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.

The 35 S percursor of the Neurospora mitochondrial large rRNA contains a 2.3-kilobase intron located towards its 3' 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' 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.

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.

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.

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.

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.

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.

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.

[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.

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'-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'-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.

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.

"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.

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.

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].