Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae

1983 ◽  
Vol 3 (9) ◽  
pp. 1625-1633
Author(s):  
D B Finkelstein ◽  
S Strausberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Plasmid Vector ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Inducible Synthesis ◽  
Beta Galactosidase

The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function. We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S. cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene. When these fusions are introduced into S. cerevisiae they direct the synthesis of unique truncated hsp90 proteins. By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site. This information allowed us to design and construct a well-defined in-frame fusion between the S. cerevisiae HSP90 gene and the Escherichia coli lacZ gene. When this fused gene is introduced into S. cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase. Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay.

scholarly journals Heat shock-regulated production of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae.

1983 ◽  
Vol 3 (9) ◽  
pp. 1625-1633 ◽  
Author(s):  
D B Finkelstein ◽  
S Strausberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Plasmid Vector ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Inducible Synthesis ◽  
Beta Galactosidase

The HSP90 gene of the yeast Saccharomyces cerevisiae encodes a heat shock-inducible protein with an Mr of 90,000 (hsp90) and unknown function. We fused DNA fragments of a known sequence (namely, either end of a 1.4-kilobase EcoRI fragment which contains the S. cerevisiae TRP1 gene) to an EcoRI site within the coding sequence of the HSP90 gene. When these fusions are introduced into S. cerevisiae they direct the synthesis of unique truncated hsp90 proteins. By determining the size and charge of these proteins we were able to deduce the translational reading frame at the (EcoRI) fusion site. This information allowed us to design and construct a well-defined in-frame fusion between the S. cerevisiae HSP90 gene and the Escherichia coli lacZ gene. When this fused gene is introduced into S. cerevisiae on a multicopy plasmid vector, it directs the heat shock-inducible synthesis of a fused protein, which is an enzymatically active beta-galactosidase. Thus, for the first time, it is possible to quantitate the heat shock response in a eucaryotic organism with a simple enzyme assay.

scholarly journals The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters.

1991 ◽  
Vol 11 (7) ◽  
pp. 3804-3813 ◽  
Author(s):  
D A Lewis ◽  
L F Bisson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Significant Proportion ◽  
Growth Defect ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Hexose Transport ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Affinity ◽  
Membrane Spanning

Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified. The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes. Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport. HXT1 was unable to complement the snf3 growth defect in low copy number. The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains. The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2. Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes. Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth. Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport.

Identification of the upstream activating sequence of MAL and the binding sites for the MAL63 activator of Saccharomyces cerevisiae

1990 ◽  
Vol 10 (7) ◽  
pp. 3797-3800
Author(s):  
B F Ni ◽  
R B Needleman
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Lacz Gene ◽  
Maltose Permease ◽  
Upstream Activating Sequence ◽  
The Subject ◽  
Dna Fragment ◽  
Mal Genes ◽  
Beta Galactosidase

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a complex of genes involved in maltose metabolism; the complex includes maltase, a maltose permease, and an activator of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. While the MAL6 locus has been the subject of numerous studies, the binding sites of the MAL63 activator have not been determined. In this study, we used Escherichia coli extracts containing the MAL63 protein to define the binding sites of the MAL63 protein in the divergently transcribed MAL61-62 promotor. When a DNA fragment containing these sites was placed upstream of a CYC1-lacZ gene, maltose induced beta-galactosidase. These sites therefore constitute an upstream activating sequence for the MAL genes.

Proline utilization in Saccharomyces cerevisiae: sequence, regulation, and mitochondrial localization of the PUT1 gene product

1987 ◽  
Vol 7 (12) ◽  
pp. 4431-4440
Author(s):  
S S Wang ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Lacz Gene ◽  
Oxygen Regulation ◽  
Reading Frame ◽  
Proline Utilization ◽  
Beta Galactosidase

The PUT1 gene of Saccharomyces cerevisiae, believed to encode proline oxidase, has been completely sequenced and contains an open reading frame capable of encoding a polypeptide of 476 amino acids in length. The amino terminus of the protein deduced from the DNA sequence has a characteristic mitochondrial import signal; two PUT1-lacZ gene fusions were constructed that produced mitochondrially localized beta-galactosidase in vivo. The transcription initiation and termination sites of the PUT1 mRNA were determined. By using a PUT1-lacZ gene fusion that makes a cytoplasmic beta-galactosidase, the regulation of the PUT1 gene was studied. PUT1 is inducible by proline, responds only slightly to carbon catabolite repression, and is not regulated by the cytochrome activator proteins HAP1 and HAP2. The PUT1 gene is under oxygen regulation; expression in anaerobically grown cells is 10-fold lower than in aerobically grown cells. Oxygen regulation is abolished when cells are respiratory deficient. PUT1 expression in a [rho-] strain grown either aerobically or anaerobically is as high as that seen in a [rho+] strain grown aerobically. Studies on PUT1 promoter deletions define a region between positions -458 and -293 from the translation initiation site that is important for full expression of the PUT1 gene and required for oxygen regulation.

