Characterization of MMS-sensitive mutants of Neurospora crassa

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 resistance 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 increase (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 histadine but have normal growth rates on minimal medium. Mutant mus-(SC10), but not mus-(SC25) and mus-(SC29), has an increased (11 ×) 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 a 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.     

Isolation and mapping of fluoropyrimidine-resistant mutants of Neurospora crassa

Summary Growth characteristics of wild type Neurospora crassa on 5-fluorouracil, 5-fluorouridine and 5-fluorodeoxyuridine and methods for selecting mutants resistant to these pyrimidine analogues are described. The mutants are mapped and characterized for resistance to the analogues. Some mutants are found to be allelic with the previously described ud-1 (fdu-1) ad uc-4 genes.Supported by S.R.C. grant GR/A/64655F. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

Biochemical studies on the Nit mutants of Neurospora crassa

Summary One allele at each of the five nit loci in Neurospora crassa together with the wild type strain have been compared on various nitrogen sources with regard to (i) their growth characteristics (ii) the level of nitrate reductase and its associated activities (reduced benzyl viologen nitrate reductase and cytochrome c reductase) (iii) the level of nitrite reductase and (iv) their ability to take up nitrite from the surrounding medium. Results are consistent with the hypothesis that nit-3 is the structural gene for nitrate reductase, nit-1 specifies in part a molybdenum containing moiety which is responsible for the nit-3 gene product dimerising to form nitrate reductase, nit-4 and nit-5 are regulator genes whose products are involved in the induction of both nitrate reductase and nitrite reductase and nit-2 codes for a generalised ammonium activated repressor protein. Studies on the induction of nitrate reductase (and its associated activities) and nitrite reductase in wild type, nit-1 and nit-3 in the presence of either nitrate or nitrite suggest that each enzyme may be regulated independently of the other and that nitrite could be true co-inducer of the assimilatory pathway. Nitrite uptake experiments with nit-2, nit-4 and nit-5 strains show that whereas nit-4 and nit-5 are freely permeable to this molecule, it is unable to enter the nit-2 mycelium.     

Partial characterization of 5-fluoropyrimidine-resistant mutants of Neurospora crassa

Summary The primary lesion in a number of 5-fluoropyrimidine resistant mutants of Neurospora crassa has been identified. ud-1 mutants, previously designated fdu-2, are deficient in nucleoside uptake and show extensive intragenic complementation. uc-4 mutants lack uracil phosphoribosyl transferase with no complementation between 23 alleles. udk mutants lack uridine kinase activity. fdu-2 mutants affect the repression of the first two de novo pyrimidine biosynthetic enzymes, have no detectable uridine kinase activity and show decreased uridine uptake. Accordingly, fdu-2 may be involved in the regulation of pyrimidine uptake, salvage and de novo synthesis.Supported by S.R.C. grant GR/A/64655F. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

Uptake and efflux of sulfate in Neurospora crassa

Sulfate efflux from an intracellular pool was observed with both wild-type and cys-11 cells of Neurospora and apparently occurs by way of the sulfate transport system. Efflux requires the presence of external sulfate or the related ions, chromate, selenate, or thiosulfate, and probably occurs by an exchange reaction. The sulfur amino acids, cysteine or methionine, do not promote sulfate efflux. The K_m for efflux is much greater than the K_m for sulfate uptake, which permits the accumulation of a considerable intracellular pool before efflux becomes significant. Substantial transmembrane movement of sulfate both influx and exit, was found to occur in azidetreated cells, although the net uptake of sulfate was abolished by this inhibitor. Both sulfate uptake and efflux are inhibited by p-chloromercuribenzoate which suggests that the sulfate permease possesses an essential sulfhydryl group.     

