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Recombination junctions of several Neurospora mitochondrial DNA (mtDNA) mutants and their revertants were identified. Their nucleotide sequences and putative secondary structures were determined in order to understand the nature of the elements involved in intramolecular recombination. Multiple deletions, involving the same portion of Neurospora mtDNA, were identified in six independently isolated mutants. A 9-nucleotide repeat element, CCCCNCCCC, was found to be involved in these and other Neurospora mitochondrial recombination events. The repeat elements were clustered as hot spots on the Neurospora mtDNA and were associated with palindromic DNA sequences. The palindromes have a potential to generate hairpin structures. A much lower free energy of the putative hairpins at the 5' end of the recombination site, and the possible formation of non-B-DNA structure by polypyrimidine tracks, may be important in the initiation of recombination. Using PCR, we found low levels of a specific mitochondrial deletion in certain Neurospora mutants. Their presence in low amounts in a population with a much larger number of normal mtDNA is unexpected. Contrary to earlier belief, this finding supports the view that deleted, smaller DNA molecules are not always suppressive relative to normal mtDNAs.

The structural and functional abnormalities in a new respiratory deficient, mitochondrial senescence mutant ER-3 of Neurospora crassa are described. The mitochondrial mutant, which grows at a rate of only 10% of that of the wild type, was found deficient in all three cytochromes, and completely lacking in cytochromes aa3. Cytochrome oxidase activity in the mutant mitochondria was only about 5% of the wild type mitochondria. However, the total whole cell respiration rate of the mutant was 33% greater than that of the wild type, while the cyanide-resistant respiration rates were equal. The results of inhibitor studies clearly demonstrate that the mutant possesses a defect in one or more components of the terminal oxidase. Electron microscopic examination of whole cell sections and subsequent morphometric analysis revealed a significant (33%) reduction in membrane surface density of mitochondrial cristae in the mutant as compared with the wild type. Results of genetic and heterokaryon analyses indicate the location of mutation (ER-3) in the mitochondrial DNA. It is concluded that the senescence mutant ER-3 possesses a defect in the terminal portion of the mitochondrial respiratory apparatus. These results are consistent with previous analyses of mitochondrial DNA populations, and support the notion that obligately aerobic eukaryotic cells deficient in mitochondrial respiration necessarily exist as a result of stable heteroplasmosis and that defects in mitochondria lead to senescence in Neurospora mutant ER-3.

Sucrose density gradient analysis of Neurospora cell free extract showed at least three distinct peaks of enzyme activity; of these, a high molecular weight enzyme was identified as DNA polymerase alpha because of its sensitivity to aphidicolin and to NEM. DNA polymerase mutants of Neurospora crassa were isolated by their resistance to aphidicolin, a specific inhibitor of the eukaryotic DNA polymerase alpha. Some mutants showed an increase in the specific activity of the enzyme. One mutant (E-2-4-1) characterized in detail showed the presence of DNA polymerase which was resistant to inhibitory action of aphidicolin in an in vitro assay. Another mutant (C-3) showed changes in the pH optimum of the enzyme activity. Genetic characterization of the mutants provided evidence for the dominance of the aphr allele controlling aphidicolin resistance and its Mendelian segregation. Some of the aphidicolin resistant mutants were found to be UV-sensitive. Neurospora wild-type and mutant genomic DNA digest was found to hybridize with a cloned yeast DNA polymerase gene. The nick translated yeast DNA polymerase gene was used to screen a genomic cosmid library of Neurospora. A putative clone containing Neurospora DNA polymerase gene has been identified. Further molecular characterization of the Neurospora DNA polymerase gene and enzyme is in progress.

Slow growing mutants of Neurospora crassa were obtained by ethidium bromide treatment of the wild type strain. A particular mutant ER-3 showed stopper phenotype accompanied by deficient cytochrome spectra. The mutant showed an altered restriction pattern of the mtDNA which indicated a deletion of 25,000 bp. The phenotype of the ethidium bromide induced mutant ER-3 seem to be related to the loss of several essential genes due to a deletion in its mtDNA.

Evidence for genetic transformation in Neurospora crassa is based on the observations that allo-DNA has a specific effect in producing transformants which is abolished by DNAase treatment and that iso-DNA is not effective in transformation. Here, unambiguous evidence for genetic transformation is provided by transfer of a temperature-sensitive inositol requirement from a donor to a recipient strain. Data provided also suggest the role of growth conditions and the involvement of a nuclease gene in the DNA uptake and transformation of N. crassa.

A simple method of replica plating has been used to isolate mutants of Neurospora crassa that have increased sensitivity to methyl methanesulfonate (MMS) and/or to histidine. Twelve mutants with increased sensitivity to MMS and one mutant with increased sensitivity to histidine showed Mendelian segregation of the mutant phenotypes. Three mutants were mapped to loci not previously associated with MMS sensitivity. Two other were allelic to the UV- and MMS-sensitive mutant, mei-3. Survival curves indicate that conidia (mutant or wild-type) survive on much higher concentrations of MMS at 25 degrees than at 37 degrees. In contrast, mycelial growth is more resistant to MMS at 37 degrees. The possibility of qualitatively different repair processes at these two temperatures is discussed.

