One-carbon metabolism in Neurospora crassa wild-type and in mutants partially deficient in serine hydroxymethyltransferase.

1. The concentrations of folate-dependent enzymes in Neurospora crassa Lindegren A wild type (FGSC no. 853), Ser-l mutant, strain H605a (FGSC no. 118), and for mutant, strain C-24 (FGSC no. 9), were compared during exponential growth on defined minimal media. Both mutants were partially lacking in serine hydroxymethyltransferase, but contained higher concentrations of 10-formyltetrahydrofolate synthetase than did the wild type. Mycelia of the mutants contained higher concentrations of these enzymes when growth media were supplemented with 1mM-glycine. In the wild-type, this glycine supplement also increased the specific activities of 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methylenetetrahydrofolate reductase. 5. During growth, total folate and polyglutamyl folate concentrations were greatest in the wild-type. Methylfolates were not detected in mutant Ser-l, and were only present in the for mutant after growth in glycine-supplemented media. Exogenous glycine increased folate concentration threefold in the wild type, mainly owing to increases in unsubstituted polyglutamyl derivatives. 3. Feeding experiments using 14C-labelled substrates showed that C1 units were generated from formate, glycine and serine in the wild type. Greater incorporation of 14C occurred when mycelia were cultured in glycine-supplemented media. Formate and serine were precursors of C1 units in the mutants, but the ability to cleave glycine was slight or lacking.     

Multiple intracellular peptidases in Neurospora crassa.

Neurospora crassa possesses multiple intracellular peptidases which display overlapping substrate specificities. They were readily detected by an in situ staining procedure for peptidases separated in polyacrylamide gels, within which the auxilliary enzyme, l-amino acid oxidase, was immobilized. Eleven different intracellular peptidases were identified by electrophoretic separation and verified by their individual patterns of substrate specificities. Most peptide substrates tested were hydrolyzed by several different peptidases. The multiple intracellular peptidases may play overlapping roles in several basic cell processes which involve peptidase activity. The amount of peptidase activity for leucylglycine present in crude extracts of cells grown under widely different conditions was relatively constant, suggesting that this enzyme may be constitutive, although alterations in the amounts of individual peptidase isozymes may occur. A single enzyme, designated peptidase II, was partially purified and obtained free from the other peptidase species. Peptidase II was found to be an aminopeptidase with activity toward many peptides of varied composition and size. It was more active with tripeptides than homologous dipeptides and showed strong activity toward methionine-containing peptides. This enzyme, with a molecular weight of about 37,000, was thermolabile at 65 degrees C and was strongly inhibited by p-hydroxymercuribenzoate, Zn(2+), Co(2+), and Mn(2+), but was insensitive to the serine protease inhibitor phenylmethylsulfonyl fluoride. Peptidase II apparently possesses an essential sulfhydryl group and may be a metalloenzyme.     

The synthesis of alkaline phosphatase in Neurospora crassa

Mutations which affect the regulation of Neurospora repressible alkaline phosphatase do so by altering the rate of de novo alkaline phosphatase synthesis. In regulatory mutants the rate of alkaline phosphatase polypeptide synthesis can vary over a 1000-fold range. Following transfer to phosphate-free medium, the wild-type cell is capable of increasing the rate of synthesis of alkaline phosphatase molecules within 30-45 min.     

Characterization of Pi-repressible enzymes secreted in culture media by Neurospora crassa wild-type cells and null-type mutants.

In wild-type mycelial cultures of Neurospora crassa under Pi-limited conditions, alkaline phosphatase, cyclic phosphodiesterases I, II, III, and IV, 5'-nucleotidase, acid and alkaline nucleases, RNase N1, and a newly detected endonuclease were secreted into the culture media. These enzymes were either not produced or were produced in very reduced levels in mutants nuc-1, -2, -3, -4, -5, -6, and -7 and cpd-4. The proteins were examined by polyacrylamide gel electrophoresis in a manner which allowed the identification of each of them.     

Urease defective mutants in Neurospora crassa

Summary A method for isolating urease mutants was developed. It is based on the use of microconidial strains with small and compact colonies. Mutants are detected by their inability to change the color of a pH indicator when they are brought in contact with a solution of urea. The assay is performed in the absence of growth conditions so that the colonies remain separate. Two isolated urease mutants are unable to grow on urea as the sole source of nitrogen, but grow as well as the wild type on other sources of nitrogen. The same two mutants give rise to different acidities in liquid growth medium. The two mutants are also genetically different (K?lmark, 1969 b). The finding that two genetically and physiologically distinct loci participate in the control of one enzyme is discussed.     

