly to endogenous RNAs in mitochondrial ribonucleoprotein particles, and the reverse transcriptase activity is rapidly and irreversibly lost when the protein is released from the endogenous RNAs by RNase digestion. Non-denaturing gel electrophoresis and activity assays show that the aI2 reverse transcriptase is associated predominantly with the excised intron RNA, while a smaller amount is associated with unspliced precursor RNA, as expected from the role of the protein in RNA splicing. Although the reverse transcriptase in wild-type yeast strains is bound tightly to endogenous RNAs, it is regulated so that it does not copy these RNAs unless a suitable DNA oligonucleotide primer or DNA target site is provided. Certain mutations in the intron-encoded protein or RNA circumvent this regulation and activate reverse transcription of endogenous RNAs in the absence of added primer. Although p62 is bound to unspliced precursor RNA in position to initiate cDNA synthesis in the 3{\textquoteright} exon, the major template for target DNA-primed reverse transcription in vitro is the reverse-spliced intron RNA, as found previously for aI1. Together, our results show that binding to intron-containing RNAs stabilizes and regulates the activity of p62.},
	keywords = {Amino Acid Sequence, Conserved Sequence, DNA Primers, DNA, Complementary, Exons, Introns, Mitochondria, Mutation, Ribonucleoproteins, RNA Precursors, RNA Splicing, RNA, Fungal, RNA-Directed DNA Polymerase, Templates, Genetic, Transcription, Genetic, Yeasts},
	issn = {0022-2836},
	doi = {10.1006/jmbi.1999.2778},
	author = {Zimmerly, S and Moran, J V and Perlman, P S and Lambowitz, A M}
}
@article {395,
	title = {How to implement a case-mix system in less than a year.},
	journal = {Manag Care Q},
	volume = {7},
	number = {1},
	year = {1999},
	month = {1999 Winter},
	pages = {64-9},
	keywords = {Certification, Diagnosis-Related Groups, Health Plan Implementation, Humans, Long-Term Care, Managed Care Programs, Medicaid, Nursing Homes, Ohio, Prospective Payment System, State Health Plans, United States},
	issn = {1064-5454},
	author = {Lambowitz, S}
}
@article {392,
	title = {A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA.},
	journal = {Mol Cell},
	volume = {4},
	number = {2},
	year = {1999},
	month = {1999 Aug},
	pages = {239-50},
	abstract = {Group II introns encode reverse transcriptases that promote RNA splicing (maturase activity) and then with the excised intron form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription (TPRT). Here, we show that the primary binding site for the maturase (LtrA) encoded by the Lactococcus lactis Ll.LtrB intron is within a region of intron domain IV that includes the start codon of the LtrA ORF. This binding is enhanced by other elements, particularly domain I and the EBS/IBS interactions, and helps position LtrA to initiate cDNA synthesis in the 3{\textquoteright} exon as occurs during TPRT. Our results suggest how the maturase functions in RNA splicing and support the hypothesis that the reverse transcriptase coding region was derived from an independent genetic element that was inserted into a preexisting group II intron.},
	keywords = {Bacterial Proteins, Base Sequence, Binding Sites, Cloning, Molecular, Codon, Initiator, DNA Transposable Elements, Escherichia coli, Exons, Introns, Kinetics, Lactococcus lactis, Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, Open Reading Frames, Recombinant Proteins, RNA Splicing, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae Proteins, Transcription, Genetic},
	issn = {1097-2765},
	author = {Wank, H and SanFilippo, J and Singh, R N and Matsuura, M and Lambowitz, A M}
}
@article {393,
	title = {RNA and protein catalysis in group II intron splicing and mobility reactions using purified components.},
	journal = {Biochemistry},
	volume = {38},
	number = {28},
	year = {1999},
	month = {1999 Jul 13},
	pages = {9069-83},
	abstract = {Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles containing only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed analysis of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a K(d) of ,
	keywords = {Bacterial Proteins, Catalysis, Deoxyribonucleases, Type II Site-Specific, DNA Transposable Elements, Enzyme Stability, Escherichia coli, Hydrogen-Ion Concentration, Introns, Lactococcus lactis, Nucleic Acid Conformation, Recombinant Proteins, Ribonucleoproteins, RNA Splicing, RNA, Bacterial, RNA-Binding Proteins, RNA-Directed DNA Polymerase, Sodium Chloride, Substrate Specificity},
	issn = {0006-2960},
	doi = {10.1021/bi982799l},
	author = {Saldanha, R and Chen, B and Wank, H and Matsuura, M and Edwards, J and Lambowitz, A M}
}
@article {396,
	title = {Group II intron mobility in yeast mitochondria: target DNA-primed reverse transcription activity of aI1 and reverse splicing into DNA transposition sites in vitro.},
	journal = {J Mol Biol},
	volume = {282},
	number = {3},
	year = {1998},
	month = {1998 Sep 25},
	pages = {505-23},
	abstract = {The retrohoming of the yeast mtDNA intron aI1 occurs by a target DNA-primed reverse transcription (TPRT) mechanism in which the intron RNA reverse splices directly into the recipient DNA and is then copied by the intron-encoded reverse transcriptase. Here, we carried out biochemical characterization of the intron-encoded reverse transcriptase and site-specific DNA endonuclease activities required for this process. We show that the aI1 reverse transcriptase has high TPRT activity in the presence of appropriate DNA target sites, but differs from the closely related reverse transcriptase encoded by the yeast aI2 intron in being unable to use artificial substrates efficiently. Characterization of TPRT products shows that the fully reverse spliced intron RNA is an efficient template for cDNA synthesis, while reverse transcription of partially reverse spliced intron RNA is impeded by the branch point. Novel features of the aI1 reaction include a prominent open-circular product in which cDNAs are incorporated at a nick at the antisense-strand cleavage site. The aI1 endonuclease activity, which catalyzes the DNA cleavage and reverse splicing reactions, is associated with ribonucleoprotein particles containing the intron-encoded protein and the excised intron RNA. As shown for the aI2 endonuclease, both the RNA and protein components are used for DNA target site recognition, but the aI1 protein has less stringent nucleotide sequence requirements for the reverse splicing reaction. Finally, perhaps reflecting this relaxed target specificity, in vitro experiments show that aI1 can reverse splice directly into ectopic mtDNA transposition sites, consistent with the previously suggested possibility that this mechanism is used for ectopic transposition of group II introns in vivo.},
	keywords = {Deoxyribonuclease I, DNA, Fungal, DNA, Mitochondrial, Gene Expression Regulation, Fungal, Introns, Mitochondria, RNA Splicing, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae, Transcription, Genetic},
	issn = {0022-2836},
	doi = {10.1006/jmbi.1998.2029},
	author = {Yang, J and Mohr, G and Perlman, P S and Lambowitz, A M}
}
@article {397,
	title = {Retrohoming of a bacterial group II intron: mobility via complete reverse splicing, independent of homologous DNA recombination.},
	journal = {Cell},
	volume = {94},
	number = {4},
	year = {1998},
	month = {1998 Aug 21},
	pages = {451-62},
	abstract = {The mobile group II intron of Lactococcus lactis, Ll.LtrB, provides the opportunity to analyze the homing pathway in genetically tractable bacterial systems. Here, we show that Ll.LtrB mobility occurs by an RNA-based retrohoming mechanism in both Escherichia coli and L. lactis. Surprisingly, retrohoming occurs efficiently in the absence of RecA function, with a relaxed requirement for flanking exon homology and without coconversion of exon markers. These results lead to a model for bacterial retrohoming in which the intron integrates into recipient DNA by complete reverse splicing and serves as the template for cDNA synthesis. The retrohoming reaction is completed in unprecedented fashion by a DNA repair event that is independent of homologous recombination between the alleles. Thus, Ll.LtrB has many features of retrotransposons, with practical and evolutionary implications.},
	keywords = {Bacterial Proteins, DNA Repair, DNA Transposable Elements, DNA, Bacterial, Escherichia coli, Evolution, Molecular, Genes, Bacterial, Introns, Lactococcus lactis, Models, Genetic, Rec A Recombinases, Recombination, Genetic, Retroelements, RNA, Bacterial, RNA, Catalytic, RNA-Directed DNA Polymerase},
	issn = {0092-8674},
	author = {Cousineau, B and Smith, D and Lawrence-Cavanagh, S and Mueller, J E and Yang, J and Mills, D and Manias, D and Dunny, G and Lambowitz, A M and Belfort, M}
}
@article {399,
	title = {A bacterial group II intron encoding reverse transcriptase, maturase, and DNA endonuclease activities: biochemical demonstration of maturase activity and insertion of new genetic information within the intron.},
	journal = {Genes Dev},
	volume = {11},
	number = {21},
	year = {1997},
	month = {1997 Nov 1},
	pages = {2910-24},
	abstract = {The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli. The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.},
	keywords = {Bacterial Proteins, Base Sequence, DNA, Mitochondrial, Endodeoxyribonucleases, Endoribonucleases, Escherichia coli, Exons, Introns, Lactococcus lactis, Nucleotidyltransferases, Oligodeoxyribonucleotides, Plasmids, RNA Splicing, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae, Substrate Specificity},
	issn = {0890-9369},
	author = {Matsuura, M and Saldanha, R and Ma, H and Wank, H and Yang, J and Mohr, G and Cavanagh, S and Dunny, G M and Belfort, M and Lambowitz, A M}
}
@article {403,
	title = {Characterization of Neurospora mitochondrial group I introns reveals different CYT-18 dependent and independent splicing strategies and an alternative 3{\textquoteright} splice site for an intron ORF.},
	journal = {RNA},
	volume = {3},
	number = {2},
	year = {1997},
	month = {1997 Feb},
	pages = {114-31},
	abstract = {The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing the N. crassa mitochondrial large rRNA intron by stabilizing the catalytically active structure of the intron core. Here, a comprehensive study of N. crassa mtDNA group I introns identified two additional introns, cob-I2 and the ND1 intron, that are dependent on CYT-18 for splicing in vitro and in vivo. The other seven N. crassa mtDNA group I introns are not CYT-18-dependent and include five that self-splice and two that do not splice under any conditions examined. Some of these introns may require maturases or other proteins for efficient splicing. All but one of the non-CYT-18-dependent introns contain large peripheral extensions of the P5 stem, related to the P5abc structure that blocks CYT-18 binding to the Tetrahymena large rRNA intron. The remaining non-CYT-18-dependent intron, cob-I1, contains a long, peripheral extension of the P9 stem, denoted P9.1, which also impedes CYT-18 binding. Detailed analysis of the CYT-18-dependent ND1 intron showed that two 3{\textquoteright} splice sites are used in vitro and in vivo. The proximal, alternative 3{\textquoteright} splice site brings the intron open reading frame, which potentially encodes a mobility endonuclease, in frame with the upstream exon, possibly providing a means of expression. Considered together, our results show that group I introns in N. crassa mitochondria use a variety of strategies involving different proteins and/or RNA structures to assist splicing, and they support the hypothesis that CYT-18 and the peripheral RNA structure P5abc are alternative evolutionary adaptations for stabilizing the active structure of the intron core.},
	keywords = {Alternative Splicing, Base Sequence, DNA, Mitochondrial, Introns, Mitochondria, Molecular Sequence Data, Neurospora crassa, Nucleic Acid Conformation, Open Reading Frames, Polymerase Chain Reaction, RNA, Fungal, Tyrosine-tRNA Ligase},
	issn = {1355-8382},
	author = {Wallweber, G J and Mohr, S and Rennard, R and Caprara, M G and Lambowitz, A M}
}
@article {400,
	title = {De novo and DNA primer-mediated initiation of cDNA synthesis by the mauriceville retroplasmid reverse transcriptase involve recognition of a 3{\textquoteright} CCA sequence.},
	journal = {J Mol Biol},
	volume = {271},
	number = {3},
	year = {1997},
	month = {1997 Aug 22},
	pages = {311-32},
	abstract = {The Mauriceville mitochondrial retroplasmid of Neurospora encodes a novel reverse transcriptase that initiates cDNA synthesis at a 3{\textquoteright} tRNA-like structure of the plasmid transcript, either de novo (i.e. without a primer) or by using the 3{\textquoteright} OH group of a DNA primer. Both the de novo and primer-mediated initiations involve recognition of structural features at the 3{\textquoteright} end of the retroplasmid transcript, which ends with a 3{\textquoteright} CCACCA. Here, detailed biochemical characterization of the retroplasmid reverse transcriptase shows that the 3{\textquoteright} CCA of the plasmid transcript is the major structural feature recognized by the reverse transcriptase for both the de novo and primer-mediated initiations. Complementarity between the DNA primer and RNA template is not required for the primer-mediated initiation, although short (1 to 3 nt) base-pairing interactions can influence both the efficiency and site of initiation near the 3{\textquoteright} end of the transcript. Single nucleotide changes in the 3{\textquoteright} CCA lead to less efficient initiation in the upstream CCA with an increased propensity to add extra "non-coded" nucleotides to the 5{\textquoteright} end of the cDNA during de novo initiation or to the 3{\textquoteright} end of the primer during primer-mediated initiation. Secondary structure features upstream of the 3{\textquoteright} CCA also influence the efficiency of initiation, but are not stringently required in vitro. Finally, we find that the retroplasmid reverse transcriptase does not efficiently use DNA primers that are base-paired to internal positions in the RNA template, nor does it use analogs of natural substrates used by non-long terminal repeat retrotransposon or retroviral reverse transcriptases. Our results indicate that the retroplasmid reverse transcriptase is uniquely adapted to initiate cDNA synthesis by recognizing a 3{\textquoteright} CCA sequence. The ability to recognize a specific template sequence is common for RNA polymerases, but unprecedented for a reverse transcriptase.},
	keywords = {Base Composition, Base Sequence, DNA Primers, DNA, Complementary, Evolution, Molecular, Molecular Sequence Data, Neurospora crassa, Nucleic Acid Conformation, Plasmids, Regulatory Sequences, Nucleic Acid, Retroelements, RNA, Fungal, RNA-Directed DNA Polymerase, Templates, Genetic},
	issn = {0022-2836},
	doi = {10.1006/jmbi.1997.1185},
	author = {Chen, B and Lambowitz, A M}
}
@article {401,
	title = {The Mauriceville retroplasmid reverse transcriptase initiates cDNA synthesis de novo at the 3{\textquoteright} end of tRNAs.},
	journal = {Mol Cell Biol},
	volume = {17},
	number = {8},
	year = {1997},
	month = {1997 Aug},
	pages = {4526-35},
	abstract = {The Mauriceville retroplasmid of Neurospora mitochondria encodes a novel reverse transcriptase that initiates cDNA synthesis de novo (i.e., without a primer) at the 3{\textquoteright} CCA of the plasmid transcript{\textquoteright}s 3{\textquoteright} tRNA-like structure (H. Wang and A. M. Lambowitz, Cell 75:1071-1081, 1993). Here, we show that the plasmid reverse transcriptase also initiates cDNA synthesis de novo at the 3{\textquoteright} end of tRNAs, leading to synthesis of a full-length cDNA copy of the tRNA. The use of tRNA templates in vivo was suggested previously by the structure of suppressive mutant plasmids that have incorporated mitochondrial tRNA sequences (R. A. Akins, R. L. Kelley, and A. M. Lambowitz, Cell 47:505-516, 1986). The in vitro experiments show that efficient de novo initiation on tRNA templates requires an unpaired 3{\textquoteright} CCA and occurs predominantly opposite position C-2 of the 3{\textquoteright} CCA sequence, the same position as in the plasmid transcript. In other reactions, the plasmid reverse transcriptase synthesizes cDNA dimers by template switching between two tRNA templates and initiates at an internal position in a tRNA by using the 3{\textquoteright} end of the tRNA as a primer. Finally, we show that template switching between the tRNA and the plasmid transcript in vitro gives rise to hybrid cDNAs of the type predicted to be intermediates in the generation of the suppressive mutant plasmids. The ability of the plasmid reverse transcriptase to initiate at the 3{\textquoteright} end of tRNAs presumably reflects the recognition of structural features similar to those of the 3{\textquoteright} tRNA-like structure of the plasmid transcript. The recognition of tRNAs or tRNA-like structures as templates for cDNA synthesis may be characteristic of primitive reverse transcriptases that evolved from RNA-dependent RNA polymerases.},
	keywords = {Cloning, Molecular, DNA, Complementary, DNA, Fungal, Models, Genetic, Mutation, Neurospora crassa, Plasmids, RNA, Transfer, Trp, RNA, Transfer, Tyr, RNA-Directed DNA Polymerase, Sequence Analysis, DNA, Templates, Genetic},
	issn = {0270-7306},
	author = {Chiang, C C and Lambowitz, A M}
}
@article {402,
	title = {Mobility of yeast mitochondrial group II introns: engineering a new site specificity and retrohoming via full reverse splicing.},
	journal = {Cell},
	volume = {88},
	number = {6},
	year = {1997},
	month = {1997 Mar 21},
	pages = {865-74},
	abstract = {The mobile group II introns aI1 and aI2 of yeast mtDNA encode endonuclease activities that cleave intronless DNA target sites to initiate mobility by target DNA-primed reverse transcription. For aI2, sense-strand cleavage occurs mainly by a partial reverse splicing reaction, whereas for aI1, complete reverse splicing occurs, leading to insertion of the linear intron RNA into double-stranded DNA. Here, we show that aI1 homing and reverse splicing depend on the EBS1 (RNA)/IBS1(DNA) pairing and that target specificity can be changed by compensatory changes in the target site and the donor intron. Using well-marked strains to follow coconversion of flanking DNA, we show that homing occurs by both RT-dependent and -independent pathways. Remarkably, in most RT-dependent events, the reverse spliced intron is the initial template for first-strand cDNA synthesis.},
	keywords = {Base Sequence, DNA, Complementary, DNA, Fungal, DNA, Mitochondrial, Endonucleases, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Fungal, Introns, Mutagenesis, Site-Directed, Plasmids, Recombination, Genetic, RNA Splicing, Yeasts},
	issn = {0092-8674},
	author = {Eskes, R and Yang, J and Lambowitz, A M and Perlman, P S}
}
@article {398,
	title = {Group II intron endonucleases use both RNA and protein subunits for recognition of specific sequences in double-stranded DNA.},
	journal = {EMBO J},
	volume = {16},
	number = {22},
	year = {1997},
	month = {1997 Nov 17},
	pages = {6835-48},
	abstract = {Group II introns use intron-encoded reverse transcriptase, maturase and DNA endonuclease activities for site-specific insertion into DNA. Remarkably, the endonucleases are ribonucleoprotein complexes in which the excised intron RNA cleaves the sense strand of the recipient DNA by reverse splicing, while the intron-encoded protein cleaves the antisense strand. Here, studies with the yeast group II intron aI2 indicate that both the RNA and protein components of the endonuclease contribute to recognition of an approximately 30 bp DNA target site. Our results lead to a model in which the protein component first recognizes specific nucleotides in the most distal 5{\textquoteright} exon region of the DNA target site (E2-21 to -11). Binding of the protein then leads to DNA unwinding, enabling the intron RNA to base pair to a 13 nucleotide DNA sequence (E2-12 to E3+1) for reverse splicing. Antisense-strand cleavage requires additional interactions of the protein with the 3{\textquoteright} exon DNA (E3+1 to +10). Our results show how enzymes can use RNA and protein subunits cooperatively to recognize specific sequences in double-stranded DNA.},
	keywords = {Base Sequence, DNA, Fungal, Electron Transport Complex IV, Endodeoxyribonucleases, Introns, Mitochondria, Models, Genetic, Molecular Sequence Data, Nucleic Acid Conformation, Ribonucleoproteins, RNA Splicing, RNA, Catalytic, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Substrate Specificity, Zinc},
	issn = {0261-4189},
	doi = {10.1093/emboj/16.22.6835},
	author = {Guo, H and Zimmerly, S and Perlman, P S and Lambowitz, A M}
}
@article {406,
	title = {Analysis of the CYT-18 protein binding site at the junction of stacked helices in a group I intron RNA by quantitative binding assays and in vitro selection.},
	journal = {J Mol Biol},
	volume = {261},
	number = {1},
	year = {1996},
	month = {1996 Aug 9},
	pages = {23-42},
	abstract = {The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. Previous studies showed that CYT-18 binds with high affinity to the P4-P6 domain of the catalytic core and that there is some additional contribution to binding from the P3-P9 domain. Here, quantitative binding assays with deletion derivatives of the N. crassa mitochondrial large rRNA intron showed that at least 70\% of the binding energy can be accounted for by the interaction of CYT-18 with the P4-P6 domain. Within this domain, P4 and P6 are required for high affinity CYT-18 binding, while the distal elements P5 and P6a may contribute indirectly by stabilizing the correct structure of the binding site in P4 and P6. CYT-18 binds to a small RNA corresponding to the isolated P4-P6 domain, but not to a permuted version of this RNA in which P4-P6 is a continuous rather than a stacked helix. Iterative in vitro selection experiments with the isolated P4-P6 domain showed a requirement for base-pairing to maintain helices P4, P6 and P6a, but indicate that P5 is subject to fewer constraints. The most strongly conserved nucleotides in the selections were clustered around the junction of the P4-P6 stacked helix, with ten nucleotides (J3/4-2,3, P4 bp -1 and 3, and P6 bp -1 and 2) found invariant in the context of the wild-type RNA structure. In vitro mutagenesis confirmed that replacement of the wild-type nucleotides at J3/4-2 and 3 or P4 bp-3 markedly decreased CYT-18 binding, reflecting either base specific contacts or indirect readout of RNA structure by the protein. Our results suggest that a major function of CYT-18 is to promote assembly of the P4-P6 domain by stabilizing the correct geometry at the junction of the P4-P6 stacked helix. The relatively large number of conserved nucleotides at the binding site suggests that the interaction of CYT-18 with group I introns is unlikely to have arisen by chance and could reflect either an evolutionary relationship between group I introns and tRNAs or interaction with a common stacked-helical structural motif that evolved separately in these RNAs.},
	keywords = {Base Sequence, Binding Sites, Cloning, Molecular, Conserved Sequence, Introns, Mitochondria, Molecular Sequence Data, Mutagenesis, Site-Directed, Neurospora crassa, Nucleic Acid Conformation, Plasmids, RNA Precursors, RNA Splicing, RNA, Catalytic, RNA, Fungal, RNA, Transfer, Tyr, Tyrosine-tRNA Ligase},
