A “Stopper” mutant of Neurospora crassa containing two populations of aberrant mitochondrial DNA

Summary [E35], an extranuclear mutant of Neurospora crassa has all the phenotypic characteristics of the stopper mutants (De Vries et al. 1980). In the present work, the mitochondrial DNA as well as the mitochondrial translation products are characterized further. The primary mutational event appears to have been the deletion of about 4 kbp from the wild-type genome. Moreover, after prolonged vegetative growth the mutant accumulates an 8-m circular mtDNA, which was demonstrated both by electronmicroscopy and by restriction enzyme analysis. Hence, the mutant contains two populations of aberrant mitochondrial DNA, the smaller of which is an amplification of the rRNA-tRNA part of the larger. We propose that the primary deletion has generated a signal in the larger DNA which can cause premature termination of replication at the deletion site, and subsequent circularization of the unfinished daughter molecule. Finally, the deleted part may contain a determinant for synthesis of a protein of 11 kDal. The function of this protein, which is not a subunit of the F₀ ATPase, is not yet known.Abbreviations (k)bp (kilo)basepairs - kDal kilodalton - mt mitochondrial     

The nucleotide excision repair epistasis group in Neurospora crassa

Summary DNA repair mutants in eucaryotes are normally assigned to three epistasis groups. Each epistasis group represents a pathway for DNA repair. The pathways are commonly designated (1) nucleotide excision repair, (2) recombination repair and (3) mutagenic repair. An excision repair epistasis group has been established in Neurospora and the mutants assigned to this group should be limited in their ability to excise pyrimidine dimers and other bulky lesions from DNA. Using a pyrimidine dimer-specific assay, we have found that all Neurospora crassa mutants assigned to the excision repair epistasis group are capable of removing pyrimidine dimers from the DNA at a rate similar to the wild-type organism.     

Bialaphos resistance as a dominant selectable marker in Neurospora crassa

Summary Conidia of Neurospora crassa are sensitive to the herbicide bialaphos at concentrations of 160 mg/1 in Westergaard's or Fries' minimal media. Plasmid pJA4 was constructed by inserting a truncated bar gene from Streptomyces hygroscopicus fused to the his-3 promoter from N. crassa into pUC19. The bar gene in plasmid pJA4 confers resistance to bialaphos when transformants are selected on a medium containing bialaphos. The bar gene can be used as an additional dominant selectable marker for transformation of fungi.     

Cloning and sequence analysis of the glucoamylase gene of Neurospora crassa

A 1.0-kb DNA fragment, corresponding to an internal region of the Neurospora crassa glucoamylase gene, gla-1, was generated from genomic DNA by the polymerase chain reaction, using oligonucleotide primers which had been deduced from the known N-terminal amino-acid sequence or from consensus regions within the aligned amino-acid sequences of other fungal glucoamylases. The fragment was used to screen an N. crassa genomic DNA library. One clone contained the gene together with flanking regions and its sequence was determined. The gene was found to code for a preproprotein of 626 amino acids, 35 of which constitute a signal and propeptide region. The protein and the gene are compared with corresponding sequences in other fungi.     

Generation of new functional mutant alleles by premeiotic disruption of the Neurospora crassa am gene

Summary In the further analysis of a cross in which the mis-sense allele, am ³, of the Neurospora crassa am (glutamate dehydrogenase) gene was present in one parent together with two ectopic wild-type gene copies, one ascus was identified in which the two ectopic copies had been inactivated by the RIP process whereas the am ³ allele continued to produce its characteristic enzyme variety in active, but heat-sensitive, form. The am ³ allele had also acquired a new HindIII restriction site. It had no detectable methylation. The mutations responsible respectively for the new restriction site and the modified enzyme properties were separated from each other, and from the original am ³ mutation, by selecting for intragenic recombination on either side of the am ³ site. In this way two new effectively wild-type alleles were generated, one characterised by its heat-sensitive and kinetically modified enzyme product and the other by a new HindIII site. These results demonstrate that the RIP phenomenon can be a source of new functional alleles.     

