Publications

1994
Chiang CC, Kennell JC, Wanner LA, Lambowitz AM. A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination. Mol Cell Biol. 14 (10) :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' 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.
Mohr G, Caprara MG, Guo Q, Lambowitz AM. A tyrosyl-tRNA synthetase can function similarly to an RNA structure in the Tetrahymena ribozyme. Nature. 370 (6485) :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.
1993
Saldanha R, Mohr G, Belfort M, Lambowitz AM. Group I and group II introns. FASEB J. 7 (1) :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.
Lambowitz AM, Belfort M. Introns as mobile genetic elements. Annu Rev Biochem. 62 :587-622.
Kennell JC, Moran JV, Perlman PS, Butow RA, Lambowitz AM. Reverse transcriptase activity associated with maturase-encoding group II introns in yeast mitochondria. Cell. 73 (1) :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' 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.
Wang H, Lambowitz AM. 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. J Biol Chem. 268 (25) :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' 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' 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.
Mohr G, Perlman PS, Lambowitz AM. Evolutionary relationships among group II intron-encoded proteins and identification of a conserved domain that may be related to maturase function. Nucleic Acids Res. 21 (22) :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.
Wang H, Lambowitz AM. The Mauriceville plasmid reverse transcriptase can initiate cDNA synthesis de novo and may be related to reverse transcriptase and DNA polymerase progenitor. Cell. 75 (6) :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' 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.
1992
Nargang FE, Pande S, Kennell JC, Akins RA, Lambowitz AM. Evidence that a 1.6 kilobase region of Neurospora mtDNA was derived by insertion of part of the LaBelle mitochondrial plasmid. Nucleic Acids Res. 20 (5) :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's mtDNA.
Wang H, Kennell JC, Kuiper MT, Sabourin JR, Saldanha R, Lambowitz AM. The Mauriceville plasmid of Neurospora crassa: characterization of a novel reverse transcriptase that begins cDNA synthesis at the 3' end of template RNA. Mol Cell Biol. 12 (11) :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' end of the plasmid transcript, which has a 3' 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' end and has a preference for transcripts having the 3' tRNA-like structure. Further, unlike retroviral reverse transcriptases, the Mauriceville plasmid reverse transcriptase begins cDNA synthesis directly opposite the 3'-terminal nucleotide of the template RNA. The ability to initiate cDNA synthesis directly at the 3' 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.
Kämper U, Kück U, Cherniack AD, Lambowitz AM. The mitochondrial tyrosyl-tRNA synthetase of Podospora anserina is a bifunctional enzyme active in protein synthesis and RNA splicing. Mol Cell Biol. 12 (2) :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.
Mohr G, Zhang A, Gianelos JA, Belfort M, Lambowitz AM. The neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core. Cell. 69 (3) :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'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.
Turcq B, Dobinson KF, Serizawa N, Lambowitz AM. A protein required for RNA processing and splicing in Neurospora mitochondria is related to gene products involved in cell cycle protein phosphatase functions. Proc Natl Acad Sci U S A. 89 (5) :1676-80.Abstract
The Neurospora crassa cyt-4 mutants have pleiotropic defects in mitochondrial RNA splicing, 5' and 3' 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.
Guo Q, Lambowitz AM. A tyrosyl-tRNA synthetase binds specifically to the group I intron catalytic core. Genes Dev. 6 (8) :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.
1991
Mohr G, Lambowitz AM. Integration of a group I intron into a ribosomal RNA sequence promoted by a tyrosyl-tRNA synthetase. Nature. 354 (6349) :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.
Schulte U, Lambowitz AM. The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase. Mol Cell Biol. 11 (3) :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.
Kubelik AR, Turcq B, Lambowitz AM. The Neurospora crassa cyt-20 gene encodes cytosolic and mitochondrial valyl-tRNA synthetases and may have a second function in addition to protein synthesis. Mol Cell Biol. 11 (8) :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.
Kittle JD, Mohr G, Gianelos JA, Wang H, Lambowitz AM. 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. Genes Dev. 5 (6) :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).
Guo QB, Akins RA, Garriga G, Lambowitz AM. Structural analysis of the Neurospora mitochondrial large rRNA intron and construction of a mini-intron that shows protein-dependent splicing. J Biol Chem. 266 (3) :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' 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' 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' splice site, either in the intron core itself or in the interaction between the core and the P1 stem.
1990
Akins RA, Lambowitz AM. Analysis of large deletions in the Mauriceville and Varkud mitochondrial plasmids of Neurospora. Curr Genet. 18 (4) :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.

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