scholarly journals A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box.

1988 ◽  
Vol 8 (11) ◽  
pp. 4634-4641 ◽  
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Gene Fusion ◽  
Regulatory Gene ◽  
Regulatory Region ◽  
Tata Box ◽  
Homologous Gene ◽  
Lacz Gene ◽  
Proline Utilization ◽  
Constitutive Mutation ◽  
Necessary And Sufficient ◽  
Utilization Pathway

Deletion analysis of the promoter of the PUT2 gene that functions in the proline utilization pathway of Saccharomyces cerevisiae identified a PUT2 upstream activation site (UAS). It is contained within a single 40-base-pair (bp) region located immediately upstream of the TATA box and is both necessary and sufficient for proline induction. When placed upstream of a CYC7-lacZ gene fusion, the 40-bp sequence conferred proline regulation on CYC7-lacZ. A 35-bp deletion within the PUT2 UAS in an otherwise intact PUT2 promoter resulted in noninducible expression of a PUT2-lacZ gene fusion. When a plasmid bearing this UAS-deleted promoter was placed in a strain carrying a constitutive mutation in the positive regulatory gene PUT3, expression of PUT2-lacZ was not constitutive but occurred at levels below those found under noninducing conditions. In heterologous as well as homologous gene fusions, the PUT2 UAS appeared to be responsible for uninduced as well as proline-induced levels of expression. Although located immediately adjacent to the PUT2 UAS, the TATA box did not appear to play a regulatory role, as indicated by the results of experiments in which it was replaced by the CYC7 TATA box. A 26-bp sequence containing this TATA box was critical to the expression of PUT2, since a deletion of this region completely abolished transcriptional activity of the gene under both inducing and noninducing conditions. Our results indicate that the PUT2 promoter has a comparatively simple structure, requiring UAS and TATA sequences as well as the PUT3 gene product (directly or indirectly) for its expression.

A regulatory region responsible for proline-specific induction of the yeast PUT2 gene is adjacent to its TATA box

1988 ◽  
Vol 8 (11) ◽  
pp. 4634-4641
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Gene Fusion ◽  
Regulatory Gene ◽  
Regulatory Region ◽  
Tata Box ◽  
Homologous Gene ◽  
Lacz Gene ◽  
Proline Utilization ◽  
Constitutive Mutation ◽  
Necessary And Sufficient ◽  
Utilization Pathway

Deletion analysis of the promoter of the PUT2 gene that functions in the proline utilization pathway of Saccharomyces cerevisiae identified a PUT2 upstream activation site (UAS). It is contained within a single 40-base-pair (bp) region located immediately upstream of the TATA box and is both necessary and sufficient for proline induction. When placed upstream of a CYC7-lacZ gene fusion, the 40-bp sequence conferred proline regulation on CYC7-lacZ. A 35-bp deletion within the PUT2 UAS in an otherwise intact PUT2 promoter resulted in noninducible expression of a PUT2-lacZ gene fusion. When a plasmid bearing this UAS-deleted promoter was placed in a strain carrying a constitutive mutation in the positive regulatory gene PUT3, expression of PUT2-lacZ was not constitutive but occurred at levels below those found under noninducing conditions. In heterologous as well as homologous gene fusions, the PUT2 UAS appeared to be responsible for uninduced as well as proline-induced levels of expression. Although located immediately adjacent to the PUT2 UAS, the TATA box did not appear to play a regulatory role, as indicated by the results of experiments in which it was replaced by the CYC7 TATA box. A 26-bp sequence containing this TATA box was critical to the expression of PUT2, since a deletion of this region completely abolished transcriptional activity of the gene under both inducing and noninducing conditions. Our results indicate that the PUT2 promoter has a comparatively simple structure, requiring UAS and TATA sequences as well as the PUT3 gene product (directly or indirectly) for its expression.

scholarly journals Evidence for Positive Regulation of the Proline Utilization Pathway in Saccharomyces cerevisiae

Genetics ◽  
1987 ◽  
Vol 117 (3) ◽  
pp. 429-435
Author(s):  
Marjorie C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Source ◽  
Sole Source ◽  
Lacz Gene ◽  
Wild Type ◽  
Proline Utilization ◽  
Constitutive Mutation ◽  
Lacz Gene Fusions ◽  
Utilization Pathway ◽  
Gene Maps

