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Query: UNIPROT:P06889 (Mol)
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The Saccharomyces cerevisiae GPA1, STE4, and STE18 genes encode products homologous to mammalian G-protein alpha, beta, and gamma subunits, respectively. All three genes function in the transduction of the signal generated by mating pheromone in haploid cells. To characterize more completely the role of these genes in mating, we have conditionally overexpressed GPA1, STE4, and STE18, using the galactose-inducible GAL1 promoter. Overexpression of STE4 alone, or STE4 together with STE18, generated a response in haploid cells suggestive of pheromone signal transduction: arrest in G1 of the cell cycle, formation of cellular projections, and induction of the pheromone-inducible transcript FUS1 25- to 70-fold. High-level STE18 expression alone had none of these effects, nor did overexpression of STE4 in a MATa/alpha diploid. However, STE18 was essential for the response, since overexpression of STE4 was unable to activate a response in a ste18 null strain. GPA1 hyperexpression suppressed the phenotype of STE4 overexpression. In addition, cells that overexpressed GPA1 were more resistant to pheromone and recovered more quickly from pheromone than did wild-type cells, which suggests that GPA1 may function in an adaptation response to pheromone.
Mol Cell Biol 1990 Feb
PMID:Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway. 210 53

The yeast GAL1 and GAL10 genes are transcribed at a remarkably low basal level when galactose is unavailable and are induced by over 4 orders of magnitude when it becomes available. Approximately six negative control elements (designated GAL operators GALO1 to GALO6) are located adjacent to or overlapping four binding sites for the transcription activator GAL4 in the GAL upstream activating sequence UASG. The negative control elements contribute to the broad range of inducibility of GAL1 and GAL10 by inhibiting two GAL4/galactose-independent activating elements (GAE1 and GAE2) in UASG. In turn, multiple GAL4-binding sites in UASG are necessary for GAL4 to overcome repression by the negative control elements under fully inducing conditions. When glucose in addition to galactose is available (repressing conditions), the ability of GAL4 to activate transcription is diminished as a result of its reduced affinity for DNA and the reduced availability of inducer. Under these conditions, the negative control elements inhibit transcriptional activation from the glucose-attenuated GAL4 sites, thus accounting at least in part for glucose repression acting in cis. A normal part of transcriptional regulation of the GAL1 and GAL10 genes, therefore, appears to involve a balance between the opposing functions of positive and negative control elements.
Mol Cell Biol 1990 Nov
PMID:Opposing regulatory functions of positive and negative elements in UASG control transcription of the yeast GAL genes. 212 31

To learn more about the variety of Ty elements capable of activating gene expression, we characterized 206 spontaneous Ty transpositions that activate the promoterless gene his3 delta 4. Most of the Ty elements appear to be full-length, although a few deleted elements were recovered. Over 95% of the insertions belong to the Ty1 family, and the rest are Ty2 elements. The excessive number of Ty1 transpositions was unexpected because there are only 2-fold more Ty1 than Ty2 elements in the yeast strains used in the selection. However, there is 20-fold more Ty1 than Ty2 RNA present in these yeast strains. This difference in RNA level explains the greater number of Ty1 verses Ty2 transpositions at his3 delta 4, because Ty elements transpose through an RNA intermediate. A similar association between the Ty transcript level and transpositional activation of his3 delta 4 is obtained in cells expressing GAL1-promoted Ty2-H556 or Ty2-917 elements, but only if the element does not contain a marker. Genetically marked Ty2-H556NEO and -917NEO elements transpose into and activate his3 delta 4 with the same efficiency as the previously characterized Ty1-H3NEO element, but are underrepresented relative to the levels of TyNEO transcript. We also found that chromosomal Ty transcripts are even more abundant than previously estimated and comprise about 1% of total cellular RNA.
Mol Gen Genet 1990 Jan
PMID:Ty RNA levels determine the spectrum of retrotransposition events that activate gene expression in Saccharomyces cerevisiae. 215 50

To determine whether the 70-kilodalton heat shock proteins of Saccharomyces cerevisiae play a role in regulating their own synthesis, we studied the effect of overexpressing the SSA1 protein on the activity of the SSA1 5'-regulatory region. The constitutive level of Ssa1p was increased by fusing the SSA1 structural gene to the GAL1 promoter. A reporter vector consisting of an SSA1-lacZ translational fusion was used to assess SSA1 promoter activity. In a strain producing approximately 10-fold the normal heat shock level of Ssa1p, induction of beta-galactosidase activity by heat shock was almost entirely blocked. Expression of a transcriptional fusion vector in which the CYC1 upstream activating sequence of a CYC1-lacZ chimera was replaced by a sequence containing a heat shock upstream activating sequence (heat shock element 2) from the 5'-regulatory region of SSA1 was inhibited by excess Ssa1p. The repression of an SSA1 upstream activating sequence by the SSA1 protein indicates that SSA1 self-regulation is at least partially mediated at the transcriptional level. The expression of another transcriptional fusion vector, containing heat shock element 2 and a lesser amount of flanking sequence, is not inhibited when Ssa1p is overexpressed. This suggests the existence of an element, proximal to or overlapping heat shock element 2, that confers sensitivity to the SSA1 protein.
Mol Cell Biol 1990 Apr
PMID:Self-regulation of 70-kilodalton heat shock proteins in Saccharomyces cerevisiae. 218 Dec 81

