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G Simchen

Publications and source records attributed to G Simchen.

At least 19 recordsLinked to original sources
PubMed1992

Interaction between the Saccharomyces cerevisiae CDC25 gene product and mammalian ras.

In order to characterize the interaction between the Saccharomyces cerevisiae Cdc25 protein and Harvey-ras (p21H-ras), we have constructed a yeast strain disrupted at the RAS1 and RAS2 loci, expressing both p21H-ras and the catalytic domain of the bovine GTPase activating protein (GAP) and containing the cdc25-2 mutation. Such a strain exhibits a temperature-sensitive phenotype. The shift to the nonpermissive temperature is accompanied by the loss of guanyl nucleotide-dependent activity of adenylylcyclase in vitro. The temperature-sensitive phenotype can be rescued by CDC25 itself, as well as by a plasmid containing a truncated SDC25 gene. In addition, wild type CDC25 significantly improves the guanyl nucleotide response observed in the background of the cdc25ts allele at the permissive temperature in a dosage-dependent manner and restores the guanyl nucleotide response at the restrictive temperature. Both CDC25 and a truncated SDC25 also restored p21H-ras-dependent guanyl nucleotide response in a strain isogenic to the one described above but containing a disrupted CDC25 locus instead of the temperature-sensitive allele. These results suggest that the S. cerevisiae Cdc25 protein interacts with p21H-ras expressed in yeast by promoting GDP-GTP exchange. It follows that the yeast system can be used for characterizing the interaction between guanyl nucleotide exchangers of Ras proteins and mammalian p21H-ras.

Adenylyl Cyclases↗
PubMed1992

Multiple sites for double-strand breaks in whole meiotic chromosomes of Saccharomyces cerevisiae.

We present a scheme for locating double-strand breaks (DSBs) in meiotic chromosomes of Saccharomyces cerevisiae, based on the separation of large DNA molecules by pulsed field gel electrophoresis. Using a rad50S mutant, in which DSBs are not processed, we show that DSBs are widely induced in S. cerevisiae chromosomes during meiosis. Some of the DSBs accumulate at certain preferred sites. We present general profiles of DSBs in chromosomes III, V, VI and VII. A map of the 12 preferred sites on chromosome III is presented. At least some of these sites correlate with known 'hot spots' for meiotic recombination. The data are discussed in view of current models of meiotic recombination and chromosome segregation.

Chromosome Aberrations↗
PubMed1992

Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein.

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

Adenylyl Cyclases↗
PubMed1990

Adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is not regulated by guanyl nucleotides.

The adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is localized to the plasma membrane of the cell. The enzyme utilizes Mn2+/ATP as substrate and free Mn2+ ions as an effector. Unlike the baker yeast Saccharomyces cerevisiae, S. pombe adenylyl cyclase does not utilize Mg2+/ATP as substrate and the activity is not stimulated by guanyl nucleotides. The optimal pH for the S. pombe adenylyl cyclase activity is 6.0. The activity dependence on ATP is cooperative with a Hill coefficient of 1.68 +/- 0.14.

Adenosine Triphosphate↗
PubMed1990

The adenylate cyclase/protein kinase cascade regulates entry into meiosis in Saccharomyces cerevisiae through the gene IME1.

Entry into meiosis in Saccharomyces cerevisiae cells is regulated by starvation through the adenylate cyclase/cAMP-dependent protein kinase (AC/PK) pathway. The gene IME1 is also involved in starvation control of meiosis. Multicopy IME1 plasmids overcome the meiotic deficiency of bcy1 and of RASval19 diploids. Double mutants ime1 cdc25 and ime1 ras2 are sporulation deficient. These results suggest that IME1 comes after the AC/PK cascade. Furthermore, the level of IME1 transcripts is affected by mutations in the AC/PK genes CDC25, CYR1 and BCY1. Moreover, the addition of cAMP to a cyr1-2 diploid suppresses IME1 transcription. The presence in a bcy1 diploid of IME1 multicopy plasmids does not cure the failure of bcy1 cells to arrest as unbudded cells following starvation and to enter the G0 state (thermotolerance, synthesis of unique G0 proteins). This indicates that the pathway downstream of the AC/PK cascade branches to control meiosis through IME1, and to control entry into G0 and cell cycle initiation, independently of IME1.