ABSTRACT A mutation has been identified that prevents Saccharomyces cerevisiae cells from growing on proline as the sole source of nitrogen, causes noninducible expression of the PUT1 and PUT2 genes, and is completely recessive. In the put3-75 mutant, the basal level of expression (ammonia as nitrogen source) of PUT1-lacZ and PUT2-lacZ gene fusions as measured by β-galactosidase activity is reduced 4- and 7-fold, respectively, compared with the wild-type strain. Normal regulation is not restored when the cells are grown on arginine as the sole nitrogen source and put3-75 cells remain sensitive to the proline analog, l-azetidine-2-carboxylic acid, indicating that the block is not at the level of transport of the inducer, proline. In a cross between the put3-75 strain and the semidominant, constitutive mutation PUT3c-68, only parental ditype tetrads were found, indicating allelism of the two mutations. Further support for allelism derives from the comparison of enzyme levels in heteroallelic and heterozygous diploid strains. The constitutive allele appears to be fully dominant to the noninducible allele but only partially dominant to the wild type, suggesting an interaction between the wild-type and PUT3c-68 gene products. The PUT3 gene maps on chromosome XI, about 5.7 cM from the centromere. The phenotypes of alleles of the PUT3 gene, either recessive and noninducible (the put3-75 phenotype) or semidominant and constitutive (the PUT3c-68 phenotype), and their pleiotropy suggest that the PUT3 gene product is a positive activator of the proline utilization pathway.

Carbon catabolite repression in Aspergillus nidulans: a review

1995 ◽  
Vol 73 (S1) ◽  
pp. 160-166 ◽  
Author(s):  
Claudio Scazzocchio ◽  
Victoria Gavrias ◽  
Beatriz Cubero ◽  
Cristina Panozzo ◽  
Martine Mathieu ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Aspergillus Nidulans ◽  
Catabolite Repression ◽  
Carbon Catabolite Repression ◽  
Regulatory Gene ◽  
Experimental Methodology ◽  
Structural Genes ◽  
General Transcription Factor ◽  
Proline Utilization ◽  
Utilization Pathway

We describe the experimental methodology that led to the discovery of the creA gene in Aspergillus nidulans. This gene codes for a transcriptional repressor mediating carbon catabolite repression in many pathways in this organism. We compare both the mode and the mechanism of action in two pathways subject to CreA-mediated repression. The genes comprising the ethanol regulon are subject to carbon catabolite repression independently of the nitrogen source, while the genes involved in proline utilization are repressed by glucose only when a repressing nitrogen source is also present. In the ethanol regulon, CreA drastically represses the expression of the positive regulatory gene alcR, thus preventing the expression of the structural genes. Direct repression of the structural genes is also existant. In the proline utilization pathway, repression operates directly at the level of the structural genes. In the ethanol regulon, CreA prevents the self-induction of alcR and the induction of the structural genes by competing with the binding of the AlcR protein. In proline gene cluster, CreA does not interfere with induction mediated by PrnA but with the activity of an unknown and more general transcription factor. Key words: carbon catabolite repression, ascomycetes, Zn fingers.

Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator

1989 ◽  
Vol 9 (11) ◽  
pp. 4696-4705
Author(s):  
J E Marczak ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Analysis ◽  
Gene Fusion ◽  
Target Genes ◽  
Constitutive Expression ◽  
Transcriptional Activator ◽  
Growth Defects ◽  
Proline Utilization ◽  
Rna Accumulation ◽  
Utilization Pathway

The enzymes of the proline utilization pathway (the products of the PUT1 and PUT2 genes) in Saccharomyces cerevisiae are coordinately regulated by proline and the PUT3 transcriptional activator. To learn more about the control of this pathway, constitutive mutations in PUT3 as well as in other regulators were sought. A scheme using a gene fusion between PUT1 (S. cerevisiae proline oxidase) and galK (Escherichia coli galactokinase) was developed to select directly for constitutive mutations affecting the PUT1 promoter. These mutations were secondarily screened for their effects in trans on the promoter of the PUT2 (delta 1-pyrroline-5-carboxylate dehydrogenase) gene by using a PUT2-lacZ (E. coli beta-galactosidase) gene fusion. Three different classes of mutations were isolated. The major class consisted of semidominant constitutive PUT3 mutations that caused PUT2-lacZ expression to vary from 2 to 22 times the uninduced level. A single dominant mutation in a new locus called PUT5 resulted in low-level constitutive expression of PUT2-lacZ; this mutation was epistatic to the recessive, noninducible put3-75 allele. Recessive constitutive mutations were isolated that had pleiotropic growth defects; it is possible that these mutations are not specific to the proline utilization pathway but may be in genes that control several pathways. Since the PUT3 gene appears to have a major role in the regulation of this pathway, a molecular analysis was undertaken. This gene was cloned by functional complementation of the put3-75 mutation. Strains carrying a complete deletion of this gene are viable, proline nonutilizing, and indistinguishable in phenotype from the original put3-75 allele. The PUT3 gene encodes a 2.8-kilobase-pair transcript that is not regulated by proline at the level of RNA accumulation. The presence of the gene on a high-copy-number plasmid did not alter the regulation of one of its target genes, PUT2-lacZ, suggesting that the PUT3 gene product is not limiting and that a titratable repressor is not involved in the regulation of this pathway.

scholarly journals Isolation of constitutive mutations affecting the proline utilization pathway in Saccharomyces cerevisiae and molecular analysis of the PUT3 transcriptional activator.