Mitochondrial adenosine triphosphatase of wild-type and poky Neurospora crassa.

We have compared the adenosine triphosphatase (ATPase) activity of mitochondria prepared from wild-type Neurospora crassa and from poky, a maternally inherited mutant known to possess defective mitochondrial ribosomes and reduced amounts of cytochromes aa3 and b. poky contains two distinct forms of mitochondrial ATPase. The first is normal in its Km for ATP, specificity for nucleotides and divalent cations, pH optimum, cold stability, and sensitivity to inhibitors (oligomycin, N,N-dicyclohexyl carbodiimide, and adenylyl imidodiphosphate). The fact that membrane-bound, cold-stable, oligomycin-sensitive ATPase activity is present in poky (with an activity of 1.93 +/- 0.03 mumol/min-mg of protein compared with 1.33 +/- 0.07 mumol/min-mg of protein in the wild-type strain) and also in chloramphenicol-grown wild-type cells suggests that products of mitochondrial protein synthesis play only a limited role in the attachment of the mitochondrial ATPase to the membrane in Neurospora. poky also contains a second form of mitochondrial ATPase, which has an activity of 1.5 +/- 0.2 mumol/min-mg of protein, is oligomycin sensitive but cold labile, and presumably is attached less firmly to the mitochondrial membrane. The two forms, added together, represent a substantial overproduction of mitochondrial ATPase by poky.     

Unsaturated fatty acid mutants of Neurospora crassa.

Unsaturated fatty acid (ufa) auxotrophs of Neurospora crassa were obtained by treatment of conidia with N-methyl-N'-nitro-N-nitrosoguanidine followed by isolation on media containing polyunsaturated fatty acids suspended in Tergitol NP-40. The 24 mutants for which reisolates were obtained from crosses with wild type were assigned to two complementation classes, ufa-1 and ufa-2, located on linkage group V. Unsaturated fatty acids with varying degrees of unsaturation, chain length, and double-bond position as well as different steric configurations were tested for growth requirements.     

Polyamine-deficient Neurospora crassa mutants and synthesis of cadaverine.

The polyamine path of Neurospora crassa originates with the decarboxylation of ornithine to form putrescine (1,4-diaminobutane). Putrescine acquires one or two aminopropyl groups to form spermidine or spermine, respectively. We isolated an ornithine decarboxylase-deficient mutant and showed the mutation to be allelic with two previously isolated polyamine-requiring mutants. We here name the locus spe-1. The three spe-1 mutants form little or no polyamines and grow well on medium supplemented with putrescine, spermidine, or spermine. Cadaverine (1,5-diaminopentane), a putrescine analog, supports very slow growth of spe-1 mutants. An arginase-deficient mutant (aga) can be deprived of ornithine by growth in the presence of arginine, because arginine feedback inhibits ornithine synthesis. Like spe-1 cultures, the ornithine-deprived aga culture failed to make the normal polyamines. However, unlike spe-1 cultures, it had highly derepressed ornithine decarboxylase activity and contained cadaverine and aminopropylcadaverine (a spermidine analog), especially when lysine was added to cells. Moreover, the ornithine-deprived aga culture was capable of indefinite growth. It is likely that the continued growth is due to the presence of cadaverine and its derivatives and that ornithine decarboxylase is responsible for cadaverine synthesis from lysine. In keeping with this, an inefficient lysine decarboxylase activity (Km greater than 20 mM) was detectable in N. crassa. It varied in constant ratio with ornithine decarboxylase activity and was wholly absent in the spe-1 mutants.     

Cyclic nucleotide phosphodiesterase activities in Neurospora crassa.

Cyclic nucleotide phosphodiesterase activities in soluble Neurospora crassa mycelial extracts were resolved into two peaks, phosphodiesterase I and II, by chromatography on DEAE-cellulose columns. Phosphodiesterase I hydrolysed cyclic AMP and cyclic GMP equally well. Phosphodiesterase II was active on cyclic GMP but scarcely active on cyclic AMP. Phosphodiesterase I was resolved by gel filtration and sucrose-density-gradient centrifugation into three peaks having molecular weights of about 57 000, 125 000 and 225 000. This suggests that this enzyme activity has at least three aggregation forms, tentatively defined as monomeric, dimeric and tetrameric. Similarly, phosphodiesterase II was resolved into two forms, having molecular weights of about 170 000 and 320 000. Evidence on the interconversion between phosphodiesterase I forms was obtained.     

