scholarly journals Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (10) ◽  
pp. 3629-3636 ◽  
Author(s):  
J Nikawa ◽  
P Sass ◽  
M Wigler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cyclic Amp ◽  
Copy Number ◽  
Growth Arrest ◽  
Yeast Cells ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
High Affinity ◽  
Cyclic Amp Phosphodiesterase

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.

Cloning and characterization of the low-affinity cyclic AMP phosphodiesterase gene of Saccharomyces cerevisiae

1987 ◽  
Vol 7 (10) ◽  
pp. 3629-3636
Author(s):  
J Nikawa ◽  
P Sass ◽  
M Wigler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cyclic Amp ◽  
Copy Number ◽  
Growth Arrest ◽  
Yeast Cells ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
High Affinity ◽  
Cyclic Amp Phosphodiesterase

Saccharomyces cerevisiae contains two genes which encode cyclic AMP (cAMP) phosphodiesterase. We previously isolated and characterized PDE2, which encodes a high-affinity cAMP phosphodiesterase. We have now isolated the PDE1 gene of S. cerevisiae, which encodes a low-affinity cAMP phosphodiesterase. These two genes represent highly divergent branches in the evolution of phosphodiesterases. High-copy-number plasmids containing either PDE1 or PDE2 can reverse the growth arrest defects of yeast cells carrying the RAS2(Val-19) mutation. PDE1 and PDE2 appear to account for the aggregate cAMP phosphodiesterase activity of S. cerevisiae. Disruption of both PDE genes results in a phenotype which resembles that induced by the RAS2(Val-19) mutation. pde1- pde2- ras1- ras2- cells are viable.

scholarly journals Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae.

1986 ◽  
Vol 83 (24) ◽  
pp. 9303-9307 ◽  
Author(s):  
P. Sass ◽  
J. Field ◽  
J. Nikawa ◽  
T. Toda ◽  
M. Wigler
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Camp Phosphodiesterase ◽  
High Affinity

Estradiol-inducible squelching and cell growth arrest by a chimeric VP16-estrogen receptor expressed in Saccharomyces cerevisiae: suppression by an allele of PDR1

1993 ◽  
Vol 13 (1) ◽  
pp. 462-472
Author(s):  
D M Gilbert ◽  
D M Heery ◽  
R Losson ◽  
P Chambon ◽  
Y Lemoine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Estrogen Receptor ◽  
Cell Growth ◽  
Copy Number ◽  
Multiple Drug Resistance ◽  
Growth Arrest ◽  
Yeast Cells ◽  
Receptor Function ◽  
Cell Growth Arrest

We have constructed and characterized a flexible system for analyzing the phenomenon of squelching and estrogen receptor function in the yeast Saccharomyces cerevisiae. The A/B region of the human estrogen receptor was replaced with the transcriptional activating domain of VP16 and expressed in yeast cells from high-copy-number plasmids. Addition of hormone resulted in an immediate inhibition of expression (squelching) of a chromosomally integrated GAL1:lacZ reporter gene and the eventual arrest of cell growth (toxicity). In order to determine whether a relationship exists between toxicity and squelching, mutations were made in this chimeric receptor (VEO) and their effects on transcriptional activation, squelching, and toxicity were compared. A direct correlation was found between mutations in VEO that reduced VP16 transactivation ability in yeast cells and those that reduced both squelching and toxicity. Surprisingly, mutations in the DNA binding domain (DBD) of VEO dramatically reduced squelching and completely relieved toxicity, suggesting a role for the DBD in squelching and strengthening the correlation between squelching and toxicity. To demonstrate the utility of this system for carrying out genetic selection, a plasmid-based yeast genomic bank was screened for genes that can relieve the toxicity of VEO by means of an elevated copy number, resulting in the repeated cloning of an allele of the PDR1 (pleiotropic drug resistance) gene. We present evidence that mutations in PDR1 can modulate the intracellular availability of estradiol by the same mechanism that leads to multiple drug resistance in yeast cells. Taken together, our results provide evidence that cell growth arrest occurs when squelching exceeds a certain threshold and that strong squelching requires both a DBD and a transcriptional activating domain. Furthermore, we show that growth arrest can provide a useful phenotype for carrying out the genetic analysis of both squelching and estrogen receptor function in yeast cells.

