Structural gene for ornithine decarboxylase in Neurospora crassa

To define the structural gene for ornithine decarboxylase (ODC) in Neurospora crassa, we sought mutants with kinetically altered enzyme. Four mutants, PE4, PE7, PE69, and PE85, were isolated. They were able to grow slowly at 25 degrees C on minimal medium but required putrescine or spermidine supplementation for growth at 35 degrees C. The mutants did not complement with one another or with ODC-less spe-1 mutants isolated in earlier studies. In all of the mutants isolated to date, the mutations map at the spe-1 locus on linkage group V. Strains carrying mutations PE4, PE7, and PE85 displayed a small amount of residual ODC activity in extracts. None of them had a temperature-sensitive enzyme. The enzyme of the PE85 mutant had a 25-fold higher Km for ornithine (5mM) than did the enzyme of wild-type or the PE4 mutant (ca. 0.2 mM). The enzyme of this mutant was more stable to heat than was the wild-type enzyme. These characteristics were normal in the mutant carrying allele PE4. The mutant carrying PE85 was able to grow well at 25 degrees C and weakly at 35 degrees C with ornithine supplementation. This mutant and three ODC-less mutants isolated previously displayed a polypeptide corresponding to ODC in Western immunoblots with antibody raised to purified wild-type ODC. We conclude that spe-1 is the structural gene for the ODC.

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REGULATION OF PHOSPHATE METABOLISM IN NEUROSPORA CRASSA: IDENTIFICATION OF THE STRUCTURAL GENE FOR REPRESSIBLE ALKALINE PHOSPHATASE

Genetics ◽

10.1093/genetics/84.2.175 ◽

1976 ◽

Vol 84 (2) ◽

pp. 175-182

Author(s):

John F Lehman ◽

Robert L Metzenberg

Keyword(s):

Alkaline Phosphatase ◽

Linkage Group ◽

Neurospora Crassa ◽

Structural Gene ◽

Type A ◽

Phosphate Metabolism ◽

Wild Type ◽

Wild Type Enzyme ◽

Group V ◽

Low Levels

ABSTRACT Five additional mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase. The mutations in these strains map at a previously assigned locus on Linkage Group V designated pho-2 (Gleason and Metzenberg 1974). The five new mutants, as well as three previously isolated by Gleason and Metzenberg (1974), were examined for the presence of cross-reacting material to antibody prepared against purified wild-type enzyme. Two of the mutants produced high levels of cross-reacting material, thus providing evidence that the pho-2 locus includes the structural gene for the repressible alkaline phosphatase. Two revertants were obtained from one of the mutants that contained cross-reacting material. Neither revertant produced an enzyme that could be distinguished physicochemically from that of wild type. A method for measuring very low levels of repressible alkaline phosphatase in crude extracts is also described.

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REGULATION OF PHOSPHATE METABOLISM IN NEUROSPORA CRASSA: ISOLATION OF MUTANTS DEFICIENT IN THE REPRESSIBLE ALKALINE PHOSPHATASE

Genetics ◽

10.1093/genetics/78.2.645 ◽

1974 ◽

Vol 78 (2) ◽

pp. 645-659

Author(s):

Mary K Gleason ◽

Robert L Metzenberg

Keyword(s):

Alkaline Phosphatase ◽

Acid Phosphatase ◽

Linkage Group ◽

Neurospora Crassa ◽

Heat Stability ◽

Phosphate Metabolism ◽

Wild Type ◽

Wild Type Enzyme ◽

Group V ◽

Suppressor Mutations

ABSTRACT Mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase, but, unlike nuc-1 and nuc-2 mutants, are able to make the repressible acid phosphatase and the repressible phosphate permease under conditions of derepression (phosphate deprivation). The new mutants, called pho-2, map in Linkage Group V, and are unlinked to the putative control mutants, nuc-1, nuc-2-pconc, and pregc. Three of the pho-2 mutants do not make detectable amounts of repressible alkaline phosphatase, but the fourth makes about 1% of the level found in wild type. The small amount of alkaline phosphatase made by this strain appears to be qualitatively similar or identical to the wild-type enzyme, as judged by electrophoretic mobility, heat stability, and titration with specific antibody to the wild-type enzyme. Several revertants of this strain have been examined in the same way, and the alkaline phosphatase of these strains also appears to be qualitatively normal. Reversion events can occur at, or near, the pho-2 locus, but also occur in at least two unlinked sites (suppressor mutations). One suppressor maps very close to nuc-1.