Neurospora crassa mutants deficient in asparagine synthetase

Neurospora crassa mutants deficient in asparagine synthetase were selected by using the procedure of inositol-less death. Complementation tests among the 100 mutants isolated suggested that their alterations were genetically allelic. Recombination analysis with strain S1007t, an asparagine auxotroph, indicated that the mutations were located near or within the asn gene on linkage group V. In vitro assays with a heterokaryon indicated that the mutation was dominant. Thermal instability of cell extracts from temperature-sensitive strains in an in vitro asparagine synthetase assay determined that the mutations were in the structural gene(s) for asparagine synthetase.     

Selection of succinic dehydrogenase mutants of Neurospora crassa.

A method is described which permits the selection of mutants of Neurospora crassa that are deficient in succinic dehydrogenase activity. The method relies on the observation that succinic dehydrogenase-deficient strains fail to reduce the dye nitrotetrazolium blue when overlaid with the dye in the presence of succinate and phenazine methosulfate. Wild-type colonies reduced the dye and turned blue, whereas mutant colonies remained colorless. In this communication we present studies of a mutant, SDH-1, isolated by this method. The mutant had 18% of the succinic dehydrogenase activity of the parent strain used in the mutation experiments as determined from the ratio of Vmax activities obtained from Lineweaver-Burk plots. The SDH-1 mutant segregated in a Mendelian manner when back-crossed to its parent strain. Succinate oxidase activity in SDH-1 was low and was markedly inhibited by adenosine 5'-diphosphate. The succinate oxidase activity of the parent strain was high and was not affected by the presence of adenosine 5'-diphosphate.     

The neutral carotenoids of wild-type and mutant strains of Neurospora crassa

The neutral carotenoids of wild-type Neurospora crassa and of carotenoid mutants at four discrete genetic loci were isolated using gradient elution chromatography on deactivated alumina columns. Carotenoids were identified by absorption spectrophotometry and thin layer cochromatography with carotenoid standards. Phytoene, phytofluene, -carotene, -carotene, neurosporene, torulene, lycopene, and 3,4-dehydrolycopene were isolated from wild type. Phytoene, phytofluene, -carotene, -carotene, neurosporene, -carotene, lycopene, and one unknown carotenoid, tentatively identified as 15,15-cis--carotene, were isolated from a yellow mutant, ylo-1. ylo-1 also contained residual carotenoids having similar absorption spectra to, but very different chromatographic behavior from, phytofluene, -carotene, -carotene, and lycopene. Albino and colored al-1 mutants contained large amounts of phytoene and only traces of other neutral carotenoids. Albino al-2 and al-3 mutants contained only traces of neutral carotenoids.     

Characterization of Neurospora crassa mutants deficient in glucosephosphate isomerase.

Two independent mutants of Neurospora crassa lacking glucosphosphate isomerase activity (gpi) were isolated. These mutants were obtained as double mutants containing the pp or T9 mutation in addition to the gpi mutation located on linkage group IV; the pp mutation caused the inability to form protoperithecium and the loss of ascospore germination, and the T9 mutation caused the alteration in glucoamylase and several growth characteristics. The gpi mutants did not grow on fructose but grew on glucose or sucrose. Growth of these mutants on glucose was stimulated by addition of fructose. The gpi mutants showed restricted colonial growth on agar media containing glucose in contrast to the normal filamentous growth of the wild-type stain.     

Protein kinase activities in Neurospora crassa

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) has been purified from human erythrocytes using a simple chromatographic procedure. Purified enzyme was obtained from individuals who were homozygous for the principal isozyme (ADA 1) as well as from individuals who were heterogyzous for the major variant (ADA 2-1). Although ADA 1 and ADA 2-1 are electrophoretically distinguishable, they have many common physical and catalytic properties. No significant differences between the two isozymic forms were found in measurements of molecular weight, catalytic activity in the presence of various substrates and inhibitors, pH optimum, turnover number, and stability in conditions of both high and low pH. ADA 2-1 was, however, substantially less stable than ADA 1 with respect to thermal denaturation. These studies support the idea that adenosine deaminase activity in erythrocytes is lower in those individuals who possess the variant form of the enzyme.     

Genetic and kinetic analysis of Neurospora crassa mtr mutants.

Allelic complementation occurs at the mtr (methyltryptophan resistance) locus. Kinetic properties of neutral amino acid transport are altered for mtr mutants.