scholarly journals The structural gene for a phosphorus-repressible phosphate permease in Neurospora crassa can complement a mutation in positive regulatory gene nuc-1.

1988 ◽  
Vol 8 (3) ◽  
pp. 1376-1379 ◽  
Author(s):  
B J Mann ◽  
R A Akins ◽  
A M Lambowitz ◽  
R L Metzenberg
Keyword(s):  
Neurospora Crassa ◽  
Structural Gene ◽  
Regulatory Gene ◽  
Cosmid Clone ◽  
Wild Type ◽  
Gene Encoding ◽  
Regulatory Locus

van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. This was unexpected because the nuc-1 host already contained a resident van+ gene. Plasmids carrying van+ complemented a nuc-2 mutation as well. Probing of RNA from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control by nuc-1+. Probing of the same RNAs with a cosmid clone, containing approximately 15 kilobases of upstream and downstream DNA, revealed no other detectable phosphorus-regulated transcripts within this 40-kilobase region of the chromosome.

The structural gene for a phosphorus-repressible phosphate permease in Neurospora crassa can complement a mutation in positive regulatory gene nuc-1

1988 ◽  
Vol 8 (3) ◽  
pp. 1376-1379
Author(s):  
B J Mann ◽  
R A Akins ◽  
A M Lambowitz ◽  
R L Metzenberg
Keyword(s):  
Neurospora Crassa ◽  
Structural Gene ◽  
Regulatory Gene ◽  
Cosmid Clone ◽  
Wild Type ◽  
Gene Encoding ◽  
Regulatory Locus

van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. This was unexpected because the nuc-1 host already contained a resident van+ gene. Plasmids carrying van+ complemented a nuc-2 mutation as well. Probing of RNA from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control by nuc-1+. Probing of the same RNAs with a cosmid clone, containing approximately 15 kilobases of upstream and downstream DNA, revealed no other detectable phosphorus-regulated transcripts within this 40-kilobase region of the chromosome.

Regulation of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) in Aspergillus nidulans

1983 ◽  
Vol 217 (1208) ◽  
pp. 243-264 ◽  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Aspergillus Nidulans ◽  
Structural Gene ◽  
Aldehyde Dehydrogenase ◽  
Gel Electrophoresis ◽  
Regulatory Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Cell Extracts

There is a single major alcohol dehydrogenase (ADH) and a single major aldehyde dehydrogenase (AldDH) in Aspergillus nidulans . Both ADH and AldDH are induced by ethanol and by acetaldehyde and both are subject to carbon catabolite repression. ADH and AldDH are necessary for the utilization of ethanol and of threonine, indicating that both compounds are utilized via acetaldehyde. ADH and AldDH each give a single major activity band on gel electrophoresis. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell extracts shows at least two similar ADH polypeptides of approximate relative molecular mass (r. m. m.) 41000 and two similar AldDH polypeptides of approximate r. m. m. 57000. The in vitro translation of mRNA from induced, carbon derepressed wild-type cells gives up to three ADH polypeptides in the r. m. m. range 39000-43000 and an AldDH polypeptide of approximate r. m. m. 57000. The mRNA from uninduced, carbon repressed wild-type cells does not direct the synthesis of the ADH and AldDH polypeptides. This indicates that the regulation of ADH and AldDH is at the level of transcription and/or post-transcriptional modification. The probable explanation of the multiple ADH polypeptides is post-transcriptional modification of the mRNA. Allyl alcohol mutants were made by using diepoxyoctane and γ-rays as mutagens. There are two classes, alcA and alcR . Neither class can utilize ethanol or threonine as a carbon source. The alcA mutants lack normal ADH and are recessive. Of the 47 alcA mutants examined 39 do not make the ADH polypeptides while eight do so. Therefore alcA is the structural gene for ADH. The two alcA mutants tested do not make functional mRNA for ADH. The alcR mutants lack both ADH and AldDH and are recessive. No alcR mutants make the ADH or the AldDH polypeptides. The three alcR mutants tested do not make functional ADH or AldDH mRNA. The mutant alcR 125 is a nonsense mutant, which establishes that alcR codes for a protein. The alcA and alcR genes are adjacent on chromosome VII and a preliminary fine-structure map of the alcA gene has been made. Three mutants that cannot utilize ethanol or threonine and have ADH, but lack AldDH, define a gene AldA on chromosome VIII. The aldA 23 mutant makes the AldDH polypeptides, the other two aldA mutants do not. Therefore aldA is probably the structural gene for AldDH. Our current hypothesis is that alcA and aldA are the structural genes for ADH and AldDH respectively and alcR is a transacting regulatory gene coding for a protein whose function is necessary for the expression of the alcA and aldA genes.

scholarly journals REGULATION OF PHOSPHATE METABOLISM IN NEUROSPORA CRASSA: IDENTIFICATION OF THE STRUCTURAL GENE FOR REPRESSIBLE ACID PHOSPHATASE

Genetics ◽  
1976 ◽  
Vol 84 (2) ◽  
pp. 183-192
Author(s):  
Robert E Nelson ◽  
John F Lehman ◽  
Robert L Metzenberg
Keyword(s):  
Acid Phosphatase ◽  
Neurospora Crassa ◽  
Structural Gene ◽  
Group Iv ◽  
Structural Genes ◽  
Wild Type ◽  
Linkage Groups ◽  
Michaelis Constant ◽  
The Right

ABSTRACT A mutant of Neurospora crassa with an altered repressible acid phosphatase has been isolated. The enzyme is much more thermolabile than that of wild type, and has an increased Michaelis constant. Tests of allelic interactions (in partial diploids) and in vitro mixing experiments were consistent with the mutation being in the structural gene for the enzyme. This gene, pho-3, was found to be located in the right arm of Linkage Group IV (LG IV). Thus, pho-3 and the structural gene for repressible alkaline phosphatase, pho-2 (LG V), map in separate linkage groups and cannot be part of the same operon. Neither of these structural genes is linked to the known regulatory genes, nuc-1 (LG I), nuc-2 (LG II), and preg (LG II).

scholarly journals Nonsense mutations of the ornithine decarboxylase structural gene of Neurospora crassa

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.