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Nonsense mutations of the ornithine decarboxylase structural gene of Neurospora crassa

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1122-1128.1987 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1122-1128

Author(s):

R H Davis ◽

L V Hynes ◽

P Eversole-Cire

Keyword(s):

Neurospora Crassa ◽

Ornithine Decarboxylase ◽

Structural Gene ◽

Wild Type Strain ◽

Normal Growth ◽

Wild Type ◽

Carboxy Terminus ◽

Nonsense Mutations ◽

Polyamine Synthesis ◽

Weak Activity

Ornithine decarboxylase (ODC) (EC 4.1.1.17) is an early enzyme of polyamine synthesis, and its activity rises quickly at the onset of growth and differentiation in most eucaryotes. Some have speculated that the enzyme protein may have a role in the synthesis of rRNA in addition to its role in catalyzing the decarboxylation of ornithine (G. D. Kuehn and V. J. Atmar, Fed. Proc. 41:3078-3083, 1982; D. H. Russell, Proc. Natl. Acad. Sci. USA 80:1318-1321, 1983). To test this possibility, we sought mutational evidence for the indispensability of the ODC protein for normal growth of Neurospora crassa. We found three new, ODC-deficient mutants that lacked ODC protein. Among these and by reversion analysis of an earlier set of mutants, we found that two ODC-deficient mutants carried nonsense mutations in the ODC structural gene, spe-1. Allele LV10 imparted a complete deficiency for enzyme activity (less than 0.006% of normal) and had no detectable ODC antigen. Allele PE4 imparted a weak activity to cells (0.1% of derepressed spe+ cultures) and encoded a lower-molecular-weight ODC subunit (Mr = 43,000) in comparison to that of the wild-type strain (Mr = 53,000). Strains carrying either mutation, like other spe-1 mutants, grew at a normal rate in exponential culture if the medium was supplemented with spermidine, the main end product of the polyamine pathway in N. crassa. Unless an antigenically silent, N-terminal fragment with an indispensable role persists in the LV10-bearing mutant, we conclude that the ODC protein has no role in the vegetative growth of this organism other than the synthesis of polyamines. The data extend earlier evidence that spe-1 is the structural gene for ODC in N. crassa. The activity found in mutants bearing allele PE4 suggests that the amino acids nearest the carboxy terminus do not contribute to the active site of the enzyme.

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Nonsense mutations of the ornithine decarboxylase structural gene of Neurospora crassa.

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1122 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1122-1128 ◽

Cited By ~ 12

Author(s):

R H Davis ◽

L V Hynes ◽

P Eversole-Cire

Keyword(s):

Neurospora Crassa ◽

Ornithine Decarboxylase ◽

Structural Gene ◽

Wild Type Strain ◽

Normal Growth ◽

Wild Type ◽

Carboxy Terminus ◽

Nonsense Mutations ◽

Polyamine Synthesis ◽

Weak Activity

Ornithine decarboxylase (ODC) (EC 4.1.1.17) is an early enzyme of polyamine synthesis, and its activity rises quickly at the onset of growth and differentiation in most eucaryotes. Some have speculated that the enzyme protein may have a role in the synthesis of rRNA in addition to its role in catalyzing the decarboxylation of ornithine (G. D. Kuehn and V. J. Atmar, Fed. Proc. 41:3078-3083, 1982; D. H. Russell, Proc. Natl. Acad. Sci. USA 80:1318-1321, 1983). To test this possibility, we sought mutational evidence for the indispensability of the ODC protein for normal growth of Neurospora crassa. We found three new, ODC-deficient mutants that lacked ODC protein. Among these and by reversion analysis of an earlier set of mutants, we found that two ODC-deficient mutants carried nonsense mutations in the ODC structural gene, spe-1. Allele LV10 imparted a complete deficiency for enzyme activity (less than 0.006% of normal) and had no detectable ODC antigen. Allele PE4 imparted a weak activity to cells (0.1% of derepressed spe+ cultures) and encoded a lower-molecular-weight ODC subunit (Mr = 43,000) in comparison to that of the wild-type strain (Mr = 53,000). Strains carrying either mutation, like other spe-1 mutants, grew at a normal rate in exponential culture if the medium was supplemented with spermidine, the main end product of the polyamine pathway in N. crassa. Unless an antigenically silent, N-terminal fragment with an indispensable role persists in the LV10-bearing mutant, we conclude that the ODC protein has no role in the vegetative growth of this organism other than the synthesis of polyamines. The data extend earlier evidence that spe-1 is the structural gene for ODC in N. crassa. The activity found in mutants bearing allele PE4 suggests that the amino acids nearest the carboxy terminus do not contribute to the active site of the enzyme.

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Enhancers of conidiation mutants in Aspergillus nidulans.