Early response and induced tolerance to cycloheximide in Neurospora crassa

Summary Incubation of Neurospora crassa mycelia with low doses of cycloheximide induces the expression of several genes. After 6 h in the presence of cycloheximide, mycelia become tolerant to further additions of the drug and the rate of protein synthesis exhibits a lower sensitivity to it. The polypeptide pattern is indicative of a stress situation.     

The function and specificity of the C-terminal tripeptide glyoxysomal targeting signal in Neurospora crassa

The function of the C-terminal tripeptide targeting signal responsible for microbody targeting in many eukaryotes has been investigated in the filamentous fugus Neurospora crassa. Using an in-vivo targeting assay that employs transformants carrying C-terminally-modified versions of the bacterial enzyme chloramphenicol acetyltransferase (CAT), it has been demonstrated that C-terminal tripeptide-dependent import occurs most efficiently in response to nutritional acetate-induction. Under these conditions Neurospora generates a specialized organelle, the glyoxysome, which carries the enzymes responsible for the glyoxylate cycle and can be distinguished from peroxisome-like microbodies that contain catalase. Moreover, several C-terminal peptides have been tested in this system to extend the tripeptide targeting consensus to A/C/G/S-H/K/Q/R-I/L/V. However, the tripeptide analogue, ARM, found at the C-terminus of the glyoxylate cycle enzyme isocitrate lyase in higher plants, does not apparently function here.     

Molecular analysis of the mitochondrial genome of Phytophthoraa

Summary The organization of the mitochondrial genomes from two morphologically similar Phytophthora isolates, P. megasperma f. sp. glycinea (Pmg) and P. megasperma f. sp. medicaginis (Pmm), and the morphologically different species, P. parasitica var. nicotianae (Ppn), has been studied. The mtDNAs are circular, and their estimated sizes are 45.3 kb, 41 kb, and 39.5 kb for Pmg, Pmm, and Ppn, respectively. Physical maps were constructed for restriction endonuclease sites. Four genes (l-rRNA, s-rRNA, oxi-2, and cob) were found to have the same order in the three mtDNAs.     

Premeiotic disruption of duplicated and triplicated copies of the Neurospora crassa am (glutamate dehydrogenase) gene

Summary Premeiotic inactivation of duplicated sequences (the RIP phenomenon of Selker et al.) was studied by tetrad analysis using ectopic copies of am ⁺ (coding for NADP-specific glutamate dehydrogenase) and a missense allele am ³, coding for a distinctive form of the enzyme, at the normal locus. In duplication crosses either both gene copies were inactivated or neither. Two inactivated am ³ derivatives were shown to have undergone methylation and numerous base-pair changes, reflected in losses and gains of restriction sites, but without sequence rearrangement. Cutting at restriction sites within the disrupted sequences was incomplete but became almost complete following growth in the presence of 5-azacytidine. In a triplication cross in which one parent carried two unlinked ectopic gene copies together with am ³ at the normal locus, premeiotic inactivation, when it occurred, tended to affect two of the three copies in any one ascus, but there were a few asci in which all three were inactivated.     

Amber nonsense mutations in regulatory and structural genes of the nitrogen control circuit of Neurospora crassa

Summary Neurospora crassa possesses a set of nitrogen-regulated enzymes whose expression requires a lifting of nitrogen catabolite repression and specific induction. The nit-2 gene is a major regulatory locus which appears to act in a positive way to turn on the expression of these nitrogen-related enzymes whereas the nit-4 gene appears to mediate nitrate induction of nitrate and nitrite reductase. The nit-3 gene specifies nitrate reductase and is subject to control by both nit-2 and nit-4. Many new nit-2, nit-3, and nit-4 mutants were isolated in order to obtain amber nonsense mutations in these loci which were suppressible by the suppressor gene, Ssu-1. A nit-2 nonsense mutant was isolated which has altered regulatory properties for control of nitrate reductase, L-amino acid oxidase, and uricase, and which may encode a truncated regulatory protein. Four nit-3 nonsense mutations were isolated, each of which completely lacks nitrate reductase activity, which is restored to markedly different levels by suppression with Ssu-1. Studies of heat activation and thermal lability of nitrate reductase suggest a qualitative alteration of the enzyme occurs in two of the Ssu-1 nit-3 strains.     

