Biomedical subjects

L J Oehlen

Publications and source records attributed to L J Oehlen.

8 recordsLinked to original sources

PubMed1996

Ste12 and Mcm1 regulate cell cycle-dependent transcription of FAR1.

The transcripts of many genes involved in Saccharomyces cerevisiae mating were found to fluctuate during the cell cycle. In the absence of a functional Ste12 transcription factor, both the levels and the cell cycle pattern of expression of these genes were affected. FUS1 and AGA1 levels, which are maximally expressed only in G1-phase cells, were strongly reduced in ste12- cells. The cell cycle transcription pattern for FAR1 was changed in ste12- cells: the gene was still significantly expressed in G2/M, but transcript levels were strongly reduced in G1 phase, resulting in a lack of Far1 protein accumulation. G2/M transcription of FAR1 was dependent on the transcription factor Mcm1, and expression of a gene with Mcm1 fused to a strong transcriptional activation domain resulted in increased levels of FAR1 transcription. The pattern of cell cycle-regulated transcription of FAR1 could involve combinatorial control of Ste12 and Mcm1. Forced G1 expression of FAR1 from the GAL1 promoter resorted the ability to arrest in response to pheromone in ste12-cells. This indicates that transcription of FAR1 in the G1 phase is essential for accumulation of the protein and for pheromone-induced cell cycle arrest.

Base Sequence↗

PubMed1995

Identification of two cell cycle regulated genes affecting the beta 1,3-glucan content of cell walls in Saccharomyces cerevisiae.

The Calcofluor white-hypersensitive mutants cwh52 and cwh53 are severely reduced in beta 1,3-glucan. CWH52 was equivalent to GAS1. CWH53 represented a new gene, located on the right arm of chromosome XII, and predicted to encode a 215 kDa protein with multiple transmembrane domains. The transcription of CWH53 was cell cycle-dependent and, similar to GAS1/CWH52, increased in late G1, indicating that the formation of beta-glucan is cell cycle-regulated. Further, in some mutant alleles of both gas1/cwh52 and cwh53 lethal concentrations of Calcofluor induced growth arrest at a specific phase of the cell cycle.

Amino Acid Sequence↗

PubMed1994

G1 cyclins CLN1 and CLN2 repress the mating factor response pathway at Start in the yeast cell cycle.

Transcriptional induction by the mating pheromone alpha-factor was monitored at different stages of the yeast cell cycle. G2/M-phase and pre-Start cells showed strong FUS1 mRNA induction, whereas in post-Start cells the signaling was reduced significantly. This reduction in signaling activity in post-Start cells was correlated with the presence of CLN1 or CLN2 transcripts and was not observed in synchronized cells lacking functional CLN1 and CLN2 genes. Activation of the Cln-Cdc28p kinase by overexpression of CLN2 from the GAL1 promoter strongly reduced FUS1 mRNA induction. CLN1 overexpression had a similar effect when the FAR1 gene, encoding a negative regulator of CLN1/2 function, was deleted. This reduction of pheromone signaling was specific for CLN1 and CLN2, as it was not observed when CLN3 was overexpressed. Inactivation of the Cln-Cdc28p kinase complex by thermal inactivation of temperature-sensitive Cdc28p prevented repression of FUS1 signaling. CLN2 overexpression suppressed the constitutive signaling and division-arrest phenotypes of cells with a disrupted gpa1 gene, indicating that the site of action for repression is downstream of the alpha-subunit (Gpa1p) of the heterotrimeric G protein. The repression at Start of pheromone signaling by Cln1-Cdc28p or Cln2-Cdc28p kinase complexes may contribute to the acquisition of pheromone resistance as cells execute Start.

Base Sequence↗

PubMed1994

Decrease in glycolytic flux in Saccharomyces cerevisiae cdc35-1 cells at restrictive temperature correlates with a decrease in glucose transport.

The glycolytic flux was investigated in the thermosensitive Saccharomyces cerevisiae adenylate cyclase mutant cdc35-1. Directly after a shift to restrictive temperature, the specific CO2 production rate increased from about 250 nmol min-1 (mg protein)-1 to more than 400 nmol min-1 (mg protein)-1, but then the CO2 production gradually fell to about 70 nmol min-1 (mg protein)-1 after 5 h. O2 consumption at restrictive temperature continued at more or less the same rate as at permissive temperature. The temperature shift in the mutant resulted in an increase in the estimated intracellular cAMP concentration from about 1.1 microM to 1.8 microM. This indicates that high cAMP levels are not sufficient for cell cycle progression and high glycolytic activity. The decrease in glycolytic activity at restrictive temperature was not paralleled by a similar decrease in the specific activity of any of the glycolytic enzymes, but correlated with a decrease in hexose transport. A drop in intracellular concentrations of the early metabolites of glycolysis further indicated a defect in transport at restrictive temperature. Our data suggest that glucose transport has a high control on glycolytic flux.