	issn = {0022-2836},
	doi = {10.1006/jmbi.1996.0439},
	author = {Saldanha, R and Ellington, A and Lambowitz, A M}
}
@article {407,
	title = {Efficient integration of an intron RNA into double-stranded DNA by reverse splicing.},
	journal = {Nature},
	volume = {381},
	number = {6580},
	year = {1996},
	month = {1996 May 23},
	pages = {332-5},
	abstract = {Some group II introns are mobile elements as well as catalytic RNAs. Introns aI1 and aI2 found in the gene COX1 in yeast mitochondria encode reverse transcriptases which promote site-specific insertion of the intron into intronless alleles ({\textquoteright}homing{\textquoteright}). For aI2 this predominantly occurs by reverse transcription of unspliced precursor RNA at a break in double-strand DNA made by an endonuclease encoded by the intron. The aI2 endonuclease involves both the excised intron RNA, which cleaves the DNA{\textquoteright}s sense strand by partial reverse splicing; and the intron-encoded reverse transcriptase which cleaves the anti-sense strand. Here we show that aI1 encodes an analogous endonuclease specific for a different target site compatible with the different exon-binding sequences of the intron RNA. Over half of aI1 undergoes complete reverse splicing in vitro, thus integrating linear intron RNA directly into the DNA. This unprecedented reaction has implications for both intron mobility and evolution, and potential genetic engineering applications.},
	keywords = {Base Sequence, DNA, DNA, Fungal, Introns, Molecular Sequence Data, Recombination, Genetic, Ribonucleoproteins, RNA Splicing, RNA, Fungal, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae},
	issn = {0028-0836},
	doi = {10.1038/381332a0},
	author = {Yang, J and Zimmerly, S and Perlman, P S and Lambowitz, A M}
}
@article {408,
	title = {A tyrosyl-tRNA synthetase protein induces tertiary folding of the group I intron catalytic core.},
	journal = {J Mol Biol},
	volume = {257},
	number = {3},
	year = {1996},
	month = {1996 Apr 5},
	pages = {512-31},
	abstract = {The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns. We have used chemical-structure mapping and footprinting to investigate the interaction of the CYT-18 protein with the N. crassa mitochondrial large subunit ribosomal RNA (mt LSU) and ND1 introns, which are not detectably self-splicing in vitro. Our results show that both these non-self-splicing introns form most of the short range pairings of the conserved group I intron secondary structure in the absence of CYT-18, but otherwise remain largely unfolded, even at high Mg2+ concentrations. The binding of CYT-18 promotes the formation of the extended helical domains P6a-P6-P4-P5 (P4-P6 domain) and P8-P3-P7-P9 (P3-P9 domain) and their interaction to form the catalytic core. In iodine-footprinting experiments, CYT-18 binding results in the protection of regions of the phosphodiester backbone expected for tertiary folding of the catalytic core, as well as additional protections that may reflect proximity of the protein. In both introns, most of the putative CYT-18 protection sites are in the P4-P6 domain, the region of the SU intron previously shown to bind CYT-18 as a separate RNA molecule, but additional sites are found in the other major helical domain in P8 and P9 in both introns and in L9 and P7.1/P7.1a in the mt LSU intron. Protease digestion of the CYT-18/intron RNA complexes results in the loss of CYT-18-induced RNA tertiary structure and splicing activity. Considered together with previous studies, or results suggest that CYT-18 binds initially to the P4-P6 region of group I introns to form a scaffold for the assembly of the P3-P9 domain, which may contain additional binding sites for the protein. A three-dimensional model structure of the CYT-18 binding site in group I introns indicates that CYT-18 interacts with the surface of the catalytic core on the side opposite the active-site cleft and may primarily recognize a specific three-dimensional geometry of the phosphodiester backbone of group I introns.},
	keywords = {Base Sequence, Introns, Molecular Sequence Data, Neurospora crassa, Nucleic Acid Conformation, Protein Conformation, Protein Folding, RNA, RNA Splicing, RNA, Ribosomal, Tyrosine-tRNA Ligase},
	issn = {0022-2836},
	doi = {10.1006/jmbi.1996.0182},
	author = {Caprara, M G and Mohr, G and Lambowitz, A M}
}
@article {404,
	title = {A tyrosyl-tRNA synthetase recognizes a conserved tRNA-like structural motif in the group I intron catalytic core.},
	journal = {Cell},
	volume = {87},
	number = {6},
	year = {1996},
	month = {1996 Dec 13},
	pages = {1135-45},
	abstract = {The Neurospora crassa mitochondrial (mt) tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns, in addition to aminoacylating tRNA(Tyr). Here, we compared the CYT-18 binding sites in the N. crassa mt LSU and ND1 introns with that in N. crassa mt tRNA(Tyr) by constructing three-dimensional models based on chemical modification and RNA footprinting data. Remarkably, superimposition of the CYT-18 binding sites in the model structures revealed an extended three-dimensional overlap between the tRNA and the group I intron catalytic core. Our results provide insight into how an RNA-splicing factor can evolve from a cellular RNA-binding protein. Further, the structural similarities between group I introns and tRNAs are consistent with an evolutionary relationship and suggest a general mechanism for the evolution of complex catalytic RNAs.},
	keywords = {Base Sequence, Binding Sites, Biological Evolution, Conserved Sequence, Introns, Molecular Sequence Data, Neurospora crassa, Nucleic Acid Conformation, Protein Conformation, Protein Structure, Tertiary, RNA Splicing, RNA, Fungal, RNA, Transfer, Tyr, Tyrosine-tRNA Ligase},
	issn = {0092-8674},
	author = {Caprara, M G and Lehnert, V and Lambowitz, A M and Westhof, E}
}
@article {405,
	title = {A tyrosyl-tRNA synthetase suppresses structural defects in the two major helical domains of the group I intron catalytic core.},
	journal = {J Mol Biol},
	volume = {262},
	number = {2},
	year = {1996},
	month = {1996 Sep 20},
	pages = {87-104},
	abstract = {The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain). To investigate how CYT-18 stabilizes the active RNA structure, we used an Escherichia coli genetic assay based on the phage T4 td intron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long-range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain. Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7. CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site. Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees. The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in the td intron. Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron{\textquoteright}s active site.},
	keywords = {Bacteriophage T4, Base Sequence, Chromosome Mapping, Escherichia coli, Introns, Kinetics, Mitochondria, Molecular Sequence Data, Mutagenesis, Site-Directed, Neurospora crassa, Nucleic Acid Conformation, Phenotype, RNA Splicing, RNA, Viral, Tyrosine-tRNA Ligase},
	issn = {0022-2836},
	doi = {10.1006/jmbi.1996.0501},
	author = {Myers, C A and Wallweber, G J and Rennard, R and Kemel, Y and Caprara, M G and Mohr, G and Lambowitz, A M}
}
@article {410,
	title = {Group II intron mobility occurs by target DNA-primed reverse transcription.},
	journal = {Cell},
	volume = {82},
	number = {4},
	year = {1995},
	month = {1995 Aug 25},
	pages = {545-54},
	abstract = {Mobile group II introns encode reverse transcriptases and insert site specifically into intronless alleles (homing). Here, in vitro experiments show that homing of the yeast mtDNA group II intron aI2 occurs by reverse transcription at a double-strand break in the recipient DNA. A site-specific endonuclease cleaves the antisense strand of recipient DNA at position +10 of exon 3 and the sense strand at the intron insertion site. Reverse transcription of aI2-containing pre-mRNA is primed by the antisense strand cleaved in exon 3 and results in cotransfer of the intron and flanking exon sequences. Remarkably, the DNA endonuclease that initiates homing requires both the aI2 reverse transcriptase protein and aI2 RNA. Parallels in their reverse transcription mechanisms raise the possibility that mobile group II introns were ancestors of nuclear non-long terminal repeat retrotransposons and telomerases.},
	keywords = {Base Sequence, DNA Primers, DNA, Complementary, DNA, Fungal, DNA, Mitochondrial, Endonucleases, Introns, Molecular Sequence Data, Plasmids, Restriction Mapping, Saccharomyces cerevisiae, Transcription, Genetic},
	issn = {0092-8674},
	author = {Zimmerly, S and Guo, H and Perlman, P S and Lambowitz, A M}
}
@article {409,
	title = {A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility.},
	journal = {Cell},
	volume = {83},
	number = {4},
	year = {1995},
	month = {1995 Nov 17},
	pages = {529-38},
	abstract = {The mobility (homing) of the yeast mitochondrial DNA group II intron al2 occurs via target DNA-primed reverse transcription at a double-strand break in the recipient DNA. Here, we show that the site-specific DNA endonuclease that makes the double-strand break is a ribonucleoprotein complex containing the al2-encoded reverse transcriptase protein and excised al2 RNA. Remarkably, the al2 RNA catalyzes cleavage of the sense strand of the recipient DNA, while the al2 protein appears to cleave the antisense strand. The RNA-catalyzed sense strand cleavage occurs via a partial reverse splicing reaction in which the protein component stabilizes the active intron structure and appears to confer preference for DNA substrates. Our results demonstrate a biologically relevant ribozyme reaction with a substrate other than RNA.},
	keywords = {Base Sequence, Deoxyribonuclease I, DNA, DNA, Antisense, Introns, Molecular Sequence Data, RNA, RNA Splicing, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae Proteins, Substrate Specificity, Yeasts, Zinc},
	issn = {0092-8674},
	author = {Zimmerly, S and Guo, H and Eskes, R and Yang, J and Perlman, P S and Lambowitz, A M}
}
@article {413,
	title = {Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymatic properties pertinent to splicing.},
	journal = {Biochemistry},
	volume = {34},
	number = {4},
	year = {1995},
	month = {1995 Jan 31},
	pages = {1275-87},
	abstract = {The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in the splicing of group I introns. Here, bacterially expressed CYT-18 protein, purified by a new procedure involving polyethyleneimine precipitation to remove tightly bound nucleic acids, was used to characterize properties pertinent to RNA splicing. Analytical ultracentrifugation and other methods showed that the CYT-18 protein is an asymmetric homodimer. The measured frictional ratio, f/fo = 1.55, corresponds to an axial ratio of 10 for a prolate ellipsoid or 12 for an oblate ellipsoid. Like bacterial TyrRSs, the CYT-18 protein exhibits half-sites reactivity, each homodimer having one active site for tyrosyl adenylation and RNA splicing. The splicing activity of CYT-18 was unaffected by aminoacylation substrates at concentrations used in aminoacylation reactions, whereas the TyrRS activity was inhibited by physiological concentrations of the splicing cofactor GTP, as well as CTP or UTP, or by low concentrations of a group I intron RNA. Kinetic measurements suggest that the binding of CYT-18 to a group I intron substrate is a two-step process, with an initial biomolecular step that is close to diffusion limited (3.24 +/- 0.03 x 10(7) M-1s-1) followed by a slower conformational change (0.54 +/- 0.07 s-1). After CYT-18 binding, splicing occurs at a rate of 0.0025 s-1, within 6-fold of the rate of self-splicing of the Tetrahymena large rRNA intron in vitro. The Kd for the complex between the CYT-18 protein and a group I intron substrate, calculated from koff/kon, was < 0.3 pM, substantially lower than determined by presumed equilibrium measurements [Guo, Q., \& Lambowitz, A. M. (1992) Genes Dev. 6, 1357-1372]. As a result of this tight binding, the CYT-18 protein functions stoichiometrically in in vitro splicing reactions due to its extremely slow dissociation from the excised intron RNA. The very tight binding of the CYT-18 protein to the intron RNA raises the possibility that specific mechanisms exist for dissociating the protein from the excised intron in vivo.},
	keywords = {Cloning, Molecular, Guanosine Triphosphate, Introns, Kinetics, Mitochondria, Neurospora crassa, Recombinant Proteins, RNA, RNA Splicing, RNA, Transfer, Tyrosine, Tyrosine-tRNA Ligase},
	issn = {0006-2960},
	author = {Saldanha, R J and Patel, S S and Surendran, R and Lee, J C and Lambowitz, A M}
}
@article {411,
	title = {Mobile group II introns of yeast mitochondrial DNA are novel site-specific retroelements.},
	journal = {Mol Cell Biol},
	volume = {15},
	number = {5},
	year = {1995},
	month = {1995 May},
	pages = {2828-38},
	abstract = {Group II introns aI1 and aI2 of the yeast mitochondrial COXI gene are mobile elements that encode an intron-specific reverse transcriptase (RT) activity. We show here that the introns of Saccharomyces cerevisiae ID41-6/161 insert site specifically into intronless alleles. The mobility is accompanied by efficient, but highly asymmetric, coconversion of nearby flanking exon sequences. Analysis of mutants shows that the aI2 protein is required for the mobility of both aI1 and aI2. Efficient mobility is dependent on both the RT activity of the aI2-encoded protein and a separate function, a putative DNA endonuclease, that is associated with the Zn2+ finger-like region of the intron reading frame. Surprisingly, there appear to be two mobility modes: the major one involves cDNAs reverse transcribed from unspliced precursor RNA; the minor one, observed in two mutants lacking detectable RT activity, appears to involve DNA level recombination. A cis-dominant splicing-defective mutant of aI2 continues to synthesize cDNAs containing the introns but is completely defective in both mobility modes, indicating that the splicing or the structure of the intron is required. Our results demonstrate that the yeast group II intron aI2 is a retroelement that uses novel mobility mechanisms.},
	keywords = {Amino Acid Sequence, Base Sequence, Crosses, Genetic, DNA, Fungal, DNA, Mitochondrial, Genes, Fungal, Genetic Markers, Introns, Molecular Sequence Data, Mutagenesis, Site-Directed, Retroelements, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae},
	issn = {0270-7306},
	author = {Moran, J V and Zimmerly, S and Eskes, R and Kennell, J C and Lambowitz, A M and Butow, R A and Perlman, P S}
}
@article {412,
	title = {The VS catalytic RNA replicates by reverse transcription as a satellite of a retroplasmid.},
	journal = {Genes Dev},
	volume = {9},
	number = {3},
	year = {1995},
	month = {1995 Feb 1},
	pages = {294-303},
	abstract = {The mitochondria of certain natural isolates of Neurospora contain both the Varkud plasmid, which encodes a reverse transcriptase, and a small unrelated RNA (VS RNA) that performs RNA-mediated self-cleavage and ligation reactions. Here, we show that VS RNA is transcribed from a VS plasmid DNA template by the Neurospora mitochondrial RNA polymerase using a promoter located immediately upstream of the RNA self-cleavage site that generates monomeric transcripts. VS RNA is then reverse transcribed by the Varkud plasmid reverse transcriptase to yield a full-length (-) strand cDNA, a predicted replication intermediate. Combined with previous genetic evidence, our results indicate that the VS plasmid replicates by reverse transcription as a satellite of the Varkud plasmid. This mode of replication, unprecedented for a satellite RNA, likely reflects the promiscuity of the Varkud plasmid reverse transcriptase, which does not require a specific primer to initiate cDNA synthesis. Our findings indicate how primitive reverse transcriptases with similar relaxed specificity could have facilitated the evolution of new retroelements.},
	keywords = {Base Sequence, Blotting, Northern, DNA, Complementary, DNA, Mitochondrial, Molecular Sequence Data, Neurospora, Plasmids, RNA, RNA, Satellite, RNA-Directed DNA Polymerase, Transcription, Genetic, Vesicular stomatitis Indiana virus},
	issn = {0890-9369},
	author = {Kennell, J C and Saville, B J and Mohr, S and Kuiper, M T and Sabourin, J R and Collins, R A and Lambowitz, A M}
}
@article {416,
	title = {The Mauriceville plasmid of Neurospora spp. uses novel mechanisms for initiating reverse transcription in vivo.},
	journal = {Mol Cell Biol},
	volume = {14},
	number = {5},
	year = {1994},
	month = {1994 May},
	pages = {3094-107},
	abstract = {The Mauriceville plasmid and the closely related Varkud plasmid of Neurospora spp. are retroelements that propagate in mitochondria. Replication appears to occur by a novel mechanism in which a monomer-length plasmid transcript having a 3{\textquoteright} tRNA-like structure ending in CCA is reverse transcribed to give a full-length minus-strand cDNA beginning at or near the 3{\textquoteright} end of the RNA. Here, we show that the plasmids are transcribed in vitro by the Neurospora mitochondrial RNA polymerase, with the major in vitro transcription start site approximately 260 bp upstream of the 5{\textquoteright} end of the plasmid transcript. The location of the transcription start site suggests that the monomer-length transcripts are generated by transcription around the plasmid combined with a site-specific RNA cleavage after the 3{\textquoteright}-CCA sequence. The 5{\textquoteright} ends of minus-strand cDNAs in ribonucleoprotein particles were analyzed to obtain insight into the mechanism of initiation of reverse transcription in vivo. A major class of minus-strand cDNAs begins opposite C2 of the 3{\textquoteright}-CCA sequence, the same site used for de novo initiation of cDNA synthesis by the plasmid reverse transcriptase in vitro. A second class of minus-strand cDNAs begins with putative primer sequences that correspond to cDNA copies of the plasmid or mitochondrial transcripts. These findings are consistent with the possibility that the plasmid reverse transcriptase initiates minus-strand cDNA synthesis in vivo both by de novo initiation and by a novel template-switching mechanism in which the 3{\textquoteright} OH of a previously synthesized cDNA is used to prime the synthesis of a new minus-strand cDNA directly at the 3{\textquoteright} end of the plasmid transcript.},
	keywords = {Base Sequence, Cloning, Molecular, DNA Primers, DNA Replication, DNA, Complementary, DNA, Fungal, DNA-Directed RNA Polymerases, Electrophoresis, Polyacrylamide Gel, Mitochondria, Molecular Sequence Data, Neurospora, Oligodeoxyribonucleotides, Plasmids, Restriction Mapping, Transcription, Genetic},
	issn = {0270-7306},
	author = {Kennell, J C and Wang, H and Lambowitz, A M}
}
@article {414,
	title = {A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination.},
	journal = {Mol Cell Biol},
	volume = {14},
	number = {10},
	year = {1994},
	month = {1994 Oct},
	pages = {6419-32},
	abstract = {The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5{\textquoteright} segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery.},
	keywords = {Base Sequence, DNA, Circular, DNA, Complementary, DNA, Mitochondrial, DNA, Recombinant, Models, Genetic, Molecular Sequence Data, Neurospora, Nucleic Acid Conformation, Polymerase Chain Reaction, Recombination, Genetic, RNA, RNA, Ribosomal, Sequence Analysis, DNA},
	issn = {0270-7306},
	author = {Chiang, C C and Kennell, J C and Wanner, L A and Lambowitz, A M}
}
@article {415,
	title = {A tyrosyl-tRNA synthetase can function similarly to an RNA structure in the Tetrahymena ribozyme.},
	journal = {Nature},
	volume = {370},
	number = {6485},
	year = {1994},
	month = {1994 Jul 14},
	pages = {147-50},
	abstract = {Group I introns are highly structured RNAs which catalyse their own splicing by guanosine-initiated transesterification reactions. Their catalytic core is generally stabilized by RNA-RNA interactions within the core and with peripheral RNA structures. Additionally, some group I introns require proteins for efficient splicing in vivo. The Neurospora CYT-18 protein, the mitochondrial tyrosyl-transfer RNA synthetase (mt TyrRS), promotes splicing of the Neurospora mitochondrial large ribosomal RNA (LSU) and other group I introns by stabilizing the catalytically active structure of the intron core. We report here that CYT-18 functions similarly to a peripheral RNA structure, P5abc, that stabilizes the catalytic core of the Tetrahymena LSU intron. The CYT-18 protein and P5abc RNA bind to overlapping sites in the intron core, inducing similar conformational changes correlated with splicing activity. Our results show that a protein can play the role of an RNA structure in a catalytic RNA, a substitution postulated for the evolution of nuclear pre-messenger RNA introns from self-splicing introns.},
	keywords = {Animals, Base Sequence, Fungal Proteins, Introns, Molecular Sequence Data, Neurospora, Nucleic Acid Conformation, Protein Binding, RNA, Catalytic, RNA, Protozoan, Tetrahymena, Tyrosine-tRNA Ligase},
	issn = {0028-0836},
	doi = {10.1038/370147a0},
	author = {Mohr, G and Caprara, M G and Guo, Q and Lambowitz, A M}
}
@article {422,
	title = {Group I and group II introns.},
	journal = {FASEB J},
	volume = {7},
	number = {1},
	year = {1993},
	month = {1993 Jan},
	pages = {15-24},
	abstract = {Group I and group II introns are two types of RNA enzymes, ribozymes, that catalyze their own splicing by different mechanisms. In this review, we summarize current information about the structures of group I and group II introns, their RNA-catalyzed reactions, the facilitation of RNA-catalyzed splicing by protein factors, and the ability of the introns to function as mobile elements. The RNA-based enzymatic reactions and intron mobility provide a framework for considering the role of primordial catalytic RNAs in evolution and the origin of introns in higher organisms.},
	keywords = {Amino Acid Sequence, Introns, Molecular Sequence Data, RNA Splicing, RNA, Catalytic},
	issn = {0892-6638},
	author = {Saldanha, R and Mohr, G and Belfort, M and Lambowitz, A M}
}
@article {421,
	title = {Introns as mobile genetic elements.},
	journal = {Annu Rev Biochem},
	volume = {62},
	year = {1993},
	month = {1993},
	pages = {587-622},
	keywords = {Base Sequence, Biological Evolution, Genes, Genes, Bacterial, Genes, Fungal, Introns, Molecular Sequence Data, Open Reading Frames, RNA Splicing},
	issn = {0066-4154},
	doi = {10.1146/annurev.bi.62.070193.003103},
	author = {Lambowitz, A M and Belfort, M}
}
@article {420,
	title = {Reverse transcriptase activity associated with maturase-encoding group II introns in yeast mitochondria.},
	journal = {Cell},
	volume = {73},
	number = {1},
	year = {1993},
	month = {1993 Apr 9},
	pages = {133-46},
	abstract = {Group II introns al1 and al2 of the yeast mtDNA cox1 gene encode reverse transcriptase-like proteins that function in RNA splicing and may play a role in intron mobility and excision. We find that ribonucleoprotein particles from yeast mitochondria contain a reverse transcriptase activity that is likely encoded by al1 and al2 and is highly specific for the introns and their flanking exons. Using a mutant strain with elevated activity, we show that the reverse transcriptase uses either excised intron RNA or cox1 pre-mRNA as template and initiates cDNA synthesis near the 3{\textquoteright} end of al2 and immediately downstream in E3. Our results suggest that introns al1 and al2 are retroelements, which encode reverse transcriptases that have adapted to function in RNA splicing.},
