scholarly journals 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.

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 Molecular cloning and regulatory analysis of the arylsulfatase structural gene of Neurospora crassa.

1989 ◽  
Vol 9 (9) ◽  
pp. 3630-3637 ◽  
Author(s):  
J V Paietta
Keyword(s):  
Enzyme Activity ◽  
Neurospora Crassa ◽  
Time Course ◽  
Constitutive Expression ◽  
Temperature Sensitive ◽  
Polymorphism Analysis ◽  
Regulatory Mutant ◽  
Coordinate Control ◽  
Arylsulfatase Activity ◽  
Regulatory Analysis

The ars-1+ gene of Neurospora crassa encodes the enzyme arylsulfatase. ars-1+ is in a group of highly regulated sulfur-related structural genes that are expressed under conditions of sulfur limitation and are under coordinate control of the cys-3+ and scon+ regulatory genes. The ars-1+ gene was cloned by chromosome walking from the qa gene cluster, using a lambda library. Cotransformation of an N. crassa ars-1 mutant with the isolated lambda clones and the benomyl resistance gene, followed by assay for arylsulfatase activity, was used to screen for the ars-1+ gene. Further confirmation that the cloned segment mapped to the ars-1+ locus was obtained by restriction-fragment-length polymorphism analysis. Northern (RNA) blot analysis showed that the ars-1+ gene was transcribed to give an mRNA of 2.3 kilobases. In wild-type cells, the ars-1+ transcript was abundant under sulfur-derepressing conditions but absent under repressing conditions. Time course analysis showed that the appearance of ars-1+ message in sulfur-derepressed cultures paralleled the appearance of arylsulfatase enzyme activity. In addition, transcription of ars-1+ was detected only under derepressing conditions in a nuclear transcription assay. In a cys-3 regulatory mutant that was unable to synthesize arylsulfatase (or other sulfur-controlled enzymes), there was no ars-1+ transcript under repressing or derepressing conditions. In a temperature-sensitive cys-3 mutant, the ars-1+ transcript was present only at the permissive growth temperature and under sulfur derepression. A negative regulatory mutant, sconc, displayed both constitutive expression of arylsulfatase enzyme activity and content of ars-1+ message.

scholarly journals Molecular cloning and analysis of the regulation of cys-14+, a structural gene of the sulfur regulatory circuit of Neurospora crassa.

1988 ◽  
Vol 8 (4) ◽  
pp. 1504-1508 ◽  
Author(s):  
J S Ketter ◽  
G A Marzluf
Keyword(s):  
Neurospora Crassa ◽  
Regulatory Gene ◽  
Constitutive Expression ◽  
Cosmid Library ◽  
Temperature Sensitive ◽  
Polymorphism Analysis ◽  
Permissive Temperature ◽  
Message Content ◽  
Regulatory Circuit ◽  
In The Wild

The cys-14+ gene encodes sulfate permease II, which is primarily expressed in mycelia. cys-14+ is one of a set of sulfur-related structural genes under the control of cys-3+ and scon+, the regulatory genes of the sulfur control circuit. We have cloned cys-14+ from a cosmid library of Neurospora crassa DNA. A restriction fragment length polymorphism analysis showed that this clone maps to the region of chromosome IV corresponding to the cys-14+ locus. Northern blot analyses were used to examine the regulated expression of the cys-14+ gene. In the wild type, a 3-kilobase cys-14+ transcript was highly expressed under sulfur-derepressing conditions but completely absent during sulfur repression. A cys-3 mutant, which cannot synthesize any of the sulfur-controlled enzymes, including sulfate permease II, did not possess any cys-14+ transcript under either condition. A cys-3 temperature-sensitive revertant completely lacked any cys-14+ mRNA at the conditional temperature but expressed the cys-14+ transcript upon derepression at the permissive temperature. Mutation of a second sulfur regulatory gene, scon(c), causes the expression of sulfur-related enzymes in a constitutive fashion; the scon(c) mutant showed a corresponding constitutive expression of cys-14+ mRNA, such that it was present even in cells subjected to sulfur repression conditions. These results show that the cys-14+ gene is regulated through the modulation of message content by the cys-3+ and scon(c) control genes in response to the sulfur levels of the cells.