Adenylyl Cyclases↗
PubMed1990

In vitro reconstitution of cdc25 regulated S. cerevisiae adenylyl cyclase and its kinetic properties.

The attenuated GTP regulation adenylyl cyclase (CDC35) lysates or membranes prepared from cells of a cdc25ts strain is enhanced 2.5- to 6-fold by mixing these lysates or membranes with lysates or membranes from a cdc35ts strain harboring wild-type CDC25. The kinetics of activation of the Saccharomyces cerevisiae adenylyl cyclase in vitro is first order, as is the activation of mammalian adenylyl cyclase. The rate of enzyme activation in the presence of non-hydrolysable analogs of GTP increases with the number of CDC25 gene copies present in the cell. When GppNHp was used the rate of activation of the cyclase in a strain harboring a multicopy plasmid of CDC25 was 7.0-fold higher than the rate in an isogenic strain with the cdc25-2 mutation. The rate of adenylyl cyclase activation from a strain with a disrupted CDC25 gene is 14.7-fold lower than the rate in an isogenic strain containing the CDC25 gene on a multicopy plasmid. The reconstitution experiments described provide direct biochemical evidence for the role of the CDC25 protein in regulating the RAS dependent adenylyl cyclase in S.cerevisiae. The reconstitution experiments and the kinetic experiments may also provide a biochemical assay for the CDC25 protein and can form the basis for its characterization. In this study we also show that adenylyl cyclase activity in ras1ras2byc1 cells is found in the soluble fraction, whereas in wild-type strain it is found in the membrane fraction. Overexpression of the gene CDC25 in the ras1ras2bcy1 strain relocalizes adenylyl cyclase activity to the membrane fraction. This finding suggests a biochemical link between CDC25 and CDC35 in the absence of RAS, in addition to its role in regulating RAS dependent adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1990

Mixed segregation of chromosomes during single-division meiosis of Saccharomyces cerevisiae.

Normal meiosis consists of two consecutive cell divisions in which all the chromosomes behave in a concerted manner. Yeast cells homozygous for the mutation cdc5, however, may be directed through a single meiotic division of a novel type. Dyad analysis of a cdc5/cdc5 strain with centromere-linked markers on four different chromosomes has shown that, in these meioses, some chromosomes within a given cell segregate reductionally whereas others segregate equationally. The choice between the two types of segregation in these meioses is made individually by each chromosome pair. Different chromosome pairs exhibit different segregation tendencies. Similar results were obtained for cells homozygous for cdc14.

Cell Division↗
PubMed1990

Centromeric regions control autonomous segregation tendencies in single-division meiosis of Saccharomyces cerevisiae.

We have previously shown that yeast cdc5 or cdc14 homozygotes can be led through a single-division meiosis in which some of the chromosomes segregate reductionally whereas others, within the same cell, segregate equationally. Chromosomes XI tend to segregate reductionally, whereas chromosomes IV tend to segregate equationally. In this report we present experiments with cdc5 homozygous strains, in which the centromeres of one or both chromosomes XI was replaced by the centromeric region from chromosome IV. Analysis of the products of single-division meioses in these strains demonstrates that the choice between reductional or equational segregation is directed by sequences in the vicinity of the centromeres. Although the choice is made separately for each individual chromosome, the analysis also reveals the existence of a system responsible for coordinated segregation of the two chromosomes of a given pair.

Cell Division↗
PubMed1989

A long region upstream of the IME1 gene regulates meiosis in yeast.

