scholarly journals Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG.

1984 ◽  
Vol 4 (11) ◽  
pp. 2467-2478 ◽  
Author(s):  
R W West ◽  
R R Yocum ◽  
M Ptashne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Transcription Initiation ◽  
Function Analysis ◽  
Lacz Gene ◽  
Chromosome 2 ◽  
Proximal Half ◽  
Base Pair Deletion ◽  
Upstream Activating Sequence ◽  
Beta Galactosidase

The GAL1 and GAL10 genes, separated by 680 base pairs and divergently transcribed on chromosome 2 of Saccharomyces cerevisiae, were separately fused to the lacZ gene of Escherichia coli so that beta-galactosidase synthesis in S. cerevisiae reflected GAL1 and GAL10 promoter function. Analysis of two sets of deletions defined a 75-base-pair sequence, located ca. midway between the transcription initiation regions of GAL1 and GAL10, that mediates GAL4-dependent induction of both genes. Deletion of various parts of this sequence (called the GAL upstream activating sequence or UASG) reduced GAL1 and GAL10 induction about equally. Sequences in the GAL10-proximal half of UASG in some sequence contexts functioned independently of sequences in the GAL1-proximal half of UASG. A 33-base-pair deletion of the GAL10-proximal half of UASG drastically reduced induction. Deletions between UASG and the GAL1 TATA box caused beta-galactosidase to be synthesized at an unexpectedly high basal level, that is, in the absence of galactose and GAL4 product. Some of these mutations also reduced the repression caused by glucose.

Saccharomyces cerevisiae GAL1-GAL10 divergent promoter region: location and function of the upstream activating sequence UASG

1984 ◽  
Vol 4 (11) ◽  
pp. 2467-2478
Author(s):  
R W West ◽  
R R Yocum ◽  
M Ptashne
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Transcription Initiation ◽  
Function Analysis ◽  
Lacz Gene ◽  
Chromosome 2 ◽  
Proximal Half ◽  
Base Pair Deletion ◽  
Upstream Activating Sequence ◽  
Beta Galactosidase

The GAL1 and GAL10 genes, separated by 680 base pairs and divergently transcribed on chromosome 2 of Saccharomyces cerevisiae, were separately fused to the lacZ gene of Escherichia coli so that beta-galactosidase synthesis in S. cerevisiae reflected GAL1 and GAL10 promoter function. Analysis of two sets of deletions defined a 75-base-pair sequence, located ca. midway between the transcription initiation regions of GAL1 and GAL10, that mediates GAL4-dependent induction of both genes. Deletion of various parts of this sequence (called the GAL upstream activating sequence or UASG) reduced GAL1 and GAL10 induction about equally. Sequences in the GAL10-proximal half of UASG in some sequence contexts functioned independently of sequences in the GAL1-proximal half of UASG. A 33-base-pair deletion of the GAL10-proximal half of UASG drastically reduced induction. Deletions between UASG and the GAL1 TATA box caused beta-galactosidase to be synthesized at an unexpectedly high basal level, that is, in the absence of galactose and GAL4 product. Some of these mutations also reduced the repression caused by glucose.

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.

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.

scholarly journals Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

1984 ◽  
Vol 4 (8) ◽  
pp. 1440-1448 ◽  
Author(s):  
M Johnston ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Base Pair ◽  
Transcription Initiation ◽  
Base Pairs ◽  
Regulation Of Expression ◽  
Notable Feature ◽  
His3 Gene

The GAL1 and GAL10 genes of Saccharomyces cerevisiae are divergently transcribed, with 606 base pairs of DNA separating their transcription initiation sites. These two genes are stringently coregulated: their expression is induced ca. 1,000-fold in cells growing on galactose and is repressed by growth on glucose. The nucleotide sequence of the region of DNA between these genes and the precise sites of transcription initiation are presented here. The most notable feature of the nucleotide sequence of this region is a 108-base-pair guanine-plus-cytosine-rich stretch of DNA located approximately in the middle of the region between GAL1 and GAL10. Analysis of the effects of mutations that alter the region between these two genes, constructed in vitro or selected in vivo, suggest that these guanine-plus-cytosine-rich sequences are required for the expression of both genes. The region of DNA between GAL1 and GAL10 is sufficient for regulation of expression of these genes: fusion of the region to the yeast HIS3 gene places HIS3 under GAL control.

scholarly journals A common element involved in transcriptional regulation of two DNA alkylation repair genes (MAG and MGT1) of Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (12) ◽  
pp. 7213-7221 ◽  
Author(s):  
W Xiao ◽  
K K Singh ◽  
B Chen ◽  
L Samson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Alkylating Agents ◽  
Consensus Sequence ◽  
Common Element ◽  
Lacz Gene ◽  
Mrna Levels ◽  
Cis Acting ◽  
Upstream Activating Sequence ◽  
Repair Genes

The Saccharomyces cerevisiae MAG gene encodes a 3-methyladenine DNA glycosylase that protects cells from killing by alkylating agents. MAG mRNA levels are induced not only by alkylating agents but also by DNA-damaging agents that do not produce alkylated DNA. We constructed a MAG-lacZ gene fusion to help identify the cis-acting promoter elements involved in regulating MAG expression. Deletion analysis defined the presence of one upstream activating sequence and one upstream repressing sequence (URS) and suggested the presence of a second URS. One of the MAG URS elements matches a decamer consensus sequence present in the promoters of 11 other S. cerevisiae DNA repair and metabolism genes, including the MGT1 gene, which encodes an O6-methylguanine DNA repair methyltransferase. Two proteins of 26 and 39 kDa bind specifically to the MAG and MGT1 URS elements. We suggest that the URS-binding proteins may play an important role in the coordinate regulation of these S. cerevisiae DNA repair genes.