Characterization of inl + transformants of Neurospora crassa obtained with a recombinant cosmid-pool

Summary We constructed a Neurospora crassa gene library in a cosmid vector and used the cosmid-pool DNA to transform an inl, rg Neurospora crassa strain to inositol prototrophy. The inl ⁺ colonies obtained in this experiment proved to be integrative type transformants. Genetic analysis revealed that the integration event occurred at or near the inl locus. In one of the transformants the inl ⁺ trait exhibited mitotic and meiotic instability. In hybridization experiments free plasmids were detected in the F₁ progeny of the transformants. We were able to recover eleven different plasmids from the F₁ progeny of the transformants. None of these plasmids proved to carry a functional copy of the inl ⁺ gene as judged by its transforming ability. Possible explanations for the observed phenomena are discussed.     

Cyclic AMP dependent,constitutive thermotolerance in the adenylate cyclase-deficient cr-1 (crisp) mutant of Neurospora crassa

Summary We investigated the heat shock response of the adenylate cyclase deficient mutant cr-1 (crisp) of Neurospora crassa. This strain was observed to be much more resistant to a lethal temperature of 50 °C than the wild type. This constitutive thermotolerance was absent in cr-1 conidiospores raised on cyclic AMP (cAMP, 2.5 mM) supplemented solid medium, but was partially restored when the conidiospores were germinated at 30 °C, a temperature which fails to induce thermotolerance in the wild-type strain. Two other crisp-like Neurospora mutants, cr-2 and cr-3 which, in contrast to cr-1, contain normal levels of cAMP, did not exhibit the thermotolerance phenomenon observed for cr-1. A cr-1, pe, fl (crisp-microconidial) strain also lacked the ability to tolerate a lethal heat treatment. Our results demonstrate that the adenylate cyclase deficient cr-1 mutant of Neu-rospora crassa expresses a constitutive thermotolerant phenotype as a consequence of its primary genetic defect: low levels of cAMP.     

Generation of new mutants of nmr,the negative-acting nitrogen regulatory gene of Neurospora crassa,by repeat induced mutation

Summary The repeat induced point mutation (RIP) phenomenon has been used to generate new mutants of nmr, the negative nitrogen regulatory gene in Neurospora crassa. The wild-type nmr gene was cotransformed along with the hygromycin B resistance gene into wild-type cells by selecting for hygromycin B resistance. Following purification of primary transformants using microconidia, crosses to wild-type. Detailed analyses of some of the progeny revealed that we had generated authentic nmr mutants at high frequency. The polymerase chain reaction was used to amplify and clone a fragment of a mutagenized nmr copy from one of the mutants. The nucleotide sequence analysis showed that 14% of the guanine residues have been converted into adenines, resulting in numerous missense and nonsense mutations. The newly created nmr mutants were found suitable for use as host strains in transformation experiments.     

Pleiotropic and differential phenotypic expression of two sn (snowflake) mutant alleles of Neurospora crassa: analysis in homokaryotic and heterokaryotic cells

Mutations sn (snowflake) JL301 and C136, in the centromere region of linkage group I in Neurospora crassa, are at 0.6–3.0 map units to the left of the os-4 locus. Strains carrying snJL301 produce very short aerial hyphae and only arthroconidia, and do not grow in high salt media. snC136 strains produce aerial hyphae, with abnormally large and rounded blastoconidia, at the top of the agar slant cultures, and revert to wild-type growth in high salt media. Studies with forced primary heterokaryons indicate that snJL301 is recessive while snC136 is a semi-dominant and gene-dose dependent allele, with respect to the wild-type. Taken together the results show that: (1) the sn mutations are allelic with a differential pleiotropic phenotype, and (2) snC136 may code for a partially functioning gene product while sn-JL301 appears to be a null allele.