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R A Singer

Publications and source records attributed to R A Singer.

At least 37 records · Page 2Linked to original sources
PubMed1993

Heat shock-mediated cell cycle blockage and G1 cyclin expression in the yeast Saccharomyces cerevisiae.

For cells of the yeast Saccharomyces cerevisiae, heat shock causes a transient inhibition of the cell cycle-regulatory step START. We have determined that this heat-induced START inhibition is accompanied by decreased CLN1 and CLN2 transcript abundance and by possible posttranscriptional changes to CLN3 (WHI1/DAF1) cyclin activity. Persistent CLN2 expression from a heterologous promoter or the CLN2-1 or CLN3-1 alleles that are thought to encode cyclin proteins with increased stability eliminated heat-induced START inhibition but did not affect other aspects of the heat shock response. Heat-induced START inhibition was shown to be independent of functions that regulate cyclin activity under other conditions and of transcriptional regulation of SWI4, an activator of cyclin transcription. Cells lacking Bcy1 function and thus without cyclic AMP control of A kinase activity were inhibited for START by heat shock as long as A kinase activity was attenuated by mutation. We suggest that heat shock mediates START blockage through effects on the G1 cyclins.

Cell Cycle↗
PubMed1993

Stationary phase in the yeast Saccharomyces cerevisiae.

Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant cells that are conditionally defective only for the resumption of proliferation from stationary phase provides evidence that stationary phase is a unique developmental state. Strains with mutations affecting entry into and survival during stationary phase have also been isolated, and the mutations have been shown to affect at least seven different cellular processes: (i) signal transduction, (ii) protein synthesis, (iii) protein N-terminal acetylation, (iv) protein turnover, (v) protein secretion, (vi) membrane biosynthesis, and (vii) cell polarity. The exact nature of the relationship between these processes and survival during stationary phase remains to be elucidated. We propose that cell cycle arrest coordinated with the ability to remain viable in the absence of additional nutrients provides a good operational definition of starvation-induced stationary phase.

Cyclic AMP↗
PubMed1992

Mitochondrial DNA loss by yeast reentry-mutant cells conditionally unable to proliferate from stationary phase.

Double-mutant cells of the budding yeast Saccharomyces cerevisiae harboring the gcs1-1 and sed1-1 mutations are conditionally defective (cold-sensitive) only for reentry into the mitotic cycle from stationary phase. If already proliferating at the permissive temperature (29 degrees C), these reentry-mutant cells continue to proliferate when transferred to the restrictive temperature of 14 degrees C, but under these conditions reentry-mutant cells lose mitochondrial DNA (mtDNA). In addition, upon exhaustion of the nutrient supply at 14 degrees C, these reentry-mutant cells entered stationary phase at a decreased cell concentration and did not accumulate the reserve carbohydrates trehalose and glycogen. Both of these deficiencies were due to the loss of mtDNA, as shown by the responses of wild-type cells also lacking mtDNA. Mitochondrial status did not affect other aspects of the reentry-mutant phenotype. Although mitochondrial activity and the accumulation of carbohydrate reserves are typical features of cells in stationary phase, the reentry-mutant phenotype reveals that neither entry into nor exit from stationary phase need involve mitochondrial function.

Cell Division↗
PubMed1991

Expression of lacZ gene fusions affects downstream transcription in yeast.

Chimeric genes containing Escherichia coli lacZ sequences are often used to characterize gene expression in yeast cells. By Northern analysis, we found that such genes produce multiple transcripts due to inefficient 3'-end formation. The same transcript pattern was found for two related chimeric genes when these genes were cloned separately into the commonly used vector, YIp5, and integrated into the yeast genome at two different locations. Each chimeric gene was composed of promoter and N-terminal coding regions from the yeast SSA1 or SSA2 genes fused in-frame to the lac operon. Transcripts were shown to initiate within the yeast promoter fragment, but transcript size indicated that 3' ends were localized to three different regions: within the lac operon near the 3' end of the lacZ gene; near a terminator region previously identified upstream of the URA3 gene in YIp5; and at the URA3 terminator region. Readthrough transcription of the URA3 promoter from upstream lac sequences decreased the basal activity of the URA3 promoter, although induced URA3 transcription levels were unaffected. This readthrough transcription also resulted in a novel, longer URA3 transcript.

Base Sequence↗
PubMed1991

A cyclin protein modulates mitosis in the budding yeast Saccharomyces cerevisiae.

