Mutagenic activity of ethylenimine in Neurospora crassa

The mutagenic activity of the monofunctional alkylating agent ethylenimine (EI) was tested with the adenine-3 (ad-3) system in a two-component heterokaryon of Neurospora crassa. The results of forward-mutation experiments showed that EI is a potent mutagen in N. crassa.Genetic analysis of EI-induced ad-3 mutants showed that the frequencies of leakiness, allelic complementation, and non-polarized complementation patterns are similar to those of ad-3 mutants induced by other alkylating agents. It seems, therefore, that in addition to multilocus deletions (which occur at low a frequency), EI-induced mutations probably include base-pair substitutions, frameshift mutations, and other types of intragenic alterations.     

Two forms of a mitochondrial endonuclease in Neurospora crassa.

Mitochondrial nuclease activity in Neurospora crassa occurs in membrane-bound and soluble forms in approximately equal proportions. These activities apparently are due to the same enzyme, which has an approximate molecular weight of 120 000. A portion of the insoluble enzyme appears to be associated with the inner mitochondrial membrane and is resistant to solubilization by detergent treatment as well as by physical disruption methods.     

Soluble adenylate cyclase activity in Neurospora crassa.

A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin.     

Microtubule dynamics and the role of molecular motors in Neurospora crassa

Live-cell imaging methods were used to study microtubule dynamics in the apical regions of leading hyphae and germ tubes of Neurospora crassa expressing beta-tubulin-GFP. Microtubule polymerization rates in hyphae of N. crassa were much faster than those previously reported in any other eukaryotic organism. In order to address the roles of motor proteins in microtubule dynamic instability in N. crassa, the microtubule-motor mutant strains, Deltankin and ro-1, were examined. Polymerization and depolymerization rates in leading hyphae of these strains were reduced by one half relative to the wild type. Furthermore, microtubules in germ tubes of wild type and microtubule-motor mutants exhibited similar dynamic characteristics as those in hyphae of mutant strains. Small microtubule fragments exhibiting anterograde and retrograde motility were present in leading hyphae of all strains and germ tubes of wild-type strains. Our data suggest that microtubule motors play important roles in regulating microtubule dynamic instability in leading hyphae but not in germ tubes.     

Amino acid substitutions in mutant forms of histidinol dehydrogenase from Neurospora crassa

Amino acid changes in the enzyme l-histidinol dehydrogenase (l-histidinol–NAD oxidoreductase, EC 1.1.1.23) have been determined between the wild-type Neurospora crassa and two temperature-sensitive mutants. Comparison was made between amino acid analyses of peptides of differing electrophoretic and chromatographic mobilities resulting from tryptic and chymotryptic digestion of protein from wild-type and mutant K26, and wild-type and mutant K445 strains, respectively. The analyses demonstrate the substitution of aspartic acid for alanine in mutant K26, and leucine for histidine in mutant K445. The effects of the resulting changes in polarity and charge are discussed in relation to the catalytic functioning of the proteins.     

Relationship between two major immunoreactive forms of arginase in Neurospora crassa.

Two major immunoreactive proteins of Mr 41,700 and 36,100 have been detected in crude mycelial extracts with polyclonal antibodies raised against arginase purified from Neurospora crassa. The latter corresponded to the protein used to obtain the antibodies. Both polypeptides were either missing or present in very low amounts in mutant strains having little or no detectable arginase activity. The relative proportion of the two species was altered in strains containing the nitrogen catabolite regulatory mutation nit-2. Peptide mapping indicated that the two species are very closely related, but several of the peptides which appeared to be identical by staining reacted differently with the antibodies. Both species were produced by in vitro translation of poly(A)+ mRNA, although the larger species was produced to a much smaller extent than was expected from its abundance in vivo. The results suggest the existence of multiple forms of arginase in N. crassa which differ in their response to nitrogen catabolite regulation.     