Molecular cloning and analysis of the regulation of cys-14+, a structural gene of the sulfur regulatory circuit of Neurospora crassa

1988 ◽  
Vol 8 (4) ◽  
pp. 1504-1508
Author(s):  
J S Ketter ◽  
G A Marzluf
Keyword(s):  
Neurospora Crassa ◽  
Regulatory Gene ◽  
Constitutive Expression ◽  
Cosmid Library ◽  
Temperature Sensitive ◽  
Polymorphism Analysis ◽  
Permissive Temperature ◽  
Message Content ◽  
Regulatory Circuit ◽  
In The Wild

The cys-14+ gene encodes sulfate permease II, which is primarily expressed in mycelia. cys-14+ is one of a set of sulfur-related structural genes under the control of cys-3+ and scon+, the regulatory genes of the sulfur control circuit. We have cloned cys-14+ from a cosmid library of Neurospora crassa DNA. A restriction fragment length polymorphism analysis showed that this clone maps to the region of chromosome IV corresponding to the cys-14+ locus. Northern blot analyses were used to examine the regulated expression of the cys-14+ gene. In the wild type, a 3-kilobase cys-14+ transcript was highly expressed under sulfur-derepressing conditions but completely absent during sulfur repression. A cys-3 mutant, which cannot synthesize any of the sulfur-controlled enzymes, including sulfate permease II, did not possess any cys-14+ transcript under either condition. A cys-3 temperature-sensitive revertant completely lacked any cys-14+ mRNA at the conditional temperature but expressed the cys-14+ transcript upon derepression at the permissive temperature. Mutation of a second sulfur regulatory gene, scon(c), causes the expression of sulfur-related enzymes in a constitutive fashion; the scon(c) mutant showed a corresponding constitutive expression of cys-14+ mRNA, such that it was present even in cells subjected to sulfur repression conditions. These results show that the cys-14+ gene is regulated through the modulation of message content by the cys-3+ and scon(c) control genes in response to the sulfur levels of the cells.

The Constitutive, Glucose-Repression-Insensitive Mutation of the Yeast MAL4 Locus Is an Alteration of the MAL43 Gene

Genetics ◽  
1987 ◽  
Vol 116 (1) ◽  
pp. 23-31
Author(s):  
Maureen J Charron ◽  
Corinne A Michels
Keyword(s):  
Glucose Repression ◽  
Regulatory Gene ◽  
Constitutive Expression ◽  
Structural Genes ◽  
Wild Type ◽  
Gene Encoding ◽  
Maltose Permease ◽  
Genes Expression ◽  
Constitutive Production ◽  
Saccharomyces Yeasts

ABSTRACT Mutations resulting in constitutive production of maltase have been identified at each of the five MAL loci of Saccharomyces yeasts. Here we examine a dominant constitutive, glucose-repression-insensitive allele of the MAL4 locus (MAL4-C). Our results demonstrate that MAL4-C is an alteration in the MAL43 gene, which encodes the positive regulator of the MAL structural genes, and that its product is trans-acting. The MAL43 gene from the MAL4-C strain was cloned and integrated into a series of nonfermenting strains lacking a functional regulatory gene but carrying copies of the maltose permease and maltase structural genes. Expression of the maltase structural gene was both constitutive and insensitive to glucose repression in these transformants. The MAL4-C allele also results in constitutive expression of the unlinked MAL12 gene (encoding maltase) in this strain. In addition, the cloned MAL43 gene was shown to be dominant to the wild-type MAL63 gene. We also show that most of the glucose repression insensitivity of strains carrying the MAL4-C allele results from alteration of MAL43.

Molecular cloning and regulatory analysis of the arylsulfatase structural gene of Neurospora crassa

1989 ◽  
Vol 9 (9) ◽  
pp. 3630-3637
Author(s):  
J V Paietta
Keyword(s):  
Enzyme Activity ◽  
Neurospora Crassa ◽  
Time Course ◽  
Constitutive Expression ◽  
Temperature Sensitive ◽  
Polymorphism Analysis ◽  
Regulatory Mutant ◽  
Coordinate Control ◽  
Arylsulfatase Activity ◽  
Regulatory Analysis

The ars-1+ gene of Neurospora crassa encodes the enzyme arylsulfatase. ars-1+ is in a group of highly regulated sulfur-related structural genes that are expressed under conditions of sulfur limitation and are under coordinate control of the cys-3+ and scon+ regulatory genes. The ars-1+ gene was cloned by chromosome walking from the qa gene cluster, using a lambda library. Cotransformation of an N. crassa ars-1 mutant with the isolated lambda clones and the benomyl resistance gene, followed by assay for arylsulfatase activity, was used to screen for the ars-1+ gene. Further confirmation that the cloned segment mapped to the ars-1+ locus was obtained by restriction-fragment-length polymorphism analysis. Northern (RNA) blot analysis showed that the ars-1+ gene was transcribed to give an mRNA of 2.3 kilobases. In wild-type cells, the ars-1+ transcript was abundant under sulfur-derepressing conditions but absent under repressing conditions. Time course analysis showed that the appearance of ars-1+ message in sulfur-derepressed cultures paralleled the appearance of arylsulfatase enzyme activity. In addition, transcription of ars-1+ was detected only under derepressing conditions in a nuclear transcription assay. In a cys-3 regulatory mutant that was unable to synthesize arylsulfatase (or other sulfur-controlled enzymes), there was no ars-1+ transcript under repressing or derepressing conditions. In a temperature-sensitive cys-3 mutant, the ars-1+ transcript was present only at the permissive growth temperature and under sulfur derepression. A negative regulatory mutant, sconc, displayed both constitutive expression of arylsulfatase enzyme activity and content of ars-1+ message.

