Uptake of Nitrite by Neurospora crassa

Like the nitrate transport system, the nitrite uptake system in Neurospora crassa is induced by either nitrate or nitrite. This induction is prevented by cycloheximide, puromycin, or 6-methyl purine. The K(m) for nitrite of the induced nitrite uptake system is 86 muM, and the V(max) is 100 mumol of nitrite per g (wet weight) per h. Nitrite uptake is inhibited by metabolic poisons such as arsenate, dinitrophenol, cyanide, and antimycin A. No repression or inhibition of the nitrite transport system by ammonia, nitrate, or Casamino Acids was observed.     

Isolation and characterization of plasma membranes from strains of Neurospora crassa with wild type morphology

A variety of commercially available cell wall hydrolytic enzyme preparations were screened alone and in various combinations for their ability to degrade the cell wall of Neurospora crassa wild type strain 1A. A combination was found which causes complete conversion of the normally filamentous germinated conidia to spherical structures in about 1.5 h. Examination of these spheroplasts by scanning electron microscopy indicated that, although they are spherical, they retain a smooth coat that can only be removed upon prolonged incubation in the enzyme mixture (about 10 h). The 10-h incubation in the enzyme mixture appears to have no obvious detrimental effects on the integrity of the plasma membrane since the activity and regulatory properties of the glucose active transport system in 10-h spheroplasts are essentially unimpaired. Importantly, plasma membranes can be isolated from the 10-h spheroplasts by an adaptation of the concanavalin A method developed previously in this laboratory for cells of the cell wall-less sl strain, which is not the case for the 1.5-h spheroplasts. The yield of plasma membrane vesicles isolated by this procedure is 18-36% as indicated by surface labeling with diazotized [125I]iodosulfanilic acid, and the preparation is less than 1% contaminated with mitochondrial protein. The chemical composition of the wild type plasma membranes is similar to that previously reported for membranes of the sl strain of Neurospora. The isolated wild type plasma membrane vesicles also exhibit all of the functional properties that have previously been demonstrated for the sl plasma membrane vesicles. The wild type vesicles catalyze MgATP-dependent electrogenic proton translocation as indicated by the concentrative uptake of [14C]SCN- and [14C]imidazole under the appropriate conditions, which indicates that they contain the plasma membrane H+-ATPase previously shown to exist in the sl plasma membranes and that they possess permeability barrier function as well. The vesicles also contain a Ca2+/H+ antiporter as evidenced by their ability to catalyze protonophore-inhibited MgATP-dependent 45Ca2+ accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of the isolated vesicles indicate that the protein composition of the wild type vesicles is roughly similar to that of the sl plasma membranes with the H+-ATPase present as a major band of Mr approximately 105,000. The wild type plasma membrane ATPase forms a phosphorylated intermediate similar to that of the sl ATPase, and the specific activity of the H+-ATPase in both wild type and sl membranes is approximately 3 mumol of Pi released/mg of protein/min.(ABSTRACT TRUNCATED AT 400 WORDS)     

Glutamate dehydrogenase from wild type Neurospora crassa; inactivation by photo-oxidation

The NADP dependent glutamate dehydrogenase from wild type Neurospora crassa is inactivated by exposure to light in the presence of the dye, Methylene Blue. Photo-oxidation appears to disturb the conformational equilibrium which controls the activity of this enzyme. Data obtained suggests that the modified group is the same as that reactive to the histidine reagent, diethylpyrocarbonate.     

Comparison of amino acid sequence and thermostability of tyrosinase from three wild type strains of Neurospora crassa

The thermostability of tyrosinase from three wild type strains of Neurospora crassa has been investigated. For this purpose a sequence comparison of two thermostable and one thermolabile tyrosinase isoenzyme was carried out. It revealed that at position 201 the thermostable enzyme forms share an aspartate residue in contrast to an asparagine residue in the thermolabile form. In addition, one of the thermostable isoenzymes displays five other substitutions. Since the relative stability of the thermostable forms as compared to the thermolabile one decreases with increasing ionic strength, the common aspartate residue is thought to bring about the additional stability of the thermostable isoenzymes by forming a salt bridge between aspartate 201 and a positively charged group of the protein. The strong pH-dependency of the thermostability with an apparent pKA of 6.6 indicates a histidinium side chain as the most likely ionic group to be involved in the salt bridge. This conjecture is also supported by measurements of the stability towards the chaotropic agent guanidinium chloride. The difference of the free energy change of denaturation delta GDH2O between the apoenzymes of a thermostable and a thermolabile isoenzyme was calculated as 2.5 kcal mol-1. Furthermore, it was shown that the copper ions of the native and the cobalt ions of Co(II)-substituted tyrosinase strongly enhance the stability of the protein as compared to its apoform.     