1989 ◽  
Vol 9 (11) ◽  
pp. 4696-4705 ◽  
Author(s):  
J E Marczak ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Analysis ◽  
Gene Fusion ◽  
Target Genes ◽  
Constitutive Expression ◽  
Transcriptional Activator ◽  
Growth Defects ◽  
Proline Utilization ◽  
Rna Accumulation ◽  
Utilization Pathway

The enzymes of the proline utilization pathway (the products of the PUT1 and PUT2 genes) in Saccharomyces cerevisiae are coordinately regulated by proline and the PUT3 transcriptional activator. To learn more about the control of this pathway, constitutive mutations in PUT3 as well as in other regulators were sought. A scheme using a gene fusion between PUT1 (S. cerevisiae proline oxidase) and galK (Escherichia coli galactokinase) was developed to select directly for constitutive mutations affecting the PUT1 promoter. These mutations were secondarily screened for their effects in trans on the promoter of the PUT2 (delta 1-pyrroline-5-carboxylate dehydrogenase) gene by using a PUT2-lacZ (E. coli beta-galactosidase) gene fusion. Three different classes of mutations were isolated. The major class consisted of semidominant constitutive PUT3 mutations that caused PUT2-lacZ expression to vary from 2 to 22 times the uninduced level. A single dominant mutation in a new locus called PUT5 resulted in low-level constitutive expression of PUT2-lacZ; this mutation was epistatic to the recessive, noninducible put3-75 allele. Recessive constitutive mutations were isolated that had pleiotropic growth defects; it is possible that these mutations are not specific to the proline utilization pathway but may be in genes that control several pathways. Since the PUT3 gene appears to have a major role in the regulation of this pathway, a molecular analysis was undertaken. This gene was cloned by functional complementation of the put3-75 mutation. Strains carrying a complete deletion of this gene are viable, proline nonutilizing, and indistinguishable in phenotype from the original put3-75 allele. The PUT3 gene encodes a 2.8-kilobase-pair transcript that is not regulated by proline at the level of RNA accumulation. The presence of the gene on a high-copy-number plasmid did not alter the regulation of one of its target genes, PUT2-lacZ, suggesting that the PUT3 gene product is not limiting and that a titratable repressor is not involved in the regulation of this pathway.

scholarly journals A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast

Genetics ◽  
1987 ◽  
Vol 117 (1) ◽  
pp. 5-12
Author(s):  
Eric Alani ◽  
Nancy Kleckner
Keyword(s):  
Gene Fusion ◽  
Enzyme Assay ◽  
Decarboxylase Activity ◽  
Lacz Gene ◽  
Coding Region ◽  
Ura3 Gene ◽  
Promoter Sequences ◽  
E Coli ◽  
New Type ◽  
Fusion Analysis

ABSTRACT We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5′-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.

Mutations Causing Constitutive Invertase Synthesis in Yeast: Genetic Interactions with snf Mutations

Genetics ◽  
1987 ◽  
Vol 115 (2) ◽  
pp. 247-253
Author(s):  
Lenore Neigeborn ◽  
Marian Carlson
Keyword(s):  
Regulatory Region ◽  
Constitutive Expression ◽  
Genetic Interactions ◽  
The Third ◽  
Recessive Mutations ◽  
Constitutive Mutation ◽  
Complementation Groups ◽  
High Level ◽  
Yeast Genetic ◽  
Very High

ABSTRACT We have selected 210 mutants able to grow on sucrose in the presence of 2-deoxyglucose. We identified recessive mutations in three major complementation groups that cause constitutive (glucose-insensitive) secreted invertase synthesis. Two groups comprise alleles of the previously identified HXK2 and REG1 genes, and the third group was designated cid1 (constitutive invertase derepression). The effect of cid1 on SUC2 expression is mediated by the SUC2 upstream regulatory region, as judged by the constitutive expression of a SUC2-LEU2-lacZ fusion in which the LEU2 promoter is under control of SUC2 upstream sequences. A cid1 mutation also causes glucose-insensitive expression of maltase. The previously isolated constitutive mutation ssn6 is epistatic to cid1, reg1 and hxk2 for very high level constitutive invertase expression. Mutations in SNF genes that prevent derepression of invertase are epistatic to cid1, reg1 and hxk2; we have previously shown that ssn6 has different epistasis relationships with snf mutations. The constitutive mutation tup1 was found to resemble ssn6 in its genetic interactions with snf mutations. These findings suggest that CID1, REG1 and HXK2 are functionally distinct from SSN6 and TUP1.

