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S I Reed

Publications and source records attributed to S I Reed.

At least 127 records · Page 7Linked to original sources
PubMed1990

G protein mutations that alter the pheromone response in Saccharomyces cerevisiae.

The GPA1 gene of Saccharomyces cerevisiae encodes a G alpha protein that couples the membrane-bound pheromone receptors to downstream elements in the mating response pathway. We have isolated seven mutant alleles of GPA1 that confer pheromone resistance: G50D (a glycine-to-aspartate change at position 50), G322E, G322R, E355K, E364K, G470D, and an E364K-G470D double mutant. All of the mutations lie within large regions that are highly conserved between Gpa1 and four other G alpha proteins; four of the changes are located in domains with proposed functions. On the basis of a gentic analysis, the pheromone-unresponsive GPA1 alleles can be divided into two classes: those that encode constitutively activated proteins and those that encode proteins unable to respond to the upstream signal. Our results support the hypothesis that the activated form of Gpa1 stimulates adaptation to pheromone.

Alleles↗
PubMed1989

An essential G1 function for cyclin-like proteins in yeast.

Cyclins were discovered in marine invertebrates based on their dramatic cell cycle periodicity. Recently, the products of three genes associated with cell cycle progression in S. cerevisiae were found to share limited homology with cyclins. Mutational elimination of the CLN1, CLN2, and DAF1/WHI1 products leads to cell cycle arrest independent of cell type, while expression of any one of the genes allows cell proliferation. Using strains where CLN1 was expressed conditionally, the essential function of Cln proteins was found to be limited to the G1 phase. Furthermore, the ability of the Cln proteins to carry out this function was found to decay rapidly upon cessation of Cln biosynthesis. The data are consistent with the hypothesis that Cln proteins activate the Cdc28 protein kinase, shown to be essential for the G1 to S phase transition in S. cerevisiae. Because of the apparent functional redundancy of these genes, DAF1/WHI1 has been renamed CLN3.

Alleles↗
PubMed1989

Conservation of mitotic controls in fission and budding yeasts.

In fission yeast, the initiation of mitosis is regulated by a control network that integrates the opposing activities of mitotic inducers and inhibitors. To evaluate whether this control system is likely to be conserved among eukaryotes, we have investigated whether a similar mitotic control operates in the distantly related budding yeast S. cerevisiae. We have found that the protein kinase encoded by the mitotic inhibitor gene wee1+ of fission yeast, which acts to delay mitosis, is able also to delay the initiation of mitosis when expressed in S. cerevisiae. The wee1+ activity is counteracted in S. cerevisiae by the gene product of MIH1, a newly identified gene capable of encoding a protein of MW 54,000, which is a structural and functional homolog of the cdc25+ mitotic inducer of fission yeast. Expression of wee1+ in a mih1- strain prevents the initiation of mitosis. These data indicate that important features of the cdc25+-wee1+ mitotic control network identified in S. pombe are conserved in S. cerevisiae, and therefore are also likely to be generally conserved among eukaryotic organisms.

Base Sequence↗
PubMed1989

A family of cyclin homologs that control the G1 phase in yeast.

Two Saccharomyces cerevisiae genes were isolated based upon their dosage-dependent rescue of a temperature-sensitive mutation of the gene CDC28, which encodes a protein kinase involved in control of cell division. CLN1 and CLN2 encode closely related proteins that also share homology with cyclins. Cyclins, characterized by a dramatic periodicity of abundance through the cell cycle, are thought to be involved in mitotic induction in animal cells. A dominant mutation in the CLN2 gene, CLN2-1, advances the G1- to S-phase transition in cycling cells and impairs the ability of cells to arrest in G1 phase in response to external signals, suggesting that the encoded protein is involved in G1 control of the cell cycle in Saccharomyces.

Amino Acid Sequence↗
PubMed1989

The Saccharomyces cerevisiae CKS1 gene, a homolog of the Schizosaccharomyces pombe suc1+ gene, encodes a subunit of the Cdc28 protein kinase complex.