For the budding yeast Saccharomyces cerevisiae the mitotic cell cycle is coordinated with cell mass at the regulatory step "start". The threshold amount of cell mass (reflected as a "critical size") necessary for "start" is proportional to nutrient quality. This relationship leads to a transient accumulation of cells at "start", termed nutrient modulation, upon enrichment of nutrient conditions. Nutrient enrichment abruptly increases the critical size needed for "start", causing the smaller cells, produced in the previous cell cycle, to be delayed at "start" while growing larger. Here we show that, in S. cerevisiae, a second cell-cycle step, at mitosis, also exhibits nutrient modulation, and is, therefore, another point of cell-cycle regulation. At both mitosis and "start", nutrient modulation was found through mutation to be regulated by the activity of the cyclin-related WHI1 (CLN3) gene product.

Culture Media↗
PubMed1991

The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles.

After the initiation of bud formation, cells of the yeast Saccharomyces cerevisiae direct new growth to the developing bud. We show here that this vectorial growth is facilitated by activity of the MYO2 gene. The wild-type MYO2 gene encodes an essential form of myosin composed of an NH2-terminal domain typical of the globular, actin-binding domain of other myosins. This NH2-terminal domain is linked by what appears to be a short alpha-helical domain to a novel COOH-terminal region. At the restrictive temperature the myo2-66 mutation does not impair DNA, RNA, or protein biosynthetic activity, but produces unbudded, enlarged cells. This phenotype suggests a defect in localization of cell growth. Measurements of cell size demonstrated that the continued development of initiated buds, as well as bud initiation itself, is inhibited. Bulk secretion continues in mutant cells, although secretory vesicles accumulate. The MYO2 myosin thus may function as the molecular motor to transport secretory vesicles along actin cables to the site of bud development.

Actins↗
PubMed1991

The G1 interval in the mammalian cell cycle: dual control by mass accumulation and stage-specific activities.

The temporal determinants of the G1 cell cycle interval were investigated using nine mammalian cell lines. In each case, cells were allowed to proliferate for many cell cycles under conditions that slowed progress through S phase without an equivalent impairment of overall mass accumulation. This disproportionate inhibition of progress through the cell cycle caused newly produced cells to be more massive than usual. Under these growth conditions, the determinants of the length of the G1 interval became evident. For two cell lines, HeLa S3 and NIH 3T3, a protracted S phase, and the resultant increase in mass, resulted in a dramatically shortened G1 interval. Thus, for these cell lines, a major portion of G1 time exists to accommodate mass accumulation needed to initiate the subsequent S phase. Nevertheless, under conditions that protracted S phase and shortened the G1 interval, cells still exhibited a measurable G1 time, reflecting the stage-specific activities within G1. One activity that may be responsible for this obligatory G1 time is the synthesis of a labile protein. For other cells studied here, protraction of S phase also caused proliferating cells to become more massive, but in these cases there was no diminution of the G1 time. For these cells, the entire G1 interval must accommodate G1-specific activities necessary to initiate a new cell cycle. A unifying view of the G1 interval recognizes the two distinct influences that determine the time spent in G1: the need to accumulate sufficient mass to initiate a new DNA-division sequence; and the stage-specific events necessary for the subsequent S phase. The length of the G1 interval is dictated by the longer of these two time-consuming activities.

Animals↗
PubMed1991

The RSF1 gene regulates septum formation in Saccharomyces cerevisiae.

Septum formation in the mitotic cell cycle of the budding yeast Saccharomyces cerevisiae occurs by conversion of the chitin ring, laid down at bud formation, into the primary septum. We show here that under certain conditions this septation is dependent on the newly identified RSF1 gene. However, cells harboring the rsf1-1 mutation accumulated in a postcytokinesis state, with delayed conversion of the chitin-rich annulus into the primary septum. This rsf1-1-mediated inhibition of septum formation only occurred under conditions of biosynthetic stress and was correlated with biosynthetically mediated inhibition of the cell-cycle regulatory step START. The RSF1 gene is distinct from the CHS2 chitin synthase gene that is responsible for septation, and thus RSF1 most likely encodes a regulator of chitin synthesis. We hypothesize that RSF1 activity facilitates septum formation during times of biosynthetic stress, to allow efficient septation even under these conditions.

Cell Cycle↗
PubMed1991

CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus.

