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.

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.

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 cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a protein with a putative leucine zipper DNA-binding element.

1989 ◽  
Vol 9 (3) ◽  
pp. 1120-1127 ◽  
Author(s):  
Y H Fu ◽  
J V Paietta ◽  
D G Mannix ◽  
G A Marzluf
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Leucine Zipper ◽  
Regulatory Gene ◽  
Temperature Sensitive ◽  
Catabolic Enzymes ◽  
Regulatory Circuit ◽  
Dna Binding Site ◽  
Dna Binding Properties ◽  
Oncogene Protein

The sulfur-regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which encode sulfur-catabolic enzymes and two major regulatory genes which govern their expression. The positive-acting cys-3 regulatory gene is required to turn on the expression of the sulfur-related enzymes, whereas the other regulatory gene, scon, acts in a negative fashion to repress the synthesis of the same set of enzymes. Expression of the cys-3 regulatory gene was found to be controlled by scon and by sulfur availability. The nucleotide sequence of the cys-3 gene was determined and can be translated to yield a protein of molecular weight 25,892 which displays significant homology with the oncogene protein Fos, yeast GCN4 protein, and sea urchin histone H1. Moreover, the putative cys-3 protein has a well-defined leucine zipper element plus an adjacent charged region which together may make up a DNA-binding site. A cys-3 mutant and a cys-3 temperature-sensitive mutant lead to substitutions of glutamine for basic amino acids within the charged region and thus may alter DNA-binding properties of the cys-3 protein.

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 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.

cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a protein with a putative leucine zipper DNA-binding element

1989 ◽  
Vol 9 (3) ◽  
pp. 1120-1127
Author(s):  
Y H Fu ◽  
J V Paietta ◽  
D G Mannix ◽  
G A Marzluf
Keyword(s):  
Dna Binding ◽  
Neurospora Crassa ◽  
Leucine Zipper ◽  
Regulatory Gene ◽  
Temperature Sensitive ◽  
Catabolic Enzymes ◽  
Regulatory Circuit ◽  
Dna Binding Site ◽  
Dna Binding Properties ◽  
Oncogene Protein

The sulfur-regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which encode sulfur-catabolic enzymes and two major regulatory genes which govern their expression. The positive-acting cys-3 regulatory gene is required to turn on the expression of the sulfur-related enzymes, whereas the other regulatory gene, scon, acts in a negative fashion to repress the synthesis of the same set of enzymes. Expression of the cys-3 regulatory gene was found to be controlled by scon and by sulfur availability. The nucleotide sequence of the cys-3 gene was determined and can be translated to yield a protein of molecular weight 25,892 which displays significant homology with the oncogene protein Fos, yeast GCN4 protein, and sea urchin histone H1. Moreover, the putative cys-3 protein has a well-defined leucine zipper element plus an adjacent charged region which together may make up a DNA-binding site. A cys-3 mutant and a cys-3 temperature-sensitive mutant lead to substitutions of glutamine for basic amino acids within the charged region and thus may alter DNA-binding properties of the cys-3 protein.

scholarly journals A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase.

1988 ◽  
Vol 106 (4) ◽  
pp. 1171-1183 ◽  
Author(s):  
T Hirano ◽  
Y Hiraoka ◽  
M Yanagida
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Gradient Centrifugation ◽  
Metaphase Plate ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
In The Wild ◽  
Spindle Elongation ◽  
Mutant Cells