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for cell cycle initiation. In an attempt to identify genes encoding proteins that interact with the Cdc28 protein kinase, high-copy plasmid suppressors of a temperature-sensitive cdc28 mutation were isolated. One such suppressor, CKS1, was found to encode an 18-kilodalton protein that shared a high degree of homology with the suc1+ protein (p13) of Schizosaccharomyces pombe (67% amino acid sequence identity). Disruption of the chromosomal CKS1 gene conferred a G1 arrest phenotype similar to that of cdc28 mutants. The presence of the 18-kilodalton Cks1 protein in yeast lysates was demonstrated by using Cks-1 specific antiserum. Furthermore, the Cks1 protein was shown to be physically associated with active forms of the Cdc28 protein kinase. These data suggest that Cks1 is an essential component of the Cdc28 protein kinase complex.

Amino Acid Sequence↗
PubMed1989

Conservation of function and regulation within the Cdc28/cdc2 protein kinase family: characterization of the human Cdc2Hs protein kinase in Saccharomyces cerevisiae.

Whereas the Cdc28 protein kinase of the budding yeast Saccharomyces cerevisiae plays an essential role in cell cycle progression during the G1 interval, a function in the progression from the G2 interval into M phase has been inferred for its homologs, including the Cdc2Hs protein kinase of humans. To better understand these apparently disparate roles, we constructed a yeast strain in which the resident CDC28 gene was replaced by its human homolog, CDC2Hs. This transgenic yeast strain was able to perform the G1 functions attributed to the Cdc28 protein kinase, including the ability to grow and divide normally, to respond to environmental signals that induce G1 arrest, and to regulate the Cdc2Hs protein kinase appropriately in response to these signals.

Cell Cycle↗
PubMed1989

Analysis of the Cdc28 protein kinase complex by dosage suppression.

In the interest of identifying components of the Cdc28 protein kinase complex, dosage suppression analysis was performed on temperature-sensitive and dominant negative CDC28 mutations. Dosage suppression is based on a rationale in which elevated expression of wild-type genes can rescue mutations in a target gene as a result of interaction between the respective encoded proteins. Three sequences capable of rescuing a temperature sensitive cdc28 mutation were isolated from a library of wild-type genomic DNA segments in the high copy vector YEp13. Two of these, named CLN1 and CLN2 were found to encode closely related proteins with homology to cyclins. The third, CKS1, encodes an 18K (K = 10(3) Mr) protein that has been shown to be a component of the Cdc28 protein kinase complex and is a homolog of the suc1+ product of fission yeast. A number of dosage suppressors of the CDC28-dn1 dominant negative mutation have been isolated. The one analyzed to date encodes a truncated subunit of the mitochondrial enzyme succinyl-CoA synthetase. The basis for suppression in this case remains to be elucidated.

Base Sequence↗
PubMed1988

Control of the yeast cell cycle is associated with assembly/disassembly of the Cdc28 protein kinase complex.

The Saccharomyces cerevisiae gene CDC28 encodes a protein kinase required for progression from G1 to S phase in the cell cycle. We present evidence that the active form of the Cdc28 protein kinase is a complex of approximately 160 kd containing an endogenous substrate, p40, and possibly other polypeptides. This complex phosphorylates p40 and exogenous histone H1 in vitro. Cell cycle arrest during G1 results in inactivation of the protein kinase accompanied by the disassembly of the complex. Furthermore, assembly of the complex is regulated during the cell cycle, reaching a maximum during G1. Partial complexes thought to be intermediates in the assembly process phosphorylate histone H1 but not p40. Addition of soluble factors to these partial complexes in vitro restores p40 phosphorylation and causes the complex to increase to the mature size. A model is presented in which p40 phosphorylation is required during G1 for cells to initiate a new cell cycle.