	keywords = {Base Sequence, DNA Transposable Elements, Exons, Introns, Mitochondria, Molecular Sequence Data, Nucleic Acid Conformation, Regulatory Sequences, Nucleic Acid, Ribonucleoproteins, RNA-Directed DNA Polymerase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic},
	issn = {0092-8674},
	author = {Kennell, J C and Moran, J V and Perlman, P S and Butow, R A and Lambowitz, A M}
}
@article {419,
	title = {Reverse transcription of the Mauriceville plasmid of Neurospora. Lack of ribonuclease H activity associated with the reverse transcriptase and possible use of mitochondrial ribonuclease H.},
	journal = {J Biol Chem},
	volume = {268},
	number = {25},
	year = {1993},
	month = {1993 Sep 5},
	pages = {18951-9},
	abstract = {The Mauriceville mitochondrial plasmid of Neurospora encodes a reverse transcriptase that synthesizes a full-length cDNA copy of the major plasmid transcript beginning directly opposite the 3{\textquoteright} end of the template RNA (Kuiper, M. T. R., and Lambowitz, A. M. (1988) Cell 55, 693-704). Here, we show that the Mauriceville plasmid reverse transcriptase has no detectable RNase H activity and that cDNAs synthesized either by the column-purified reverse transcriptase or by the endogenous reverse transcriptase in purified ribonucleoprotein particles remain in a full-length duplex with the template RNA. The column-purified Mauriceville plasmid reverse transcriptase initiates cDNA synthesis by using short DNA primers, which remain attached to the 5{\textquoteright} end of the (-) strand DNA (Wang, H., Kennell, J. C., Kuiper, M. T. R., Sabourin, J. R., Saldanha, R., and Lambowitz, A. M. (1992) Mol. Cell. Biol. 12, 5131-5144). We find that these primer DNAs can be precisely removed by S1 nuclease digestion of the initial cDNA.RNA duplex, suggesting a mechanism whereby this structure may contribute to primer removal in vivo. Finally, we show that Neurospora mitochondria contain an endogenous RNase H activity, which is present in mitochondrial ribonucleoprotein particle preparations prior to their purification. This mitochondrial RNase H can degrade the endogenous plasmid transcript in ribonucleoprotein particles in vitro and could play a similar role in vivo. The finding that the Mauriceville plasmid reverse transcriptase, which is believed to be a primitive enzyme, has no detectable RNase H activity is consistent with the hypothesis that retroviral reverse transcriptases acquired their RNase H domains from a gene encoding a cellular RNase H.},
	keywords = {Amino Acid Sequence, DNA, Magnesium, Manganese, Mitochondria, Molecular Sequence Data, Neurospora, Plasmids, Ribonuclease H, Ribonucleoproteins, RNA, Fungal, RNA-Directed DNA Polymerase, Single-Strand Specific DNA and RNA Endonucleases, Templates, Genetic},
	issn = {0021-9258},
	author = {Wang, H and Lambowitz, A M}
}
@article {418,
	title = {Evolutionary relationships among group II intron-encoded proteins and identification of a conserved domain that may be related to maturase function.},
	journal = {Nucleic Acids Res},
	volume = {21},
	number = {22},
	year = {1993},
	month = {1993 Nov 11},
	pages = {4991-7},
	abstract = {Many group II introns encode reverse transcriptase-like proteins that potentially function in intron mobility and RNA splicing. We compared 34 intron-encoded open reading frames and four related open reading frames that are not encoded in introns. Many of these open reading frames have a reverse transcriptase-like domain, followed by an additional conserved domain X, and a Zn(2+)-finger-like region. Some open reading frames have lost conserved sequence blocks or key amino acids characteristic of functional reverse transcriptases, and some lack the Zn(2+)-finger-like region. The open reading frames encoded by the chloroplast tRNA(Lys) genes and the related Epifagus virginiana matK open reading frame lack a Zn(2+)-finger-like region and have only remnants of a reverse transcriptase-like domain, but retain a readily identifiable domain X. Several findings lead us to speculate that domain X may function in binding of the intron RNA during reverse transcription and RNA splicing. Overall, our findings are consistent with the hypothesis that all of the known group II intron open reading frames evolved from an ancestral open reading frame, which contained reverse transcriptase, X, and Zn(2+)-finger-like domains, and that the reverse transcriptase and Zn(2+)-finger-like domains were lost in some cases. The retention of domain X in most proteins may reflect an essential function in RNA splicing, which is independent of the reverse transcriptase activity of these proteins.},
	keywords = {Amino Acid Sequence, Binding Sites, Biological Evolution, Chloroplasts, Conserved Sequence, Endoribonucleases, Introns, Molecular Sequence Data, Nucleotidyltransferases, Open Reading Frames, Plants, Proteins, RNA, Transfer, Lys, Sequence Homology, Amino Acid},
	issn = {0305-1048},
	author = {Mohr, G and Perlman, P S and Lambowitz, A M}
}
@article {417,
	title = {The Mauriceville plasmid reverse transcriptase can initiate cDNA synthesis de novo and may be related to reverse transcriptase and DNA polymerase progenitor.},
	journal = {Cell},
	volume = {75},
	number = {6},
	year = {1993},
	month = {1993 Dec 17},
	pages = {1071-81},
	abstract = {We show that the reverse transcriptase (RT) encoded by the Mauriceville mitochondrial plasmid of Neurospora closely resembles viral RNA-dependent RNA polymerases in initiating cDNA synthesis opposite the penultimate C residue of a 3{\textquoteright} tRNA-like structure and has the unprecedented ability for a DNA polymerase to initiate DNA synthesis at a specific site in a natural template without a primer. The Mauriceville plasmid enzyme can also use DNA or RNA primers in a manner suggesting how a primitive RT could have evolved from an RNA-dependent RNA polymerase into retroviral and other types of RTs. The characteristics of the Mauriceville plasmid RT suggest that it may be related to the progenitor of present-day RTs and DNA polymerases.},
	keywords = {Base Sequence, Cloning, Molecular, DNA Replication, DNA, Complementary, DNA, Fungal, DNA-Directed DNA Polymerase, Mitochondria, Molecular Sequence Data, Neurospora crassa, Oligodeoxyribonucleotides, Plasmids, Polymerase Chain Reaction, RNA-Directed DNA Polymerase},
	issn = {0092-8674},
	author = {Wang, H and Lambowitz, A M}
}
@article {426,
	title = {Evidence that a 1.6 kilobase region of Neurospora mtDNA was derived by insertion of part of the LaBelle mitochondrial plasmid.},
	journal = {Nucleic Acids Res},
	volume = {20},
	number = {5},
	year = {1992},
	month = {1992 Mar 11},
	pages = {1101-8},
	abstract = {The LaBelle mitochondrial plasmid hybridizes to a small region of the mtDNA of different Neurospora species. Here, we show that the region of homology encompasses 1385 bp of plasmid sequence and 1649 bp of mtDNA sequence. Several findings--that the region of homology is not found in the mtDNAs of other organisms, that it includes the C-terminus of the ORF encoding the plasmid DNA polymerase, and that the ORF sequence in the mtDNA is interrupted by insertions--suggest that the region was part of the plasmid that integrated into mtDNA prior to the divergence of Neurospora species. Since the LaBelle plasmid has been found in only one Neurospora strain, we infer that the plasmid was lost subsequently from most strains. The LaBelle plasmid is transcribed by the host Neurospora mitochondrial RNA polymerase and the major promoter is located upstream of the long ORF, within the region of homology to mtDNA. A promoter used for the transcription of the mitochondrial small rRNA is found at a corresponding position in Neurospora mtDNA and may have been acquired via integration of the plasmid sequence. Our results provide evidence that an autonomous infectious element may contribute to sequences that functionally constitute an organism{\textquoteright}s mtDNA.},
	keywords = {Base Sequence, Blotting, Southern, DNA Transposable Elements, DNA, Mitochondrial, Molecular Sequence Data, Neurospora, Plasmids, Promoter Regions, Genetic, RNA, Ribosomal, Sequence Homology, Nucleic Acid},
	issn = {0305-1048},
	author = {Nargang, F E and Pande, S and Kennell, J C and Akins, R A and Lambowitz, A M}
}
@article {423,
	title = {The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3{\textquoteright} end of template RNA.},
	journal = {Mol Cell Biol},
	volume = {12},
	number = {11},
	year = {1992},
	month = {1992 Nov},
	pages = {5131-44},
	abstract = {The Mauriceville and Varkud plasmids are retroid elements that propagate in the mitochondria of some Neurospora spp. strains. Previous studies of endogenous reactions in ribonucleoprotein particle preparations suggested that the plasmids use a novel mechanism of reverse transcription that involves synthesis of a full-length minus-strand DNA beginning at the 3{\textquoteright} end of the plasmid transcript, which has a 3{\textquoteright} tRNA-like structure (M. T. R. Kuiper and A. M. Lambowitz, Cell 55:693-704, 1988). In this study, we developed procedures for releasing the Mauriceville plasmid reverse transcriptase from mitochondrial ribonucleoprotein particles and partially purifying it by heparin-Sepharose chromatography. By using these soluble preparations, we show directly that the Mauriceville plasmid reverse transcriptase synthesizes full-length cDNA copies of in vitro transcripts beginning at the 3{\textquoteright} end and has a preference for transcripts having the 3{\textquoteright} tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3{\textquoteright}-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3{\textquoteright} end of template RNAs may also be relevant to the mechanisms of reverse transcription used by LINEs, group II introns, and other non-long terminal repeat retroid elements.},
	keywords = {Base Sequence, DNA, Fungal, Mitochondria, Molecular Sequence Data, Neurospora crassa, Nucleic Acid Conformation, Plasmids, Polymerase Chain Reaction, Retroviridae, Ribonucleoproteins, RNA, Fungal, RNA, Transfer, RNA-Directed DNA Polymerase, Solubility, Substrate Specificity, Templates, Genetic},
	issn = {0270-7306},
	author = {Wang, H and Kennell, J C and Kuiper, M T and Sabourin, J R and Saldanha, R and Lambowitz, A M}
}
@article {428,
	title = {The mitochondrial tyrosyl-tRNA synthetase of Podospora anserina is a bifunctional enzyme active in protein synthesis and RNA splicing.},
	journal = {Mol Cell Biol},
	volume = {12},
	number = {2},
	year = {1992},
	month = {1992 Feb},
	pages = {499-511},
	abstract = {The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by the nuclear gene cyt-18, functions not only in aminoacylation but also in the splicing of group I introns. Here, we isolated the cognate Podospora anserina mt tyrRS gene, designated yts1, by using the N. crassa cyt-18 gene as a hybridization probe. DNA sequencing of the P. anserina gene revealed an open reading frame (ORF) of 641 amino acids which has significant similarity to other tyrRSs. The yts1 ORF is interrupted by two introns, one near its N terminus at the same position as the single intron in the cyt-18 gene and the other downstream in a region corresponding to the nucleotide-binding fold. The P. anserina yts1+ gene transformed the N. crassa cyt-18-2 mutant at a high frequency and rescued both the splicing and protein synthesis defects. Furthermore, the YTS1 protein synthesized in Escherichia coli was capable of splicing the N. crassa mt large rRNA intron in vitro. Together, these results indicate that YTS1 is a bifunctional protein active in both splicing and protein synthesis. The P. anserina YTS1 and N. crassa CYT-18 proteins share three blocks of amino acids that are not conserved in bacterial or yeast mt tyrRSs which do not function in splicing. One of these blocks corresponds to the idiosyncratic N-terminal domain shown previously to be required for splicing activity of the CYT-18 protein. The other two are located in the putative tRNA-binding domain toward the C terminus of the protein and also appear to be required for splicing. Since the E. coli and yeast mt tyrRSs do not function in splicing, the adaptation of the Neurospora and Podospora spp. mt tyrRSs to function in splicing most likely occurred after the divergence of their common ancestor from yeast.},
	keywords = {Amino Acid Sequence, Ascomycota, Base Sequence, Cloning, Molecular, DNA, Mitochondrial, Fungal Proteins, Mitochondria, Molecular Sequence Data, Neurospora crassa, RNA Splicing, Sequence Alignment, Tyrosine-tRNA Ligase},
	issn = {0270-7306},
	author = {K{\"a}mper, U and K{\"u}ck, U and Cherniack, A D and Lambowitz, A M}
}
@article {425,
	title = {The neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core.},
	journal = {Cell},
	volume = {69},
	number = {3},
	year = {1992},
	month = {1992 May 1},
	pages = {483-94},
	abstract = {The Neurospora CYT-18 protein, a tyrosyl-tRNA synthetase, which functions in splicing group I introns in mitochondria, promotes splicing of mutants of the distantly related bacteriophage T4 td intron. In an in vivo assay, wild-type CYT-18 protein expressed in E. coli suppressed mutations in the td intron{\textquoteright}s catalytic core. CYT-18-suppressible mutations were also suppressed by high Mg2+ or spermidine in vitro, suggesting they affect intron structure. Both the N- and C-terminal domains of CYT-18 are required for efficient splicing, but CYT-18 with a large C-terminal truncation retains some activity. Our results indicate that CYT-18 interacts with conserved structural features of group I introns, and they provide direct evidence that a protein promotes splicing by stabilizing the catalytically active structure of the intron RNA.},
	keywords = {Base Sequence, Fungal Proteins, Genes, Suppressor, Genes, Viral, Introns, Molecular Sequence Data, Molecular Structure, Neurospora crassa, RNA Splicing, RNA, Viral, Spermidine, T-Phages, Tyrosine-tRNA Ligase, Viral Structural Proteins},
	issn = {0092-8674},
	author = {Mohr, G and Zhang, A and Gianelos, J A and Belfort, M and Lambowitz, A M}
}
@article {427,
	title = {A protein required for RNA processing and splicing in Neurospora mitochondria is related to gene products involved in cell cycle protein phosphatase functions.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {89},
	number = {5},
	year = {1992},
	month = {1992 Mar 1},
	pages = {1676-80},
	abstract = {The Neurospora crassa cyt-4 mutants have pleiotropic defects in mitochondrial RNA splicing, 5{\textquoteright} and 3{\textquoteright} end processing, and RNA turnover. Here, we show that the cyt-4+ gene encodes a 120-kDa protein with significant similarity to the SSD1/SRK1 protein of Saccharomyces cerevisiae and the DIS3 protein of Schizosaccharomyces pombe, which have been implicated in protein phosphatase functions that regulate cell cycle and mitotic chromosome segregation. The CYT-4 protein is present in mitochondria and is truncated or deficient in two cyt-4 mutants. Assuming that the CYT-4 protein functions in a manner similar to the SSD1/SRK1 and DIS3 proteins, we infer that the mitochondrial RNA splicing and processing reactions defective in the cyt-4 mutants are regulated by protein phosphorylation and that the defects in the cyt-4 mutants result from failure to normally regulate this process. Our results provide evidence that RNA splicing and processing reactions may be regulated by protein phosphorylation.},
	keywords = {Amino Acid Sequence, Base Sequence, Cell Cycle, Cloning, Molecular, DNA, Fungal, DNA, Mitochondrial, Fungal Proteins, Genes, Fungal, Mitochondrial Proteins, Molecular Sequence Data, Neurospora crassa, Phosphoprotein Phosphatases, Restriction Mapping, RNA Processing, Post-Transcriptional, RNA, Fungal, Sequence Alignment},
	issn = {0027-8424},
	author = {Turcq, B and Dobinson, K F and Serizawa, N and Lambowitz, A M}
}
@article {424,
	title = {A tyrosyl-tRNA synthetase binds specifically to the group I intron catalytic core.},
	journal = {Genes Dev},
	volume = {6},
	number = {8},
	year = {1992},
	month = {1992 Aug},
	pages = {1357-72},
	abstract = {The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in splicing group I introns in mitochondria. Here, we show that CYT-18 binds strongly to diverse group I introns that have minimal sequence homology and recognizes highly conserved structural features of the catalytic core of these introns. Inhibition experiments indicate that the intron RNA and tRNA(Tyr) compete for the same or overlapping binding sites in the CYT-18 protein. Considered together with functional analysis, our results indicate that the CYT-18 protein promotes splicing by binding to the intron core and stabilizing it in a conformation required for catalytic activity. Furthermore, the specific binding of the synthetase suggests that the group I intron catalytic core has structural similarities to tRNAs, which could reflect either convergent evolution or an evolutionary relationship between group I introns and tRNAs.},
	keywords = {Base Sequence, Binding Sites, Biological Evolution, Introns, Mitochondria, Molecular Sequence Data, Neurospora, Nucleic Acid Conformation, Plasmids, RNA, RNA Splicing, RNA, Ribosomal, Sequence Homology, Nucleic Acid, Tyrosine-tRNA Ligase},
	issn = {0890-9369},
	author = {Guo, Q and Lambowitz, A M}
}
@article {429,
	title = {Integration of a group I intron into a ribosomal RNA sequence promoted by a tyrosyl-tRNA synthetase.},
	journal = {Nature},
	volume = {354},
	number = {6349},
	year = {1991},
	month = {1991 Nov 14},
	pages = {164-7},
	abstract = {Group I and II introns are mobile elements that propagate by insertion into different genes. Some introns of both types self-splice in vitro by transesterification reactions catalysed by the intron RNA. These transesterifications are reversible, and it has been suggested that reverse splicing followed by reverse transcription and recombination with genomic DNA may be a mechanism for intron transposition. In vivo the splicing of many, if not all, group I and II introns requires protein factors, which may facilitate correct folding of the intron RNAs. Here we show that the Neurospora mitochondrial large rRNA intron, a group I intron that is not self-splicing in vitro, undergoes reverse splicing in a reaction promoted by the CYT-18 protein, the Neurospora mitochondrial tyrosyl-tRNA synthetase, which is required for splicing the intron in vivo. In contrast to known RNA-catalysed reverse splicing reactions, this protein-assisted reverse splicing is sufficiently rapid to compete with forward splicing at low RNA concentrations under physiologically relevant conditions, including high GTP and low Mg2+ concentrations. Our results indicate that proteins that promote splicing could contribute to intron mobility by promoting reverse splicing in vivo.},
	keywords = {Base Sequence, DNA Transposable Elements, DNA, Fungal, DNA, Mitochondrial, DNA, Ribosomal, Introns, Molecular Sequence Data, Neurospora crassa, Polymerase Chain Reaction, RNA Splicing, RNA, Ribosomal, Tyrosine-tRNA Ligase},
	issn = {0028-0836},
	doi = {10.1038/354164a0},
	author = {Mohr, G and Lambowitz, A M}
}
@article {432,
	title = {The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase.},
	journal = {Mol Cell Biol},
	volume = {11},
	number = {3},
	year = {1991},
	month = {1991 Mar},
	pages = {1696-706},
	abstract = {The LaBelle-1b strain of Neurospora intermedia contains a 4.1-kb closed-circular mitochondrial plasmid DNA, which encodes a single long open reading frame of 1,151 amino acids reported to have sequence similarity to reverse transcriptases. Here, we show that the LaBelle strain contains a novel DNA polymerase activity that is highly specific for the endogenous LaBelle plasmid DNA in nucleoprotein particles and can be distinguished from the mitochondrial DNA polymerase by several characteristics. Photolabeling experiments indicate that the LaBelle-specific DNA polymerase activity is associated with a polypeptide of 120 kDa, which is in good agreement with the size predicted for the protein encoded by the LaBelle plasmid open reading frame (132 kDa). This 120-kDa polypeptide is found only in the LaBelle strain that contains the mitochondrial plasmid, and it cosegregates with mitochondria in sexual crosses, suggesting that it is encoded by the plasmid. The LaBelle-specific DNA polymerase efficiently uses the artificial DNA substrates, poly(dA)-oligo(dT) and poly(dC)-oligo(dG), but despite its reported sequence similarity to reverse transcriptases, it has very low activity with analogous RNA substrates, poly(rA)-oligo(dT), poly(rC)-oligo(dG), or poly(rCm)-oligo(dG). Considered together with the previous sequence comparisons, our results suggest that the LaBelle plasmid encodes a novel DNA polymerase, which was derived from a protein that was at one time a reverse transcriptase but lost its ability to use RNA templates. This DNA polymerase now presumably functions in replication of the plasmid. Our results constitute the first biochemical evidence for a DNA polymerase activity associated with a mitochondrial plasmid. Further, they may provide insight into the evolution of DNA polymerases from reverse transcriptases, as presumably occurred in the course of evolution following the transition from the so-called RNA world to the present DNA world.},
	keywords = {Amino Acid Sequence, Blotting, Southern, DNA, Fungal, DNA-Directed DNA Polymerase, Genes, Fungal, Mitochondria, Molecular Sequence Data, Neurospora, Nucleoproteins, Plasmids, Restriction Mapping, RNA-Directed DNA Polymerase},
	issn = {0270-7306},
	author = {Schulte, U and Lambowitz, A M}
}
@article {430,
	title = {The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis.},
	journal = {Mol Cell Biol},
	volume = {11},
	number = {8},
	year = {1991},
	month = {1991 Aug},
	pages = {4022-35},
	abstract = {The cyt-20-1 mutant of Neurospora crassa is a temperature-sensitive, cytochrome b- and aa3-deficient strain that is severely deficient in both mitochondrial and cytosolic protein synthesis (R.A. Collins, H. Bertrand, R.J. LaPolla, and A.M. Lambowitz, Mol. Gen. Genet. 177:73-84, 1979). We cloned the cyt-20+ gene by complementation of the cyt-20-1 mutation and found that it contains a 1,093-amino-acid open reading frame (ORF) that encodes both the cytosolic and mitochondrial valyl-tRNA synthetases (vaIRSs). A second mutation, un-3, which is allelic with cyt-20-1, also results in temperature-sensitive growth, but not in gross deficiencies in cytochromes b and aa3 or protein synthesis. The un-3 mutant had also been reported to have pleiotropic defects in cellular transport process, resulting in resistance to amino acid analogs (M.S. Kappy and R.L. Metzenberg, J. Bacteriol. 94:1629-1637, 1967), but this resistance phenotype is separable from the temperature sensitivity in crosses and may result from a mutation in a different gene. The 1,093-amino-acid ORF encoding vaIRSs is the site of missense mutations resulting in temperature sensitivity in both cyt-20-1 and un-3 and is required for the transformation of both mutants. The opposite strand of the cyt-20 gene encodes an overlapping ORF of 532 amino acids, which may also be functional but is not required for transformation of either mutant. The cyt-20-1 mutation in the vaIRS ORF results in severe deficiencies of both mitochondrial and cytosolic vaIRS activities, whereas the un-3 mutation does not appear to result in a deficiency of these activities or of mitochondrial or cytosolic protein synthesis sufficient to account for its temperature-sensitive growth. The phenotype of the un-3 mutant raises the possibility that the vaIRS ORF has a second function in addition to protein synthesis.},