Neurospora crassa mitochondria contain two forms of a 4'-phosphopantetheine-modified protein

When Neurospora crassa was labeled with [14C]pantothenic acid during growth, the mitochondrial fraction contained two bands of radioactivity of Mr 19,000 and 22,000 by sodium dodecyl sulfate gel electrophoresis. The 19-kDa band was converted to the 22-kDa band by four treatments which are characteristic of the cleavage of a thioester bond: dithiothreitol and 2-mercaptoethanol at basic but not neutral pH, alkaline methanolysis, sodium borohydride in tetrahydrofuran, and hydroxylamine at neutral pH. Mitochondrial subfractionation indicated that the 22-kDa form was preferentially associated with the soluble fraction while the 19-kDa form was found in all fractions. Several properties of the mitochondrial protein were similar to the Escherichia coli acyl carrier protein: Mr on sodium dodecyl sulfate gels, decreased electrophoretic mobility under deacylating conditions, isoelectric point, and covalent attachment of 4'-phosphopantetheine. The 19- and 22-kDa bands may therefore represent acylated and deacylated forms of a mitochondrial acyl carrier protein.     

Microtubule dynamics and the role of molecular motors in Neurospora crassa.

Live-cell imaging methods were used to study microtubule dynamics in the apical regions of leading hyphae and germ tubes of Neurospora crassa expressing beta-tubulin-GFP. Microtubule polymerization rates in hyphae of N. crassa were much faster than those previously reported in any other eukaryotic organism. In order to address the roles of motor proteins in microtubule dynamic instability in N. crassa, the microtubule-motor mutant strains, Deltankin and ro-1, were examined. Polymerization and depolymerization rates in leading hyphae of these strains were reduced by one half relative to the wild type. Furthermore, microtubules in germ tubes of wild type and microtubule-motor mutants exhibited similar dynamic characteristics as those in hyphae of mutant strains. Small microtubule fragments exhibiting anterograde and retrograde motility were present in leading hyphae of all strains and germ tubes of wild-type strains. Our data suggest that microtubule motors play important roles in regulating microtubule dynamic instability in leading hyphae but not in germ tubes.     

Conidiation in Neurospora crassa

Summary Conidiation in Neurospora crassa has been studied in vivo by time-lapse microphotography and shown to be most generally (in aerial, dry conditions) a budding-fission process. Such a two-phase process is characterized by an initial basifugal budding of proconidial elements which are then secondarily separated as maturing conidia by interconidial septa. Dry macroconidia of Neurospora are thus blasto-arthrospores, i.e. blastospores basifugally budded on conidiophores and secondarily disarticulated from the proconidial chain as arthrosporal elements. Inception and median splitting of the interconidial septum have been electron microphotographed.In the vegetative hyphae, ethanol dehydrogenase has been cytochemically detected by oxidative assay and demonstrates a dense, uniform distribution of activity except at the hyphal tips. In the conidiating hyphae, the ethanol dehydro-genase becomes less dense in distribution, especially in the budding apices. Cytochrome oxidase activity, localized in the mitochondria, is confined in the subapical zone of vegetative hyphae while at the initiation of conidiation it becomes dispersed throughout the proconidial buds.     

Photoperiodism in Neurospora crassa

Plants and animals use day or night length for seasonal control of reproduction and other biological functions. Overwhelming evidence suggests that this photoperiodic mechanism relies on a functional circadian system. Recent progress has defined how flowering time in plants is regulated by photoperiodic control of output pathways, but the underlying mechanisms of photoperiodism remain to be described. The authors investigate photoperiodism in a genetic model system for circadian rhythms research, Neurospora crassa. They find that both propagation and reproduction respond systematically to photoperiod. Furthermore, a nonreproductive light-regulated function is also enhanced under certain photoperiodic conditions. All of these photoperiodic responses require a functional circadian clock, in that they are absent in a clock mutant. Night break experiments show that measuring night length is one of the mechanisms used for photoperiod assessment. This represents the first formal report of photoperiodism in the fungi.     

Changes in enzyme activity of germinating conidia of Neurospora crassa

The conidia of wild-type Neurospora crassa are shown to have a drastically lower activity for three enzymes of the isoleucine-valine pathway—acetohydroxy acid synthetase, dihydroxy acid dehydratase, and aminotransferase—than the actively growing mycelium. Lower activity was also found in the conidia for ornithine transcarbamylase and aspartate transcarbamylase. Lower activity (10- to 100-fold) was found for the overall synthesis of valine from pyruvate in the conidia as compared to the mycelium as expected.In addition it is also apparent that the distribution of the isoleucine-valine enzymes is different in conidia from the mycelium as regards activity in the mitochondria as compared to the cytosol. In conidia their activity in the mitochondria is lower than in the cytosol, but the opposite holds in the mycelium. These differences are also reflected in the overall activity.Cycloheximide inhibits the increase in total activity of the acetohydroxy acid synthetase and the dehydratase during germination of the conidia.