Cell Cycle↗
PubMed1988

The histidine-221 to tyrosine substitution in v-mos abolishes its biological function and its protein kinase activity.

The viral mos gene encodes a cytoplasmic transforming protein termed p37mos. Evidence gathered from a number of experimental approaches is consistent with p37mos having a serine/threonine protein kinase activity. To gain further understanding of the p37mos-associated biochemical activity, we constructed a mutation in the v-mos gene by oligonucleotide-directed mutagenesis yielding a histidine to tyrosine substitution at residue 221 in p37mos. Based upon nucleotide sequences, the histidine residue at the corresponding position is conserved in all the serine/threonine protein kinases from yeast to man, and is absent in protein-tyrosine kinases. The mutant p37mos (Tyr-221) was expressed in yeast and assayed for kinase activity. The mutant protein was inactive as judged by a loss of autophosphorylation activity in vitro, thus providing further support for the conclusion that p37mos is a protein kinase. When the mutant v-mos gene was introduced into a retroviral vector, pDD102, and assayed for focus-forming ability on NIH/3T3 cells, it was found to be inactive at both 37 and 30 degrees. In contrast, the wild-type v-mos had transforming activity at both temperatures. These results extend our earlier findings on the correlation between transforming ability and protein kinase activity. A histidine to tyrosine substitution at the corresponding position of the v-mos protein and the yeast CDC28 gene product causes a similar effect on the kinase activity. Therefore, this residue and/or the sequence near the N-terminal side of the conserved predicted phosphate transfer domain, near the middle of the complete catalytic domain, might be specifically involved in the catalytic activity of serine/threonine protein kinases in general.

Amino Acid Sequence↗
PubMed1988

Dominant negative protein kinase mutations that confer a G1 arrest phenotype.

The CDC28 gene of Saccharomyces cerevisiae encodes a protein kinase that is required for passage through the G1 phase of the cell cycle. We have used an inducible promoter fused to the CDC28 coding sequence to isolate conditionally dominant mutant alleles of CDC28. Overexpression of these dominant alleles causes arrest in the G1 phase of the cell cycle but permits the distinctive asymmetric growth that is characteristic of recessive temperature-sensitive cdc28 mutants. The dominant alleles encode products with no detectable protein kinase activity, and their phenotypic effects can be suppressed by simultaneous overproduction of the wild-type protein. DNA sequence analysis showed that the mutant site in at least one of the dominant alleles is in a residue that is highly conserved among protein kinases. These properties are best understood if the dominant mutation results in the catalytic inactivation of the protein kinase but still allows the binding of another component needed for CDC28 function. By this model, high levels of the mutant protein arrest cell division by denying the wild-type protein access to this other component. Suppressors that may encode this other component have been isolated on high-copy-number plasmids.

Amino Acid Sequence↗
PubMed1988

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

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.

Agglutinins↗
PubMed1988

Invariant phosphorylation of the Saccharomyces cerevisiae Cdc28 protein kinase.

The phosphorylation level of the Saccharomyces cerevisiae Cdc28 protein remained invariant under conditions that resulted in cell cycle arrest in the G1 phase and loss of Cdc28-specific protein kinase activity when the activity was assayed in vitro. These results are in contrast to the proposed regulation of the homologous Cdc2 protein kinase of Schizosaccharomyces pombe.

Antigen-Antibody Complex↗
PubMed1987

Dual regulation of the yeast CDC28-p40 protein kinase complex: cell cycle, pheromone, and nutrient limitation effects.

A 40 kd polypeptide that coprecipitates with the CDC28 gene product in immune complexes is specifically phosphorylated by the CDC28 protein kinase. Using this reaction, we detect activity only in extracts from dividing G1 phase cells. Exit from G1 by entry into S phase or the preconjugatory state induced by mating pheromone correlates with loss of p40 phosphorylation activity. Inactive extracts from cdc28 mutants complement extracts from cells arrested in S or M phase, suggesting that non-G1 cells are deficient in an exchangeable activating factor. Stationary and pheromone-treated cultures are rich in this exchangeable factor, but possess an inactive kinase that is not activated by complementation. cAMP-deficient mutants resemble stationary cells.