A temperature-sensitive mutant nuc2-663 of the fission yeast Schizosaccharomyces pombe specifically blocks mitotic spindle elongation at restrictive temperature so that nuclei in arrested cells contain a short uniform spindle (approximately 3-micron long), which runs through a metaphase plate-like structure consisting of three condensed chromosomes. In the wild-type or in the mutant cells at permissive temperature, the spindle is fully extended approximately 15-micron long in anaphase. The nuc2' gene was cloned in a 2.4-kb genomic DNA fragment by transformation, and its complete nucleotide sequence was determined. Its coding region predicts a 665-residues internally repeating protein (76.250 mol wt). By immunoblots using anti-sera raised against lacZ-nuc2+ fused proteins, a polypeptide (designated p67; 67,000 mol wt) encoded by nuc2+ is detected in the wild-type S. pombe extracts; the amount of p67 is greatly increased when multi-copy or high-expression plasmids carrying the nuc2+ gene are introduced into the S. pombe cells. Cellular fractionation and Percoll gradient centrifugation combined with immunoblotting show that p67 cofractionates with nuclei and is enriched in resistant structure that is insoluble in 2 M NaCl, 25 mM lithium 3,5'-diiodosalicylate, and 1% Triton but is soluble in 8 M urea. In nuc2 mutant cells, however, soluble p76, perhaps an unprocessed precursor, accumulates in addition to insoluble p67. The role of nuc2+ gene may be to interconnect nuclear and cytoskeletal functions in chromosome separation.

nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain

1990 ◽  
Vol 10 (3) ◽  
pp. 1056-1065
Author(s):  
Y H Fu ◽  
G A Marzluf
Keyword(s):  
Nucleotide Sequence ◽  
Dna Binding ◽  
Neurospora Crassa ◽  
Zinc Finger ◽  
Regulatory Gene ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Catabolic Enzymes ◽  
Regulatory Circuit ◽  
Putative Zinc Finger

The nitrogen regulatory circuit of Neurospora crassa consists of a set of unlinked structural genes which specify various nitrogen catabolic enzymes plus control genes and metabolic effectors which regulate their expression. The positive-acting nit-2 regulatory gene is required to turn on the expression of the nitrogen catabolic enzymes during conditions of nitrogen limitation. The complete nucleotide sequence of the nit-2 gene was determined. The nit-2 mRNA is 4.3 kilobases long and has a long nontranslated sequence at both its 5' and 3' ends. The nit-2 gene nucleotide sequence can be translated to yield a protein containing 1,036 amino acid residues with a molecular weight of approximately 110,000. Deletion analyses demonstrated that approximately 21% of the NIT2 protein at its carboxy terminus can be removed without loss of function. The nit-2 protein contains a single putative Cys2/Cys2 zinc finger domain which appears to function in DNA binding and which has striking homology to a mammalian trans-acting factor, GF-1.

scholarly journals SwoHp, a Nucleoside Diphosphate Kinase, Is Essential in Aspergillus nidulans

2003 ◽  
Vol 2 (6) ◽  
pp. 1169-1177 ◽  
Author(s):  
Xiaorong Lin ◽  
Cory Momany ◽  
Michelle Momany
Keyword(s):  
Aspergillus Nidulans ◽  
Elevated Temperatures ◽  
Nucleoside Diphosphate Kinase ◽  
Temperature Sensitive ◽  
Transferase Activity ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Cell Extracts ◽  
In The Wild ◽  
Chromosome Ii

ABSTRACT The temperature-sensitive swoH1 mutant of Aspergillus nidulans was previously identified in a screen for mutants with defects in polar growth. In the present work, we found that the swoH1 mutant swelled, lysed, and did not produce conidia during extended incubation at the restrictive temperature. When shifted from the permissive to the restrictive temperature, swoH1 showed the temperature-sensitive swelling phenotype only after 8 h at the higher temperature. The swoH gene was mapped to chromosome II and cloned by complementation of the temperature-sensitive phenotype. The sequence showed that swoH encodes a homologue of nucleoside diphosphate kinases (NDKs) from other organisms. Deletion experiments showed that the swoH gene is essential. A hemagglutinin-SwoHp fusion complemented the mutant phenotype, and the purified fusion protein possessed phosphate transferase activity in thin-layer chromatography assays. Sequencing of the mutant allele showed a predicted V83F change. Structural modeling suggested that the swoH1 mutation would lead to perturbation of the NDK active site. Crude cell extracts from the swoH1 mutant grown at the permissive temperature had ∼20% of the NDK activity seen in the wild type and did not show any decrease in activity when assayed at higher temperatures. Though the data are not conclusive, the lack of temperature-sensitive NDK activity in the swoH1 mutant raises the intriguing possibility that the SwoH NDK is required for growth at elevated temperatures rather than for polarity maintenance.

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