scholarly journals Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling.

1988 ◽  
Vol 8 (6) ◽  
pp. 2484-2493 ◽  
Author(s):  
K Y Jahng ◽  
J Ferguson ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G Protein ◽  
Genomic Library ◽  
Negative Impact ◽  
Receptor Gene ◽  
Temperature Sensitive ◽  
Specific Cell ◽  
Protein Activity ◽  
Mating Pheromone ◽  
Pheromone Receptor

Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to genes encoding the alpha subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpa1 mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G1, deposition of mating-specific cell surface agglutinins, and induction of pheromone-specific mRNAs, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.

Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling

1988 ◽  
Vol 8 (6) ◽  
pp. 2484-2493
Author(s):  
K Y Jahng ◽  
J Ferguson ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
G Protein ◽  
Genomic Library ◽  
Negative Impact ◽  
Receptor Gene ◽  
Temperature Sensitive ◽  
Specific Cell ◽  
Protein Activity ◽  
Mating Pheromone ◽  
Pheromone Receptor

Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPA1 (also known as SCG1), recently shown to be highly homologous to genes encoding the alpha subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpa1 mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G1, deposition of mating-specific cell surface agglutinins, and induction of pheromone-specific mRNAs, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.

scholarly journals Mot3, a Zn Finger Transcription Factor That Modulates Gene Expression and Attenuates Mating Pheromone Signaling in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 879-892 ◽  
Author(s):  
Anatoly V Grishin ◽  
Michael Rothenberg ◽  
Maureen A Downs ◽  
Kendall J Blumer
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Signaling Pathway ◽  
G Protein ◽  
Binding Sites ◽  
Dependent Manner ◽  
Pheromone Response ◽  
Mating Pheromone ◽  
Pheromone Signaling ◽  
Yeast Genes

Abstract In the yeast Saccharomyces cerevisiae, mating pheromone response is initiated by activation of a G protein- and mitogen-activated protein (MAP) kinase-dependent signaling pathway and attenuated by several mechanisms that promote adaptation or desensitization. To identify genes whose products negatively regulate pheromone signaling, we screened for mutations that suppress the hyperadaptive phenotype of wild-type cells overexpressing signaling-defective G protein β subunits. This identified recessive mutations in MOT3, which encodes a nuclear protein with two Cys2-His2 Zn fingers. MOT3 was found to be a dosage-dependent inhibitor of pheromone response and pheromone-induced gene expression and to require an intact signaling pathway to exert its effects. Several results suggested that Mot3 attenuates expression of pheromone-responsive genes by mechanisms distinct from those used by the negative transcriptional regulators Cdc36, Cdc39, and Mot2. First, a Mot3-lexA fusion functions as a transcriptional activator. Second, Mot3 is a dose-dependent activator of several genes unrelated to pheromone response, including CYC1, SUC2, and LEU2. Third, insertion of consensus Mot3 binding sites (C/A/T)AGG(T/C)A activates a promoter in a MOT3-dependent manner. These findings, and the fact that consensus binding sites are found in the 5′ flanking regions of many yeast genes, suggest that Mot3 is a globally acting transcriptional regulator. We hypothesize that Mot3 regulates expression of factors that attenuate signaling by the pheromone response pathway.

Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway

1990 ◽  
Vol 10 (2) ◽  
pp. 510-517
Author(s):  
G M Cole ◽  
D E Stone ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Transduction ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Mating Pheromone ◽  
Gal1 Promoter ◽  
Gamma Subunits ◽  
Adaptation Response ◽  
High Level

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.

Cloning of a human S-phase cell cycle gene: use of transient expression for screening

1987 ◽  
Vol 7 (2) ◽  
pp. 775-779
Author(s):  
A Fainsod ◽  
G Diamond ◽  
M Marcus ◽  
F H Ruddle
Keyword(s):  
Cell Cycle ◽  
Genomic Library ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
S Phase ◽  
Phase Cell ◽  
Cell Cycle Gene ◽  
Temperature Sensitive ◽  
Specific Cell ◽  
Restrictive Temperature

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.

A cloning strategy for G-protein-coupled hormone receptors: the ovine beta 1-adrenergic receptor

1995 ◽  
Vol 7 (3) ◽  
pp. 521 ◽  
Author(s):  
JF Padbury ◽  
YT Tseng ◽  
JA Waschek
Keyword(s):  
G Protein ◽  
Sequence Homology ◽  
Hormone Receptors ◽  
Genomic Library ◽  
Receptor Gene ◽  
G Protein Coupled Receptors ◽  
Transcriptional Level ◽  
Fetal Life ◽  
Single Clone ◽  
G Protein Coupled

Regulation of beta 1-adrenergic receptors is unusual in developing animals. For example, glucocorticoid-and thyroid hormone-responsiveness for several genes is seen in animals treated during fetal life but beta 1-responsiveness is not seen until after birth. In order to investigate this at the transcriptional level, the ovine beta 1 receptor gene was cloned from a sheep genomic library. An approach using high-stringency screening with cDNA probes and oligonucleotides from regions of human and rat genes conserved but unique to the beta 1 receptor but not to other seven transmembrane, G-protein-coupled receptors. Over 800,000 clones were screened from which 40-50 positive clones were identified by each of the probes. There was, however, only a single clone which was recognized by each of the probes. A 5-kb insert was subcloned and shown to contain sequences which hybridized to each of the probes. Using the restriction map of the rat beta 1 receptor, a 1.0-kb Pst1 internal fragment was further subcloned for sequence identification. Confirmation of this fragment as the ovine beta 1 receptor was based on homology of the beta 1 receptor from other species and tissue distribution of mRNA. Nucleotide sequence homology was 93% with the human beta 1 receptor and 84% with rat. Amino acid sequence homology was > 75% and approached 100% in the transmembrane regions. The approach described represents a practical approach to cloning and identification of hormone receptors from the highly homologous members of the seven-transmembrane, G-protein-coupled receptors.

scholarly journals PTA1, an essential gene of Saccharomyces cerevisiae affecting pre-tRNA processing.