scholarly journals Correlation of the physical and genetic maps of the centromeric region of the right arm of linkage group III of Neurospora crassa.

Genetics ◽  
1994 ◽  
Vol 136 (4) ◽  
pp. 1297-1306
Author(s):  
C R Davis ◽  
R R Kempainen ◽  
M S Srodes ◽  
C R McClung
Keyword(s):  
Linkage Group ◽  
Neurospora Crassa ◽  
Fragment Length Polymorphism ◽  
Centromeric Region ◽  
Recombination Frequency ◽  
Genetic Maps ◽  
Polymorphism Analysis ◽  
Group Iii ◽  
Sib Selection ◽  
The Right

Abstract We have cloned three linked genes serine-1 (ser-1), proline-1 (pro-1) and acetate-2 (ace-2) that lie near the centromere on the right arm of linkage group III (LGIIIR) of Neurospora crassa. The ser-1 gene was cloned by sib selection. A chromosomal walk that spans 205 kilobases (kb) was initiated from ser-1. Complementation analysis with clones isolated during the walk allowed identification of the pro-1 and ace-2 genes. Restriction fragment length polymorphism analysis has confirmed the localization of ser-1, pro-1 and ace-2 to the centromeric region of LGIIIR. Genetically, we measured 1% recombination between ser-1 and pro-1 and 2% recombination between pro-1 and ace-2. Physical distances for these intervals were 114 kb from ser-1 to pro-1 and 36 kb from pro-1 to ace-2. Thus, for the pro-1 to ace-2 interval we calculate a physical/genetic correlation of 18 kb/map unit (mu) whereas, in the immediately adjacent, centromere-proximal interval from ser-1 to pro-1, we calculate 114 kb/mu. This provides evidence for a centromere effect, a decrease in recombination frequency as one approaches the centromere.

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.

Identification of a second trans-acting gene controlling maltose fermentation in Saccharomyces carlsbergensis

1986 ◽  
Vol 6 (8) ◽  
pp. 2757-2765
Author(s):  
R A Dubin ◽  
E L Perkins ◽  
R B Needleman ◽  
C A Michels
Keyword(s):  
Structural Gene ◽  
Regulatory Gene ◽  
Constitutive Expression ◽  
Gene Mutations ◽  
Gene Products ◽  
Structural Genes ◽  
Maltose Permease ◽  
Maltose Fermentation ◽  
New Gene

Maltose fermentation in Saccharomyces spp. requires the presence of a dominant MAL locus. The MAL6 locus has been cloned and shown to encode the structural genes for maltose permease (MAL61), maltase (MAL62), and a positively acting regulatory gene (MAL63). Induction of the MAL61 and MAL62 gene products requires the presence of maltose and the MAL63 gene. Mutations within the MAL63 gene produce nonfermenting strains unable to induce the two structural gene products. Reversion of these mal63 nonfermenters to maltose fermenters nearly always leads to the constitutive expression of maltase and maltose permease, and constitutivity is always linked to MAL6. We demonstrated that for one such revertant, strain C2, constitutivity did not require the MAL63 gene, since deletion disruption of this gene did not affect the constitutive expression of the structural genes. In addition, constitutivity was trans acting. Deletion disruption of the MAL6-linked structural genes for maltase and maltose permease in this strain did not affect the constitutive expression of a second, unlinked maltase structural gene. We isolated new maltose-fermenting revertants of a nonfermenting strain which carried a deletion disruption of the MAL63 gene. All 16 revertants isolated expressed maltase constitutively. In one revertant studied in detail, strain R10, constitutive expression was demonstrated to be linked to MAL6, semidominant, trans acting, and residing outside the MAL63-MAL61-MAL62 genes. From these studies we propose the existence of a second trans-acting regulatory gene at the MAL6 locus. We call this new gene MAL64. We mapped the MAL64 gene 2.3 centimorgans to the left of MAL63. The role of the MAL64 gene product in maltose fermentation is discussed.

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