The cell cycle of the budding yeast Saccharomyces cerevisiae has been investigated through the study of conditional cdc mutations that specifically affect cell cycle performance. Cells bearing the cdc68-1 mutation (J. A. Prendergast, L. E. Murray, A. Rowley, D. R. Carruthers, R. A. Singer, and G. C. Johnston, Genetics 124:81-90, 1990) are temperature sensitive for the performance of the G1 regulatory event, START. Here we describe the CDC68 gene and present evidence that the CDC68 gene product functions in transcription. CDC68 encodes a 1,035-amino-acid protein with a highly acidic and serine-rich carboxyl terminus. The abundance of transcripts from several unrelated genes is decreased in cdc68-1 mutant cells after transfer to the restrictive temperature, while at least one transcript, from the HSP82 gene, persists in an aberrant fashion. Thus, the cdc68-1 mutation has both positive and negative effects on gene expression. Our findings complement those of Malone et al. (E. A. Malone, C. D. Clark, A. Chiang, and F. Winston, Mol. Cell. Biol. 11:5710-5717, 1991), who have independently identified the CDC68 gene (as SPT16) as a transcriptional suppressor of delta-insertion mutations. Among transcripts that rapidly become depleted in cdc68-1 mutant cells are those of the G1 cyclin genes CLN1, CLN2, and CLN3/WHI1/DAF1, whose activity has been previously shown to be required for the performance of START. The decreased abundance of cyclin transcripts in cdc68-1 mutant cells, coupled with the suppression of cdc68-1-mediated START arrest by the CLN2-1 hyperactive allele of CLN2, shows that the CDC68 gene affects START through cyclin gene expression.

Amino Acid Sequence↗
PubMed1990

Size selection identifies new genes that regulate Saccharomyces cerevisiae cell proliferation.

A centrifugation procedure to enrich for enlarged cells has been used to isolate temperature-sensitive cdc mutants of the yeast Saccharomyces cerevisiae. Among these mutants are strains containing mutations that arrest proliferation at the regulatory step start. These new start mutations define two previously unidentified genes, CDC67 and CDC68, and reveal that a previously identified gene, DNA33 (here termed CDC65), can harbour start mutations. Each new start mutation permits significant biosynthetic activity after transfer of mutant cells to the non-permissive temperature. The cdc68-1 start mutation causes arrest of cell proliferation without inhibition of mating ability, while the cdc65-1 and cdc67-1 mutations inhibit zygote formation and successful conjugation. The identification of new start genes by a novel selection procedure suggests that the catalog of genes that influence start is large.

Cell Division↗
PubMed1990

Genetic assessment of stationary phase for cells of the yeast Saccharomyces cerevisiae.

Starvation of cells of the yeast Saccharomyces cerevisiae causes cessation of proliferation and acquisition of characteristic physiological properties. The stationary-phase state that results represents a unique developmental state, as shown by a novel conditional phenotype (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987): mutant cells cannot proliferate at the restrictive temperature when stimulated to reenter the mitotic cell cycle from stationary phase but are unaffected and continue proliferation indefinitely if transferred to the restrictive temperature during exponential growth. We have exploited this reentry mutant phenotype to demonstrate that the same stationary-phase state is generated by nitrogen, sulfur, or carbon starvation and by the cdc25-1 mutation, which conditionally impairs the cyclic AMP-mediated signal transduction pathway. We also show that heat shock, a treatment that elicits physiological perturbations associated with stationary phase, does not cause cells to enter stationary phase. The physiological properties associated with stationary phase therefore do not result from residence in stationary phase but from the stress conditions that bring about stationary phase.

Cell Cycle↗
PubMed1990

Thermotolerance is independent of induction of the full spectrum of heat shock proteins and of cell cycle blockage in the yeast Saccharomyces cerevisiae.

Cells of the yeast Saccharomyces cerevisiae are known to acquire thermotolerance in response to the stresses of starvation or heat shock. We show here through the use of cell cycle inhibitors that blockage of yeast cells in the G1, S, or G2 phases of the mitotic cell cycle is not a stress that induces thermotolerance; arrested cells remained as sensitive to thermal killing as proliferating cells. These G1- or S-phase-arrested cells were unimpaired in the acquisition of thermotolerance when subjected to a mild heat shock by incubation at 37 degrees C. One cell cycle inhibitor, o-phenanthroline, did in fact cause cells to become thermotolerant but without induction of the characteristic pattern of heat shock proteins. Thermal induction of heat shock protein synthesis was unaffected; the o-phenanthroline-treated cells could still synthesize heat shock proteins upon transfer to 37 degrees C. Use of a novel mutant conditionally defective only for the resumption of proliferation from stationary phase (M. A. Drebot, G. C. Johnston, and R. A. Singer, Proc. Natl. Acad. Sci. USA 84:7948-7952, 1987) indicated that o-phenanthroline inhibition produces a stationary-phase arrest, a finding which is consistent with the increased thermotolerance and regulated cessation of proliferation exhibited by the inhibited cells. These findings show that the acquired thermotolerance of cells is unrelated to blockage of the mitotic cell cycle or to the rapid synthesis of the characteristic spectrum of heat shock proteins.