Regulation of expression and activity of the yeast transcription factor ADR1

1988 ◽  
Vol 8 (5) ◽  
pp. 1868-1876
Author(s):  
H Blumberg ◽  
T A Hartshorne ◽  
E T Young
Keyword(s):  
Carbon Source ◽  
Fusion Protein ◽  
Glucose Repression ◽  
Regulatory Gene ◽  
Lacz Gene ◽  
Mrna Levels ◽  
Multicopy Plasmid ◽  
Regulation Of Expression ◽  
Yeast Saccharomyces Cerevisiae ◽  
Beta Galactosidase

Disruption of ADR1, a positive regulatory gene in the yeast Saccharomyces cerevisiae, abolished derepression of ADH2 but did not affect glucose repression of ADH2 or cell viability. The ADR1 mRNA was 5 kilobases long and had an unusually long leader containing 509 nucleotides. ADR1 mRNA levels were regulated by the carbon source in a strain-dependent fashion. beta-Galactosidase levels measured in strains carrying an ADR1-lacZ gene fusion paralleled ADR1 and ADR1-lacZ mRNA levels, indicating a lack of translational regulation of ADR1 mRNA. ADH2 was regulated by the carbon source to the same extent in all strains examined and showed complete dependence on ADR1 as well. The expression of ADR1 mRNA and an ADR1-beta-galactosidase fusion protein during glucose repression suggested that the activity of the ADR1 protein is regulated at the posttranslational level to properly regulate ADH2 expression. The ADR1-beta-galactosidase fusion protein was able to activate ADH2 expression during glucose repression but showed significantly higher levels of activation upon derepression. A similar result was obtained when ADR1 was present on a multicopy plasmid. These results suggest that low-level expression of ADR1 is required to maintain glucose repression of ADH2 and are consistent with the hypothesis that ADR1 is regulated at the posttranslational level.

The HXT1 gene product of Saccharomyces cerevisiae is a new member of the family of hexose transporters

1991 ◽  
Vol 11 (7) ◽  
pp. 3804-3813 ◽  
Author(s):  
D A Lewis ◽  
L F Bisson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Significant Proportion ◽  
Growth Defect ◽  
Lacz Gene ◽  
Multicopy Plasmid ◽  
Hexose Transport ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Affinity ◽  
Membrane Spanning

Two novel genes affecting hexose transport in the yeast Saccharomyces cerevisiae have been identified. The gene HXT1 (hexose transport), isolated from plasmid pSC7, was sequenced and found to encode a hydrophobic protein which is highly homologous to the large family of sugar transporter proteins from eucaryotes and procaryotes. Multicopy expression of the HXT1 gene restored high-affinity glucose transport to the snf3 mutant, which is deficient in a significant proportion of high-affinity glucose transport. HXT1 was unable to complement the snf3 growth defect in low copy number. The HXT1 protein was found to contain 12 putative membrane-spanning domains with a central hydrophilic domain and hydrophilic N- and C-terminal domains. The HXT1 protein is 69% identical to GAL2 and 66% identical to HXT2, and all three proteins were found to have a putative leucine zipper motif at a consensus location in membrane-spanning domain 2. Disruption of the HXT1 gene resulted in loss of a portion of high-affinity glucose and mannose transport, and wild-type levels of transport required both the HXT1 and SNF3 genes. Unexpectedly, expression of beta-galactosidase activity by using a fusion of the lacZ gene to the HXT1 promoter in a multicopy plasmid was maximal during lag and early exponential phases of growth, decreasing approximately 100-fold upon further entry into exponential growth. Deletion analysis of pSC7 revealed the presence of another gene (called ORF2) capable of suppressing the snf3 null mutant phenotype by restoring high-affinity glucose transport and increased low-affinity transport.

scholarly journals Regulation of expression and activity of the yeast transcription factor ADR1.

1988 ◽  
Vol 8 (5) ◽  
pp. 1868-1876 ◽  
Author(s):  
H Blumberg ◽  
T A Hartshorne ◽  
E T Young
Keyword(s):  
Carbon Source ◽  
Fusion Protein ◽  
Glucose Repression ◽  
Regulatory Gene ◽  
Lacz Gene ◽  
Mrna Levels ◽  
Multicopy Plasmid ◽  
Regulation Of Expression ◽  
Yeast Saccharomyces Cerevisiae ◽  
Beta Galactosidase