Meiosis and sporulation in yeast are subject to two types of regulation. The first depends on environmental conditions. The second depends on a genetic pathway which involves the control of the positive regulatory gene IME1 by RME1, which is in turn controlled by the MAT locus. The presence of IME1 on a multicopy plasmid enables cells to undergo meiosis regardless of their genotype at MAT or RME1. We show here that a multicopy plasmid carrying IME1 also enables meiosis, regardless of the environment. Therefore, both kinds of regulation appear to act through IME1. Furthermore, the behavior of multicopy plasmids carrying various segments from the IME1 region suggests that the region upstream of IME1 contains both positive and negative regulatory sites. Control of IME1 by the environment and by the MAT pathway both act through negative regulatory sites.

Cloning, Molecular↗
PubMed1989

Rapid intracellular alkalinization of Saccharomyces cerevisiae MATa cells in response to alpha-factor requires the CDC25 gene product.

The alpha-factor mating pheromone induces a transient intracellular alkalinization of MATa cells within minutes after exposure to the pheromone, and is the earliest biochemical event that can be identified subsequent to the exposure. Dissipation of the pheromone induced pH gradient, using 2,4-dinitrophenol or sodium orthovanadate, does not inhibit the biological response of the yeast to the pheromone such as mating and 'schmoo' formation. These findings suggest that the pheromone mediated pH change per se is not a part of the transmembrane signalling but rather the consequence of a biochemical reaction triggered by the alpha-pheromone interaction with its receptor and may have a permissive effect on the pheromonal response. The cdc25ts mutation causes MATa cells to become nonresponsive to alpha-factor subsequent to a shift to the restrictive temperature, suggesting that the CDC25 gene product participates in the pheromone response pathway.

Cell Cycle Proteins↗
PubMed1989

Genetic regulation of differentiation towards meiosis in the yeast Saccharomyces cerevisiae.

Normally, meiosis and sporulation in Saccharomyces cerevisiae occur only in diploid strains and only when the cells are exposed to starvation conditions. Diploidy is determined by the mating-type system (the genes MAT, RME1, IME1), whereas the starvation signal is transmitted through the adenylate cyclase - protein kinase pathway (the genes CDC25, RAS2, CDC35 (CYR1), BCY1, TPK1, TPK2, TPK3). The two regulatory pathways converge at the gene IME1, which is a positive regulator of meiosis and whose early expression in sporulating cells correlates with the initiation of meiosis. Sites upstream (5') of IME1 appear to mediate in the repression of the gene by repressors originating from both the mating-type and the cyclase--kinase pathways.

Adenylyl Cyclases↗
PubMed1979

Morphogenesis of the synapton during yeast meiosis.

The formation of the synapton (synaptonemal complex) was followed by an electron microscopic examination of large samples of Saccharomyces cerevisiae cells at various stages of meiosis. Three temperature-sensitive mutants were used, cdc4, cdc5 and cdc7, which undergo a slow but normal meiosis at 25 degrees C. At the restrictive temperature of 34 degrees C, cdc4 and cdc5 arrest at an advanced enough stage of meiosis to allow the study of synapton morphogenesis. Based on the frequencies of nuclear structures, we describe the formation of the central region and central elements of the synapton in the dense body, which may be part of the nucleolus. This process occurs during early meiotic stages, concomittantly with recombination commitment and premeiotic DNA replication. Mature synaptons usually appear after premeiotic S, at the pachytene stage, and later disappear. A possible intermediate stage in this disappearance is found in arrested cdc5 cells, which contain paired lateral elements without central elements. Following the frequencies of spindle plaque configurations, we conclude that the plaques in meiosis duplicate once at the beginning of the main DNA replication, as is also observed prior to mitosis. In contrast to mitotic cells, however, meiotic plaques remain duplicated for a long period, until the synaptons disappear, and only then separate from each other to form a spindle. During late stages of the first meiotic division, the outer plates of the spindle plaques thicken, to duplicate later and give the second division spindles. The characteristically thick outer plate may have a role in the formations of the ascopore wall.

Meiosis↗
PubMed1978

Meiotic recombination and DNA synthesis in a new cell cycle mutant of Saccharomyces cerevisiae.

Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.

Cell Cycle↗
PubMed1978

Cell cycle mutants.

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Aspergillus nidulans↗