Isolation of a new keto-acid, produced from epsilon-N-trimethyllysine by Neurospora crassa]

A new epsilon-N-trimethyllysine metabolite has been isolated from the mycelium of Neurospora crassa. The labelled compound produced from incubations in vivo and in vitro from epsilon-N-trimethyl ([14-C]H3)L-lysine has been identified as 2-keto-epsilon-N-trimethyl-hexanoic acid by reducing its 2,4-dinitrophenyl hydrazone back to epsilon-N-trimethyllysine by hydrogenolysis in a Parr bomb. Analyses on TLC and in four different ion exchange chromatographic systems show the appearance of a ninhydrin positive product having the same Rf and the same retention time as epsilon-N-trimethyllysine; it contains more than 85% of the radioactivity of the 2,4-dinitrophenylhydrazone of the keto acid.     

Isolation of Neurospora crassa bradytrophs.

A method was developed for the isolation of Neurospora bradytrophs. The bradytrophs (representing lesions in a number of pathways) were resistant to DL-p-fluorophenylalanine when growing in a leaky fashion but were sensitive when grown in the presence of their stimulating supplement.     

Isolation and characterization of a laccase-derepressed mutant of Neurospora crassa.

Laccase from the ascomycete Neurospora crassa is an inducible secretory enzyme. Production of this enzyme is repressed in vegetative cultures but can be induced by treatment with low concentrations of cycloheximide. Isolation and characterization of a derepressed mutant, the lah-1 mutant, that is capable of producing laccase in vegetative cultures without induction by cycloheximide are described. The lah-1 mutation is mapped between nit-2 and leu-3 on linkage group I, and it behaved as a recessive mutation in a forced heterokaryon. No differences were detected biochemically or immunologically between the laccase protein produced by the lah-1 mutant in the absence of cycloheximide and that induced with cycloheximide in the wild-type strain. This suggests that both laccases (66 kilodaltons) are products of the same structural gene. Relative amounts of laccase in the culture filtrate of the lah-1 mutant were much higher than those induced with cycloheximide in the wild-type strain, demonstrating high efficiency of the lah-1 mutant in production and secretion of laccase. The time course of laccase production by the lah-1 mutant revealed that expression of 66-kilodalton laccase was repressed in conidia and derepressed during vegetative mycelial growth. This suggests that a multiple regulatory mechanism is involved in the production and/or maturation of Neurospora laccase. The lah-1 mutant may be useful for identifying genes that regulate expression of the laccase gene in N. crassa.     

Isolation of putrescine-requiring mutants of Neurospora crassa

Two auxotrophs of Neurospora crassa have been isolated that give a positive growth response to putrescine, spermidine or spermine. One of the mutants is deficient in ornithine decarboxylase activity and has been designated put-1. Both mutants map on linkage group VR, fail to complement and are infertile when crossed to one another, indicating that they are probably alleles. A putrescine auxotroph is incapable of suppressing a pro-4 mutant. The isolation of the mutants confirms that putrescine is an essential factor for the normal growth of the organism, and is synthesized via a single pathway in Neurospora.     

Isolation, Mapping, and Characterization of Trehalaseless Mutants of Neurospora crassa

Mutant strains of Neurospora crassa that lack trehalase and are unable to grow on trehalose were isolated, and the gene (tre) was positioned on the right arm of linkage group I. Maltase and beta-galactosidase activities are almost identical in tre(-) strains, whereas that of invertase was reduced by more than half and those of acid phosphatase and amylase were somewhat increased. Heterocaryons between standard and trehalaseless strains yield less than one-tenth the activity of the former. In addition, strains with duplications heterozygous for trehalase produce less than 1% of the activity of the standard strain. An inhibitor of trehalase has been found in tre(-) strains; its sensitivity to heat and proteolysis, and its nondialyzability suggest that this substance is a protein. The mig gene, which determines the rate of migration of trehalase on acrylamide gels, has been shown to be less than 1 map unit away from the tre gene.     