scholarly journals Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (10) ◽  
pp. 3502-3512 ◽  
Author(s):  
M C Brandriss ◽  
K A Krzywicki
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Regulatory Sequences ◽  
Lacz Gene ◽  
Amino Terminal ◽  
Proline Utilization ◽  
The Matrix ◽  
Matrix Compartment ◽  
P5c Dehydrogenase ◽  
Beta Galactosidase

delta 1-Pyrroline-5-carboxylate (P5C) dehydrogenase, the second enzyme in the proline utilization (Put) pathway of Saccharomyces cerevisiae and the product of the PUT2 gene, was localized to the matrix compartment by a mitochondrial fractionation procedure. This result was confirmed by demonstrating that the enzyme had limited activity toward an externally added substrate that could not penetrate the inner mitochondrial membrane (latency). To learn more about the nature of the import of this enzyme, three gene fusions were constructed that carried 5'-regulatory sequences through codons 14, 124, or 366 of the PUT2 gene ligated to the lacZ gene of Escherichia coli. When these fusions were introduced into S. cerevisiae either on multicopy plasmids or stably integrated into the genome, proline-inducible beta-galactosidase was made. The shortest gene fusion, PUT2-lacZ14, caused the production of a high level of beta-galactosidase that was found exclusively in the cytoplasm. The PUT2-lacZ124 and PUT2-lacZ366 fusions made lower levels of beta-galactosidases that were mitochondrially localized. Mitochondrial fractionation and protease-protection experiments showed that the PUT2-lacZ124 hybrid protein was located exclusively in the matrix, while the PUT2-lacZ366 hybrid was found in the matrix as well as the inner membrane. Thus, the amino-terminal 124 amino acids of P5C dehydrogenase carries sufficient information to target and deliver beta-galactosidase to the matrix compartment. The expression of the longer hybrids had deleterious effects on cell growth; PUT2-lacZ366-containing strains failed to grow on proline as the sole source of nitrogen. In the presence of the longest hybrid beta-galactosidase, the wild-type P5C dehydrogenase was still properly localized in the matrix compartment, but its activity was reduced. The nature of the effects of these hybrid proteins on cell growth is discussed.

Upstream elements repress premature expression of an Aspergillus developmental regulatory gene

1990 ◽  
Vol 10 (9) ◽  
pp. 4912-4919
Author(s):  
T H Adams ◽  
W E Timberlake
Keyword(s):  
Base Pair ◽  
Transcription Initiation ◽  
Regulatory Gene ◽  
Developmentally Appropriate ◽  
Lacz Gene ◽  
Developmentally Regulated ◽  
Dna Fragment ◽  
Flanking Region ◽  
Spatial Specificity ◽  
Similar Elements

The Aspergillus nidulans abaA gene regulates intermediate steps in asexual reproductive development and is itself developmentally regulated. An 822-base-pair DNA fragment from the abaA 5'-flanking region is sufficient to drive developmentally appropriate expression of the Escherichia coli lacZ gene. Deletion analysis showed that this fragment contains elements that repress transcription in vegetative cells and immature conidiophores and that activate transcription later during development. A 45-base-pair region encompassing the major and minor abaA transcription initiation sites contains directly repeated sequences related to the mammalian initiator (Inr) element (S. T. Smale and D. Baltimore, Cell 57:103-113, 1989). This element or sequences in the untranslated leader were sufficient for correct transcription initiation and for measurable developmental induction. Similar elements were present at or near the initiation sites of other developmentally regulated genes. We propose that the temporal and spatial specificity of expression of these genes results from modulation of the activity of Inr elements.

Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (10) ◽  
pp. 3502-3512
Author(s):  
M C Brandriss ◽  
K A Krzywicki
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Regulatory Sequences ◽  
Lacz Gene ◽  
Amino Terminal ◽  
Proline Utilization ◽  
The Matrix ◽  
Matrix Compartment ◽  
P5c Dehydrogenase ◽  
Beta Galactosidase

delta 1-Pyrroline-5-carboxylate (P5C) dehydrogenase, the second enzyme in the proline utilization (Put) pathway of Saccharomyces cerevisiae and the product of the PUT2 gene, was localized to the matrix compartment by a mitochondrial fractionation procedure. This result was confirmed by demonstrating that the enzyme had limited activity toward an externally added substrate that could not penetrate the inner mitochondrial membrane (latency). To learn more about the nature of the import of this enzyme, three gene fusions were constructed that carried 5'-regulatory sequences through codons 14, 124, or 366 of the PUT2 gene ligated to the lacZ gene of Escherichia coli. When these fusions were introduced into S. cerevisiae either on multicopy plasmids or stably integrated into the genome, proline-inducible beta-galactosidase was made. The shortest gene fusion, PUT2-lacZ14, caused the production of a high level of beta-galactosidase that was found exclusively in the cytoplasm. The PUT2-lacZ124 and PUT2-lacZ366 fusions made lower levels of beta-galactosidases that were mitochondrially localized. Mitochondrial fractionation and protease-protection experiments showed that the PUT2-lacZ124 hybrid protein was located exclusively in the matrix, while the PUT2-lacZ366 hybrid was found in the matrix as well as the inner membrane. Thus, the amino-terminal 124 amino acids of P5C dehydrogenase carries sufficient information to target and deliver beta-galactosidase to the matrix compartment. The expression of the longer hybrids had deleterious effects on cell growth; PUT2-lacZ366-containing strains failed to grow on proline as the sole source of nitrogen. In the presence of the longest hybrid beta-galactosidase, the wild-type P5C dehydrogenase was still properly localized in the matrix compartment, but its activity was reduced. The nature of the effects of these hybrid proteins on cell growth is discussed.

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