Adenosine↗
PubMed1990

Induction of yeast histone genes by stimulation of stationary-phase cells.

In the cell cycle of the budding yeast Saccharomyces cerevisiae, expression of the histone genes H2A and H2B of the TRT1 and TRT2 loci is regulated by the performance of "start," the step that also regulates the cell cycle. Here we show that histone production is also subject to an additional form of regulation that is unrelated to the mitotic cell cycle. Expression of histone genes, as assessed by Northern (RNA) analysis, was shown to increase promptly after the stimulation, brought about by fresh medium, that activates stationary-phase cells to reenter the mitotic cell cycle. The use of a yeast mutant that is conditionally blocked in the resumption of proliferation at a step that is not part of the mitotic cell cycle (M.A. Drebot, G.C. Johnston, and R.A. Singer, Proc. Natl. Acad. Sci. 84:7948, 1987) showed that this increased gene expression that occurs upon stimulation of stationary-phase cells took place in the absence of DNA synthesis and without the performance of start. This stimulation-specific gene expression was blocked by the mating pheromone alpha-factor, indicating that alpha-factor directly inhibits expression of these histone genes, independently of start.

Cell Cycle↗
PubMed1990

Regulation of proliferation by the budding yeast Saccharomyces cerevisiae.

Mutations in the budding yeast Saccharomyces cerevisiae define regulatory activities both for the mitotic cell cycle and for resumption of proliferation from the quiescent stationary-phase state. In each case, the regulation of proliferation occurs in the prereplicative interval that precedes the initiation of DNA replication. This regulation is particularly responsive to the nutrient environment and the biosynthetic capacity of the cell. Mutations in components of the cAMP-mediated effector pathway and in components of the biosynthetic machinery itself affect regulation of proliferation within the mitotic cell cycle. In the extreme case of nutrient starvation, cells cease proliferation and enter stationary phase. Mutations in newly defined genes prevent stationary-phase cells from reentering the mitotic cell cycle, but have no effect on proliferating cells. Thus stationary phase represents a unique developmental state, with requirements to resume proliferation that differ from those for the maintenance of proliferation in the mitotic cell cycle.

Cell Cycle↗
PubMed1989

rRNA transcription initiation is decreased by inhibitors of the yeast cell cycle control step "start".

Inhibitors of the "start" regulatory step in the cell cycle of the yeast Saccharomyces cerevisiae are known by indirect studies to perturb RNA metabolism. We have investigated these effects further and show here by a pulse-labeling and quantitative hybridization procedure that pre-rRNA transcription was substantially decreased by five inhibitors of start but was transiently stimulated by the mating pheromone alpha-factor. Thus in contrast to the effects of the other start inhibitors, the inhibition of start by alpha-factor is unrelated to this aspect of biosynthetic activity. Mating factor treatment also stimulated the synthesis rate of poly(A)+ RNA. The start inhibitors o-phenanthroline and L-ethionine inhibited pre-rRNA transcription with little effect on poly(A)+ RNA synthesis rates. Northern analysis showed that all inhibitors of start also inhibited pre-rRNA transcript cleavage, a process that has been dissociated from the inhibition of start. Most inhibitors also affected ATP pool size. One inhibitor, o-phenanthroline, markedly induced the general control response.

Adenosine Triphosphate↗
PubMed1989

Heat shock causes transient inhibition of yeast rRNA gene transcription.

The heat-shock response in the yeast Saccharomyces cerevisiae includes transiently decreased production of the full-size pre-rRNA transcript. Here we have used quantitative hybridization of pulse-labeled RNA to cloned, immobilized sequences derived from the external transcribed spacer of yeast rDNA, coupled with determinations of relative changes in ATP pool specific activities, to show that the heat shock associated with the transfer of growing cells from 23 to 36 degrees C caused decreased transcription of the rRNA genes. This decrease in hybridization to DNA sequences complementary to the immediate 5' end of the pre-rRNA transcript suggests that the decreased transcription reflects decreased initiation of pre-rRNA transcription.

Adenosine Triphosphate↗