Cytology of recessive sexual-phase mutants from wild strains of Neurospora crassa.

Wild-collected strains of Neurospora crassa harbor recessive mutations that are expressed in the sexual phase when homozygous. Thirty-two representative mutants that produced barren perithecia were examined cytologically. Six of these mutants failed to form asci. Of the remaining 26, chromosome pairing was disturbed in 12 and meiosis was disturbed at pachytene or diplotene in 5. Seven mutants showed normal meiosis I but then diverged from the normal sequence, and two showed perithecial beak abnormalities. In many mutants, ascus development and nuclear divisions continued after the initial defect, albeit abnormally. Nuclear divisions were often delayed, essentially uncoupling them from other ascus events such as the formation of enlarged spindle pole body plaques, ascospore wall membranes, and spore delimitation. All 32 mutants were recessive and none showed obvious morphological abnormalities during vegetative growth. This phenotype contrasts sharply with that of numerous laboratory-induced ascus mutants, which are frequently expressed pleiotropically in the vegetative phase and several are dominant in the sexual phase.     

Electron microscopy of selectively stimulated microconidiogenesis in wild type Neurospora crassa

Summary Microconidiogenesis in Neurospora crassa wild type is selectively stimulated by iodoacetate. The ultrastructural features of various stages of microconidiogenesis have been studied. The conidiogenous cells or phialides first bear a protuberance which then erupts into a conspicuous collarette surrounding, above a lamellated cushion, the successively budded, often elongated microconidia.     

Occurrence in wild strains of Neurospora crassa of genes controlling genetic recombination.

Each of the main laboratory wild stocks of N. crassa carries one of two alleles at the rec-1 and rec-2 loci and one of three at the rec-3 locus. The constitutions of the stocks are given in Fig. 1. Some of those conserved are evidently not the originals. The third rec-3 gene (rec-3L), found in Lindegren A, controls recombination at the am-1 locus to a level between that of rec-3+ and rec-3, the relative levels being 1 : 8 : 25. At the his-2 locus rec-3L is indistinguishable from rec-3+ in its level of control. This proves that there are minor differences between the control (con) genes, near to am-1 and his-2, which recognize products of rec-3 genes. Further, this is the first clear evidence, though indirect, that the binding sites for products of rec genes are situated in the chromosome regions where recombination is modulated.     

Fine-scale mapping in Neurospora crassa by using genome-wide knockout strains

Fine-scale genetic mapping is often hindered by the lack of adequate markers surrounding the locus of interest. In the filamentous ascomycete Neurospora crassa, the genome has been sequenced and an effort has been made to generate genome-wide deletion strains for the entire gene set. Accordingly, the hygromycin-resistant marker in each deletion strain can be used as a mapping locus in a classical three-point cross, along with the mapping target and a standard marker. We have demonstrated the feasibility of this fine-scale mapping approach in N. crassa by refining the location of r(Sk-2).     

Characteristics of a glycerol utilization mutant of Neurospora crassa.

A mutant of Neurospora crassa able to grow on liquid minimal glycerol medium without evidence of conidiation and with high cell yields has been isolated and shown to be allelic to ff-1. The glycerol-specific induction of glycerokinase and glycerol-3-phosphate dehydrogenase was similar in both wild-type and mutant cells, although higher specific activities as well as higher glycerokinase cross-reacting material levels were found in fully induced mutant cells. After growth in minimal glycerol medium there is a significant reduction in wild-type cells of the activities of both pyruvate dehydrogenase and dihydrolipoyl transacetylase. This evidence indicates a relationship between the conditional acetate requirement by wild-type cells grown on glycerol medium and the levels of the pyruvate dehydrogenase complex.