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.

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.

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' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' 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' splice site are base-paired to an internal, 5' 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' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.

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' 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.

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.

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.

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.

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.

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' ends and a single major 3' end. The major 5' and 3' 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.

[ 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'-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' end of the rRNA. This deletion apparently results in synthesis of aberrant 19S rRNAs that are missing 38-45 nucleotides from their 5' ends. We propose that the 4-base-pair deletion is the primary defect in [ poky ] and other group I extranuclear mutants.

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' 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' 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' end of the short intron is about 285 nucleotides downstream from the 5' 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).

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.

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.

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.

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.

Neurospora mtDNA contains a repetitive, 18 nucleotide palindromic sequence (5'-CCCTGCAGTACTGCAGGG-3') 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.

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.

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' splice site; that the intron loop and the central hairpin are different configurations of sequences at or near the 5' 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.

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.

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.