Adenylyl Cyclases↗

PubMed1993

Inactivation of the CDC25 gene product in Saccharomyces cerevisiae leads to a decrease in glycolytic activity which is independent of cAMP levels.

In the budding yeast Saccharomyces cerevisiae cyclic AMP (cAMP) can influence the activity of key enzymes in carbohydrate metabolism through modulation of the activity of cAMP-dependent protein kinase. One of the components involved in cAMP production is the CDC25 gene product, which can activate the RAS/adenylate cyclase pathway by promoting the exchange of guanine nucleotides bound to RAS. In two yeast strains carrying different thermosensitive alleles of the CDC25 gene, cAMP levels respond differently to an increase in growth temperature from 23 degrees C (permissive) to 36 degrees C (restrictive). In strain OL86 (cdc25-5) the estimated intracellular concentration of cAMP dropped after transfer to restrictive temperature whereas in strain ts321 (cdc25-1) the cAMP level rose under the same conditions. Despite the differences in cAMP levels the glycolytic flux in the two mutants responded in a very similar way to the shift from permissive to restrictive temperature; after the increase in the incubation temperature, the specific glycolytic flux in both cdc25-1 and cdc25-5 initially increased from about 300 nmol min-1 (mg protein)-1 to about 500 nmol min-1 (mg protein)-1 (presumably mainly as a consequence of the increase in temperature), but then gradually fell to 100-200 nmol min-1 (mg protein)-1. A similar pattern of CO2 production to that found in the two cdc25 mutants was also observed for several other thermosensitive mutants displaying a Start-II type of G1 arrest. In contrast, in a wild-type strain and in strains giving a Start-I type of G1 arrest, CO2 production did not drop after a temperature shift. The specific activities of glycolytic enzymes in the two cdc25 mutants did not show much change after the temperature shift, indicating that the decrease in glycolytic flux was not caused by a decrease in the activity of any of the glycolytic enzymes. Our data show that, at least in long-term regulation, the cAMP levels per se are not likely to be a prime factor controlling glycolytic flux.

Alleles↗

PubMed1991

Changes in the activities of key enzymes of glycolysis during the cell cycle in yeast: a rectification.

Activities of glycolytic enzymes were determined in elutriation fractionated cultures of Saccharomyces cerevisiae grown on different carbon sources. Almost pure fractions of single cells at the G1 state of cell division were obtained for some of the growth conditions tested, whereas other stages were enriched in particular fractions. Specific activities of glucose-6-phosphate dehydrogenase and alcohol dehydrogenase were found to be constant during the cell cycle, as reported by van Doorn et al. (1988a), Journal of Bacteriology 170, 4808-4815, and (1988b), Journal of General Microbiology 134, 785-790. In contrast to the earlier reports, the activities of hexokinase, phosphofructokinase, pyruvate kinase and trehalase were also constant in different states of the cell cycle. For hexokinase and phosphofructokinase it was shown that the apparent specific activity in a cell-free extract strongly diminished when extracts contained less that 0.5-1 mg protein ml-1. In the experiments of van Doorn et al. (1988a) the protein content of the outer fractions was up to 20 times lower than that of the central fractions, suggesting an alternative explanation for the observed changes in enzyme activities during the cell cycle. Therefore, we want to rectify the observations presented by van Doorn et al. (1988a), and conclude that the activities of the glycolytic enzymes do not vary greatly during the cell cycle of S. cervisiae.

Cell Cycle↗

PubMed1990

Changes in the incorporation of carbon derived from glucose into cellular pools during the cell cycle of Saccharomyces cerevisiae.

The rate of incorporation of 14C derived from [U-14C]glucose into cells of Saccharomyces cerevisiae X2180(1B) was investigated as a function of the cell cycle. After pulse-labelling of exponentially growing populations, centrifugal elutriation was used to isolate various cell fractions of increasing cell size, representing successive stages of the cell cycle. The total amount of 14C incorporated per cell was found to increase continuously during the cell cycle along with cellular protein content and Coulter counter cell volume. This pattern supports the model of exponential cell growth. In order to evaluate changes in intracellular carbon flow during the cell cycle, chemical extraction procedures were used to obtain four cellular fractions enriched in either low-molecular-mass components, lipid material, polysaccharides or proteins. The distribution of 14C among these cellular fractions varied during successive stages of the cell cycle, indicating cell-cycle-dependent fluctuations in intracellular carbon flow. During the G1 phase the flow of 14C into the low-molecular-mass pool increased markedly; concurrently, the rate of incorporation into the polysaccharide-enriched pool decreased.

Carbon↗

PubMed1988

Changes in activities of several enzymes involved in carbohydrate metabolism during the cell cycle of Saccharomyces cerevisiae.

Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.

Alcohol Dehydrogenase↗