	keywords = {Amino Acid Sequence, Base Sequence, Cloning, Molecular, Codon, Cosmids, Cytosol, DNA, Fungal, Genes, Bacterial, Mitochondria, Molecular Sequence Data, Neurospora crassa, Oligonucleotide Probes, Open Reading Frames, Phenotype, Plasmids, Restriction Mapping, Saccharomyces cerevisiae, Sequence Homology, Nucleic Acid, Valine-tRNA Ligase},
	issn = {0270-7306},
	author = {Kubelik, A R and Turcq, B and Lambowitz, A M}
}
@article {431,
	title = {The Neurospora mitochondrial tyrosyl-tRNA synthetase is sufficient for group I intron splicing in vitro and uses the carboxy-terminal tRNA-binding domain along with other regions.},
	journal = {Genes Dev},
	volume = {5},
	number = {6},
	year = {1991},
	month = {1991 Jun},
	pages = {1009-21},
	abstract = {Neurospora mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by nuclear gene cyt-18, functions in splicing of group I introns in mitochondria. Here, we overproduced functional cyt-18 protein in Escherichia coli and purified it to near homogeneity. The purified protein has splicing and tyrRS activities similar to those of cyt-18 protein isolated from mitochondria and is by itself sufficient to splice the mitochondrial large rRNA intron in vitro. Structure-function relationships in the cyt-18 protein were analyzed by in vitro mutagenesis. We confirmed that a small amino-terminal domain not found in bacterial tyrRSs is required for splicing activity, but not tyrRS activity. Two linker insertion mutations, which disrupt the predicted ATP-binding site, completely inhibit tyrRS activity but leave substantial splicing activity. Finally, deletions or linker insertion mutations in the putative carboxy-terminal tRNA-binding domain inhibit both tyrRS and splicing activities, although some have differential effects on the two activities. Our results show that the normal catalytic activity of the cyt-18 protein is not required for splicing and are consistent with the hypothesis that the protein functions by binding to the precursor RNA and facilitating formation of the correct RNA structure. Regions required for splicing are distributed throughout the cyt-18 protein and overlap, but are not identical to, regions required for tyrRS activity. The finding that the putative carboxy-terminal tRNA-binding domain is required for both tyrRS and splicing activities suggests that the mechanism for binding the intron has similarities to the mechanism for binding tRNA(Tyr).},
	keywords = {Adenosine Triphosphate, Amino Acid Sequence, Base Sequence, Cloning, Molecular, DNA, Fungal, Genes, Fungal, Introns, Mitochondria, Molecular Sequence Data, Mutagenesis, Insertional, Neurospora crassa, Restriction Mapping, RNA Splicing, RNA, Fungal, RNA, Transfer, Tyrosine-tRNA Ligase},
	issn = {0890-9369},
	author = {Kittle, J D and Mohr, G and Gianelos, J A and Wang, H and Lambowitz, A M}
}
@article {433,
	title = {Structural analysis of the Neurospora mitochondrial large rRNA intron and construction of a mini-intron that shows protein-dependent splicing.},
	journal = {J Biol Chem},
	volume = {266},
	number = {3},
	year = {1991},
	month = {1991 Jan 25},
	pages = {1809-19},
	abstract = {The gene encoding the Neurospora mitochondrial large rRNA contains a single group I intron of 2.3 kilobases that is not self-splicing in vitro. We showed previously that the splicing of this intron in vivo and in vitro is dependent on the Neurospora cyt-18 protein, mitochondrial tyrosyl-tRNA synthetase. In the present work, we carried out further structural analysis of the intron and constructed mutant derivatives of it in order to identify features that are either required for splicing or prevent it from self-splicing. Previous studies showed that the intron contains a large hairpin structure near the 5{\textquoteright} splice site. By mapping RNase III cleavage sites, we identified this hairpin structure as an extended P2 stem. We construct a mini-intron of 388 nucleotides by deleting the 426-amino acid intron open reading frame, most of the 5{\textquoteright} intron hairpin, and all of L8. This mini-intron shows the same protein-dependent splicing as the full length intron, but is still not self-splicing. Further deletions, which remove all of P2 or all or part of P4, P6, P7, or P9, inactivate splicing, suggesting that an intact group I intron core structure is required. Strengthening the P1, P10, or P9.0 pairings did not enable the mini-intron to self-splice. Our findings indicate that the inability of the mitochondrial large rRNA intron to self-splice reflects deficiency of a structure or activity required for cleavage at the 5{\textquoteright} splice site, either in the intron core itself or in the interaction between the core and the P1 stem.},
	keywords = {Base Sequence, DNA Mutational Analysis, DNA, Fungal, DNA, Mitochondrial, DNA, Ribosomal, Endoribonucleases, Genes, Guanosine, Guanosine Triphosphate, Hydrogen Bonding, Introns, Molecular Sequence Data, Molecular Structure, Neurospora crassa, Regulatory Sequences, Nucleic Acid, Restriction Mapping, Ribonuclease III, Ribosomal Proteins, RNA Splicing, Structure-Activity Relationship},
	issn = {0021-9258},
	author = {Guo, Q B and Akins, R A and Garriga, G and Lambowitz, A M}
}
@article {434,
	title = {Analysis of large deletions in the Mauriceville and Varkud mitochondrial plasmids of Neurospora.},
	journal = {Curr Genet},
	volume = {18},
	number = {4},
	year = {1990},
	month = {1990 Nov},
	pages = {365-9},
	abstract = {The Mauriceville and Varkud mitochondrial plasmids are closely related, closed-circular DNAs (3.6 and 3.7 kb, respectively) that have characteristics of mtDNA introns and retroid elements. Both plasmids contain a 710 amino acid open reading frame (ORF) that encodes an 81 kDa protein having reverse transcriptase activity. Here, we analyzed two mutant plasmids, V5-36 and M3-24, that have undergone relatively large deletions (approximately 0.35 and 0.5 kb, respectively). Both deletions occur downstream of the long ORF in a non-coding region of the plasmids that contains a direct repeat of 160 bp and a cluster of five PstI-palindromes, a repetitive sequence element in Neurospora mtDNA. In V5-36, the deletion end points are at the bases of two hairpin structures that are centered around PstI-palindromes and flank the deleted region. In M3-24, the deletion junction contains an extra T-residue that is not encoded in the plasmid. In both plasmids, the deletion end points do not correspond to homologous or directly repeated sequences of more than one nucleotide, whose pairing could account for the deletion junction. The characteristics of the deletion end points can be accounted for either by illegitimate recombination, possibly following double strand breaks at cruciform structures, or by interruption of reverse transcription followed by reinitiation downstream. The finding that the deletions encompass the 160 bp direct repeat and all five PstI-palindromes indicates that neither are required for propagation of the plasmids and supports the hypothesis that PstI-palindromes are selfish DNA elements that inserted into a nonessential region of the plasmid.},
	keywords = {Amino Acid Sequence, Base Sequence, Chromosome Deletion, DNA, Fungal, DNA, Mitochondrial, Introns, Molecular Sequence Data, Neurospora, Nucleic Acid Conformation, Open Reading Frames, Plasmids, Recombination, Genetic, Repetitive Sequences, Nucleic Acid, Restriction Mapping, Transcription, Genetic},
	issn = {0172-8083},
	author = {Akins, R A and Lambowitz, A M}
}
@article {436,
	title = {Function of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing requires an idiosyncratic domain not found in other synthetases.},
	journal = {Cell},
	volume = {62},
	number = {4},
	year = {1990},
	month = {1990 Aug 24},
	pages = {745-55},
	abstract = {Neurospora mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), which is encoded by nuclear gene cyt-18, functions in splicing group I introns. Analysis of intragenic partial revertants of the cyt-18-2 mutant and in vitro mutants of the cyt-18 protein expressed in E. coli showed that splicing activity of the cyt-18 protein is dependent on a small N-terminal domain that has no homolog in bacterial or yeast mt TyrRSs. This N-terminal splicing domain apparently acts together with other regions of the protein to promote splicing. Our findings support the hypothesis that idiosyncratic sequences in aminoacyl-tRNA synthetase may function in processes other than aminoacylation. Furthermore, they suggest that splicing activity of the Neurospora mt TyrRs was acquired after the divergence of Neurospora and yeast, and they demonstrate one mechanism whereby splicing factors may evolve from cellular RNA binding proteins.},
	keywords = {Amino Acid Sequence, Amino Acyl-tRNA Synthetases, Base Sequence, Genes, Fungal, Mitochondria, Molecular Sequence Data, Molecular Weight, Mutation, Neurospora, Neurospora crassa, Phenotype, Protein Conformation, RNA Splicing, Spectrum Analysis, Structure-Activity Relationship, Tyrosine, Tyrosine-tRNA Ligase},
	issn = {0092-8674},
	author = {Cherniack, A D and Garriga, G and Kittle, J D and Akins, R A and Lambowitz, A M}
}
@article {438,
	title = {Identification of Neurospora mitochondrial promoters and analysis of synthesis of the mitochondrial small rRNA in wild-type and the promoter mutant [poky].},
	journal = {J Biol Chem},
	volume = {265},
	number = {8},
	year = {1990},
	month = {1990 Mar 15},
	pages = {4515-26},
	abstract = {Using an in vitro transcription assay, we previously identified promoters in Neurospora mtDNA at the 5{\textquoteright} ends of the genes encoding the mitochondrial (mt) small and large rRNAs and cob pre-mRNA. Here, we identified two additional promoters in mtDNA restriction fragment EcoRI-6, 3.8 and 5.5 kilobases upstream of the 5{\textquoteright} end of the mt small rRNA. By comparing the two new promoters with the three identified previously, we derived a modified promoter consensus sequence (AT-rich)15-27TTAG(A/T)RR(G/T)(G/C)N(A/T). The mt small rRNA in Neurospora is transcribed from at least two promoters, a major promoter at the 5{\textquoteright} end of the small rRNA and one or both of the newly identified promoters in EcoRI-6. The latter gives a series of putative pre-rRNAs that contain 5{\textquoteright} end extensions of various sizes. The 5{\textquoteright} ends of a number of these RNAs map at or near hairpin structures. The [poky] mutant, which is grossly deficient in the mt small rRNA, has a 4-base pair deletion in the major promoter at the 5{\textquoteright} end of the mt small rRNA. The residual small rRNAs in [poky] appear to be synthesized via the upstream promoter(s), but are missing 37-44 nucleotides from their 5{\textquoteright} ends, indicating either that pre-rRNAs are processed abnormally or that abnormal 5{\textquoteright} RNA ends are unstable. The effect of the promoter mutation in [poky] on other transcripts suggests that the mt small rRNA is cotranscribed with downstream genes encoding tRNAs, coIII and ND6. Seven nonallelic nuclear suppressors of [poky] result in increased concentrations of the mt small rRNA and pre-rRNAs, but do not restore the ability to synthesize small rRNAs having the correct 5{\textquoteright} ends. The suppressor mutations could act by increasing transcription, processing, or stability of the mt small rRNA or its precursors. The suppressors provide a genetic approach for identifying components that affect transcription and processing of the mt small rRNA.},
	keywords = {Base Sequence, Cloning, Molecular, Deoxyribonuclease EcoRI, DNA, Fungal, DNA, Mitochondrial, Escherichia coli, Molecular Sequence Data, Mutation, Neurospora, Neurospora crassa, Nucleic Acid Hybridization, Promoter Regions, Genetic, Restriction Mapping, RNA, RNA Precursors, RNA, Fungal, RNA, Ribosomal, Sequence Homology, Nucleic Acid, Transcription, Genetic},
	issn = {0021-9258},
	author = {Kubelik, A R and Kennell, J C and Akins, R A and Lambowitz, A M}
}
@article {437,
	title = {Identification of the reverse transcriptase encoded by the Mauriceville and Varkud mitochondrial plasmids of Neurospora.},
	journal = {J Biol Chem},
	volume = {265},
	number = {12},
	year = {1990},
	month = {1990 Apr 25},
	pages = {6936-43},
	abstract = {The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) that have characteristics of mtDNA introns and retroid elements. The plasmids contain a single long open reading frame (710 amino acids), whose amino-terminal half has structural similarity to reverse transcriptases. Using antibodies against synthetic peptides and trpE fusion proteins, we detected an 81-kDa protein encoded by this open reading frame in mitochondrial preparations from the plasmid-containing strains. This 81-kDa protein cosegregates with reverse transcriptase activity in sexual crosses and comigrates with reverse transcriptase activity in sodium dodecyl sulfate-polyacrylamide gels, where it can be assayed after renaturation of the protein. In glycerol gradients under nondenaturing conditions, the reverse transcriptase activity sediments at approximately 145 kDa, close to the value expected for a dimer of the 81-kDa protein. The 81-kDa protein represents most of the 710-amino acid open reading frame, but may be missing some amino acids at the amino terminus. The regions upstream and downstream of the putative reverse transcriptase domain lack sequences characteristic of gag, protease, RNase H, or integrase domains found in other retroid elements. The plasmid-encoded 81-kDa protein seems to be a novel type of reverse transcriptase that may provide insight into the evolution of these enzymes.},
	keywords = {Cloning, Molecular, DNA, Mitochondrial, Escherichia coli, Mitochondria, Molecular Weight, Neurospora, Neurospora crassa, Plasmids, Restriction Mapping, Ribonucleoproteins, RNA-Directed DNA Polymerase, Species Specificity},
	issn = {0021-9258},
	author = {Kuiper, M T and Sabourin, J R and Lambowitz, A M}
}
@article {435,
	title = {Involvement of aminoacyl-tRNA synthetases and other proteins in group I and group II intron splicing.},
	journal = {Trends Biochem Sci},
	volume = {15},
	number = {11},
	year = {1990},
	month = {1990 Nov},
	pages = {440-4},
	abstract = {Group I and group II introns catalyse their own splicing, but depend on protein factors for efficient splicing in vivo. Some of these proteins, termed maturases, are encoded by the introns themselves and may also function in intron mobility. Other proteins are encoded by host chromosomal genes and include aminoacyl-tRNA synthetases and various proteins that function in protein synthesis. The splicing factors identified thus far appear to be idiosyncratic, even in closely related organisms. We suggest that some of these protein-assisted splicing reactions evolved relatively recently, possibly reflecting the recent dispersal of the introns themselves.},
	keywords = {Amino Acyl-tRNA Synthetases, Biological Evolution, Introns, Neurospora, RNA Splicing, Saccharomyces cerevisiae},
	issn = {0968-0004},
	author = {Lambowitz, A M and Perlman, P S}
}
@article {446,
	title = {Characterization of mutant mitochondrial plasmids of Neurospora spp. that have incorporated tRNAs by reverse transcription.},
	journal = {Mol Cell Biol},
	volume = {9},
	number = {2},
	year = {1989},
	month = {1989 Feb},
	pages = {678-91},
	abstract = {The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, closed-circular DNAs (3.6 and 3.7 kilobases, respectively) whose nucleotide sequences and genetic organization suggest relationships to mitochondrial introns and retroelements. We have characterized nine suppressive mutants of these plasmids that outcompete mitochondrial DNA and lead to impaired growth. All nine suppressive plasmids contain small insertions, corresponding to or including a mitochondrial tRNA (tRNATrp, tRNAGly, or tRNAVal) or a tRNA-like sequence. The insertions are located at the position corresponding to the 5{\textquoteright} end of the major plasmid transcript or 24 nucleotides downstream near a cognate of the sequence at the major 5{\textquoteright} RNA end. The structure of the suppressive plasmids suggests that the tRNAs were inserted via an RNA intermediate. The 3{\textquoteright} end of the wild-type plasmid transcript can itself be folded into a secondary structure which has tRNA-like characteristics, similar to the tRNA-like structures at the 3{\textquoteright} ends of plant viral RNAs. This structure may play a role in replication of the plasmids by reverse transcription. Major transcripts of the suppressive plasmids begin at the 5{\textquoteright} end of the inserted mitochondrial tRNA sequence and are present in 25- to 100-fold-higher concentrations than are transcripts of wild-type plasmids. Mapping of 5{\textquoteright} RNA ends within the inserted mtDNA sequences identifies a short consensus sequence (PuNPuAG) which is present at the 5{\textquoteright} ends of a subset of mitochondrial tRNA genes. This sequence, together with sequences immediately upstream in the plasmids, forms a longer consensus sequence, which is similar to sequences at transcription initiation sites in Neurospora mitochondrial DNA. The suppressive behavior of the plasmids is likely to be directly related to the insertion of tRNAs leading to overproduction of plasmid transcripts.},
	keywords = {DNA Mutational Analysis, DNA Transposable Elements, DNA, Fungal, DNA, Mitochondrial, Genes, Fungal, Molecular Sequence Data, Neurospora, Plasmids, Restriction Mapping, RNA, Fungal, RNA, Transfer, RNA-Directed DNA Polymerase, Suppression, Genetic, Transcription, Genetic},
	issn = {0270-7306},
	author = {Akins, R A and Kelley, R L and Lambowitz, A M}
}
@article {440,
	title = {Cloning and analysis of the Neurospora crassa gene for cytochrome c heme lyase.},
	journal = {J Biol Chem},
	volume = {264},
	number = {30},
	year = {1989},
	month = {1989 Oct 25},
	pages = {17897-906},
	abstract = {The cyt-2-1 mutant of Neurospora crassa is deficient in cytochromes aa3 and c and in cytochrome c heme lyase activity (Mitchell, M.B., Mitchell, H.K., and Tissieres, A. (1953) Proc. Natl. Acad. Sci. U.S.A. 39, 606-613; Nargang, F.E., Drygas, M.E., Kwong, P.L., Nicholson, D.W., and Neupert, W. (1988) J. Biol. Chem. 263, 9388-9394). By rescue of the slow growth character of the cyt-2-1 mutant, we have cloned the cyt-2+ gene from a N. crassa genomic library using sib selection. Analysis of the DNA sequence of the cyt-2+ gene revealed an open reading frame of 346 amino acids that has homology to the yeast cytochrome c heme lyase. The open reading frame is interrupted by two short introns. Codon usage and Northern hybridization analysis suggest that the cyt-2 gene is expressed at low levels. The cyt-2-1 mutant allele was cloned from a partial cyt-2-1 gene bank using the wild-type gene as a probe. Sequence analysis of the mutant gene revealed a 2-base (CT) deletion that alters the reading frame for 21 codons before generating an early stop codon in the protein-coding sequence. It was previously suggested that the cyt-2-1 mutation inactivates one of two regulatory circuits controlling the production of cytochrome aa3. The finding that the cyt-2-1 mutation affects the coding sequence for cytochrome c heme lyase provides a direct explanation for the deficiency of cytochrome c in the mutant and suggests that the lack of cytochrome aa3 is a regulatory response to the deficiency of cytochrome c.},
	keywords = {Amino Acid Sequence, Base Sequence, Cloning, Molecular, Escherichia coli, Genes, Fungal, Lyases, Molecular Sequence Data, Mutation, Neurospora, Neurospora crassa, Polymorphism, Restriction Fragment Length, Restriction Mapping, RNA Splicing, Saccharomyces cerevisiae, Sequence Homology, Nucleic Acid},
	issn = {0021-9258},
	author = {Drygas, M E and Lambowitz, A M and Nargang, F E}
}
@article {442,
	title = {Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.},
	journal = {Mol Cell Biol},
	volume = {9},
	number = {9},
	year = {1989},
	month = {1989 Sep},
	pages = {3603-13},
	abstract = {We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5{\textquoteright} ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5{\textquoteright} ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5{\textquoteright}-TTAGARA(T/G)G(T/G)ARTRR-3{\textquoteright}, all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5{\textquoteright}-TTAGARR(T/G)R(T/G)A-3{\textquoteright}, was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5{\textquoteright} end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99\%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.},
	keywords = {Base Sequence, DNA, Fungal, DNA, Mitochondrial, Genes, Fungal, Molecular Sequence Data, Mutation, Neurospora, Neurospora crassa, Promoter Regions, Genetic, RNA, Fungal, RNA, Ribosomal, Transcription, Genetic},
	issn = {0270-7306},
	author = {Kennell, J C and Lambowitz, A M}
}
@article {439,
	title = {A group II intron in the Neurospora mitochondrial coI gene: nucleotide sequence and implications for splicing and molecular evolution.},
	journal = {Nucleic Acids Res},
	volume = {17},
	number = {22},
	year = {1989},
	month = {1989 Nov 25},
	pages = {9087-99},
	abstract = {The temperature-sensitive Neurospora nuclear mutant cyt18-1 is deficient in splicing many Group I mitochondrial introns when grown at its non-permissive temperature; however, splicing of intron 1 in the coI gene of the Adiopodoume (formerly called North Africa) strain is unaffected (R.A. Collins and A.M. Lambowitz, J. Mol. Biol. 184: 413-428, 1985). Here we show that coI intron 1 is a typical Group II intron, the only one identified to date in Neurospora. The differential effect of the cyt18-1 mutation suggests that splicing of certain introns could be regulated independently of others by nuclear-encoded proteins. The intron contains a long open reading frame (ORF) resembling that of the Neurospora Mauriceville mitochondrial plasmid. The intron and plasmid ORFs share unusual features of codon usage that suggest both evolved outside of the Neurospora mitochondrial genetic system.},
	keywords = {Amino Acid Sequence, Base Sequence, Biological Evolution, Exons, Genes, Fungal, Introns, Molecular Sequence Data, Mutation, Neurospora, Neurospora crassa, Nucleic Acid Conformation, Plasmids, Restriction Mapping, RNA Splicing, Sequence Homology, Nucleic Acid},
	issn = {0305-1048},
	author = {Field, D J and Sommerfield, A and Saville, B J and Collins, R A}
}
@article {444,
	title = {Immunological identification of the alternative oxidase of Neurospora crassa mitochondria.},
	journal = {Mol Cell Biol},
	volume = {9},
	number = {3},
	year = {1989},
	month = {1989 Mar},
	pages = {1362-4},