Yeast (Saccharomyces cerevisiae) histone mRNA synthesis is tightly regulated to the S phase of the cell division cycle as a result of both transcriptional and posttranscriptional regulation. We focused on the role of posttranscriptional control in histone H2B1 gene (HTB1) regulation and studied a portion of the HTB1 message required for cell-cycle-specific accumulation. The 3' end of the HTB1 gene containing a 17-amino-acid coding sequence and entire noncoding sequence was fused to the bacterial neomycin phosphotransferase II gene (neo) under control of the GAL1 promoter. The expression of the endogenous and chimeric HTB1 genes was analyzed during the yeast cell cycle. As yeast cells entered a synchronous cell cycle following release from alpha-factor arrest, the level of GAL1-promoter-controlled neo-HTB1 message increased approximately 12-fold during S phase and dropped to basal level when the cells left S phase. This indicates that the 3' end of the HTB1 mRNA is capable of conferring cycle-specific regulation on a heterologous message. Deletion analysis of the 3' end showed that the signal for cell cycle control of HTB1 mRNA includes contiguous coding and noncoding sequences surrounding the stop codon. This differs from the situation in mammalian cells, whose posttranscriptional regulation of histone genes is mediated through a short sequence containing a stem-loop structure near the very terminus of the untranslated 3' end.
Mol Cell Biol 1990 Jun
PMID:Coding and noncoding sequences at the 3' end of yeast histone H2B mRNA confer cell cycle regulation. 218 95

GAL4I, GAL4II, and GAL4III are three forms of the yeast transcriptional activator protein that are readily distinguished on the basis of electrophoretic mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphorylation accounts for the reduced mobility of the slowest-migrating form, GAL4III, which is found to be closely associated with high-level GAL/MEL gene expression (L. Mylin, P. Bhat, and J. Hopper, Genes Dev. 3:1157-1165, 1989). Here we show that GAL4II, like GAL4III, can be converted to GAL4I by phosphatase treatment, suggesting that in vivo GAL4II is derived from GAL4I by phosphorylation. We found that cells which overproduced GAL4 under conditions in which it drove moderate to low levels of GAL/MEL gene expression showed only forms GAL4I and GAL4II. To distinguish which forms of GAL4 (GAL4I, GAL4II, or both) might be responsible for transcription activation in the absence of GAL4III, we performed immunoblot analysis on UASgal-binding-competent GAL4 proteins from four gal4 missense mutants selected for their inability to activate transcription (M. Johnston and J. Dover, Proc. Natl. Acad. Sci. USA 84:2401-2405, 1987; Genetics 120;63-74, 1988). The three mutants with no detectable GAL1 expression did not appear to form GAL4II or GAL4III, but revertants in which GAL4-dependent transcription was restored did display GAL4II- or GAL4III-like electrophoretic species. Detection of GAL4II in a UASgal-binding mutant suggests that neither UASgal binding nor GAL/MEL gene activation is required for the formation of GAL4II. Overall, our results imply that GAL4I may be inactive in transcriptional activation, whereas GAL4II appears to be active. In light of this work, we hypothesize that phosphorylation of GAL4I makes it competent to activate transcription.
Mol Cell Biol 1990 Sep
PMID:Phosphorylated forms of GAL4 are correlated with ability to activate transcription. 220 97

Expression of the GAL1 gene in Saccharomyces cerevisiae is strongly repressed by growth on glucose. We show that two sites within the GAL1 promoter mediate glucose repression. First, glucose inhibits transcription activation by GAL4 protein through UASG. Second, a promoter element, termed URSG, confers glucose repression independently of GAL4. We have localized the URSG sequences responsible for glucose repression to an 87-base-pair fragment located between UASG and the TATA box. Promoters deleted for small (20-base-pair) segments that span this sequence are still subject to glucose repression, suggesting that there are multiple sequences within this region that confer repression. Extended deletions across this region confirm that it contains at least two and possibly three URSG elements. To identify the gene products that confer repression upon UASG and URSG, we have analyzed glucose repression mutants and found that the GAL83, REG1, GRR1, and SSN6 genes are required for repression mediated by both UASG and URSG. In contrast, GAL82 and HXK2 are required only for UASG repression. A mutation designated urr1-1 (URSG repression resistant) was identified that specifically relieves URSG repression without affecting UASG repression. In addition, we observed that the SNF1-encoded protein kinase is essential for derepression of both UASG and URSG. We propose that repression of UASG and URSG is mediated by two independent pathways that respond to a common signal generated by growth on glucose.
Mol Cell Biol 1990 Sep
PMID:Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae. 220 2