scholarly journals SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (1) ◽  
pp. 505-510 ◽  
Author(s):  
R B Wilson ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cyclic Amp ◽  
Nitrogen Starvation ◽  
Carbon Sources ◽  
Its Sequence ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
Rich Media ◽  
Minimal Media ◽  
The Right

sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity. We have cloned SRA5 by complementation. It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase. Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP. sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.

scholarly journals Estradiol-inducible squelching and cell growth arrest by a chimeric VP16-estrogen receptor expressed in Saccharomyces cerevisiae: suppression by an allele of PDR1.

1993 ◽  
Vol 13 (1) ◽  
pp. 462-472 ◽  
Author(s):  
D M Gilbert ◽  
D M Heery ◽  
R Losson ◽  
P Chambon ◽  
Y Lemoine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Estrogen Receptor ◽  
Cell Growth ◽  
Copy Number ◽  
Multiple Drug Resistance ◽  
Growth Arrest ◽  
Yeast Cells ◽  
Receptor Function ◽  
Cell Growth Arrest

We have constructed and characterized a flexible system for analyzing the phenomenon of squelching and estrogen receptor function in the yeast Saccharomyces cerevisiae. The A/B region of the human estrogen receptor was replaced with the transcriptional activating domain of VP16 and expressed in yeast cells from high-copy-number plasmids. Addition of hormone resulted in an immediate inhibition of expression (squelching) of a chromosomally integrated GAL1:lacZ reporter gene and the eventual arrest of cell growth (toxicity). In order to determine whether a relationship exists between toxicity and squelching, mutations were made in this chimeric receptor (VEO) and their effects on transcriptional activation, squelching, and toxicity were compared. A direct correlation was found between mutations in VEO that reduced VP16 transactivation ability in yeast cells and those that reduced both squelching and toxicity. Surprisingly, mutations in the DNA binding domain (DBD) of VEO dramatically reduced squelching and completely relieved toxicity, suggesting a role for the DBD in squelching and strengthening the correlation between squelching and toxicity. To demonstrate the utility of this system for carrying out genetic selection, a plasmid-based yeast genomic bank was screened for genes that can relieve the toxicity of VEO by means of an elevated copy number, resulting in the repeated cloning of an allele of the PDR1 (pleiotropic drug resistance) gene. We present evidence that mutations in PDR1 can modulate the intracellular availability of estradiol by the same mechanism that leads to multiple drug resistance in yeast cells. Taken together, our results provide evidence that cell growth arrest occurs when squelching exceeds a certain threshold and that strong squelching requires both a DBD and a transcriptional activating domain. Furthermore, we show that growth arrest can provide a useful phenotype for carrying out the genetic analysis of both squelching and estrogen receptor function in yeast cells.

SRA5 encodes the low-Km cyclic AMP phosphodiesterase of Saccharomyces cerevisiae

1988 ◽  
Vol 8 (1) ◽  
pp. 505-510
Author(s):  
R B Wilson ◽  
K Tatchell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cyclic Amp ◽  
Nitrogen Starvation ◽  
Carbon Sources ◽  
Its Sequence ◽  
Phosphodiesterase Activity ◽  
Camp Phosphodiesterase ◽  
Rich Media ◽  
Minimal Media ◽  
The Right

sra5 mutations in Saccharomyces cerevisiae were previously shown to suppress the inefficient growth of ras2 strains on nonfermentable carbon sources and to result in deficient low-Km cyclic AMP (cAMP) phosphodiesterase activity. We have cloned SRA5 by complementation. It maps to the right arm of chromosome XV, tightly linked to PRT1, and its sequence matches the sequence of PDE2, encoding the low-Km cAMP phosphodiesterase. Disruptions of SRA5 allowed ras1 ras2 strains to grow either on rich media supplemented with cAMP or on minimal media without exogenous cAMP. sra5 strains failed to survive prolonged nitrogen starvation in the presence of exogenous cAMP.

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