	abstract = {Neurospora crassa mitochondria use a branched electron transport system in which one branch is a conventional cytochrome system and the other is an alternative cyanide-resistant, hydroxamic acid-sensitive oxidase that is induced when the cytochrome system is impaired. We used a monoclonal antibody to the alternative oxidase of the higher plant Sauromatum guttatum to identify a similar set of related polypeptides (Mr, 36,500 and 37,000) that was associated with the alternative oxidase activity of N. crassa mitochondria. These polypeptides were not present constitutively in the mitochondria of a wild-type N. crassa strain, but were produced in high amounts under conditions that induced alternative oxidase activity. Under the same conditions, mutants in the aod-1 gene, with one exception, produced apparently inactive alternative oxidase polypeptides, whereas mutants in the aod-2 gene failed to produce these polypeptides. The latter findings support the hypothesis that aod-1 is a structural gene for the alternative oxidase and that the aod-2 gene encodes a component that is required for induction of alternative oxidase activity. Finally, our results indicate that the alternative oxidase is highly conserved, even between plant and fungal species.},
	keywords = {Antibodies, Monoclonal, Electron Transport, Genes, Genes, Fungal, Mitochondria, Molecular Weight, Mutation, Neurospora, Neurospora crassa, Oxidoreductases},
	issn = {0270-7306},
	author = {Lambowitz, A M and Sabourin, J R and Bertrand, H and Nickels, R and McIntosh, L}
}
@article {445,
	title = {Infectious introns.},
	journal = {Cell},
	volume = {56},
	number = {3},
	year = {1989},
	month = {1989 Feb 10},
	pages = {323-6},
	keywords = {DNA Transposable Elements, Introns, RNA Splicing},
	issn = {0092-8674},
	author = {Lambowitz, A M}
}
@article {443,
	title = {Involvement of tyrosyl-tRNA synthetase in splicing of group I introns in Neurospora crassa mitochondria: biochemical and immunochemical analyses of splicing activity.},
	journal = {Mol Cell Biol},
	volume = {9},
	number = {5},
	year = {1989},
	month = {1989 May},
	pages = {2089-104},
	abstract = {We reported previously that mitochondrial tyrosyl-tRNA synthetase, which is encoded by the nuclear gene cyt-18 in Neurospora crassa, functions in splicing several group I introns in N. crassa mitochondria (R. A. Akins and A. M. Lambowitz, Cell 50:331-345, 1987). Two mutants in the cyt-18 gene (cyt-18-1 and cyt-18-2) are defective in both mitochondrial protein synthesis and splicing, and an activity that splices the mitochondrial large rRNA intron copurifies with a component of mitochondrial tyrosyl-tRNA synthetase. Here, we used antibodies against different trpE-cyt-18 fusion proteins to identify the cyt-18 gene product as a basic protein having an apparent molecular mass of 67 kilodaltons (kDa). Both the cyt-18-1 and cyt-18-2 mutants contain relatively high amounts of inactive cyt-18 protein detected immunochemically. Biochemical experiments show that the 67-kDa cyt-18 protein copurifies with splicing and synthetase activity through a number of different column chromatographic procedures. Some fractions having splicing activity contain only one or two prominent polypeptide bands, and the cyt-18 protein is among the few, if not only, major bands in common between the different fractions that have splicing activity. Phosphocellulose columns resolve three different forms or complexes of the cyt-18 protein that have splicing or synthetase activity or both. Gel filtration experiments show that splicing activity has a relatively small molecular mass (peak at 150 kDa with activity trailing to lower molecular masses) and could correspond simply to dimers or monomers, or both, of the cyt-18 protein. Finally, antibodies against different segments of the cyt-18 protein inhibit splicing of the large rRNA intron in vitro. Our results indicate that both splicing and tyrosyl-tRNA synthetase activity are associated with the same 67-kDa protein encoded by the cyt-18 gene. This protein is a key constituent of splicing activity; it functions directly in splicing, and few, if any, additional components are required for splicing the large rRNA intron.},
	keywords = {Amino Acyl-tRNA Synthetases, Genes, Fungal, Introns, Mitochondria, Mutation, Neurospora, Neurospora crassa, RNA Splicing, RNA, Fungal, Tyrosine-tRNA Ligase},
	issn = {0270-7306},
	author = {Majumder, A L and Akins, R A and Wilkinson, J G and Kelley, R L and Snook, A J and Lambowitz, A M}
}
@article {441,
	title = {Mutations in nuclear gene cyt-4 of Neurospora crassa result in pleiotropic defects in processing and splicing of mitochondrial RNAs.},
	journal = {Genetics},
	volume = {123},
	number = {1},
	year = {1989},
	month = {1989 Sep},
	pages = {97-108},
	abstract = {The nuclear cyt-4 mutants of Neurospora crassa have been shown previously to be defective in splicing the group I intron in the mitochondrial large rRNA gene and in 3{\textquoteright} end synthesis of the mitochondrial large rRNA. Here, Northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial RNA processing pathways, including those for cob, coI, coII and ATPase 6 mRNAs, as well as mitochondrial tRNAs. Defects in these pathways include inhibition of 5{\textquoteright} and 3{\textquoteright} end processing, accumulation of aberrant RNA species, and inhibition of splicing of both group I introns in the cob gene. The various defects in mitochondrial RNA synthesis in the cyt-4-1 mutant cannot be accounted for by deficiency of mitochondrial protein synthesis or energy metabolism, and they suggest that the cyt-4-1 mutant is defective in a component or components required for processing and/or turnover of a number of different mitochondrial RNAs. Defective splicing of the mitochondrial large rRNA intron in the cyt-4-1 mutant may be a secondary effect of failure to synthesize pre-rRNAs having the correct 3{\textquoteright} end. However, a similar explanation cannot be invoked to account for defective splicing of the cob pre-mRNA introns, and the cyt-4-1 mutation may directly affect splicing of these introns.},
	keywords = {Blotting, Northern, Electrophoresis, Polyacrylamide Gel, Introns, Mitochondria, Mutation, Neurospora, Neurospora crassa, Polymorphism, Restriction Fragment Length, Restriction Mapping, RNA, RNA Probes, RNA Splicing, Transcription, Genetic},
	issn = {0016-6731},
	author = {Dobinson, K F and Henderson, M and Kelley, R L and Collins, R A and Lambowitz, A M}
}
@article {450,
	title = {A 3{\textquoteright} splice site mutation in a nuclear gene encoding a mitochondrial ribosomal protein in Neurospora crassa.},
	journal = {J Biol Chem},
	volume = {263},
	number = {6},
	year = {1988},
	month = {1988 Feb 25},
	pages = {2848-52},
	abstract = {We showed previously that the cyt-21+ gene of Neurospora crassa encodes a mitochondrial ribosomal protein homologous to Escherichia coli ribosomal protein S-16 (Kuiper, M. T. R., Akins, R. A., Holtrop, M., de Vries, H., and Lambowitz, A. M. (1988) J. Biol. Chem. 263, 2840-2847). A mutation in this gene, cyt-21-1, results in deficiency of mitochondrial small ribosomal subunits and small rRNA (Collins, R. A., Bertrand, H., LaPolla, R. J., and Lambowitz, A. M. (1979) Mol. Gen. Genet. 177, 73-84). In the present work, cloning and sequencing of the cyt-21-1 mutant allele show that it contains a single dG to dA transition at the 3{\textquoteright} splice site AG of the first intron in the protein coding region. This mutation leads to inactivation of the normal 3{\textquoteright} splice site and activation of a cryptic 3{\textquoteright} splice site, 15 nucleotides downstream. The use of this cryptic splice site results in an in-frame deletion of 5 amino acids from the cyt-21 protein. Comparison of mutant and wild-type mitochondrial small ribosomal subunit proteins showed one protein, S-24, with an altered electrophoretic mobility, consistent with the predicted deletion. The mutant ribosomal protein is still capable of binding to mitochondrial small ribosomal subunits, but results in abnormal mitochondrial ribosome assembly.},
	keywords = {Alleles, Amino Acid Sequence, Base Sequence, Endonucleases, Genes, Fungal, Mutation, Neurospora, Neurospora crassa, Ribosomal Proteins, RNA, Fungal, Single-Strand Specific DNA and RNA Endonucleases},
	issn = {0021-9258},
	author = {Kuiper, M T and Holtrop, M and Vennema, H and Lambowitz, A M and de Vries, H}
}
@article {451,
	title = {Isolation and analysis of the Neurospora crassa Cyt-21 gene. A nuclear gene encoding a mitochondrial ribosomal protein.},
	journal = {J Biol Chem},
	volume = {263},
	number = {6},
	year = {1988},
	month = {1988 Feb 25},
	pages = {2840-7},
	abstract = {The Neurospora crassa nuclear mutant cyt-21-1 (originally 297-24; Pittenger, T.H., and West, D.J. (1979) Genetics 93, 539-555) has a defect leading to gross deficiency of mitochondrial small ribosomal subunits. Here, we have cloned the cyt-21+ gene from a N. crassa genomic library, using the sib selection procedure (Akins, R. A., and Lambowitz, A. M. (1985) Mol. Cell Biol. 5, 2272-2278). The genomic clone contains a short split gene encoding a basic protein of 107 amino acid residues. This protein shows strong homology to Escherichia coli ribosomal protein S-16. Comparison of mutant and wild-type mitochondrial ribosomal proteins (Kuiper, M. T. R., Holtrop, M., Vennema, H., Lambowitz, A. M., and de Vries, H. (1988) J. Biol. Chem. 263, 2848-2852) indicates that the cyt-21 gene encodes N. crassa mitochondrial ribosomal protein S-24. The expression of the cyt-21+ gene is regulated such that the level of the putative cyt-21+ mRNA is increased about 5-fold when mitochondrial protein synthesis is inhibited. We suggest that this reflects part of a general mechanism for coordinately activating Neurospora nuclear genes that encode mitochondrial constituents in response to impaired mitochondrial function. This is the first report of the cloning and characterization of a mitochondrial ribosomal protein gene from N. crassa.},
	keywords = {Amino Acid Sequence, Base Sequence, DNA Restriction Enzymes, Genes, Fungal, Mitochondria, Molecular Sequence Data, Mutation, Neurospora, Neurospora crassa, Nucleic Acid Hybridization, Ribosomal Proteins, Transcription, Genetic},
	issn = {0021-9258},
	author = {Kuiper, M T and Akins, R A and Holtrop, M and de Vries, H and Lambowitz, A M}
}
@article {449,
	title = {The structural gene for a phosphorus-repressible phosphate permease in Neurospora crassa can complement a mutation in positive regulatory gene nuc-1.},
	journal = {Mol Cell Biol},
	volume = {8},
	number = {3},
	year = {1988},
	month = {1988 Mar},
	pages = {1376-9},
	abstract = {van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. This was unexpected because the nuc-1 host already contained a resident van+ gene. Plasmids carrying van+ complemented a nuc-2 mutation as well. Probing of RNA from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control by nuc-1+. Probing of the same RNAs with a cosmid clone, containing approximately 15 kilobases of upstream and downstream DNA, revealed no other detectable phosphorus-regulated transcripts within this 40-kilobase region of the chromosome.},
	keywords = {Cloning, Molecular, Gene Expression Regulation, Genes, Genes, Fungal, Genes, Regulator, Genetic Complementation Test, Membrane Transport Proteins, Mutation, Neurospora, Neurospora crassa, Nucleic Acid Hybridization, Phosphate Transport Proteins, Phosphates, Phosphorus, Plasmids, RNA, Fungal, Transcription, Genetic, Vanadates},
	issn = {0270-7306},
	author = {Mann, B J and Akins, R A and Lambowitz, A M and Metzenberg, R L}
}
@article {447,
	title = {A novel reverse transcriptase activity associated with mitochondrial plasmids of Neurospora.},
	journal = {Cell},
	volume = {55},
	number = {4},
	year = {1988},
	month = {1988 Nov 18},
	pages = {693-704},
	abstract = {The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related DNA elements whose nucleotide sequences and genetic organization suggest relationships to retrotransposons and mitochondrial introns. Both plasmids potentially encode a reverse transcriptase-like protein of 710 amino acids. We show that mitochondria from the Mauriceville and Varkud strains contain a reverse transcriptase activity highly specific for endogeneous plasmid RNA in RNP preparations. The reverse transcriptase synthesizes full-length minus-strand DNA beginning at the 3{\textquoteright} end of the plasmid transcript, which has tRNA-like characteristics similar to the 3{\textquoteright} ends of plant viral RNAs. Our results suggest that the plasmids use a novel mechanism of reverse transcription, which may have evolved to utilize tRNA-like structures at the 3{\textquoteright} ends of self-replicating RNAs. This mechanism may be ancestral to the standard retroviral mechanism.},
	keywords = {DNA Transposable Elements, DNA, Mitochondrial, Endonucleases, Introns, Kinetics, Neurospora, Nucleic Acid Conformation, Plasmids, RNA-Directed DNA Polymerase, Single-Strand Specific DNA and RNA Endonucleases},
	issn = {0092-8674},
	author = {Kuiper, M T and Lambowitz, A M}
}
@article {448,
	title = {Nucleotide sequence of the Varkud mitochondrial plasmid of Neurospora and synthesis of a hybrid transcript with a 5{\textquoteright} leader derived from mitochondrial RNA.},
	journal = {J Mol Biol},
	volume = {204},
	number = {1},
	year = {1988},
	month = {1988 Nov 5},
	pages = {1-25},
	abstract = {The Mauriceville and Varkud mitochondrial plasmids of Neurospora are closely related, closed circular DNAs (3.6 and 3.7 kb, respectively; 1 kb = 10(3) bases or base-pairs), whose characteristics suggest relationships to mitochondrial DNA introns and retrotransposons. Here, we characterized the structure of the Varkud plasmid, determined its complete nucleotide sequence and mapped its major transcripts. The Mauriceville and Varkud plasmids have more than 97\% positional identity. Both plasmids contain a 710 amino acid open reading frame that encodes a reverse transcriptase-like protein. The amino acid sequence of this open reading frame is strongly conserved between the two plasmids (701/710 amino acids) as expected for a functionally important protein. Both plasmids have a 0.4 kb region that contains five PstI palindromes and a direct repeat of approximately 160 base-pairs. Comparison of sequences in this region suggests that the Varkud plasmid has diverged less from a common ancestor than has the Mauriceville plasmid. Two major transcripts of the Varkud plasmid were detected by Northern hybridization experiments: a full-length linear RNA of 3.7 kb and an additional prominent transcript of 4.9 kb, 1.2 kb longer than monomer plasmid. Remarkably, we find that the 4.9 kb transcript is a hybrid RNA consisting of the full-length 3.7 kb Varkud plasmid transcript plus a 5{\textquoteright} leader of 1.2 kb that is derived from the 5{\textquoteright} end of the mitochondrial small rRNA. This and other findings suggest that the Varkud plasmid, like certain RNA viruses, has a mechanism for joining heterologous RNAs to the 5{\textquoteright} end of its major transcript, and that, under some circumstances, nucleotide sequences in mitochondria may be recombined at the RNA level.},
	keywords = {Amino Acid Sequence, Base Sequence, DNA, Fungal, Mitochondria, Molecular Sequence Data, Neurospora, Nucleic Acid Hybridization, Plasmids, Repetitive Sequences, Nucleic Acid, Restriction Mapping, RNA, Fungal, RNA, Ribosomal, Transcription, Genetic},
	issn = {0022-2836},
	author = {Akins, R A and Grant, D M and Stohl, L L and Bottorff, D A and Nargang, F E and Lambowitz, A M}
}
@article {453,
	title = {Molecular cloning and characterization of the cys-3 regulatory gene of Neurospora crassa.},
	journal = {Mol Cell Biol},
	volume = {7},
	number = {7},
	year = {1987},
	month = {1987 Jul},
	pages = {2506-11},
	abstract = {The regulatory gene cys-3+ controls the synthesis of a number of enzymes involved in sulfur metabolism. cys-3 mutants show a multiple loss of enzymes in different pathways of sulfur metabolism. The cys-3+ gene was isolated by transformation of an aro-9 qa-2 cys-3 inl strain with a clone bank followed by screening with the "sib selection" method. The library used (pRAL1) contained inserts of Sau3a partial digest fragments of about 9 kilobases as well as the Neurospora qa-2+ gene. Double selection for qa-2+ and cys-3+ function was carried out. The transformants obtained with the isolated cys-3+ clone show recovery of the enzyme activities associated with the cys-3 mutation (e.g., arylsulfatase and sulfate permease). Restriction fragment length polymorphism experiments confirmed the identity of the clone, mRNA studies with Northern blots show that the expression of the cys-3+ gene is inducible. In contrast to cys-3+, the cys-3 (P22) mutant gene was not expressed at a higher level under sulfur-derepressed conditions.},
	keywords = {Cloning, Molecular, Gene Expression Regulation, Genes, Fungal, Genes, Regulator, Neurospora, Neurospora crassa, Polymorphism, Restriction Fragment Length, Sulfur, Transformation, Genetic},
	issn = {0270-7306},
	author = {Paietta, J V and Akins, R A and Lambowitz, A M and Marzluf, G A}
}
@article {452,
	title = {A protein required for splicing group I introns in Neurospora mitochondria is mitochondrial tyrosyl-tRNA synthetase or a derivative thereof.},
	journal = {Cell},
	volume = {50},
	number = {3},
	year = {1987},
	month = {1987 Jul 31},
	pages = {331-45},
	abstract = {The nuclear cyt-18 mutants of Neurospora crassa are defective in splicing a number of group I introns in mitochondria. Here, cloning and sequencing of the cyt-18 gene show that it contains an open reading frame having significant homology to bacterial tyrosyl-tRNA synthetases. Biochemical and genetic experiments lead to the conclusions that the cyt-18 gene encodes mitochondrial tyrosyl-tRNA synthetase, that mutations in this gene inhibit splicing directly, and that mitochondrial tyrosyl-tRNA synthetase or a derivative of this protein is related to the soluble activity that functions in splicing the mitochondrial large rRNA intron and possibly other group I introns. Analysis of partial revertants provides direct evidence that the cyt-18 gene encodes a protein or proteins with two activities, splicing and aminoacylation, that can be partially separated by mutation. Our findings may be relevant to the evolution of introns and splicing mechanisms in eukaryotes.},
	keywords = {Amino Acid Sequence, Amino Acyl-tRNA Synthetases, Base Sequence, Chromatography, Cloning, Molecular, DNA, Mitochondrial, Genes, Fungal, Introns, Mitochondria, Mutation, Neurospora, RNA Splicing, Sequence Homology, Nucleic Acid, Tyrosine-tRNA Ligase},
	issn = {0092-8674},
	author = {Akins, R A and Lambowitz, A M}
}
@article {459,
	title = {Mitochondrial plasmids of Neurospora and other filamentous fungi.},
	journal = {Basic Life Sci},
	volume = {40},
	year = {1986},
	month = {1986},
	pages = {83-92},
	keywords = {DNA Restriction Enzymes, DNA, Mitochondrial, Fungi, Neurospora, Plasmids, Species Specificity},
	issn = {0090-5542},
	author = {Lambowitz, A M and Akins, R A and Kelley, R L and Pande, S and Nargang, F E}
}
@article {458,
	title = {Classification of Histoplasma capsulatum isolates by restriction fragment polymorphisms.},
	journal = {J Bacteriol},
	volume = {165},
	number = {3},
	year = {1986},
	month = {1986 Mar},
	pages = {813-8},
	abstract = {Twenty isolates of the dimorphic, pathogenic fungus Histoplasma capsulatum were divided into three classes based on comparisons of restriction enzyme digests of their mitochondrial DNA and rDNA. The majority of isolates, including most North American strains and the African H. capsulatum var. duboisii variants, belong to class 2. Isolates from Central America and South America make up class 3. The attenuated Downs strain is the only member of class 1.},
	keywords = {Africa, Animals, Central America, DNA Restriction Enzymes, DNA, Fungal, DNA, Mitochondrial, Histoplasma, Humans, North America, Nucleic Acid Hybridization, RNA, Fungal, RNA, Ribosomal, South America},
	issn = {0021-9193},
	author = {Vincent, R D and Goewert, R and Goldman, W E and Kobayashi, G S and Lambowitz, A M and Medoff, G}
}
@article {454,
	title = {Correlation between pathogenicity and temperature sensitivity in different strains of Histoplasma capsulatum.},
	journal = {J Clin Invest},
	volume = {78},
	number = {6},
	year = {1986},
	month = {1986 Dec},
	pages = {1638-47},
	abstract = {We compared the mycelial to yeast transitions of the Downs strain of Histoplasma capsulatum (low level of virulence) with those of G184A and G222B, two more virulent strains having different levels of pathogenicity for mice. When the morphological transitions are initiated by a temperature shift from 25 degrees to 37 degrees C, all three strains undergo similar physiological changes, but these are less severe in G184A and G222B than in the Downs strain. The transitions from mycelial to yeast morphology in both of the more virulent strains are also one-third more rapid than in Downs. We also find that the differences in temperature sensitivity of the three strains can be correlated with the temperature required for complete uncoupling of oxidative phosphorylation. The differences in sensitivity to elevated temperatures extend to the growth of yeast cells of all three strains. Considered together, our results suggest that sensitivity to elevated temperatures may be a key factor accounting for differences in virulence and that uncoupling of oxidative phosphorylation may be the primary event in the morphological transition in all three strains.},
	keywords = {Adenosine Triphosphate, Animals, Carbonyl Cyanide m-Chlorophenyl Hydrazone, Electron Transport, Histoplasma, Mice, Mice, Inbred AKR, Oxygen Consumption, Temperature, Virulence},
	issn = {0021-9738},
	doi = {10.1172/JCI112757},
	author = {Medoff, G and Maresca, B and Lambowitz, A M and Kobayashi, G and Painter, A and Sacco, M and Carratu, L}
}
@article {457,
	title = {Mechanism of recognition of the 5{\textquoteright} splice site in self-splicing group I introns.},
	journal = {Nature},
	volume = {322},
	number = {6074},
	year = {1986},
	month = {1986 Jul 3-9},
	pages = {86-9},
	abstract = {Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5{\textquoteright} splice site precedes cleavage at the 3{\textquoteright} splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5{\textquoteright} exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5{\textquoteright} splice site are base-paired to an internal, 5{\textquoteright} exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5{\textquoteright} splice site and, following the first step of splicing, to hold the 5{\textquoteright} exon in place for exon ligation.},
	keywords = {Animals, Base Sequence, Cytochrome b Group, Neurospora, Nucleic Acid Conformation, Nucleic Acid Precursors, Physarum, RNA Precursors, RNA Splicing, RNA, Messenger, RNA, Ribosomal, Tetrahymena},
	issn = {0028-0836},
	doi = {10.1038/322086a0},
	author = {Garriga, G and Lambowitz, A M and Inoue, T and Cech, T R}
}
@article {455,
	title = {Mitochondrial plasmids of Neurospora: integration into mitochondrial DNA and evidence for reverse transcription in mitochondria.},
	journal = {Cell},
	volume = {47},
	number = {4},
	year = {1986},
	month = {1986 Nov 21},
	pages = {505-16},