Isolation and characterization of the Neurospora crassa endoplasmic reticulum

The endoplasmic reticulum from Neurospora crassa was identified by monitoring the activity of the putative enzyme marker phosphatidylcholine glyceride transferase. After differential centrifugation of a cell homogenate, phosphatidylcholine glyceride transferase activity initially copurified with plasma membrane H+-ATPase. However, isopycnic centrifugation of the whole-cell homogenate on a linear sucrose gradient separated the two enzyme activities into different fractions. The lighter membrane fraction exhibited characteristics that have been associated with the endoplasmic reticulum in other organisms: (i) the inclusion of magnesium caused this light membrane fraction to shift to a higher density on the gradient; (ii) it was highly enriched in cytochrome c reductase, an endoplasmic reticulum marker in other systems; and (iii) the morphology of the light fraction with and without added magnesium was clearly distinguishable from that of the plasma membrane fraction by electron microscopy. A reinvestigation of the location of chitin synthetase confirmed its association with the plasma membrane fraction even after separation of the lighter fractions.     

Isolation and characterization of Neurospora crassa plasma membranes.

The isolation and characterization of plasma membranes from a cell wall-less mutant of Neurospora crassa are described. The plasma membranes are stabilized against fragmentation and vesiculation by treatment of intact cells with concanavalin A just prior to lysis. After lysis, the concanavalin A-stabilized plasma membrane ghosts are isolated by low speed centrifugation techniques and the purified ghosts subsequently converted to vesicles by removal of the bulk of the concanavalin A. The yield of ghosts is about 50% whereas the yield of vesicles is about 20%. The isolated plasma membrane vesicles have a characteristically high sterol to phospholipid ratio, Mg2+-dependent ATPase activity and (Na+ plus K+)-stimulated Mg2+ATPase activity. Only traces of succinate dehydrogenase and 5'-nucleotidase are present in the plasma membrane preparations.     

Isolation and Characterization of Low-Kynureninase Mutants of Neurospora crassa

Two kynureninase activities are known in Neurospora crassa, one of which (kynureninase I) is inducible, the other (kynureninase II) being constitutive. A method is described for the isolation of low-kynureninase mutants of N. crassa. When grown on an inducer, the mutants show significantly less kynureninase I activity compared with wild type, whereas constitutive kynureninase II activity is unaffected. Since a low level of kynureninase I activity remains in the mutants examined, the mutations may be in a regulatory gene or genes. Other experiments are described concerning the molecular weights of the two enzymes and the intracellular localization and specificity of kynureninase II.     

Isolation and characterization of cadmium-resistant mutants of Neurospora crassa

This study identified and characterized four cadmium-resistant mutants of Neurospora crassa. One of these mutants maps to linkage group II and the other three map to linkage group VII, whereas a naturally occurring resistant trait in a strain from Japan resides at a distinct but unmapped locus. Transport of cadmium into Neurospora cells occurs by more than a single uptake system and involves both energy-dependent and -independent components. The resistant mutants transport cadmium in the same manner as does the cadmium-sensitive wild-type strain. Cadmium resistance in these mutants does not appear to result from an increase in cytosolic heat-stable cadmium-binding proteins. Cadmium does not induce the typical heat-shock response in conidia. Under various growth conditions, each of the mutants exhibited morphological alterations, possibly involving the cell wall or plasma membrane.     

Isolation and characterization of guanine auxotrophs in Neurospora crassa

An ad-9 strain of Neurospora crassa was mutagenized with ethylmethane sulfonate (5%) and selected for guanine auxotrophy. The resultant double adenine plus guanine mutant was backcrossed with wild type and a single guanine auxotroph was isolated from the progeny. In vitro assays indicated that the mutant had GMP synthetase activity comparable with wild type, but was completely lacking of IMP dehydrogenase activity. The guanine requirement can therefore be explained by the mutant's inability to convert IMP to XMP. Another guanosine auxotroph was able to adapt and grow on minimal medium after 3 days. This mutant had GMP synthetase activity comparable with wild type but had only 10% of the IMP dehydrogenase activity of wild type, which may possibly explain its ability to grow on minimal medium after 3 days. It was confirmed that the two isolates are not allelic by crossing the two and recovering 25% wild-type progeny. Out isolate must therefore be designated gua-2.     

Isolation and properties of tripolyphosphatase from Neurospora crassa]

Homogenous preparation of tripolyphosphatase from Neurospora crassa is obtained. The enzyme is found to consist of two equal subunits with molecular weight of 40 000 and to have pH optimum 7.0 and temperature optimum 50 degrees C. Bivalent metal ions are required for its catalytical activity, the hest activators being Co2+, Mg2+ and Mn2+. Strict specificity of the enzyme to tripolyphosphate is demonstrated, Km being 5.9-10(-4) M. The enzyme hydrolyses tripolyphosphate to equimolar mixture of ortho- and pyrophosphate. The enzyme activity depends on orthophosphate and pyrophosphate concentrations in the incubation medium.     