Adenylyl Cyclases↗
PubMed1987

Subcellular localization of a protein kinase required for cell cycle initiation in Saccharomyces cerevisiae: evidence for an association between the CDC28 gene product and the insoluble cytoplasmic matrix.

The product of the Saccharomyces cerevisiae gene CDC28, a protein kinase required for initiation of the cell division cycle, was localized within yeast cells. By using immunofluorescence methods, the CDC28 product was shown to be primarily cytoplasmic in distribution. The gene product was localized largely to the particulate fraction by differential centrifugation after mechanical disruption in aqueous buffers. The particulate association was not affected by the presence of nonionic detergent. To refine this localization further, a procedure was developed for the preparation of yeast cytoplasmic matrices which resemble the cytoskeletons of vertebrate cells on the basis of methodology, immunochemistry, and gross ultrastructure. A portion of the CDC28 product was found to be tightly associated with these detergent-insoluble cytoplasmic matrices by both immunofluorescence and immunoblotting procedures. Although, for technical reasons, precise quantitation was not possible, it is estimated that a minimum of 2-15% of the total CDC28 product pool is involved in the association with the insoluble matrix. Alcohol dehydrogenase, a soluble cytoplasmic protein, was found not to be associated with the cytoplasmic matrices at any detectable level, whereas, in contrast, approximately 10-40% of the total cellular actin, a bonafide cytoskeletal protein, was present in these structures. The proportion of CDC28 gene product associated with the particulate fraction, and perhaps the insoluble matrix, appears to be substantially decreased during the preparation of spheroplasts.

Cell Compartmentation↗
PubMed1987

Temperature-sensitive lethal pseudorevertants of ste mutations in Saccharomyces cerevisiae.

A procedure was devised to isolate mutations that could restore conjugational competence to temperature sensitive ste mutants and simultaneously confer temperature-sensitive lethal growth phenotypes. Three such mutations, falling into two complementation groups, were identified on the basis of suppression of ste5 alleles. These same mutations were later shown to be capable of suppressing ste4 and ste7 alleles. Five mutations in a single complementation group were isolated as suppressors of ste2 alleles. None of the mutations described in this study conferred a homogeneous cell cycle arrest phenotype, and all were shown to define complementation groups distinct from those previously identified in studies of cell division cycle (cdc) mutations. In no instance did pseudoreversion appear to be achieved by mutational G1 arrest of ste mutant cells. Instead, it is proposed that the mutations restore conjugation by reestablishing the normal pheromone response.

Alleles↗
PubMed1986

Nucleotide sequence of the yeast cell division cycle start genes CDC28, CDC36, CDC37, and CDC39, and a structural analysis of the predicted products.

The nucleotide sequences of the yeast cell division cycle start genes CDC36, CDC37, and CDC39 are presented. An open reading frame corresponding in size and mapped position to the mRNA for each gene was revealed. These sequences, as well as that of the CDC28 gene, were analyzed for the presence of consensus sequences postulated to be transcriptional or translational signals, or to be involved in mRNA processing. In addition, the predicted protein products of the four genes were subjected to a number of structural and statistical analyses including codon usage bias analysis, secondary structure analysis and hydropathicity analysis.

Amino Acid Sequence↗
PubMed1986

Moloney murine sarcoma virus encoded p37mos expressed in yeast has protein kinase activity.

We describe the expression of the Moloney murine sarcoma virus env-mos protein (p37mos) in yeast under the control of the yeast GAL1 promoter. Consistent with our previous results concerning the p37mos protein kinase made in virus infected mouse cells, p37mos produced in yeast possesses autophosphorylation activity in an immune complex kinase assay using anti-mos (37-35) serum.

Gene Expression Regulation↗