	abstract = {The Mauriceville (3.6 kb) and Varkud (3.8 kb) mitochondrial plasmids of Neurospora are closely related, closed circular DNAs whose nucleotide sequences and genetic organization suggest relationships to mitochondrial introns and retrotransposons. Here we isolated mutants whose growth is impaired as a result of malevolent behavior of these plasmids. All 12 mutants contain variant plasmids that are suppressive relative to mtDNA, and ten also contain defective mtDNAs. All the suppressive plasmids contain small insertions, generally including a mitochondrial tRNA sequence, at or near the major 5{\textquoteright} RNA start site. The structure of the suppressive plasmids suggests that they were generated via an RNA intermediate and a reverse transcription step. At least three of the mutants contain defective mtDNAs into which mitochondrial plasmid sequences have integrated. Sequences at the plasmid-mtDNA junctions are also consistent with integration via an RNA intermediate.},
	keywords = {Chromosome Mapping, DNA Restriction Enzymes, DNA, Circular, DNA, Fungal, DNA, Mitochondrial, Mutation, Neurospora, Neurospora crassa, Plasmids, RNA, Fungal, RNA, Transfer, Suppression, Genetic, Transcription, Genetic},
	issn = {0092-8674},
	author = {Akins, R A and Kelley, R L and Lambowitz, A M}
}
@article {456,
	title = {Protein-dependent splicing of a group I intron in ribonucleoprotein particles and soluble fractions.},
	journal = {Cell},
	volume = {46},
	number = {5},
	year = {1986},
	month = {1986 Aug 29},
	pages = {669-80},
	abstract = {The group I intron in the Neurospora mitochondrial large rRNA gene is not self-splicing in vitro. Here, we show that this intron can be spliced from 35S pre-rRNA in RNPs or from deproteinized 35S pre-rRNA or in vitro transcripts by a soluble activity that is present in mitochondrial lysates and can be released from RNPs. Splicing occurs by the same guanosine-initiated transesterification mechanism characteristic of self-splicing group I introns, but is absolutely dependent upon proteins that are presumably required for correct folding of the pre-rRNA. The soluble splicing activity is not simply associated with large subunit ribosomal proteins. Nuclear mutant cyt18-1, which is defective in splicing a number of group I introns in vivo, is grossly deficient in the soluble splicing activity. Our results suggest that the cyt18 gene encodes or regulates a component of an activity that functions in splicing group I introns in Neurospora mitochondria.},
	keywords = {Base Sequence, Fungal Proteins, Mitochondria, Neurospora, Neurospora crassa, Nucleic Acid Conformation, Nucleic Acid Precursors, Ribonucleoproteins, RNA, RNA Precursors, RNA Splicing, RNA, Fungal, RNA, Ribosomal},
	issn = {0092-8674},
	author = {Garriga, G and Lambowitz, A M}
}
@article {460,
	title = {General method for cloning Neurospora crassa nuclear genes by complementation of mutants.},
	journal = {Mol Cell Biol},
	volume = {5},
	number = {9},
	year = {1985},
	month = {1985 Sep},
	pages = {2272-8},
	abstract = {We have developed a sib selection procedure for cloning Neurospora crassa nuclear genes by complementation of mutants. This procedure takes advantage of a modified N. crassa transformation procedure that gives as many as 10,000 to 50,000 stable transformants per microgram of DNA with recombinant plasmids containing the N. crassa qa-2+ gene. Here, we describe the use of the sib selection procedure to clone genes corresponding to auxotrophic mutants, nic-1 and inl. The identities of the putative clones were confirmed by mapping their chromosomal locations in standard genetic crosses and using restriction site polymorphisms as genetic markers. Because we can obtain very high N. crassa transformation frequencies, cloning can be accomplished with as few as five subdivisions of an N. crassa genomic library. The sib selection procedure should, for the first time, permit the cloning of any gene corresponding to an N. crassa mutant for which an appropriate selection can be devised. Analogous procedures may be applicable to other filamentous fungi before the development of operational shuttle vectors.},
	keywords = {Cloning, Molecular, DNA, Recombinant, Genes, Fungal, Genetic Complementation Test, Genetic Vectors, Neurospora, Neurospora crassa, Plasmids, Selection, Genetic, Transformation, Genetic},
	issn = {0270-7306},
	author = {Akins, R A and Lambowitz, A M}
}
@article {461,
	title = {RNA splicing in Neurospora mitochondria. Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1.},
	journal = {J Mol Biol},
	volume = {184},
	number = {3},
	year = {1985},
	month = {1985 Aug 5},
	pages = {413-28},
	abstract = {cyt18-1 (299-9) is a nuclear mutant of Neurospora crassa that has been shown to have a temperature-sensitive defect in splicing the mitochondrial large rRNA intron. In the present work, we investigate the effect of the cyt18-1 mutation on splicing of mitochondrial mRNA introns. Two genes were studied in detail; the cytochrome b (cob) gene, which contains two introns, and a "long form" of the cytochrome oxidase subunit I (coI) gene, which contains four introns. We found that splicing of both cob introns and splicing of at least two of the coI introns are strongly inhibited in the mutant, whereas splicing of coI intron 1, which is excised as a 2.6 X 10(3) base circle, is relatively unaffected. The rRNA intron and both cob introns are group I introns, whereas the circular coI intron may belong to another structural class. Control experiments showed that the degree of inhibition of splicing is greater in the mutant than can be accounted for by severe inhibition of mitochondrial protein synthesis. Finally, experiments in which mutant cells were shifted from 25 degrees C to 37 degrees C showed that splicing of the large rRNA precursor and splicing of the coI mRNA precursor are inhibited with similar kinetics. Considered together, our results suggest that the cyt18 gene encodes a trans-acting component that is required for the splicing of group I mitochondrial DNA introns or some subclass thereof. Since Neurospora cob intron 1 has been shown to be self-splicing in vitro, defective splicing of this intron in cyt18-1 indicates that an essentially RNA-catalyzed splicing reaction must be facilitated by a trans-acting factor, presumably a protein, in vivo.},
	keywords = {Electrophoresis, Agar Gel, Microscopy, Electron, Mutation, Neurospora crassa, Nucleic Acid Hybridization, Nucleic Acid Precursors, RNA, RNA Precursors, RNA Splicing, RNA, Fungal, RNA, Messenger, Temperature, Transcription, Genetic},
	issn = {0022-2836},
	author = {Collins, R A and Lambowitz, A M}
}
@article {465,
	title = {Characterization of deletion derivatives of an autonomously replicating Neurospora plasmid.},
	journal = {Nucleic Acids Res},
	volume = {12},
	number = {15},
	year = {1984},
	month = {1984 Aug 10},
	pages = {6169-78},
	abstract = {We previously described two plasmids that replicate autonomously in both Neurospora and E. coli (Stohl and Lambowitz, Proc. Natl. Acad. Sci., U.S.A. 80, 1058-1062, 1983). One plasmid, pALS1, consists of the Neurospora ga-2+ gene (3 kb Hind III-fragment), the mitochondrial plasmid from N. intermedia strain P405-Labelle, and E. coli plasmid pBR325. The other, pALS2, is a putative deletion derivative of pALS1 that lacks most or all of the Labelle insert and that was repeatedly recovered from Neurospora transformants. We have now sequenced the region encompassing the deletion in five pALS2 plasmids isolated independently in two different laboratories. All five plasmids are identical in this region and completely lack the Labelle insert. We have also characterized an additional deletion derivative that retains a small (approximately 0.5 kb) segment of the Labelle insert. The results for pALS2 suggest that pBR325 plus the ga-2+ segment constitute a Neurospora replicon.},
	keywords = {Base Sequence, Chromosome Deletion, DNA Replication, DNA, Mitochondrial, Neurospora, Neurospora crassa, Plasmids, Replicon},
	issn = {0305-1048},
	author = {Stohl, L L and Akins, R A and Lambowitz, A M}
}
@article {464,
	title = {The DNA sequence and genetic organization of a Neurospora mitochondrial plasmid suggest a relationship to introns and mobile elements.},
	journal = {Cell},
	volume = {38},
	number = {2},
	year = {1984},
	month = {1984 Sep},
	pages = {441-53},
	abstract = {We have determined the complete 3581 bp sequence of the mitochondrial plasmid from Neurospora crassa strain Mauriceville-1c. The plasmid contains a long open reading frame that is expressed in its major transcript and could encode a hydrophilic protein of 710 amino acids. Two characteristics of the plasmid--codon usage and the presence of conserved sequence elements--suggest that it is related to Group I mtDNA introns. The major transcripts of the plasmid are approximately full-length, colinear RNAs that have heterogenous 5{\textquoteright} ends and a single major 3{\textquoteright} end. The major 5{\textquoteright} and 3{\textquoteright} ends are adjacent and slightly overlapping. The Mauriceville plasmid may belong to a class of genetic elements that were or are the progenitors of mtDNA introns.},
	keywords = {Base Sequence, Codon, DNA Transposable Elements, DNA, Fungal, DNA, Mitochondrial, Genes, Neurospora, Neurospora crassa, Plasmids, Protein Biosynthesis, Transcription, Genetic},
	issn = {0092-8674},
	author = {Nargang, F E and Bell, J B and Stohl, L L and Lambowitz, A M}
}
@article {466,
	title = {The [poky] mutant of Neurospora contains a 4-base-pair deletion at the 5{\textquoteright} end of the mitochondrial small rRNA.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {81},
	number = {12},
	year = {1984},
	month = {1984 Jun},
	pages = {3791-5},
	abstract = {[ poky ] and other group I extranuclear mutants of Neurospora crassa are characterized by gross deficiencies of mitochondrial small ribosomal subunits and small (19S) rRNA. Blot-hybridization and other experiments suggest that the 19S rRNA (2.0 kilobases) is synthesized via precursors that contain 5{\textquoteright}-end extensions. The ratio of precursors to mature rRNA is higher in [ poky ] and other group I mutants than in wild type, indicating that the defect involves impaired processing and/or instability of 19S rRNA. [ poky ] and other group I mutants contain a 4-base-pair deletion in the coding sequence for the mitochondrial small rRNA, just downstream from what would normally be the 5{\textquoteright} end of the rRNA. This deletion apparently results in synthesis of aberrant 19S rRNAs that are missing 38-45 nucleotides from their 5{\textquoteright} ends. We propose that the 4-base-pair deletion is the primary defect in [ poky ] and other group I extranuclear mutants.},
	keywords = {Base Sequence, Chromosome Deletion, DNA, Mitochondrial, Mutation, Neurospora, Neurospora crassa, Nucleic Acid Conformation, RNA Processing, Post-Transcriptional, RNA, Ribosomal},
	issn = {0027-8424},
	author = {Akins, R A and Lambowitz, A M}
}
@article {467,
	title = {RNA splicing in Neurospora mitochondria: nuclear mutants defective in both splicing and 3{\textquoteright} end synthesis of the large rRNA.},
	journal = {Cell},
	volume = {36},
	number = {3},
	year = {1984},
	month = {1984 Mar},
	pages = {623-34},
	abstract = {We have identified nuclear mutants of Neurospora that are defective in splicing the mitochondrial large rRNA and that accumulate unspliced pre-rRNA (35S RNA). In cyt-4 mutants, the unspliced pre-rRNA contains short 3{\textquoteright} end extensions (110 nucleotides) that are not present in pre-rRNAs from the other mutants. This and other characteristics suggest that the cyt-4 mutants may be primarily defective in 3{\textquoteright} end synthesis and the RNA splicing defect occurs secondarily as a result of impaired RNA folding. The cyt-4 mutants also accumulate a "short" intron RNA and small exon RNAs that may reflect aberrant RNA cleavages. The 5{\textquoteright} end of the short intron is about 285 nucleotides downstream from the 5{\textquoteright} splice site at or near the base of the "central hairpin", a putative intermediate in folding of the pre-rRNA. Furthermore, the aberrant cleavage sites are immediately after a six nucleotide sequence (GAUAAU) homologous to the final splice junction (GAU/AAC).},
	keywords = {Base Sequence, Cell Nucleus, Chromosome Mapping, DNA, Mitochondrial, Genes, Regulator, Neurospora, Neurospora crassa, RNA Processing, Post-Transcriptional, RNA Splicing, RNA, Ribosomal},
	issn = {0092-8674},
	author = {Garriga, G and Bertrand, H and Lambowitz, A M}
}
@article {462,
	title = {RNA splicing in neurospora mitochondria: self-splicing of a mitochondrial intron in vitro.},
	journal = {Cell},
	volume = {39},
	number = {3 Pt 2},
	year = {1984},
	month = {1984 Dec},
	pages = {631-41},
	abstract = {We have used Neurospora nuclear mutant cyt-18-1, which accumulates a number of unspliced mitochondrial precursor RNAs, to identify rapidly mitochondrial introns that are self-splicing in vitro. Incubation of deproteinized whole mitochondrial RNA from the mutant with 32P-GTP resulted in strong labeling of a 1.3 kb RNA, subsequently identified as cytochrome b (cob) intron 1, and weaker labeling of additional RNAs. Self-splicing of cob intron 1, including precise cleavage and ligation, was confirmed using an in vitro transcript synthesized from the SP6 promoter. The in vitro splicing reaction was shown to be analogous to that for the Tetrahymena nuclear rRNA intron. Since splicing of cob intron 1 is inhibited in a recessive nuclear mutant, we infer that this essentially RNA-catalyzed splicing reaction must be facilitated by a protein in vivo.},
	keywords = {Base Sequence, Cytochrome b Group, DNA Restriction Enzymes, DNA, Mitochondrial, Genes, Genes, Fungal, Guanosine Triphosphate, Mitochondria, Molecular Weight, Neurospora, Neurospora crassa, Nucleic Acid Conformation, Nucleic Acid Hybridization, Plasmids, RNA Processing, Post-Transcriptional, RNA Splicing, RNA, Messenger, Transcription, Genetic},
	issn = {0092-8674},
	author = {Garriga, G and Lambowitz, A M}
}
@article {463,
	title = {Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.},
	journal = {Mol Cell Biol},
	volume = {4},
	number = {10},
	year = {1984},
	month = {1984 Oct},
	pages = {2041-51},
	abstract = {We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am132 host strain which contains a deletion encompassing the cloned fragment. Every one of 33 transformants tested showed extreme meiotic instability: less than 1 or 2\% am+ progeny were obtained in initial or successive backcrosses between am+ transformants and am132 or in intercrosses between am+ progeny. Furthermore, am+ progeny from backcrosses gave a high proportion of auxotrophic (am) mitotic segregants during vegetative growth. These results indicate that the am+ character is not stably integrated into chromosomal DNA in any of the transformants tested. Nuclear DNAs from six transformants were analyzed by Southern hybridization. All six transformants contained sequences homologous to pJR2. Four showed restriction fragments expected for unmodified pJR2, but most showed additional bands. Southern blots of undigested DNAs showed that the plasmid sequences are present predominantly in high-molecular-weight form (larger than 20 kilobases). Southern blots showed that auxotrophic (am) progeny from a backcross to am132 had lost restriction bands corresponding to free plasmid but retained additional bands, apparently integrated into chromosomal DNA in a nonfunctional manner. Considered together, these results are most reasonably interpreted as follows: recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, the free plasmids are present in oligomeric or modified form or both, and plasmid sequences also integrate at multiple sites in the deletion host but in a nonfunctional manner. An alternate interpretation--that tandem repeats of the plasmid are integrated into chromosomal DNA but eliminated during meiosis--cannot be completely excluded. However, stable integration of the am gene can be obtained under a variety of other conditions, viz., using the am gene cloned in a phage lambda vector (J. A. Kinsey and J. A. Rambosek, Mol. Cell. Biol. 4:117-122, 1984), using derivatives of pJR2, or using pJR2 to transform a frameshift mutant.},
	keywords = {Deoxyribonuclease EcoRI, DNA Replication, DNA Restriction Enzymes, DNA Transposable Elements, DNA, Recombinant, Glutamate Dehydrogenase, Neurospora, Neurospora crassa, Nucleic Acid Hybridization, Plasmids, Transformation, Genetic},
	issn = {0270-7306},
	author = {Grant, D M and Lambowitz, A M and Rambosek, J A and Kinsey, J A}
}
@article {469,
	title = {A colony filter-hybridization procedure for the filamentous fungus Neurospora crassa.},
	journal = {Anal Biochem},
	volume = {134},
	number = {1},
	year = {1983},
	month = {1983 Oct 1},
	pages = {82-5},
	abstract = {A colony filter-hybridization procedure for the filamentous fungus Neurospora crassa has been developed. The procedure is sensitive enough to detect Escherichia coli plasmid pBR322 DNA integrated into chromosomal DNA in a Neurospora transformant. Thus, it should facilitate the isolation of nuclear genes by plasmid-rescue procedures.},
	keywords = {Base Sequence, DNA, Bacterial, DNA, Fungal, Escherichia coli, Filtration, Hybridization, Genetic, Neurospora, Neurospora crassa, Plasmids},
	issn = {0003-2697},
	author = {Stohl, L L and Lambowitz, A M}
}
@article {474,
	title = {Construction of a shuttle vector for the filamentous fungus Neurospora crassa.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {80},
	number = {4},
	year = {1983},
	month = {1983 Feb},
	pages = {1058-62},
	abstract = {We have constructed a recombinant plasmid, pALS-1, that replicates autonomously in both Neurospora and Escherichia coli. pALS-1 consists of the mitochondrial plasmid from Neurospora strain P405-Labelle, the Neurospora qa-2+ gene, and E. coli plasmid pBR325. pALS-1 transforms the Neurospora qa-2+ gene at frequencies 5- to 10-fold higher than those for plasmids that transform mainly by integration. When E. coli was transformed with DNA from Neurospora transformants, we recovered not only pALS-1 but also a smaller plasmid, pALS-2, which had undergone deletion of most and possibly all Labelle sequences, and the immediately flanking sequences in pBR325. pALS-2 also appears to replicate autonomously in Neurospora, but less efficiently than does pALS-1. Southern blots show that free pALS-1 and pALS-2 are present in nuclear and cytosolic (supernatant from high-speed centrifugation) fractions of Neurospora transformants and that small, variable proportions of the plasmids also can be detected in mitochondria. pALS-1 and pALS-2 constitute putative shuttle vectors for Neurospora.},
	keywords = {DNA, Circular, DNA, Mitochondrial, Genetic Vectors, Neurospora, Neurospora crassa, Plasmids, Transformation, Genetic},
	issn = {0027-8424},
	author = {Stohl, L L and Lambowitz, A M}
}
@article {472,
	title = {A family of repetitive palindromic sequences found in Neurospora mitochondrial DNA is also found in a mitochondrial plasmid DNA.},
	journal = {J Biol Chem},
	volume = {258},
	number = {7},
	year = {1983},
	month = {1983 Apr 10},
	pages = {4257-60},
	abstract = {Neurospora mtDNA contains a repetitive, 18 nucleotide palindromic sequence (5{\textquoteright}-CCCTGCAGTACTGCAGGG-3{\textquoteright}) that contains two closely spaced PstI sites (CTGCAG) in the arms of the palindrome (Yin, S., Heckman, J., and RajBhandary, U. L. (1981) Cell 26, 325-332). In the present study, DNA sequence analysis was carried out to determine whether PstI palindromes are present in an apparently distinct genetic element, the 3.6-kilobase mitochondrial plasmid from Neurospora crassa strain Mauriceville-1c (FGSC 2225). The plasmid contains a cluster of closely spaced PstI sites extending over a 0.4-kilobase region (Collins, R. A., Stohl, L. L., Cole, M. D., and Lambowitz, A. M. (1981) Cell 24, 443-452). The DNA sequence shows that the cluster consists of eight PstI sites organized in five palindromic elements. Two of the elements are identical with the canonical sequence found in mtDNA, whereas the remaining three elements differ from the canonical sequence by a few nucleotides. The occurrence of the PstI palindromes in two otherwise unrelated DNA species is consistent with the hypothesis that they are related to mobile DNA sequences that either propagate or were once capable of propagating within mitochondria.},
	keywords = {Base Sequence, Cloning, Molecular, DNA Restriction Enzymes, DNA, Mitochondrial, Escherichia coli, Neurospora, Neurospora crassa, Plasmids, Repetitive Sequences, Nucleic Acid},
	issn = {0021-9258},
	author = {Nargang, F E and Bell, J B and Stohl, L L and Lambowitz, A M}
}
@article {471,
	title = {Possible relationship of morphogenesis in pathogenic fungus, Histoplasma capsulatum, to heat shock response.},
	journal = {Nature},
	volume = {303},
	number = {5920},
	year = {1983},
	month = {1983 Jun 30},
	pages = {806-8},
	abstract = {Histoplasma capsulatum, like many other fungal pathogens, is dimorphic: it exists as mycelia in the soil and yeast in animal hosts. Because only the yeast phase is parasitic, factors which affect morphogenesis have been of interest for understanding and controlling pathogenicity. In culture, the mycelial to yeast transition of H. capsulatum is induced by a temperature shift from 25 to 37 degrees C (ref. 1). The transition occurs over several days and is accompanied by marked changes in metabolic processes, including respiration and cysteine metabolism. Here, we show that the triggering event for these morphological and biochemical changes is a rapid decline in intracellular ATP levels that follows uncoupling of oxidative phosphorylation when mycelia are shifted from 25 to 37 degrees C. We also show that respiration in the yeast phase is coupled at 37 degrees C and thus that the morphological transition may be viewed as a heat shock followed by cellular adaptation to higher temperature.},
	keywords = {Adaptation, Physiological, Adenosine Triphosphate, Histoplasma, Hot Temperature, Morphogenesis, Oxidative Phosphorylation},
	issn = {0028-0836},
	author = {Lambowitz, A M and Kobayashi, G S and Painter, A and Medoff, G}
}
@article {473,
	title = {RNA splicing in neurospora mitochondria: structure of the unspliced 35S precursor ribosomal RNA detected by psoralen cross-linking.},
	journal = {Cell},
	volume = {32},
	number = {2},
	year = {1983},
	month = {1983 Feb},
	pages = {397-407},
	abstract = {The structure of the unspliced 35S precursor rRNA of Neurospora mitochondria was studied by psoralen photochemical cross-linking. The results show that when the 35S RNA is cross-linked in ribonucleoprotein particles (RNPs) under appropriate conditions, the predominant configuration is a 2.2 kb intron loop which brings opposite splice sites into proximity; that the predominant secondary structural feature in the free RNA is a relatively large hairpin (length = 0.105 kb) in the center of the molecule at or near the 5{\textquoteright} splice site; that the intron loop and the central hairpin are different configurations of sequences at or near the 5{\textquoteright} splice site; and that the intron loop is stabilized by protein components of RNPs. Based on the structures detected by psoralen photochemical cross-linking, we propose a mechanism for the splicing of the Neurospora mitochondrial precursor rRNA. We propose further that certain features of this mechanism may be relevant to the splicing of other RNAs, including eucaryotic mRNAs.},
	keywords = {Base Sequence, Cross-Linking Reagents, Mitochondria, Neurospora, Nucleic Acid Conformation, Nucleic Acid Precursors, Ribonucleoproteins, RNA Splicing, RNA, Fungal, RNA, Ribosomal, Trioxsalen},
	issn = {0092-8674},
	author = {Wollenzien, P L and Cantor, C R and Grant, D M and Lambowitz, A M}
}