Molecular cloning and analysis of the scon-2 negative regulatory gene of Neurospora crassa

1990 ◽  
Vol 10 (10) ◽  
pp. 5207-5214
Author(s):  
J V Paietta
Keyword(s):  
Neurospora Crassa ◽  
Regulatory Gene ◽  
Constitutive Expression ◽  
Regulatory System ◽  
Polymorphism Analysis ◽  
Structural Genes ◽  
Gene Encoding ◽  
Coordinate Control ◽  
Mrna Content ◽  
Sib Selection

The sulfur regulatory system of Neurospora crassa is composed of a group of highly regulated structural genes (e.g., the gene encoding arylsulfatase) that are under coordinate control of scon+ (sulfur controller) negative and cys-3+ positive regulatory genes. In scon-1 (previously designated sconC) and scon-2 mutants, there is constitutive expression of sulfur structural genes regardless of the sulfur level available to the cells. The scon-2+ gene was cloned by sib selection screening of a cosmid-based gene library. The screening was based on the use of chromate, a toxic sulfate analog, which is transported into scon-2 cells grown on high sulfur but is not transported into cells that have regained normal sulfur regulation. Restriction fragment length polymorphism analysis was used to confirm that the cloned segment mapped to the proper chromosomal location. In wild-type cells, Northern (RNA) blot analysis showed that a 2.6-kilobase scon-2+ transcript was present at a substantial level only under sulfur-derepressing conditions. Kinetic analysis showed that scon-2+ mRNA content increased as the cells became sulfur starved. Further, scon-2+ RNA was detectable in a nuclear transcription assay only under derepressing conditions. In scon-1, the levels of scon-2+ mRNA were found to be constitutive. In the cys-3 regulatory mutant, there was a reduced level of scon-2+ transcript. cys-3+ and ars-1+ mRNAs were present under both derepressing and repressing conditions in the scon-2 mutant. Repeat-induced point mutation-generated scon-2 mutants were identical in phenotype to the known mutant.

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

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.

scholarly journals Structural gene for ornithine decarboxylase in Neurospora crassa

1985 ◽  
Vol 5 (6) ◽  
pp. 1301-1306
Author(s):  
P Eversole ◽  
J J DiGangi ◽  
T Menees ◽  
R H Davis
Keyword(s):  
Linkage Group ◽  
Neurospora Crassa ◽  
Ornithine Decarboxylase ◽  
Structural Gene ◽  
Minimal Medium ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Group V

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.

scholarly journals Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

1989 ◽  
Vol 9 (11) ◽  
pp. 4631-4644 ◽  
Author(s):  
C M Chow ◽  
R L Metzenberg ◽  
U L Rajbhandary
Keyword(s):  
Amino Acid ◽  
Neurospora Crassa ◽  
Structural Gene ◽  
Nuclear Gene ◽  
Trna Synthetase ◽  
Chromosomal Mapping ◽  
Cdna Clones ◽  
Sequence Information ◽  
Wild Type ◽  
Apparent Lack

We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by S1 nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the leu-5 mutation had been mapped before. We show that the leu-5 strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the leu-5 strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in leu-5 extracts, the leu-5 strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.

Autogenous regulation of the positive regulatory qa-1F gene in Neurospora crassa

1985 ◽  
Vol 5 (12) ◽  
pp. 3593-3599
Author(s):  
V B Patel ◽  
N H Giles
Keyword(s):  
Neurospora Crassa ◽  
Gene Cluster ◽  
Dna Sequences ◽  
Regulatory Gene ◽  
Quinic Acid ◽  
The Other ◽  
Wild Type ◽  
Low Level ◽  
Activator Protein ◽  
Autogenous Regulation

In Neurospora crassa, the qa-1F regulatory gene positively controls transcription of all genes in the quinic acid (qa) gene cluster. qa-1F is transcribed at a low, uninduced level but is subject to strong (50-fold), autogenous regulation as well as to control by the negative regulatory gene, qa-1S, and the inducer quinic acid. Cloned qa-1F DNA sequences hybridize to two related mRNAs of 2.9 and 3.0 kilobases. When wild-type (qa-1F+) cultures are transferred to inducing conditions, qa-1F mRNA increases for 4 h, remains somewhat level, and decreases after 8 to 10 h. That this control is autogenous, i.e., that the qa-1F gene controls the synthesis of its own mRNA, is indicated by the presence of approximately the same low level of qa-1F mRNA in poly(A)+ RNA from noninducible qa-1F- mutant cultures under inducing conditions as that observed in uninduced wild-type cultures. The qa-1S gene also regulates the transcription of qa-1F, since a qa-1S- mutant, whether in noninducing or inducing conditions, contains a level of qa-1F mRNA that corresponds to the low level observed in uninduced wild-type cultures. These results corroborate the hypothesis (M. E. Case and N. H. Giles, Proc. Natl. Acad. Sci. USA 72:553-557, 1975; V. B. Patel, M. Schweizer, C. C. Dykstra, S. R. Kushner, and N. H. Giles, Proc. Natl. Acad. Sci. USA 78:5783-5787, 1981; L. Huiet, Proc. Natl. Acad. Sci. USA 81:1174-1178, 1984) that the qa-1F gene encodes an activator protein and acts positively in controlling transcription of itself and the other qa genes.

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