Unstable mitochondrial DNA in natural-death nuclear mutants of Neurospora crassa.

The natural-death mutant of Neurospora crassa has an accelerated senescence phenotype caused by a recessive mutation, nd, in a nuclear gene that is located in linkage group I. An examination of mitochondrial functions, however, revealed that the mutant has phenotypic and molecular defects similar to those commonly associated with maternally transmitted fungal senescence syndromes, including (i) deficiencies in cytochromes aa3 and b; (ii) a deficit in small subunits of mitochondrial ribosomes, and hence defective mitochondrial protein synthesis; and (iii) accumulation of gross rearrangements, including large deletions, in the mitochondrial chromosome of vegetatively propagated cells. These traits indicate that the nd+ allele codes for a function that is essential for stable maintenance of the mitochondrial chromosome, possibly a protein involved in replication, repair, or recombination.     

Cloning and characterization of centromeric DNA from Neurospora crassa.

The centromere locus from linkage group VII of Neurospora crassa has been cloned, characterized, and physically mapped. The centromeric DNA is contained within a 450-kb region that is recombination deficient, A+T-rich, and contains repetitive sequences. Repetitive sequences from within this region hybridize to a family of repeats located at or near centromeres in all seven linkage groups of N. crassa. Genomic Southern blots and sequence analysis of these repeats revealed a unique centromere structure containing a divergent family of centromere-specific repeats. The predominantly transitional differences between copies of the centromere-specific sequence repeats and their high A+T content suggest that their divergence was mediated by repeat-induced point (RIP) mutations.     

Molecular Characterization of the Mitochondrial DNA of a New Stopper Mutant Er-3 of Neurospora Crassa

An ethidium bromide-induced stopper mutant of Neurospora crassa is characterized at the molecular level. The mutant has two populations of mitochondrial DNA: a defective predominant mutant molecule and a basal level of the wild-type molecule. The aberrant DNA resulted after a 25-kbp deletion from the wild-type mitochondrial chromosome, which included major genes such as cytb, co1 and oli2. The deletion endpoints are located in the second intron of the ND5 gene, and in a sequence 250 nucleotides upstream of the co2 gene. The recombination has taken place between two nine nucleotide repeats CCCCGCCCC, one of which is close to a PstI palindrome at its 5' end. Thus the mutant ER-3 differs from all the other stopper mutants described previously in the extent and location of the deletions in the mtDNA.     

The E35 stopper mutant of Neurospora crassa: precise localization of deletion endpoints in mitochondrial DNA and evidence that the deleted DNA codes for a subunit of NADH dehydrogenase.

Two types of defective mitochondrial DNA molecules with large deletions (5 kbp and 40 kbp) have previously been identified in the stopper mutant, E35, of Neurospora crassa. The junction fragments spanning the deletion endpoints have now been cloned and sequenced, and their sequences compared with those of the corresponding wild-type fragments. We show that both types of defective mitochondrial DNAs result from deletions of sequences flanked by short direct repeats, which are themselves parts of larger inverted repeat sequences. In every case, the short direct repeat sequences consist of a run of pyrimidines in one strand and purines in the other. We also report the sequence of a 2151-bp HindIII fragment, which is deleted in both of the defective mitochondrial DNAs. Besides the previously identified gene for a methionine tRNA, the 2151-bp DNA sequence contains an open reading frame with the potential to code for a hydrophobic protein 583 amino acids long. This hydrophobic protein has three blocks of significant homology with proteins coded by URF2 found in other mitochondrial genomes. Since the mammalian mitochondrial URF2 has recently been shown to code for a subunit of NADH dehydrogenase, part of the DNA sequence missing in the E35 stopper mutant of N. crassa may also code for a subunit of NADH dehydrogenase.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. 2. Localization of genes by interspecific hybridization in strain ade7-50h- and cloning of the genome in small fragments

A series of 18 small overlapping restriction fragments has been cloned, covering the complete mitochondrial genome of Schizosaccharomyces pombe. By hybridizing mitochondrial gene probes from Saccharomyces cerevisiae and Neurospora crassa with restriction fragments of Schizosaccharomyces pombe mitochondrial DNA, the following homologous genes were localized on the mitochondrial genome of S. pombe: cob, cox1, cox2 and cox3, ATPase subunit 6 and 9 genes, the large rRNA gene and both types of open reading frames occurring in mitochondrial introns of various ascomycetes. The region of the genome, hybridizing with cob exon probes is separated by an intervening sequence of about 2500 bp, which is homologous with the first two introns of the cox1 gene in Saccharomyces cerevisiae (class II introns according to Michel et al. 1982). Similarly, in the cox1 homologous region, which covers about 4000 bp, two regions were detected hybridizing with class I intron probes, suggesting the existence of two cox1 introns in Schizosaccharomyces pombe. Hybridization with several specific exon probes with a determined order has revealed that cob, cox1, cox3 and the large rRNA gene are all transcribed from the same DNA strand. The low intensities of hybridization signals suggest a large evolutionary distance between Schizosaccharomyces pombe and Saccharomyces cerevisiae or Neurospora crassa mitochondrial genes. Considering the length of the mitochondrial DNA of Schizosaccharomyces pombe (about 19.4 kbp) and the expected length of the localized genes and intron sequences there is enough space left for encoding the expected set of tRNAs and the small rRNA gene. The existence of leader-, trailer-, ori- and spacer sequences or further unassigned reading frames is then restricted to a total length of about 3000 bp only.     

Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

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.     

Intramolecular recombination as a source of mitochondrial chromosome heteromorphism in Neurospora

Approximately 20% of the genome is missing and an equivalent amount is under-represented in the mitochondrial chromosome population of a stopper (stp) mutant of Neurospora crassa during the stopped phase of its cyclical growth pattern. At this stage, a 21 kb (7.2 mu) circular molecule, one-third the length of the normal chromosome, is the predominant form. A complementary 43 kb (14.6 mu) circle appears upon resumption of growth. The circles arise by reciprocal recombination at or near directly repeated tRNAmet sequences and the frequency of the two depends upon their rates of replication from replication origins unique to each of them. The 7.2 and 14.6 mu circles are the most frequent of the heteromorphic derivatives found within wild-type mitochondrial chromosome populations.     

Molecular cloning of a Neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes.

The albino-3 (al-3) gene of Neurospora crassa, which probably encodes the carotenoid biosynthetic enzyme geranylgeranyl pyrophosphate synthetase, was cloned. The N. crassa triple mutant al-3 qa-2 aro-9 was transformed to qa-2+ with mixtures of plasmids bearing N. crassa DNA inserts, and the transformants were screened for the al-3+ phenotype. One al-3+ qa-2+ transformant (AL3-1) was examined in detail and shown to contain intact vector sequences integrated into the N. crassa genome. The vector and some flanking sequences were recovered from AL3-1 after restriction, ligation, and selection of chloramphenicol-resistant transformants of Escherichia coli. The flanking sequences were subsequently used to detect the al-3-containing plasmid in the mixture of about 1,800 plasmids. Restriction fragment length polymorphism mapping was carried out to confirm the identity of the cloned fragment. The level of the al-3 mRNA was shown to be increased 15-fold in light-induced (compared with that in dark-grown) wild-type mycelia. The light-dependent increase in al-3 mRNA levels was not observed in presumed regulatory mutant (white collar) strains.     

Genomic analysis of a virulent and a less virulent strain of the entomopathogenic fungus Beauveria bassiana, using restriction fragment length polymorphisms.

The genomic DNA of two strains of the entomopathogenic fungus Beauveria bassiana, strain GK2016, a "wild type" (virulent), and strain GK2051, a less virulent mutant to grasshoppers, was digested with 12 restriction endonucleases. Gel electrophoresis conditions were established to show restriction fragment length patterns visually in the digested DNA stained with ethidium bromide. The less virulent mutant was generated by ultraviolet illumination of conidiospores at a 95% lethal dose. Both strains of the fungi were identical in morphology as well as in 16 of 22 API-ZYM kit enzyme assays. Differences in levels of total enzyme activity were observed for esterase, esterase-lipase, beta-galactosidase, chitinase, and protease. A Neurospora crassa beta-tubulin gene (heterologous gene) and two homologous DNA probes (pJK16 and pJK18) hybridized to several specific DNA bands in B. bassiana strain GK2016 but not in strain GK2051. Strain GK2051 gave different restriction fragment length pattern when compared with its parent strain. Taken together, the data show restriction fragment length differences between the genomic DNA of the two strains, including the loss of some DNA sequences from the mutant strain, which may be involved in pathogenicity. Finally, B. bassiana GK2016 contains a beta-tubulin gene with at least partial homology to that of N. crassa.     

The frost gene of Neurospora crassa is a homolog of yeast cdc1 and affects hyphal branching via manganese homeostasis

The Neurospora crassa mutant frost has a hyperbranching phenotype that can be corrected by adding Ca(2+), suggesting that characterization of this gene might clarify the mechanism of Ca(2+)-dependent tip growth. The wild-type allele was cloned by sib selection using protoplasts from arthroconidia. RFLP analysis revealed that the cloned DNA fragment mapped to the fr locus. The nucleotide sequence of genomic and cDNA was determined. The deduced amino acid sequence showed homology to the Saccharomyces cerevisiae CDC1 protein, implicated in manganese homeostasis. The fr mutant was sensitive to Mn(2+), and a revertant allele whose product differs by one amino acid was tolerant to Mn(2+). Mn(2+) depletion induced the wild-type strain to hyperbranch, resulting in a morphology similar to that of fr. The fr mutant was also sensitive to calcineurin inhibitors. These results suggest that fr is involved in Mn(2+) homeostasis and point to a role for Mn(2+) in Neurospora branching.     