@article {475,
	title = {Structural variations and optional introns in the mitochondrial DNAs of Neurospora strains isolated from nature.},
	journal = {Plasmid},
	volume = {9},
	number = {1},
	year = {1983},
	month = {1983 Jan},
	pages = {53-70},
	abstract = {Mitochondrial DNAs from ten wild-type Neurospora crassa, Neurospora intermedia, and Neurospora sitophila strains collected from different geographical areas were screened for structural variations by restriction enzyme analysis. The different mtDNAs show much greater structural diversity, both within and among species, than had been apparent from previous studies of mtDNA from laboratory N. crassa strains. The mtDNAs range in size from 60 to 73 kb, and both the smallest and largest mtDNAs are found in N. crassa strains. In addition, four strains contain intramitochondrial plasmid DNAs that do not hybridize with the standard mtDNA. All of the mtDNA species have a basically similar organization. A 25-kb region that includes the rRNA genes and most tRNA genes shows very strong conservation of restriction sites in all strains. The 2.3-kb intron found in the large rRNA gene in standard N. crassa mtDNAs is present in all strains examined, including N. intermedia and N. sitophila strains. The size differences between the different mtDNAs are due to insertions or deletions that occur outside of the rRNA-tRNA region. Restriction enzyme and heteroduplex mapping suggest that four of these insertions are optional introns in the gene encoding cytochrome oxidase subunit I. Mitochondrial DNAs from different wild-type strains contain zero, one, three, or four of these introns.},
	keywords = {Chromosome Mapping, DNA Restriction Enzymes, DNA Transposable Elements, DNA, Fungal, DNA, Mitochondrial, Neurospora, Neurospora crassa, Protein Biosynthesis, RNA, Fungal, RNA, Ribosomal, Species Specificity},
	issn = {0147-619X},
	author = {Collins, R A and Lambowitz, A M}
}
@article {470,
	title = {Sulfhydryl induced respiratory "shunt" pathways and their role in morphogenesis in the fungus, Histoplasma capsulatum.},
	journal = {J Biol Chem},
	volume = {258},
	number = {13},
	year = {1983},
	month = {1983 Jul 10},
	pages = {8223-30},
	abstract = {When the mycelial to yeast transition of the dimorphic fungus Histoplasma capsulatum is induced by a temperature shift from 25 to 37 degrees C, the activities of the cytochrome system and the alternate oxidase decrease in parallel over the first 24 to 40 h (stage 1 of the transition). The decrease in activity of the cytochrome system is correlated with extensive decreases in the amounts of cytochromes b, c, and aa3, assayed spectrophotometrically. After 40 h, the cells enter a dormant phase (stage 2 of the transition) and cysteine or other sulfhydryl-containing compounds are required to reactivate mitochondrial respiration. This reactivation is due to the establishment of shunt pathways which bypass blocked segments of the electron transport system. The "shunt" pathways operate normally in mycelia grown at 25 degrees C, but are shut down during the transition, possibly because of depletion of intracellular cysteine. The longstanding observation that cysteine is required to progress beyond the initial stages of the morphological transition may be due, at least in part, to the reactivation of these "shunt" pathways.},
	keywords = {Cytochromes, Electron Transport, Flavoproteins, Histoplasma, Kinetics, Mitochondria, Morphogenesis, Oxygen Consumption},
	issn = {0021-9258},
	author = {Sacco, M and Medoff, G and Lambowitz, A M and Kumar, B V and Kobayashi, G S and Painter, A}
}
@article {468,
	title = {RNA splicing in Neurospora mitochondria. The large rRNA intron contains a noncoded, 5{\textquoteright}-terminal guanosine residue.},
	journal = {J Biol Chem},
	volume = {258},
	number = {24},
	year = {1983},
	month = {1983 Dec 25},
	pages = {14745-8},
	abstract = {The 35 S percursor of the Neurospora mitochondrial large rRNA contains a 2.3-kilobase intron located towards its 3{\textquoteright} end. The intron RNA is excised in a single cleavage-ligation reaction and is detectable in mitochondria by Northern hybridization experiments. We now show (i) that the free intron RNA is a full-length linear molecule, and (ii) that it, like the Tetrahymena nuclear rRNA intron, contains an extra, noncoded guanosine residue at its 5{\textquoteright} end. The latter finding suggests that the Neurospora mitochondrial large rRNA may be spliced via a phosphoester transfer mechanism similar to that proposed for the "self-splicing" Tetrahymena intron.},
	keywords = {Base Sequence, Guanosine, Microscopy, Electron, Mitochondria, Neurospora, RNA Splicing, RNA, Ribosomal, Tetrahymena},
	issn = {0021-9258},
	author = {Garriga, G and Lambowitz, A M}
}
@article {478,
	title = {Characterization of two new plasmid DNAs found in mitochondria of wild-type Neurospora intermedia strains.},
	journal = {Nucleic Acids Res},
	volume = {10},
	number = {5},
	year = {1982},
	month = {1982 Mar 11},
	pages = {1439-58},
	abstract = {Mitochondria from two Neurospora intermedia strains (P4O5-Labelle and Fiji N6-6) were found to contain plasmid DNAs in addition to the standard mitochondrial DNA species. The plasmid DNAs consist of monomeric circles (4.1-4.3 kbp and 5.2-5.3 kbp for Labelle and Fiji, respectively) and oligomers in which monomers are organized as head-to-tail repeats. DNA-DNA hybridization experiments showed that the plasmids have no substantial sequence homology to mtDNA, to each other, or to a previously characterized mitochondrial plasmid from N. crassa strain Mauriceville-lc (Collins et al. Cell 24, 443-452, 1981). The intramitochondrial location of the plasmids was established by cell fractionation and nuclease protection experiments. In sexual crosses, the plasmids showed strict maternal inheritance, the same as Neurospora mitochondrial DNA. The plasmids may represent a novel class of mitochondrial genetic elements.},
	keywords = {Base Composition, DNA Restriction Enzymes, DNA, Mitochondrial, Microscopy, Electron, Mitochondria, Neurospora, Neurospora crassa, Nucleic Acid Hybridization, Plasmids, Repetitive Sequences, Nucleic Acid},
	issn = {0305-1048},
	author = {Stohl, L L and Collins, R A and Cole, M D and Lambowitz, A M}
}
@article {476,
	title = {Mitochondrial ribosome assembly in Neurospora. Structural analysis of mature and partially assembled ribosomal subunits by equilibrium centrifugation in CsCl gradients.},
	journal = {J Cell Biol},
	volume = {95},
	number = {1},
	year = {1982},
	month = {1982 Oct},
	pages = {267-77},
	abstract = {In Neurospora, one protein associated with the mitochondrial small ribosomal subunit (S-5, Mr 52,000) is synthesized intramitochondrially and is assumed to be encoded by mtDNA. When mitochondrial protein synthesis is inhibited, either by chloramphenicol or by mutation, cells accumulate incomplete mitochondrial small subunits (CAP-30S and INC-30S particles) that are deficient in S-5 and several other proteins. To gain additional insight into the role of S-5 in mitochondrial ribosome assembly, the structures of Neurospora mitochondrial ribosomal subunits, CAP-30S particles, and INC-30S particles were analyzed by equilibrium centrifugation in CsCl gradients containing different concentrations of Mg+2. The results show (a) that S-5 is tightly associated with small ribosomal subunits, as judged by the fact that it is among the last proteins to be dissociated in CsCl gradients as the Mg+2 concentration is decreased, and (b) that CAP-30S and INC-30S particles, which are deficient in S-5, contain at most 12 proteins that are bound as tightly as in mature small subunits. The CAP-30S particles isolated from sucrose gradients contain a number of proteins that appear to be loosely bound, as judged by dissociation of these proteins in CsCl gradients under conditions in which they remain associated with mature small subunits. The results suggest that S-5 is required for the stable binding of a subset of small subunit ribosomal proteins.},
	keywords = {Centrifugation, Isopycnic, Chloramphenicol, DNA, Mitochondrial, Mitochondria, Morphogenesis, Neurospora, Neurospora crassa, Protein Binding, Ribosomal Proteins, Ribosomes, RNA, Ribosomal},
	issn = {0021-9525},
	author = {Lapolla, R J and Lambowitz, A M}
}
@article {477,
	title = {RNA splicing in Neurospora mitochondria. Characterization of new nuclear mutants with defects in splicing the mitochondrial large rRNA.},
	journal = {Cell},
	volume = {29},
	number = {2},
	year = {1982},
	month = {1982 Jun},
	pages = {517-26},
	abstract = {In Neurospora, the gene encoding the mitochondrial large (25S) ribosomal RNA contains an intervening sequence of 2.3 kb. We have identified eight nuclear mutants that are defective in splicing the mitochondrial large ribosomal RNA and that accumulate unspliced precursor RNA. These mutants identify three different nuclear genes required for the same mitochondrial RNA splicing reaction. Some of the mutants have unique phenotypic characteristics (for example, accumulation of an unusual intron RNA) that may provide insight into specific aspects of mitochondrial RNA splicing. Mutations at one locus, cyt4, are subject to partial phenotypic suppression by the electron-transport inhibitor antimycin. This phenomenon suggests that at least one component required for mitochondrial RNA splicing is regulated such that its synthesis or activity is increased in response to impairment of electron transport.},
	keywords = {Antimycin A, Base Sequence, Cell Nucleus, DNA, Mitochondrial, Electron Transport, Gene Expression Regulation, Mitochondria, Mutation, Neurospora, RNA Splicing, RNA, Ribosomal, RNA, Transfer},
	issn = {0092-8674},
	author = {Bertrand, H and Bridge, P and Collins, R A and Garriga, G and Lambowitz, A M}
}
@article {483,
	title = {Characterization of a novel plasmid DNA found in mitochondria of N. crassa.},
	journal = {Cell},
	volume = {24},
	number = {2},
	year = {1981},
	month = {1981 May},
	pages = {443-52},
	abstract = {We have identified a plasmid DNA that is found within mitochondria of wild-type N. crassa strain Mauriceville-1c (FGSC $\#$2225), but that shows no detectable sequence homology with mitochondrial DNA. The plasmid DNA consists of an oligomeric series of circular molecules of monomer length 3.6 kb. There are two unusual clusters of restriction enzyme sites, one consisting of six Eco RI sites in a 1 kb region, and the other of five or more Pst I sites in a 0.4 kb region. RNA gel transfer hybridization experiments show a major transcript 3.3 to 3.4 kb long, close to the monomer length of the plasmid. The latter finding implies that the plasmid DNA contains specific sites for the initiation and termination of transcription.},
	keywords = {Base Sequence, DNA Restriction Enzymes, DNA, Mitochondrial, Gene Expression Regulation, Molecular Weight, Neurospora, Neurospora crassa, Plasmids, Transcription, Genetic},
	issn = {0092-8674},
	author = {Collins, R A and Stohl, L L and Cole, M D and Lambowitz, A M}
}
@article {479,
	title = {Characterization of a variant Neurospora crassa mitochondrial DNA which contains tandem reiterations of a 1.9 kb sequence.},
	journal = {Curr Genet},
	volume = {4},
	number = {2},
	year = {1981},
	month = {1981 Nov},
	pages = {131-3},
	abstract = {We have identified a second variant of Neurospora mtDNA which contains tandem, head-to-tail repeats of sequences at the boundary of Eco RI-4 and 6. This region may contain a. major replication origin of Neurospora mtDNA.},
	issn = {0172-8083},
	doi = {10.1007/BF00365691},
	author = {Collins, R A and Lambowitz, A M}
}
@article {480,
	title = {Mitochondrial ribosome assembly in Neurospora crassa. Purification of the mitochondrially synthesized ribosomal protein, S-5.},
	journal = {J Biol Chem},
	volume = {256},
	number = {13},
	year = {1981},
	month = {1981 Jul 10},
	pages = {7064-7},
	abstract = {In Neurospora, one mitochondrial ribosomal protein (S-5, Mr = 52,000) is synthesized intramitochondrially and is presumably encoded by mitochondrial DNA. We have developed a rapid method for the purification of S-5 which takes advantage of its high affinity for carboxymethyl-Sepharose in the presence of 6 M urea. Using this method, S-5, at purity greater than 95\%, can be prepared by column chromatography in a single batch elution step. The amino acid composition of S-5 was determined. Judged by the contents of hydrophilic and basic amino acids, S-5 is more similar to Escherichia coli and yeast ribosomal proteins than to other mitochondrial translation products which are hydrophobic membrane proteins.},
	keywords = {Amino Acids, DNA, Mitochondrial, Macromolecular Substances, Mitochondria, Molecular Weight, Neurospora, Neurospora crassa, Ribosomal Proteins, Ribosomes},
	issn = {0021-9258},
	author = {Lapolla, R J and Lambowitz, A M}
}
@article {485,
	title = {A novel extranuclear mutant of Neurospora with a temperature-sensitive defect in mitochondrial protein synthesis and mitochondrial ATPase.},
	journal = {Mol Gen Genet},
	volume = {181},
	number = {1},
	year = {1981},
	month = {1981},
	pages = {13-9},
	abstract = {[C93] is a novel, extranuclear mutant of Neurospora crassa which has a normal mitochondrial phenotype when grown at 25 degrees, but which is deficient in cytochromes b and aa3 when grown at 37 degrees (Pittenger and West 1979). In the present work, the phenotype of [C93] was characterized in greater detail. When [C93] is grown at 37 degrees, the rate of mitochondrial protein synthesis is decreased to approximately 25\% that of wild type; the ratio of mitochondrial small to large ribosomal subunits is decreased to 1:4 and mitochondrial small subunits are deficient in the mitochondrially-synthesized protein, S-5. The mitochondrial ribosome assembly defects in 37 degrees-grown [C93] resemble those in chloramphenicol-treated wild-type cells and could merely be a consequence of the decreased rate of mitochondrial protein synthesis. Analysis of mitochondrial translation products by SDS gel electrophoresis suggests that 37 degrees-grown [C93] is grossly deficient in the 19,000 Mr subunit of the oligomycin-sensitive ATPase relative to other mitochondrially-synthesized proteins. The ATPase defect was not found in other extranuclear or nuclear mutants deficient in mitochondrial protein synthesis. These data and additional evidence suggest that the primary defect in [C93] may be in the assembly of the ATPase complex. The possible connection between the ATPase defect and the deficiency of mitochondrial protein synthesis is discussed.},
	keywords = {Adenosine Triphosphatases, Cytochrome b Group, Cytochromes, Electron Transport Complex IV, Electrophoresis, Polyacrylamide Gel, Fungal Proteins, Mitochondria, Mutation, Neurospora, Neurospora crassa, Oligomycins, Ribosomes, Temperature},
	issn = {0026-8925},
	author = {Collins, R A and Bertrand, H and Lapolla, R J and Lambowitz, A M}
}
@article {482,
	title = {Ribonucleic acid splicing in Neurospora Mitochondria: secondary structure of the 35S ribosomal precursor ribonucleic acid investigated by digestion with ribonuclease III and by electron microscopy.},
	journal = {Biochemistry},
	volume = {20},
	number = {10},
	year = {1981},
	month = {1981 May 12},
	pages = {2836-42},
	abstract = {In Neurospora, the gene encoding the mitochondrial large (25S) ribosomal ribonucleic acid (rRNA) contains an intervening sequence of approximately 2.3 kilobases (kb). We have identified two temperature-sensitive mutants (289-67 and 299-9) which are defective in a factor encoded by a nuclear gene but required for the splicing of 25S RNA. When grown at the nonpermissive temperature (37 degrees C), the mutants accumulate a novel 35S RNA (5.2-5.6 kb) which is related to the natural precursor of 25S RNA and which has been shown to be a collinear transcript of the 25S RNA gene including the intervening sequence. In the present work, the secondary structure of 35S RNA was investigated by digestion with ribonuclease III and by electron microscopy of the RNA spread under partially denaturing conditions. Ribonuclease III cleaves 35S RNA predominantly at a central site or sites near the 5{\textquoteright}-intron-exon boundary and produces fragments which correspond roughly to half-molecules (2.5-3 kb). Electron microscopy of 35S RNA shows a relatively large, central hairpin (180 +/- 45 nucleotides), which presumably corresponds to the central ribonuclease III site, and few other secondary structure features. Both experimental approaches indicate that the large hairpin is not present in 35S RNA. From this finding and from the location of the hairpin near the 5{\textquoteright}-intron-exon boundary in 35S RNA, we infer that its formation requires intron sequences. 35S RNA from the mutants can be isolated as a ribonucleoprotein particle associated with almost the full complement of large subunit ribosomal proteins. The 35S RNA in such particles can be cleaved by ribonuclease III at the central site(s), consistent with the idea that the central hairpin is accessible to RNA-processing enzymes in vivo.},
	keywords = {DNA Restriction Enzymes, DNA, Mitochondrial, Endonucleases, Microscopy, Electron, Neurospora, Neurospora crassa, Nucleic Acid Conformation, Nucleic Acid Hybridization, Nucleic Acid Precursors, Oligoribonucleotides, Ribonuclease III, Ribonucleases, RNA Precursors, RNA, Fungal, RNA, Ribosomal},
	issn = {0006-2960},
	author = {Grimm, M F and Cole, M D and Lambowitz, A M}
}
@article {481,
	title = {Role of cysteine in regulating morphogenesis and mitochondrial activity in the dimorphic fungus Histoplasma capsulatum.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {78},
	number = {7},
	year = {1981},
	month = {1981 Jul},
	pages = {4596-600},
	abstract = {Three stages can be distinguished in the temperature-induced mycelial-to-yeast phase transition of Histoplasma capsulatum. Stage one is characterized by a progressive decrease in the respiration rate and in the intracellular concentrations of cysteine and other amino acids. By stage two, respiration has ceased completely and free cysteine has fallen to low levels. Exogenous cysteine is required during the second stage for activation of mitochondrial respiration (stage three) and completion of the morphological transition. Mitochondria isolated from cells in the second stage show no respiration with NADH, succinate, or other substrates unless they are first incubated with cysteine. In addition, a novel, cytosolic cysteine oxidase appears during the latter part of the second stage. In stage three, the respiration rate rises, intracellular concentrations of free cysteine and other amino acids increase to levels characteristic of yeast, and the morphological transition is completed. The results support the idea that alterations in cysteine metabolism play a key role in this differentiation process.},
	keywords = {Cell Differentiation, Cysteine, Cystine, Histoplasma, Mitochondria, Morphogenesis, Oxygen Consumption},
	issn = {0027-8424},
	author = {Maresca, B and Lambowitz, A M and Kumar, V B and Grant, G A and Kobayashi, G S and Medoff, G}
}
@article {484,
	title = {Transcripts and processing patterns for the ribosomal RNA and transfer RNA region of Neurospora crassa mitochondrial DNA.},
	journal = {J Biol Chem},
	volume = {256},
	number = {4},
	year = {1981},
	month = {1981 Feb 25},
	pages = {2027-34},
	keywords = {Base Sequence, DNA, Mitochondrial, Genes, Microscopy, Electron, Molecular Weight, Neurospora, Neurospora crassa, Nucleic Acid Conformation, Nucleic Acid Hybridization, RNA, Ribosomal, RNA, Transfer, Transcription, Genetic},
	issn = {0021-9258},
	author = {Green, M R and Grimm, M F and Goewert, R R and Collins, R A and Cole, M D and Lambowitz, A M and Heckman, J E and Yin, S and RajBhandary, U L}
}
@article {486,
	title = {Deletion mutants of Neurospora crassa mitochondrial DNA and their relationship to the "stop-start" growth phenotype.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {77},
	number = {10},
	year = {1980},
	month = {1980 Oct},
	pages = {6032-6},
	abstract = {"Stoppers" are a class of Neurospora crassa extranuclear mutants characterized by gross deficiencies of cytochromes b and aa3 and an unusual growth phenotype which involves irregular periods of growth andnongrowth. In the present work, mtDNAs from all four stopper mutants were found to contain deletions or insertions detectable by restriction enzyme analysis. [stp] mtDNA consists predominantly of defective molecules which retain a 16-megadalton segment (EcoRI-1, -4, and -6) of wild-type mtDNA (40 megadaltons). The other stopper mutants show smaller alterations: [stp A18t]-618, a 0.35-kilobase deletion in EcoRI-7b; [stp B2]-651, a 4-kilobase insertion in EcoRI-2; and [stp A]-574, a 5-kilobase deletion in EcoRI-2 and -10. Based on these results, we propose that "stop-start" growth results from competition between certain defective mtDNAs which have a tendency to predominate and low concentrations of less defective mtDNA species which must be retained to sustain growth. Three additional extranuclear mutants ("nonstoppers") have also been found to contain deletions in mtDNA. Remarkably, the defective mtDNA species in two of these mutants ([poky]H1-10 and [SG-3]-551) retain different sizes (18 and 13 megadlatons, respectively) of the same region retained in [stp] mtDNA (i.e., EcoRI-1, -4, and -6). The findings suggest that production of defective mtDNAs in Neurospora is nonrandom with a preferred mechanism leading to retention of this segment. It may be significant that the retained segment contains both mitochondrial rRNA genes and most mitochondrial tRNA genes. These deletion mutants may provide a tool for genetic mapping of Neurospora mtDNA.},
	keywords = {DNA, Mitochondrial, Genes, Membrane Proteins, Mutation, Neurospora, Neurospora crassa, Phenotype, RNA, Ribosomal},
	issn = {0027-8424},
	author = {Bertrand, H and Collins, R A and Stohl, L L and Goewert, R R and Lambowitz, A M}
}
@article {489,
	title = {The 32 S RNA of Neurospora crassa mitochondria is not a precursor of the mitochondrial ribosomal RNAs.},
	journal = {J Mol Biol},
	volume = {134},
	number = {3},
	year = {1979},
	month = {1979 Nov 5},
	pages = {667-72},
	keywords = {Mitochondria, Molecular Weight, Neurospora, Neurospora crassa, RNA, Fungal, RNA, Ribosomal},
	issn = {0022-2836},
	author = {Grimm, M F and Lambowitz, A M}
}
@article {492,
	title = {Defective splicing of mitochondrial rRNA in cytochrome-deficient nuclear mutants of Neurospora crassa.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {76},
	number = {6},
	year = {1979},
	month = {1979 Jun},
	pages = {2635-9},
	abstract = {Recent studies have shown that the gene encoding the large (25 S) mitochondrial rRNA of Neurospora crassa contains an intervening sequence of 2-2.5 kilobases that is not present in the mature 25S mitochondrial rRNA. Earlier studies had provided evidence that mitochondrial rRNAs in Neurospora are synthesized via a 32S precursor RNA that contains sequences for both the mature 19S and 25S RNA species. The present work shows that the intervening sequence is not present in 32S RNA. However, we have identified two temperature-sensitive nuclear mutants that fail to excise the intervening sequence at the nonpermissive temperature (37 degrees C). When grown at 37 degrees C, the mutants show decreased ratios of 25S to 19S RNA and accumulate a novel 35S RNA that appears to consist of 25S RNA plus most or all of the intervening sequence. The mutants are allelic but can be distinguished in temperature shift-up experiments, mitochondrial rRNA processing turning off more rapidly in one than in the other. These mutants should provide powerful new tools for studying RNA processing in eukaryotic cells.},