Isolation and characterization of nuclei from Neurospora crassa.

A procedure was developed for isolating nuclei from either the conidial or germinated conidial growth phase of Neurospora crassa. A frozen conidial suspension was lysed by passage through a French pressure cell, and the nuclei were freed from the broken cells by repeated homogenization in an Omni-Mixer. Pure nuclei were obtained from the crude nuclear fraction by density banding in a Ludox gradient. The final nuclear yield was 20 to 30%. The nuclei had a deoxyribonucleic acid (DNA):ribonucleic acid (RNA):protein ratio of 1:3.5:7 and were active in RNA synthesis. The nuclei, stained with the DNA stain 4,6-diamidino-2-phenylindole, appeared under fluorescence microscopy as bright blue spheres, 1 micron in diameter, essentially free from cytoplasmic attachments. Chromatin extracted from the nuclei in a 70 to 75% yield by dissociation with 2 M sodium chloride and 5 M urea had a DNA:RNA:protein ratio of 1:1.05:1.7. Chromatin reconstituted from this preparation exhibited a level of RNA polymerase template activity lower than that of pure Neurospora DNA, but the maximum level of reconstitution obtained was only 10%. Fractionation of Neurospora chromatin on hydroxylapatite separated the histones from the chromatin acidic proteins. The normal complement of histone proteins was present in both the reconstituted and dissociated chromatin preparations. The acidic protein fraction exhibited a variety of bands on sodium dodecyl sulfate gel electrophoresis ranging in molecular weight from 15,000 to 70,000. The gel pattern was much more complex for total dissociated chromatin than for reconstituted chromatin.     

Isolation and characterization of MMS-sensitive mutants of Neurospora crassa

Seven different mutants that show high sensitivity to MMS killing were isolated and mapped at different loci. One group, mms-(SA1), mms-(SA2) and mms-(SA6), showed high sensitivity to MMS but not to UV or gamma-rays. Another group, mms-(SA4) and mms-(SA5), showed extremely high sensitivity to UV and MMS. And mms-(SA3) and mms-(SA7) were moderately sensitive to both UV and MMS. Mms-(SA4) and mms-(SA1) were identified as alleles of uvs-2 and mus-7, respectively, which had been previously isolated. The mms-(SA1), mms-(SA6) and mms-(SA7) strains were barren in homozygous crosses, and the mms-(SA5) strain was barren in heterozygous crosses. The mms-(SA1), mms-(SA3) and mms-(SA5) strains showed high sensitivity to histidine. In summary, at least two new loci involved in the repair of MMS damage have been identified. The possibility that some of these new mutants are in new repair pathways is suggested.     

Calcium as a branching signal in Neurospora crassa

The divalent cation ionophore A23187 was found to induce apical branching in Neurospora crassa. Optimal effects were obtained by treatment with 0.1 mM ionophore for 30 min. Branching first became manifest during or shortly after treatment; successive rounds of branching could be observed at later times. Calcium starvation of the mycelium markedly reduced its subsequent response to the ionophore, whereas starvation for other divalent cations had no detectable effect. The branching response was markedly reduced in the presence of 10 to 30 mM cyclic AMP or derivatives thereof.     

Isolation of telomere DNA from Neurospora crassa.

The most distal known gene on Neurospora crassa linkage group VR, his-6, was cloned. A genomic walk resulted in isolation of the telomere at VR. It was obtained from a library in which the endmost nucleotides of the chromosome had not been removed by nuclease treatment before being cloned, and mapping indicates that the entire chromosome end has probably been cloned. Sequences homologous to the terminal 2.5 kilobases of DNA from VR from these Oak Ridge N. crassa strains are found at other sites in the genome. To characterize these sites, I crossed an Oak Ridge-derived his-6 strain with a wild-type strain of different genetic background (Mauriceville) and characterized the hybridization patterns seen in the progeny. It appears that the sequences homologous to the VR terminus are found at genetically different sites in the two parental strains, and no hybridization to the VR telomere from Mauriceville was detected. The other genomic copies identified in the Oak Ridge parent were not telomeres. I suggest that any repeating sequence blocks found immediately adjacent to the VR terminus in Oak Ridge strains must be small and that the repeating element identified in that background may be an N. crassa transposable element integrated near the the chromosome end at VR.