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.     

Neurospora crassa ribosomal DNA: sequence of internal transcribed spacer and comparison with N. intermedia and N. sitophila

Using [32P]DNA probes from a clone containing 17S, 5.8S and 26S rRNA of Neurospora crassa, the remainder of the repeat unit (RU) for ribosomal DNA (rDNA) has been cloned. Combining restriction analysis of the cloned DNA and restriction digests of genomic DNA, the RU was found to be 8.7 kb. The nucleotide sequence was determined for the internal transcribed spacer (ITS) regions one and two, for 5.8S rRNA and for portions of 17S and 26S rRNAs immediately flanking the ITS regions, and compared to the corresponding region of Saccharomyces carlsbergensis. In addition, a comparative restriction analysis of two other Neurospora species was performed using twelve restriction endonucleases. Genomic DNA blots of rDNA from N. intermedia and N. sitophila revealed rDNA RUs of 8.4 kb. The majority of differences in restriction patterns were confined to sequences outside the mature rRNA regions. However, one SmaI recognition site was found in 26S rRNA of N. crassa and N. sitophila but not in N. intermedia.     

Cloning of methylated transforming DNA from Neurospora crassa in Escherichia coli.

An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N. crassa genomic DNA library. Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. Analyses of a N. crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues. This methylation was shown to be responsible for our inability to recover transformants in standard strains of E. coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems. Restriction of methylated DNA in E. coli may explain the general failure to recover vector or transforming sequences from N. crassa transformants.     

Identification and comparative analysis of the large subunit mitochondrial ribosomal proteins of Neurospora crassa

The mitochondrial ribosome (mitoribosome) has highly evolved from its putative prokaryotic ancestor and varies considerably from one organism to another. To gain further insights into its structural and evolutionary characteristics, we have purified and identified individual mitochondrial ribosomal proteins of Neurospora crassa by mass spectrometry and compared them with those of the budding yeast Saccharomyces cerevisiae. Most of the mitochondrial ribosomal proteins of the two fungi are well conserved with each other, although the degree of conservation varies to a large extent. One of the N. crassa mitochondrial ribosomal proteins was found to be homologous to yeast Mhr1p that is involved in homologous DNA recombination and genome maintenance in yeast mitochondria.     

The ribosomal RNA genes on Neurospora crassa mitochondrial DNA are adjacent.

Hybridization of separated 24 S and 17 S ribosomal RNA from Neurospora crassa mitochondrial ribosomes to restriction fragments of mitochondrial DNA leads to the conclusion that the large and small ribosomal RNA are adjacent on the restriction endonuclease cleavage map of the DNA. The distance between the two genes is estimated at 900 basepairs. This result is consistent with the existence of a ribosomal precursor RNA in N. crassa mitochondria and is in contrast to the situation in yeast, where the ribosomal genes are far apart on the mitochondrial DNA. The position of the ribosomal RNA genes on the cleavage map of N. crassa mtDNA provides a start for ordering the Hind III restriction fragments.     

Recombination and Replication of Plasmid-like Derivatives of a Short Section of the Mitochondrial Chromosome of Neurospora Crassa

The 21-kbp mitochondrial chromosome of the stp-ruv strain of Neurospora crassa undergoes regional amplification yielding plasmid-like supercoiled circles varying in size from subunit length to very high multimers. A comparison of the base sequence of the five plasmids studied, with the region of the chromosome from which they were derived, indicated that the amplified chromosomal segments were determined by a recombination-excision process near or within two structurally distinctive regions. One of these, consisting of nearly uninterrupted strings of Cs and Gs straddling tandem PstI site direct repeats, could form an extended hairpin loop with only a few mismatches. It was found at or near the 5' exchange point of all of the plasmids. An extended 35-bp sequence containing 17-bp direct repeats was the primary 3' site of exchange. Base sequence changes were found in the vicinity of exchange points. Most notable of these was a G insertion and T to C transition within a section of the 5' region likely to form a hairpin loop, suggesting the involvement of a mismatch repair-like mechanism in the recombination process. The sequence, TATATAGACATATA, was identified as a likely candidate for the site of replication initiation. A nearly identical sequence was found common to all of the corresponding plasmids of Podospora anserina and was reported near the presumed replication origin of the Drosophila yakuba mitochondrial chromosome. A search of GenBank revealed a remarkable association of the consensus sequence, TATATAGAXATATA, with the plus strand of organelle DNA.