scholarly journals Determination of amino acids misincorporation in recombinant protein by mass spectrometry

2013 ◽  
Vol 42 (1) ◽  
pp. 11-19 ◽  
Author(s):  
MZ Alam ◽  
L Regioneiri ◽  
MAS Santos
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Recombinant Protein ◽  
Gene Fusion ◽  
Aminoglycoside Antibiotic ◽  
Protein Functionality ◽  
Lacz Gene ◽  
Host Organism ◽  
E Coli

The synthesis of protein according to genetic code of a gene determines the basis of life and a stable proteome is necessary for cell homeostatis. However, errors occur naturally during translation of protein from its mRNA, which varies from 10-3 to 10-4 per codon. These errors are more frequent in recombinant protein overexpressed in heterologous hosts and affect protein functionality. The increasing amount of nonfunctional protein is often related to mistranslation of a gene under stress. In the present study, Saccharomyces cerevisiae as a host organism to overexpress E. coli lacZ gene fusion with GST to quantify misincorporation of amino acid in GST-? galactosidase recombinant protein. The yeast was treated with various stressors such as ethanol, chromium (CrO3), and aminoglycoside antibiotic - geneticin (G418) to induce protein aggregation. The misincorporation of amino acids was studied in soluble protein fractions by mass-spectrometry to determine how much misincorporation occur. We found that under experimental stress conditions the misincorporation of amino acids ranges from 5.6 ×10-3 to 8 × 10-3, which represents 60-80 fold higher than reported level. DOI: http://dx.doi.org/10.3329/bjas.v42i1.15760 Bang. J. Anim. Sci. 2013. 42 (1): 11-19

scholarly journals Molecular genetics of cryptopleurine resistance in Saccharomyces cerevisiae: expression of a ribosomal protein gene family.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 719-730
Author(s):  
A G Paulovich ◽  
J R Thompson ◽  
J C Larkin ◽  
Z Li ◽  
J L Woolford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Gene Fusion ◽  
Null Allele ◽  
Ribosomal Subunit ◽  
Null Alleles ◽  
Protein Synthesis Inhibitor ◽  
Lacz Gene ◽  
Ribosomal Subunits ◽  
Cry1 Gene

Abstract The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-delta 1::URA3 null allele is viable, cryptopleurine sensitive (CryS), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage lambda, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-delta 2::TRP1 cry2-delta 1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into CryR yeast of genotype cry1 CRY2 confers a CryS phenotype. Transformation of these CryR yeast with CRY2 on a low copy CEN plasmid does not confer a CryS phenotype. (2) Haploids containing the cry1-delta 2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-delta 1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of beta-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the CryR phenotype of cry2 mutants is only expressed in strains containing a cry1-delta null allele.

A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs

Development ◽  
1991 ◽  
Vol 112 (3) ◽  
pp. 807-811 ◽  
Author(s):  
K. Schughart ◽  
C.J. Bieberich ◽  
R. Eid ◽  
F.H. Ruddle
Keyword(s):  
Gene Expression ◽  
Transgenic Mouse ◽  
Reporter Gene ◽  
Developmental Stages ◽  
Regulatory Region ◽  
Homeobox Gene ◽  
Regulatory Elements ◽  
Body Region ◽  
Lacz Gene ◽  
Mouse Lines

To characterize cis-acting regulatory elements of the murine homeobox gene, Hox-2.2, transgenic mouse lines were generated that contained the LacZ reporter gene under the control of different fragments from the presumptive Hox-2.2 promoter. A promoter region of 3600 base pairs (bp) was identified, which reproducibly directed reporter gene expression into specific regions of developing mouse embryos. At 8.5 days postcoitum (p.c.) reporter gene activity was detected in posterior regions of the lateral mesoderm and, in subsequent developmental stages, expression of the LacZ gene was restricted to specific regions of the developing limb buds and the mesenchyme of the ventrolateral body region. This pattern of Hox-2.2-LacZ expression was found in all transgenic embryos that have been generated with the 3.6 kb promoter fragment (two founder embryos and embryos from five transgenic lines). In addition, embryos from two transgenic mouse lines expressed the reporter gene at low levels in the developing central nervous system (CNS). Our results are consistent with the idea that in addition to their presumptive role in CNS and vertebrae development, Hox-2.2 gene products are involved in controlling pattern formation in developing limbs.

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