We have identified a consensus operator sequence, YYYATTGTTCTC, which mediates the repression imposed by the ROX1 factor upon the members of the hypoxic gene regulon, which includes ANB1, HEM13, COX5b, and CYC7. The members of the regulon were repressed with widely varying stringency, and the variation was correlated with the number and fidelity of operator sequences observed. ANB1 had two operators operating with unequal efficiency, each containing two copies of the operator sequence. Synthetic operator sequences introduced into an operator deletion were effective as monomers but much more so as dimers, consistent with cooperativity. The native operators both imposed ROX1 repression on the GAL1 gene, in either orientation, but the synthetic operators did not, indicating that the sequence context may be important. The repression and activation of ANB1 are independent spatially and functionally, since deletion of the operators did not reduce expression and since both the operator and activation regions functioned separately in the GAL1 UAS. The ANB1 UAS was constitutive, containing several elements distributed over a 300-bp region. There were two dT-rich segments, one of 51 bp and one of 165 bp, the latter capable of activating transcription by itself. Flanking segments containing GRF2 (REB1) and ABF1 (GF1) sites may contribute to activation but were not essential. The UAS showed a strongly preferred orientation.
Mol Cell Biol 1990 Nov
PMID:A hypoxic consensus operator and a constitutive activation region regulate the ANB1 gene of Saccharomyces cerevisiae. 223 24

Two signals are required for meiosis and spore formation in the yeast Saccharomyces cerevisiae: starvation and the MAT products a1 and alpha 2, which determine the a/alpha cell type. These signals lead to increased expression of the IME1 (inducer of meiosis) gene, which is required for sporulation and sporulation-specific gene expression. We report here the sequence of the IME1 gene and the consequences of IME1 expression from the GAL1 promoter. The deduced IME1 product is a 360-amino-acid protein with a tyrosine-rich C-terminal region. Expression of PGAL1-IME1 in vegetative a/alpha cells led to moderate accumulation of four early sporulation-specific transcripts (IME2, SPO11, SPO13, and HOP1); the transcripts accumulated 3- to 10-fold more after starvation. Two sporulation-specific transcripts normally expressed later (SPS1 and SPS2) did not accumulate until PGAL1-IME1 strains were starved, and the intact IME1 gene was not activated by PGAL1-IME1 expression. In a or alpha cells, which lack alpha 2 or a1, expression of PGAL1-IME1 led to the same pattern of IME2 and SPO13 expression as in a/alpha cells, as measured with ime2::lacZ and spo13::lacZ fusions. Thus, in wild-type strains, the increased expression of IME1 in starved a/alpha cells can account entirely for cell type control, but only partially for nutritional control, of early sporulation-specific gene expression. PGAL1-IME1 expression did not cause growing cells to sporulate but permitted efficient sporulation of amino acid-limited cells, which otherwise sporulated poorly. We suggest that IME1 acts primarily as a positive regulator of early sporulation-specific genes and that growth arrest is an independent prerequisite for execution of the sporulation program.
Mol Cell Biol 1990 Dec
PMID:Role of IME1 expression in regulation of meiosis in Saccharomyces cerevisiae. 224 50

Bacteriophage T7 RNA polymerase and derivatives that contain the nuclear localization signal (NLS) from simian virus 40 T antigen (J. J. Dunn, B. Krippl, K. Bernstein, H. Westphal, and F. W. Studier, Gene 68:259-266, 1988) were expressed in Saccharomyces cerevisiae under the control of the inducible GAL1 promoter. As determined by indirect immunofluorescence, T7 RNA polymerase lacking the NLS remained mostly in the cytoplasm, whereas the protein containing the NLS localized to the nucleus. T7 RNA polymerase containing a mutated NLS remained mostly cytoplasmic. Hybrid proteins containing the NLS near the amino terminus were enzymatically active in the yeast cell, initiating transcription selectively at a T7 promoter placed in yeast chromosomal or plasmid DNA and stopping at a specific T7 terminator. At limiting enzyme concentrations, 5 to 10 times as much target RNA was produced when the polymerase contained the NLS, presumably because more enzyme reached the nucleus. Although substantial amounts of intact mRNA accumulated, no translation of target mRNAs in yeast cells was detected.
Mol Cell Biol 1990 Jan
PMID:Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes. 240 41


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