Genetics ◽

10.1093/genetics/137.1.79 ◽

1994 ◽

Vol 137 (1) ◽

pp. 79-85

Author(s):

D H Gems ◽

A J Clutterbuck

Keyword(s):

Linkage Group ◽

Aspergillus Nidulans ◽

Double Mutant ◽

Slight Modification ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Wild Type ◽

Group V ◽

Group I

Abstract Mutants at a number of loci, designated sthenyo, have been isolated as enhancers of the oligoconidial mutations at the medA locus. Two loci have been mapped: sthA on linkage group I, and sthB on linkage group V. Two probable alleles have been identified at each locus but two further mutants were unlinked to either sthA or sthB. Neither sthA nor sthB mutants have conspicuous effects on morphology on their own, nor could the sthA1 sthB2 double mutant be distinguished from wild type. Mutants at both loci also interact with the temperature-sensitive brlA42 mutant at the permissive temperature to give a phenotype described as "Abacoid." sthA1 also induces a slight modification of the phenotype of an abaA mutant. We conclude that sthenyo genes act mainly at the phialide stage of conidiation. We also describe the isolation of new medA mutants arising spontaneously as outgrowths on brlA42 colonies.

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COMPLEMENTATION ANALYSIS OF LINKED CIRCADIAN CLOCK MUTANTS OF NEUROSPORA CRASSA

Genetics ◽

10.1093/genetics/82.1.9 ◽

1976 ◽

Vol 82 (1) ◽

pp. 9-17 ◽

Cited By ~ 3

Author(s):

Jerry F Feldman ◽

Marian N Hoyle

Keyword(s):

Linkage Group ◽

Circadian Clock ◽

Neurospora Crassa ◽

Period Length ◽

Complementation Analysis ◽

Wild Type ◽

The Right

ABSTRACT A fourth mutant of Neurospora crassa, designated frq-4, has been isolated in which the period length of the circadian conidiation rhythm is shortened to 19.3 ± 0.3 hours. This mutant is tightly linked to the three previously isolated frq mutants, and all four map to the right arm of linkage group VII about 10 map units from the centromere. Complementation tests suggest, but do not prove, that all four mutations are allelic, since each of the four mutants is co-dominant with the frq + allele—i.e., heterokaryons have period lengths intermediate between the mutant and wild-type—and since heterokaryons between pairs of mutants also have period lengths intermediate between those of the two mutants.

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Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa

Molecular and Cellular Biology ◽

10.1128/mcb.12.4.1568-1577.1992 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1568-1577

Author(s):

J V Paietta

Keyword(s):

Gene Expression ◽

Neurospora Crassa ◽

Structural Gene ◽

Control Point ◽

Regulatory Protein ◽

Negative Regulator ◽

Temperature Sensitive ◽

Cys3 Protein

The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. The N. crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant. Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis. The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions. In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.

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Isolation of telomere DNA from Neurospora crassa

Molecular and Cellular Biology ◽

10.1128/mcb.7.9.3168-3177.1987 ◽

1987 ◽

Vol 7 (9) ◽

pp. 3168-3177

Author(s):

M G Schechtman

Keyword(s):

Linkage Group ◽

Neurospora Crassa ◽

Genetic Background ◽

Type Strain ◽

Wild Type Strain ◽

The Other ◽

Wild Type ◽

Oak Ridge ◽

Entire Chromosome ◽

Different Genetic Background

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.

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SOME GENETIC AND PHYSIOLOGICAL CHARACTERISTICS OF UREASE-DEFECTIVE STRAINS OF NEUROSPORA CRASSA

Canadian Journal of Genetics and Cytology ◽

10.1139/g71-043 ◽

1971 ◽

Vol 13 (2) ◽

pp. 256-269 ◽

Cited By ~ 11

Author(s):

Philip Haysman ◽

H. Branch Howe Jr.

Keyword(s):

Linkage Group ◽

Nitrogen Source ◽

Urease Activity ◽

Temperature Sensitive ◽

Sole Nitrogen Source ◽

Group V ◽

Group I ◽

Mutant Strains

Fourteen mutant strains have been isolated which differ from wild type with respect to urease activity: Seven strains lack detectable activity in vivo and in vitro, B1, C21 and allele 601, D1 and allele D3, D2, and W2, and fail to grow with urea as sole nitrogen source; seven have activity in vivo and varying amounts in vitro, A7, E3, E7, R2, S3, and temperature-sensitive strains C5 and K3. Strains D1 and D3 are allelic to Kolmark's ure-1; W2, to Kolmark's ure-2. Strain D2, which is either allelic or closely linked to ure-1, complements none of the strains lacking urease activity nor three of the strains having defective activity, and may be a regulatory mutant. Strains B1, C21 and allele 601 represent two previously unreported loci in linkage group I. Only two of the seven swains having defective urease activity have been mapped, A7 and S3, and have been assigned to linkage group V. These seven strains grow readily on Vogel's medium modified by having urea as sole nitrogen source but not on W-M medium similarly modified; growth is restricted on modified Vogel's medium as well, however, if the initial concentration of nitrogen, as urea, is suitably adjusted to exceed that of phosphorus.

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