Disruption of ADR1, a positive regulatory gene in the yeast Saccharomyces cerevisiae, abolished derepression of ADH2 but did not affect glucose repression of ADH2 or cell viability. The ADR1 mRNA was 5 kilobases long and had an unusually long leader containing 509 nucleotides. ADR1 mRNA levels were regulated by the carbon source in a strain-dependent fashion. beta-Galactosidase levels measured in strains carrying an ADR1-lacZ gene fusion paralleled ADR1 and ADR1-lacZ mRNA levels, indicating a lack of translational regulation of ADR1 mRNA. ADH2 was regulated by the carbon source to the same extent in all strains examined and showed complete dependence on ADR1 as well. The expression of ADR1 mRNA and an ADR1-beta-galactosidase fusion protein during glucose repression suggested that the activity of the ADR1 protein is regulated at the posttranslational level to properly regulate ADH2 expression. The ADR1-beta-galactosidase fusion protein was able to activate ADH2 expression during glucose repression but showed significantly higher levels of activation upon derepression. A similar result was obtained when ADR1 was present on a multicopy plasmid. These results suggest that low-level expression of ADR1 is required to maintain glucose repression of ADH2 and are consistent with the hypothesis that ADR1 is regulated at the posttranslational level.

scholarly journals Identification of the upstream activating sequence of MAL and the binding sites for the MAL63 activator of Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (7) ◽  
pp. 3797-3800 ◽  
Author(s):  
B F Ni ◽  
R B Needleman
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Binding Sites ◽  
Lacz Gene ◽  
Maltose Permease ◽  
Upstream Activating Sequence ◽  
The Subject ◽  
Dna Fragment ◽  
Mal Genes ◽  
Beta Galactosidase

Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a complex of genes involved in maltose metabolism; the complex includes maltase, a maltose permease, and an activator of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. While the MAL6 locus has been the subject of numerous studies, the binding sites of the MAL63 activator have not been determined. In this study, we used Escherichia coli extracts containing the MAL63 protein to define the binding sites of the MAL63 protein in the divergently transcribed MAL61-62 promotor. When a DNA fragment containing these sites was placed upstream of a CYC1-lacZ gene, maltose induced beta-galactosidase. These sites therefore constitute an upstream activating sequence for the MAL genes.

scholarly journals Proline utilization in Saccharomyces cerevisiae: sequence, regulation, and mitochondrial localization of the PUT1 gene product.

1987 ◽  
Vol 7 (12) ◽  
pp. 4431-4440 ◽  
Author(s):  
S S Wang ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Lacz Gene ◽  
Oxygen Regulation ◽  
Reading Frame ◽  
Proline Utilization ◽  
Beta Galactosidase

The PUT1 gene of Saccharomyces cerevisiae, believed to encode proline oxidase, has been completely sequenced and contains an open reading frame capable of encoding a polypeptide of 476 amino acids in length. The amino terminus of the protein deduced from the DNA sequence has a characteristic mitochondrial import signal; two PUT1-lacZ gene fusions were constructed that produced mitochondrially localized beta-galactosidase in vivo. The transcription initiation and termination sites of the PUT1 mRNA were determined. By using a PUT1-lacZ gene fusion that makes a cytoplasmic beta-galactosidase, the regulation of the PUT1 gene was studied. PUT1 is inducible by proline, responds only slightly to carbon catabolite repression, and is not regulated by the cytochrome activator proteins HAP1 and HAP2. The PUT1 gene is under oxygen regulation; expression in anaerobically grown cells is 10-fold lower than in aerobically grown cells. Oxygen regulation is abolished when cells are respiratory deficient. PUT1 expression in a [rho-] strain grown either aerobically or anaerobically is as high as that seen in a [rho+] strain grown aerobically. Studies on PUT1 promoter deletions define a region between positions -458 and -293 from the translation initiation site that is important for full expression of the PUT1 gene and required for oxygen regulation.

Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1590-1598
Author(s):  
M Patterson ◽  
R A Sclafani ◽  
W L Fangman ◽  
J Rosamond
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genomic Dna ◽  
Genetic Recombination ◽  
Multiple Roles ◽  
Predicted Amino Acid Sequence ◽  
Cell Cycle Gene ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Reading Frame ◽  
Genomic Insert

The product of the CDC7 gene of Saccharomyces cerevisiae appears to have multiple roles in cellular physiology. It is required for the initiation of mitotic DNA synthesis. While it is not required for the initiation of meiotic DNA replication, it is necessary for genetic recombination during meiosis and for the formation of ascospores. It has also been implicated in an error-prone DNA repair pathway. Plasmids capable of complementing temperature-sensitive cdc7 mutations were isolated from libraries of yeast genomic DNA in the multicopy plasmid vectors YRp7 and YEp24. The complementing activity was localized within a 3.0-kilobase genomic DNA fragment. Genetic studies that included integration of the genomic insert at or near the CDC7 locus and marker rescue of four cdc7 alleles proved that the cloned fragment contains the yeast chromosomal CDC7 gene. The RNA transcript of CDC7 is about 1,700 nucleotides. Analysis of the nucleotide sequence of a 2.1-kilobase region of the cloned fragment revealed the presence of an open reading frame of 1,521 nucleotides that is presumed to encode the CDC7 protein. Depending on which of two possible ATG codons initiates translation, the calculated size of the CDC7 protein is 58.2 or 56 kilodaltons. Comparison of the predicted amino acid sequence of the CDC7 gene product with other known protein sequences suggests that CDC7 encodes a protein kinase.

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