	keywords = {Base Sequence, Cell Nucleus, Cytochromes, DNA Restriction Enzymes, DNA, Mitochondrial, Mitochondria, Molecular Weight, Mutation, Neurospora, Neurospora crassa, RNA, Ribosomal},
	issn = {0027-8424},
	author = {Mannella, C A and Collins, R A and Green, M R and Lambowitz, A M}
}
@article {496,
	title = {Mitochondrial ribosome assembly in Neurospora crassa: mutants with defects in mitochondrial ribosome assembly.},
	journal = {Mol Gen Genet},
	volume = {177},
	number = {1},
	year = {1979},
	month = {1979},
	pages = {73-84},
	keywords = {Chloramphenicol, Cytochromes, DNA, Mitochondrial, Mutation, Neurospora, Neurospora crassa, Phenotype, Ribosomal Proteins, Ribosomes, RNA},
	issn = {0026-8925},
	author = {Collins, R A and Bertrand, H and Lapolla, R J and Lambowitz, A M}
}
@article {491,
	title = {Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins.},
	journal = {J Cell Biol},
	volume = {82},
	number = {1},
	year = {1979},
	month = {1979 Jul},
	pages = {17-31},
	abstract = {Recent results with Neurospora crassa show that one protein (S-5, mol wt 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria (Lambowitz et al. 1976. J. Mol. Biol. 107:223-253). In the present work, Neurospora mit ribosomal proteins were analyzed by two-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 MKCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI greater than 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol was found to rapidly inhibit the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits (LaPolla and Lambowitz. 1977. J. Mol. 116: 189-205). Two-dimensional gel analysis shows that the more slowly sedimentaing CAP-30S particles are deficient in S-5 and in several other proteins, whereas these proteins are present in normal concentrations in mature small subunits from the same cells. Because S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol, the results tentatively suggest that S-5 plays a role in the assembly of mit small subunits. In addition, the results are consistent with the idea that S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and six additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. However, [poky] mit small subunits are deficient in S-5 and also contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].},
	keywords = {Chloramphenicol, Electrophoresis, Polyacrylamide Gel, Mitochondria, Mutation, Neurospora, Neurospora crassa, Ribosomal Proteins},
	issn = {0021-9525},
	author = {Lambowitz, A M and Lapolla, R J and Collins, R A}
}
@article {495,
	title = {Preparation and analysis of mitochondrial ribosomes.},
	journal = {Methods Enzymol},
	volume = {59},
	year = {1979},
	month = {1979},
	pages = {421-33},
	keywords = {Cell Fractionation, Fungal Proteins, Mitochondria, Neurospora, Polyribosomes, Protein Biosynthesis, Ribonucleases, Ribosomes, RNA, Ultracentrifugation},
	issn = {0076-6879},
	author = {Lambowitz, A M}
}
@article {493,
	title = {Respiration in the yeast and mycelial phases of Histoplasma capsulatum.},
	journal = {J Bacteriol},
	volume = {138},
	number = {2},
	year = {1979},
	month = {1979 May},
	pages = {647-9},
	abstract = {Respiration in the yeast and mycelial phases of Histoplasma capsulatum proceeds via a cytochrome system and an alternate oxidase, both present constitutively. The mycelial cytochrome system is distinguished by an additional partial shunt around the antimycin-sensitive site.},
	keywords = {Antimycin A, Cyanides, Cytochromes, Histoplasma, Hydroxamic Acids, Oxygen Consumption, Salicylamides, Temperature},
	issn = {0021-9193},
	author = {Maresca, B and Lambowitz, A M and Kobayashi, G S and Medoff, G}
}
@article {494,
	title = {Transmission of mitochondrial deoxyribonucleic acid in Neurospora crassa sexual crosses.},
	journal = {J Bacteriol},
	volume = {137},
	number = {3},
	year = {1979},
	month = {1979 Mar},
	pages = {1449-51},
	abstract = {Neurospora crassa mitochondrial deoxyribonucleic acid shows strict uniparental inheritance in sexual crosses, with a notable absence of mixtures and recombinant types that appear frequently in heteroplasmons.},
	keywords = {Conjugation, Genetic, Crosses, Genetic, DNA, DNA Restriction Enzymes, Mitochondria, Neurospora, Neurospora crassa},
	issn = {0021-9193},
	author = {Mannella, C A and Pittenger, T H and Lambowitz, A M}
}
@article {490,
	title = {Unidirectional gene conversion associated with two insertions in neurospora crassa mitochondrial DNA.},
	journal = {Genetics},
	volume = {93},
	number = {3},
	year = {1979},
	month = {1979 Nov},
	pages = {645-54},
	abstract = {The mitochondrial phenotype of [poky] and other extranuclear Neurospora mutants is known to predominate over that of wild type in heteroplasmons. In the present work, we have investigated the interaction between wild-type and [poky] mtDNAs using as many as four physical markers to distinguish the two types of mtDNAs. Two insertions, one of 1200 bp in Eco RI-5 and the other 50 bp in Eco RI-9, are identified as sites of high frequency, unidirectional gene conversion leading to their spread through mtDNA populations in heteroplasmons. However, the transmission of the [poky] mutation does not appear to be correlated with the transmission of either of these insertions or of other physical markers. The possibility that other loci of nonreciprocal recombination might be responsible for the "dominance" of Neurospora extranuclear mutants is discussed.},
	keywords = {Cell Nucleus, DNA Transposable Elements, DNA, Bacterial, DNA, Mitochondrial, Heterozygote, Mutation, Neurospora, Neurospora crassa, Phenotype, Recombination, Genetic},
	issn = {0016-6731},
	author = {Mannella, C A and Lambowitz, A M}
}
@article {488,
	title = {Binding of mitochondrial ribosomal proteins to a mitochondrial ribosomal precursor RNA containing a 2.3-kilobase intron.},
	journal = {J Biol Chem},
	volume = {254},
	number = {22},
	year = {1979},
	month = {1979 Nov 25},
	pages = {11746-50},
	abstract = {In Neurospora, the gene encoding the mitochondrial large subunit (25 S) rRNA contains an intervening sequence of approximately 2.3 kilobases. We have recently identified two temperature-sensitive, nuclear mutants (289-67 and 299-9) which are defective in splicing of the 25 S RNA. When grown at the nonpermissive temperature (37 degrees C), the mutants show decreased ratios of 25 S/19 S RNA and accumulate a novel 35 S RNA which appears to be a continuous transcript of the 25 S RNA gene, including the intervening sequence. In the present work, mitochondrial ribonucleoprotein particles present in the 50 S subunit peak from wild type and mutant 299-9 were analyzed by equilibrium centrifugation in CsCl gradients containing 25 mM MgCl2. The results show that 35 S RNA can be isolated as part of a ribonucleoprotein particle associated with nearly all of the large subunit ribosomal proteins. However, the particles appear to be less stable in CsCl gradients and more sensitive to nucleolytic degradation than particles derived from mature large subunits. Our results indicate that binding of ribosomal proteins to 35 S RNA could precede removal of the intron, but that removal of the intron may be required to achieve stable protein binding.},
	keywords = {Base Sequence, DNA Restriction Enzymes, Genes, Macromolecular Substances, Mitochondria, Molecular Weight, Neurospora, Neurospora crassa, Protein Binding, Ribosomal Proteins, RNA, Ribosomal, Transcription, Genetic},
	issn = {0021-9258},
	author = {Lapolla, R J and Lambowitz, A M}
}
@article {487,
	title = {Characterization of variant Neurospora crassa mitochondrial DNAs which contain tandem reiterations.},
	journal = {Cell},
	volume = {18},
	number = {4},
	year = {1979},
	month = {1979 Dec},
	pages = {1197-207},
	abstract = {Two variant mtDNA types ((types IIa and HI-10) have been identified in individual subcultures of the extra-nuclear [poky] mutant of Neurospora crassa. Eco RI digests of type IIa mtDNA are characterized by an extra band, alpha, Mr = 1.4 Mdal, which arises from tandemly inserted reiterations of a 1.4 Mdal sequence. Restriction enzyme analysis and Southern hybridization experiments show: that the 1.4 Mdal repeats are located at the junction of Eco RI-4 and -6, that the repeats contain sequences ordinarily present in Eco RI-4 and -6, that the repeats are oriented head-to-tail and that the number of repeats per molecule (n) varies from n = 0 to n = 8, with about half of the molecules containing no repeats. The 1.4 Mdal repeats appear to be actively mained in type IIa mtDNA populations as a result of a specific alteration in mtDNA. Data are presented which suggest that this alteration may be located near small deletions and/or sequence changes in Eco RI-3 and -10, fragments almost exactly opposite the site of the repeats on the genome. The second variant, HI-10 mtDNA, arose in a heteroplasmic strain in which type IIa mtDNA was one component. The most striking feature of HI-10 mtDNA is the up to 5-fold amplification of an 18 Mdal segment extending from Eco RI-4 (the site of the 1.4 Mdal repeats) through the rRNA genes. Eco RI digests show that HI-10 possesses characteristic features of type IIa mtDNA, including the 1.4 Mdal repeats and the alteration in Eco RI-10. HI-10 mtDNA also shows a novel Eco RI fragment, beta, Mr = 2.9 Mdal. The variant Neurospora mtDNAs may be generated by mechanisms analogous to those which give rise to defective mtDNAs of yeast petite mutants. The possible consequences of defective mtDNAs in obligately aerobic organisms are discussed.},
	keywords = {Base Sequence, DNA Restriction Enzymes, DNA, Fungal, DNA, Mitochondrial, Mutation, Neurospora, Neurospora crassa, Nucleic Acid Hybridization, Recombination, Genetic},
	issn = {0092-8674},
	author = {Mannella, C A and Goewert, R R and Lambowitz, A M}
}
@article {497,
	title = {Interaction of wild-type and poky mitochondrial DNA in heterokaryons of Neurospora.},
	journal = {Biochem Biophys Res Commun},
	volume = {80},
	number = {3},
	year = {1978},
	month = {1978 Feb 14},
	pages = {673-9},
	keywords = {Base Sequence, DNA Replication, DNA Restriction Enzymes, DNA, Mitochondrial, Extrachromosomal Inheritance, Genes, Dominant, Neurospora, Neurospora crassa, Phenotype, Recombination, Genetic},
	issn = {0006-291X},
	author = {Mannella, C A and Lambowitz, A}
}
@article {498,
	title = {Mitochondrial ribosome assembly in Neurospora crassa. Chloramphenicol inhibits the maturation of small ribosomal subunits.},
	journal = {J Mol Biol},
	volume = {116},
	number = {2},
	year = {1977},
	month = {1977 Oct 25},
	pages = {189-205},
	keywords = {Chloramphenicol, Electrophoresis, Polyacrylamide Gel, Mitochondria, Neurospora, Neurospora crassa, Ribosomal Proteins, Ribosomes, RNA, Ribosomal},
	issn = {0022-2836},
	author = {Lapolla, R J and Lambowitz, A M}
}
@article {499,
	title = {Mitochondrial ribosome assembly in Neurospora. Preparation of mitochondrial ribosomal precursor particles, site of synthesis of mitochondrial ribosomal proteins and studies on the poky mutant.},
	journal = {J Mol Biol},
	volume = {107},
	number = {3},
	year = {1976},
	month = {1976 Nov 5},
	pages = {223-53},
	keywords = {Cell Fractionation, Chloramphenicol, Electrophoresis, Polyacrylamide Gel, Mitochondria, Molecular Weight, Mutation, Neurospora, Neurospora crassa, Protein Biosynthesis, Ribosomal Proteins, Ribosomes, Species Specificity},
	issn = {0022-2836},
	author = {Lambowitz, A M and Chua, N H and Luck, D J}
}
@article {500,
	title = {Studies on the poky mutant of eurospora crassa. Fingerprint analysis of mitochondrial ribosomal RNA.},
	journal = {J Biol Chem},
	volume = {251},
	number = {10},
	year = {1976},
	month = {1976 May 25},
	pages = {3081-95},
	abstract = {Base sequence and methylation of mitochondrial ribosomal RNAs from wild type and poky strains of Neurospora crassa were compared to determine whether a mutational lesion exists in poky 19 S RNA. At the outset, new procedures were developed for the isolation of intact nucleic acids from Neurospora mitochondria based on the substitution of Ca2+ for Mg2+ in the isolation media to inhibit mitochondrial nuclease activity. Using these procedures, intact and highly purified 32P-labeled ribosomal RNAs were extracted from purified mitochondrial ribosomal subunits of wild type and poky and compared using three complementary fingerprinting systems: two-dimensional electrophoresis of T1 plus phosphatase digests and homochromatography of T1 and pancreatic RNase digests. In supplementary experiments, 32P-labeled wild type RNA was co-fingerprinted with 32P-labeled poky and ratios of 32P/33P radioactivity were determined in each fragment to detect possible differences in stoichiometry. In addition, levels and patterns of methylated nucleotides were compared using procedures based on in vivo labeling with [methyl-3H]methionine and [32P]orthophosphate. In all these experiments, no difference was detected between wild type and poky in base sequence or methylation of either 19 S or 25 S RNA. Levels of methylation of Neurospora mitochondrial ribosomal RNAs were extremely low (less than 0.1\% of the nucleotides), and results based on fingerprint analysis and DEAE-cellulose chromatography of alkaline hydrolysates of the [3H]methyl-labeled RNA suggested that 25 S RNA contains two ribose methylations, while 19 S RNA contains no methylated nucleotides.},
	keywords = {Calcium, Electrophoresis, Cellulose Acetate, Magnesium, Methionine, Methylation, Mitochondria, Molecular Weight, Mutation, Neurospora, Neurospora crassa, Oligoribonucleotides, Phosphoric Monoester Hydrolases, Ribonucleases, RNA, Ribosomal},
	issn = {0021-9258},
	author = {Lambowitz, A M and Luck, D J}
}
@article {501,
	title = {Letter: Methylation of mitochondrial RNA species in the wild-type and poky strains of Neurospora crassa.},
	journal = {J Mol Biol},
	volume = {96},
	number = {1},
	year = {1975},
	month = {1975 Jul 25},
	pages = {207-14},
	keywords = {Chromatography, DEAE-Cellulose, Cytoplasm, Electrophoresis, Polyacrylamide Gel, Methionine, Methyltransferases, Mitochondria, Mutation, Neurospora, Neurospora crassa, RNA, S-Adenosylmethionine, Species Specificity},
	issn = {0022-2836},
	author = {Lambowitz, A M and Luck, D J}
}
@article {503,
	title = {The b-cytochromes of plant mitochondria. A spectrophotometric and potentiometric study.},
	journal = {J Biol Chem},
	volume = {249},
	number = {8},
	year = {1974},
	month = {1974 Apr 25},
	pages = {2428-40},
	keywords = {Adenosine Triphosphate, Anaerobiosis, Animals, Antimycin A, Citric Acid Cycle, Cold Temperature, Cytochromes, Electron Transport, Energy Transfer, Mitochondria, NAD, Oxidation-Reduction, Oxygen Consumption, Plant Cells, Plants, Polarography, Potentiometry, Species Specificity, Spectrophotometry, Succinates, Sulfites},
	issn = {0021-9258},
	author = {Lambowitz, A M and Bonner, W D}
}
@article {505,
	title = {The interaction of redox mediators with the {\textquoteright}second phosphorylation site{\textquoteright}; significance for the cytochrome bT hypothesis.},
	journal = {FEBS Lett},
	volume = {40},
	number = {1},
	year = {1974},
	month = {1974 Mar 15},
	pages = {149-53},
	keywords = {Adenosine Triphosphatases, Animals, Antimycin A, Cytochromes, Dinitrophenols, Hydrogen-Ion Concentration, Kinetics, Mitochondria, Liver, Oligomycins, Oxidation-Reduction, Oxidative Phosphorylation, Phenazines, Potentiometry, Rats},
	issn = {0014-5793},
	author = {Wikstr{\"o}m, M K and Lambowitz, A M}
}
@article {504,
	title = {On the lack of ATP-induced midpoint potential shift for cytochrome b-566 in plant mitochondria.},
	journal = {Proc Natl Acad Sci U S A},
	volume = {71},
	number = {4},
	year = {1974},
	month = {1974 Apr},
	pages = {1183-7},
	abstract = {Recently proposed mechanisms of site II energy transduction that assign a key role to cytochrome b-566 are based on the finding that the apparent midpoint potential of b-566 in animal mitochondria increases by more than 250 mV upon addition of ATP [Chance et al. (1970) Proc. Nat. Acad. Sci. USA 66, 1175-1182]. However, since it has never been shown that the redox mediators used in the midpoint potential measurements equilibrate directly with b-566, the observed midpoint potential shift could merely reflect reversed electron transport. In mung bean mitochondria, the apparent midpoint potential of b-566 is known to be unaffected by addition of ATP [Dutton and Storey (1971) Plant Physiol. 47, 282-288]. In the present work, mung bean b-566 is shown to undergo an ATP-induced reduction similar to that observed for b-566 in animal mitochondria. However, in mung bean mitochondria the reduction is found to be rapidly relaxed by addition of redox mediator (phenazine methosulfate, PMS) and concomitantly PMS causes a marked, antimycinsensitive stimulation of ATPase activity. These results suggest that the ATP-induced reduction in mung bean mitochondria is due to reversed electron transport and that PMS can effectively short-circuit reversed electron transport in this system, bringing it close to equilibrium. Moreover, since mung bean and animal b-566 are identical in all other respects tested, the results support the idea that the apparent midpoint potential shift in animal mitochondria is also merely due to reversed electron transport, and that the mediators are now not effective enough to bring the system to equilibrium.},
	keywords = {Adenosine Triphosphatases, Adenosine Triphosphate, Cytochromes, Electron Transport, Hydrolysis, Mitochondria, Mitochondria, Liver, Oxidation-Reduction, Phenazines, Plant Cells, Plants, Potentiometry, Spectrophotometry},
	issn = {0027-8424},
	author = {Lambowitz, A M and Bonner, W D and Wikstr{\"o}m, M K}
}
@article {502,
	title = {The mitochondrial beta-cytochromes of the wild type and poky strains of Neurospora crassa. Evidence for a component reduced only by dithionite.},
	journal = {J Biol Chem},
	volume = {249},
	number = {9},
	year = {1974},
	month = {1974 May 10},
	pages = {2886-90},
	keywords = {Cold Temperature, Cytochromes, Mitochondria, Mutation, Neurospora, Neurospora crassa, Oxidation-Reduction, Potentiometry, Species Specificity, Spectrophotometry, Sulfites},
	issn = {0021-9258},
	author = {Lambowitz, A M and Bonner, W D}
}
@article {506,
	title = {The effect of antimycin on the b-cytochromes of plant mitochondria. Oxidation-reduction behavior of cytochrome b-566.},
	journal = {Biochem Biophys Res Commun},
	volume = {52},
	number = {3},
	year = {1973},
	month = {1973 Jun 8},
	pages = {703-11},
	keywords = {Aerobiosis, Anaerobiosis, Antimycin A, Cytochromes, Electron Transport, Mitochondria, Oxidation-Reduction, Oxidative Phosphorylation, Plant Cells, Potentiometry, Spectrophotometry, Succinate Dehydrogenase, Temperature, Time Factors, Uncoupling Agents},
	issn = {0006-291X},
	author = {Lambowitz, A M and Bonner, W D}
}
@article {510,
	title = {The electron transport components of wild type and poky strains of Neurospora crassa.},
	journal = {J Biol Chem},
	volume = {247},
	number = {5},
	year = {1972},
	month = {1972 Mar 10},
	pages = {1536-45},
	keywords = {Adenine Nucleotides, Cytochromes, Electron Transport, Electron Transport Complex IV, Flavoproteins, Genetics, Microbial, Mitochondria, Mutation, NAD, NADP, Neurospora, Neurospora crassa, Oxidation-Reduction, Oxygen Consumption, Spectrophotometry, Succinates, Temperature},
	issn = {0021-9258},
	author = {Lambowitz, A M and Slayman, C W and Slayman, C L and Bonner, W D}
}
@article {509,
	title = {Electron transport in Neurospora mitochondria. Studies on wild type and poky.},
	journal = {J Biol Chem},
	volume = {247},
	number = {15},
	year = {1972},
	month = {1972 Aug 10},
	pages = {4850-8},
	keywords = {Aerobiosis, Amobarbital, Antimycin A, Ascorbic Acid, Citric Acid Cycle, Cyanides, Electron Transport, Electron Transport Complex IV, Hydroxamic Acids, Ketoglutaric Acids, Kinetics, Malates, Mitochondria, Mutation, NAD, Neurospora, Neurospora crassa, Oxidative Phosphorylation, Oxidoreductases, Oxygen Consumption, Plant Proteins, Polarography, Pyruvates, Rotenone, Salicylates, Succinates},
	issn = {0021-9258},
	author = {Lambowitz, A M and Smith, E W and Slayman, C W}
}
@article {507,
	title = {Effect of pyrrolnitrin on electron transport and oxidative phosphorylation in mitochondria isolated from Neurospora crassa.},
	journal = {J Bacteriol},
	volume = {112},
	number = {2},
	year = {1972},
	month = {1972 Nov},
	pages = {1020-2},
	abstract = {Pyrrolnitrin, at low concentrations, uncouples oxidative phosphorylation in Neurospora mitochondria. At higher concentrations, pyrrolnitrin inhibits electron transport both in the flavine region and through cytochrome oxidase.},
	keywords = {Aniline Compounds, Antifungal Agents, Antimycin A, Cyanides, Electrodes, Electron Transport, Mitochondria, NAD, Neurospora, Neurospora crassa, Oxidative Phosphorylation, Oxidoreductases, Oxygen Consumption, Spectrophotometry, Succinate Dehydrogenase, Succinates, Uncoupling Agents},
	issn = {0021-9193},
	author = {Lambowitz, A M and Slayman, C W}
}
@article {508,
	title = {Oxidative phosphorylation in Neurospora mitochondria. Studies on wild type, poky, and chloramphenicol-induced wild type.},
	journal = {J Biol Chem},
	volume = {247},
	number = {15},
	year = {1972},
	month = {1972 Aug 10},
	pages = {4859-65},
	keywords = {Aniline Compounds, Ascorbic Acid, Chloramphenicol, Citrates, Hydroxamic Acids, Ketoglutaric Acids, Mitochondria, Mutation, NAD, Neurospora, Neurospora crassa, Oxidation-Reduction, Oxidative Phosphorylation, Pyruvates, Succinates},
	issn = {0021-9258},
	author = {Lambowitz, A M and Smith, E W and Slayman, C W}
}
@article {511,
	title = {Cyanide-resistant respiration in Neurospora crassa.},
	journal = {J Bacteriol},
	volume = {108},
	number = {3},
	year = {1971},
	month = {1971 Dec},
	pages = {1087-96},
	abstract = {Cell respiration in wild type and poky was studied as part of a long-term investigation of cyanide-resistant respiration in Neurospora. Respiration in wild type proceeds via a cytochrome chain which is similar to that of higher organisms; it is sensitive to antimycin A or cyanide. Poky, on the other hand, respires by means of two alternative oxidase systems. One of these is analogous to the wild-type cytochrome chain in that it can be inhibited by antimycin A or cyanide; this system accounts for as much as 15\% of the respiration of poky f(-) and 34\% of the respiration of poky f(+). The second oxidase system is unaffected by antimycin A or cyanide at concentrations which inhibit the cytochrome chain maximally. It can, however, be specifically inhibited by salicyl hydroxamic acid. The cyanide-resistant oxidase is not exclusive to poky, but is also present in small quantities in wild type grown under ordinary circumstances. These quantities may be greatly increased (as much as 20-fold) by growing wild type in the presence of antimycin A, cyanide, or chloramphenicol.},
	keywords = {Aerobiosis, Antimycin A, Chloramphenicol, Culture Media, Cyanides, Cytochromes, Electrodes, Electron Transport, Electron Transport Complex IV, Genetics, Microbial, Hydroxamic Acids, Models, Chemical, Mutation, Neurospora, Neurospora crassa, Oxygen Consumption, Polarography},
	issn = {0021-9193},
	author = {Lambowitz, A M and Slayman, C W}
}