Ribosomal DNA inheritance and recombination in Neurospora crassa

Summary The genetic segregation of ribosomal DNA (rDNA) in Neurospora crassa was analyzed by exploiting restriction fragment length polymorphisms in the nontranscribed spacer (NTS) sequences of nine laboratory wild-type strains and wild-collected strains. In an analysis of random spore progeny from seven crosses, and of ordered tetrads from two of those crosses the rDNA was shown to be inherited in a simple, stable Mendelian fashion, exhibiting an approximately 1:1 ratio of the two parental rDNA types. No meiotic recombinants were detected among the progeny, indicating that non-sister-chromatid crossing over is highly suppressed in the rDNA region. The basis for this suppression of meiotic recombination is not known.     

A ras homologue of Neurospora crassa regulates morphology

In order to study the role of signal transduction pathways in the regulation of morphology in Neurospora crassa, we cloned and characterized a ras homologue, termed NC-ras2. The predicted protein product of this gene is composed of 229 amino acid residues and contains all the consensus sequences shared by the ras protein family. The gene is located in linkage group V. An NC-ras2 disruptant showed morphological characteristics very similar to those of the smco7 mutant, which also maps to linkage group V. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in the NC-ras2 gene, which is predicted to result in the truncation of the protein product. Introduction into the smco7 mutant of an NC-ras2 clone yielded stable transformants with a wild-type phenotype. The smco7 mutant exhibited very slow hyphal growth and the rate of conidial formation was approximately one two-hundredth of wild type. The smco7 mutation causes both the changes in the pattern of hyphal growth and the defects in cell wall synthesis. Both the diameter and the length of the apical compartment were shorter in the hyphae of the smco7 mutant. These results suggest that NC-ras2 is identical to smco7, and that the signal transduction pathway mediated by the NC-ras2 protein regulates the apical growth of hyphae, cell wall synthesis, and conidial formation in N. crassa. Received: 1 October 1996 / Accepted: 9 December 1996     

Regulation of synthesis of ribosomal protein in Neurospora crassa

In Neurospora crassa during a nutritional shift-down transition of growth, the synthesis of rRNA is for about 2 h largely inhibited and the rate of protein synthesis is only partially reduced (by about 25 %). During this period the relative rate of synthesis of individual ribosomal proteins, measured irrespectively of their incorporation into ribosomes, is reduced by 70–80%. The ribosomal proteins synthesized during the shift are stable. Thus, the synthesis of ribosomal proteins appears in N. crassa to be coordinately regulated with that of rRNA.     

Gene order in the qa gene cluster of Neurospora crassa

Summary Four different types of crosses have been used to establish the order of the four genes in the qa gene cluster of Neurospora crassa, which encode the following proteins involved in the inducible catabolism of quinic acid: a regulatory (activator) protein (qa-1), catabolic dehydroquinase (qa-2), quinate dehydrogenase (qa-3), and dehydroshikimate dehydrase (qa-4). The four crosses involved (1) the ordering of the four qa genes relative to the closely-linked me-7 locus; (2) the ordering of the three other qa genes relative to a qa-1 ^S mutant; (3) the use of a three factor cross-qa-3xqa-4 qa-2 and (4) the use of four factor crosses-qa-1 ^S xqa-3 qa-4 qa-2. The results of all four types of crosses agree in establishing an apparently definitive proximal to distal order, within the right arm of linkage group VII, i.e., qa-1 qa-3 qa-4 qa-2 me-7.The significance of a definitive establisment of the gene order within the qa cluster for an understanding of the organization and mechanism of genetic regulation in this cluster is discussed.     

In vitro restoration of nitrate reductase: Investigation of Aspergillus nidulans and Neurospora crassa nitrate reductase mutants