1992 ◽  
Vol 12 (9) ◽  
pp. 3843-3856 ◽  
Author(s):  
J P O'Connor ◽  
C L Peebles
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Gene Product ◽  
Genomic Library ◽  
Essential Gene ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Putative Open Reading Frame ◽  
Reading Frame ◽  
Trna Processing ◽  
Chromosome I

We have identified an essential Saccharomyces cerevisiae gene, PTA1, that affects pre-tRNA processing. PTA1 was initially defined by a UV-induced mutation, pta1-1, that causes the accumulation of all 10 end-trimmed, intron-containing pre-tRNAs and temperature-sensitive but osmotic-remedial growth. pta1-1 does not appear to be an allele of any other known gene affecting pre-tRNA processing. Extracts prepared from pta1-1 strains had normal pre-tRNA splicing endonuclease activity. pta1-1 was suppressed by the ochre suppressor tRNA gene SUP11, indicating that the pta1-1 mutation creates a termination codon within a protein reading frame. The PTA1 gene was isolated from a genomic library by complementation of the pta1-1 growth defect. Episome-borne PTA1 directs recombination to the pta1-1 locus. PTA1 has been mapped to the left arm of chromosome I near CDC24; the gene was sequenced and could encode a protein of 785 amino acids with a molecular weight of 88,417. No other protein sequences similar to that of the predicted PTA1 gene product have been identified within the EMBL or GenBank data base. Disruption of PTA1 near the carboxy terminus of the putative open reading frame was lethal. Possible functions of the PTA1 gene product are discussed.

scholarly journals The Asexual Yeast Candida glabrata Maintains Distinct a and α Haploid Mating Types

2008 ◽  
Vol 7 (5) ◽  
pp. 848-858 ◽  
Author(s):  
Héloïse Muller ◽  
Christophe Hennequin ◽  
Julien Gallaud ◽  
Bernard Dujon ◽  
Cécile Fairhead
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Type Strain ◽  
Candida Glabrata ◽  
Sexual Cycle ◽  
Mating Types ◽  
Differential Splicing ◽  
Mating Pheromone ◽  
Pheromone Receptor ◽  
Mating Pheromones ◽  
Key Genes

ABSTRACT The genome of the type strain of Candida glabrata (CBS138, ATCC 2001) contains homologs of most of the genes involved in mating in Saccharomyces cerevisiae, starting with the mating pheromone and receptor genes. Only haploid cells are ever isolated, but C. glabrata strains of both mating types are commonly found, the type strain being MATα and most other strains, such as BG2, being MAT a. No sexual cycle has been documented for this species. In order to understand which steps of the mating pathway are defective, we have analyzed the expression of homologs of some of the key genes involved as well as the production of mating pheromones and the organism's sensitivity to artificial pheromones. We show that cells of opposite mating types express both pheromone receptor genes and are insensitive to pheromones. Nonetheless, cells maintain specificity through regulation of the α1 and α2 genes and, more surprisingly, through differential splicing of the a 1 transcript.

scholarly journals Temperature-sensitive cdc7 mutations of Saccharomyces cerevisiae are suppressed by the DBF4 gene, which is required for the G1/S cell cycle transition.

Genetics ◽  
1992 ◽  
Vol 131 (1) ◽  
pp. 21-29 ◽  
Author(s):  
K Kitada ◽  
L H Johnston ◽  
T Sugino ◽  
A Sugino
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Genomic Library ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Allele Specific ◽  
Cell Cycle Transition ◽  
Mutant Cells

Abstract When present on a multicopy plasmid, a gene from a Saccharomyces cerevisiae genomic library suppresses the temperature-sensitive cdc7-1 mutation. The gene was identified as DBF4, which was previously isolated by complementation in dbf4-1 mutant cells and is required for the G1----S phase progression of the cell cycle. DBF4 has an open reading frame encoding 695 amino acid residues and the predicted molecular mass of the gene product is 80 kD. The suppression is allele-specific because a CDC7 deletion is not suppressed by DBF4. Suppression is mitosis-specific and the sporulation defect of cdc7 mutations is not suppressed by DBF4. Conversely, CDC7 on a multicopy plasmid suppresses the dbf4-1, -2, -3 and -4 mutations but not dbf4-5 and DBF4 deletion mutations. Furthermore, cdc7 mutations are incompatible with the temperature-sensitive dbf4 mutations. These results suggest that the CDC7 and DBF4 polypeptides interact directly or indirectly to permit initiation of yeast chromosome replication.

Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway

1990 ◽  
Vol 10 (6) ◽  
pp. 2966-2972
Author(s):  
M de Barros Lopes ◽  
J Y Ho ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
G Protein ◽  
G1 Arrest ◽  
Restrictive Temperature ◽  
Gene Products ◽  
Pheromone Response ◽  
Mating Pheromone ◽  
Pheromone Response Pathway ◽  
Mutational Activation

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.

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