Several MMS-sensitive mutants of Neurospora crassa were compared with the wild-type strain for their relative sensitivities to UV, X-ray, and histidine. They were also compared for the frequency of spontaneous mutation at the loci which confer assistance to p-fluorophenylalanine. The mutants were also examined for possible defects in meiotic behavior in homozygous crosses and for any change in the inducible DNA salvage pathways (as indicated by their ability to utilize DNA as the sole phosphate source in the growth medium). On the basis of these characterizations, the present MMS-sensitive mutants of Neurospora can be placed into three groups. The first group includes three mutants, mus-(SC3), mus-(SC13), and mus-(SC28). These are slow growers, insensitive to histidine with no apparent meiotic defects and may have reduced frequency of spontaneous mutation. In addition, their mycelial growth is sensitive to MMS but the conidial viability following MMS, UV or X-ray treatment appears normal or only slightly more sensitive than the wild-type. The second group includes only one mutant, mus-(SC15); its mycelial growth is very sensitive to MMS but the conidial survival following treatment with MMS or UV appears normal; however, the conidial survival following exposure to X-ray is significantly reduced. This mutant shows an increased (more than 10-fold) frequency of spontaneous mutation, but behaves normal like the wild-type with respect to fertility, growth rate and insensitivity to histidine. The third group includes mutants mus-(SC10), mus-(SC25), and mus-(SC29). These mutants are very sensitive to UV, X-rays and MMS and to histidine but have normal growth rates on minimal medium. Mutant mus-(SC10), but not mus-(SC25) and mus-(SC29), has an increased (11 X) frequency of spontaneous mutation. On the basis of data presented, the MMS sensitivity of the first group of mutants cannot be ascertained to arise from a defect in the DNA repair pathways; instead, it may stem from altered cell permeability or other pleotropic effects of the mus mutations. However, it can be suggested that the second and third group of mus mutants may indeed result from a defect in the DNA repair pathways controlled by the mus genes; this conclusion is based on their cross-sensitivity to the number of DNA-damaging agents such as MMS, UV and/or X-ray, high frequencies of spontaneous mutation (mutator effects) and defects in meiotic behavior.

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 effect of purified wild-type RNA (allo-RNA) on genetic reversion of inositol-requiring mutant 89601 of Neurospora crassa is described. The mutant (inos minus) strain, on treatment with the wild-type RNA preparation, was found to revert to wild type (inos+) in significant numbers. RNA from the mutant (iso-RNA) and allo-RNA digested by RNase were ineffective in causing genetic reversion at the inositol locus. The allo-RNA-induced revertants were stable and showed a Mendelian transmission of the inos+ character.

An enzyme, galactosyltransferase, able to catalyze the formation of galactose polymers was detected in cell-free extracts of a wild type strain of Neurospora crassa. Enzyme activity was found in both the supernatant and the particle fractions after centrifugation at 100,000 X g. The enzyme assayed in the 100,000 X g supernatant showed a 4fold difference in specific activity as compared to that found in the particle fraction.

Inositol-independent (inos(+)) revertants of Neurospora induced in inositol-requiring mutants by treatment with wild-type DNA in previous studies were found to be stable and to grow well in the absence of inositol. Genetic data presented in this paper show that a major proportion of these induced revertants rarely transmitted the inositol independence character (inos(+)) to their sexual progeny. Non-Mendelian transmission of the transformed character (inos(+)) was also found to occur in some of the sexual progeny in subsequent generations. These genetic data support the idea that the transforming DNA pieces carrying the genetic information (called exosomes) are not readily integrated into the host genome. It is suggested that elimination of most exosomes during meiosis causes a loss of the genetic information and leads to non-Mendelian transmission of the induced revertant character (inos(+)).

Neurospora glycogen synthetase (EC 2.4.1.11) occurs in 100,000 x g particles. The two forms (glucose-6-phosphate dependent-independent) of glycogen synthetase were solubilized and separated by digitonin treatment of the 100,000 x g particles. Glucan synthetase activity of Neurospora was found only in a cell-wall preparation. These two enzymes have been characterized in relation to the paramorphogenic action of sorbose. Sorbose-grown cultures showed a marked decrease in the specific activity of both enzymes, as compared to sucrose-grown wild-type cultures. Sorbose inhibited the activity of the wild-type enzymes both in vivo and in vitro. In the presence of 5 mM sorbose incorporation of [(14)C]glucose from [(14)C]uridinediphosphate glucose into glycogen by the dependent form of glycogen synthetase was completely inhibited. Thus, the paramorphogenic action of sorbose seems to result from its inhibition of these enzymes of cell-wall biosynthesis. Activities of the enzymes from the sorbose-resistant mutant, patch, were not affected by sorbose either in vivo or in vitro.

Biochemical and genetic evidence is presented that the rg-1 and rg-2 genes control the structure of the two isozymes of phosphoglucomutase (Phosphoglucomutase I and II) in Neurospora silophila. Results of kinetic and gel filtration studies show that the two phosphoglucomutases may exist in vitro either as separate or as a single but complex molecular species depending on the ionic concentration. The complex phosphoglucomutase molecule is suggested to be the physiologically active form of the enzyme. The change in the enzyme structure is discussed in relation to the morphological change in the ragged mutant strains.