Extracts of Aspergillus nidulans wild type (bi-1) and the nitrate reductase mutant niaD-17 were active in the in vitro restoration of NADPH-dependent nitrate reductase when mixed with extracts of Neurospora crassa, nit-1. Among the A. nidulans cnx nitrate reductase mutants tested, only the molybdenum repair mutant, cnxE-14 grown in the presence of 10⁻³ M Na₂MoO₄ was active in the restoration assay.Aspergillus extracts contained an inhibitor(s) which was measured by the decrease in NADPH-dependent nitrate reductase formed when extracts of Rhodospirillum rubrum and N. crassa, nit-1 were incubated at room temperature. The inhibition by extracts of A. nidulans, bi-1, cnxG-4 and cnxH-3 was a linear function of time and a logarithmic function of the protein concentration in the extract.The molybdenum content of N. crassa wild type and nit-1 mycelia were found to be similar, containing approx. 10 μg molybdenum/mg dry mycelium. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The enzyme purified from wild-type N. crassa contained more than 1 mol of molybdenum per mol of enzyme, whereas the enzyme purified from nit-1 contained negligible amounts of molybdenum.     

An efficient method for gene disruption in Neurospora crassa

The frequency with which transforming DNA undergoes homologous recombination at a chromosomal site can be quite low in some fungal systems. In such cases, strategies for gene disruption or gene replacement must either select against ectopic integration events or provide easy screening to identify homologous site, double-crossover insertion events. A protocol is presented for efficient isolation of Neurospora crassa strains carrying a definitive null allele in a target gene. The protocol relies on the presence of a selectable marker flanking a disrupted plasmid-borne copy of the gene, and in the case presented led to a seven-fold enrichment for putative homologous site replacement events. In addition, a polymerase chain reaction assay is utilized for rapid identification of homologous recombinants among the remaining candidates. This protocol was used to identify 3 isolates, out of 129 primary transformants, which have a disruption in the Neurospora ccg-1 gene. The method should be applicable to a variety of fungal systems in which two selectable markers can be expressed, including those in which homologous recombination rates are too low to allow easy identification of homologous site insertions by the more traditional molecular method of Southern analysis. In addition to disrupting target genes for the purpose of generating null mutations, this method is useful for the targeting of reporter gene fusions to a native chromosomal site for the purpose of studying gene regulation.     

Analysis of a case of mutagen specificity in Neurospora crassa

Summary When ultraviolet irradiation of doubly auxotrophic conidia was preceded or followed by weakly mutagenic doses of DEB, the frequency of adenine-reversions was increased above additivity, while that of inositol-reversions was additive or—usually—was decreased below additivity. These interactions did not affect completed revertants nor were they due to plating interactions between potential revertants and the non-mutant background cells. The interaction was stronger when DEB was given as pretreatment than when it was given as post-treatment. During the DEB-treatment, sensitivity to interaction increased from the low effect observed with post-treatment to the higher one typical for pretreatment. Irradiation towards the end of the treatment period gave the same interaction as irradiation of treated and washed cells. In post-treatment experiments, the irradiated cells retained their capacity for interaction with DEB undiminished for at least on hour. In pretreatment experiments, the washed cells retained their capacity for interaction with UV over at least 16 minutes. After 2 hours, interaction was diminished; after 4 hours, it had disappeared.These results suggest a number of conclusions. (a) Interaction is mainly or wholly due to the effect of DEB on UV-induced mutations. (b) Interaction does not occur at the level of the primary lesions in DNA but at some later step in mutagenesis. (c) The mechanism of interaction is not the same for the two types of reversion. (d) The enhanced frequency of adenine-reversions is possibly due to inhibition of a repair enzyme by DEB. (e) The decreased frequency of inositol-reversions does not appear to be due to inositol-less death, but does seem connected with some specific phenotypic feature of inositol-reversions.     

An inducible acetate transport system in Neurospora crassa conidia

Neurospora crassa conidia possess an active transport system for the uptake of acetate. This system was characterized as: (a) energy dependent; (b) taking place against a concentration gradient; (c) saturating at higher substrate concentrations and (d) competitively inhibited by propionate.Activity of the acetate transport system can be further enhanced by preincubating conidia in 1 mM acetate medium for 180 min (the inducible transport system). The conidial system and the inducible system have similar properties. The development of the inducible transport was dependent on RNA and protein synthesis. A genetic control of this system was further confirmed by isolating a mutant (acp⁻ⁱ acetate permease, inducible) that fails to develop the inducible transport system.