Identification and genetic analysis of Schizosaccharomyces pombe cDNAs that suppress deletion of IRA1 in Saccharomyces cerevisiae

Heather Matviw, Gang Yu, Dallan Young

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Ira1 is a negative regulator of Ras proteins in Saccharomyces cerevisiae. Deletion of IRA1 leads to constitutive activation of the Ras/cyclic AMP (cAMP) pathway, which results in several phenotypes including sensitivity to heatshock (HS) treatment. We have identified eight Schizosaccharomyces pombe cDNAs that, when overexpressed, suppress the HS-sensitive phenotype associated with the deletion of IRA1 in S. cerevisiae. To determine where these cDNAs act, we tested their ability to suppress other mutations that activate the Ras/cAMP pathway in S. cerevisiae. Two of the cDNA clones, pPSI1 and pPSI2, failed to suppress the HS-sensitive phenotype induced by the activating RAS2Val19 mutation. Clone pPSI2 encodes Gap1/Sar1, a Sz. pombe homologue of Ira1, which has been previously identified. Three of the six RAS2Val19 suppressors could suppress the deletion of PDE1 and PDE2, the cAMP phosphodiesterase (Pde)-encoding genes, suggesting that they act downstream from adenylyl cyclase (Cyr). The remaining three clones, pPSI3, pPSI6 and pPSI7, encode proteins that may suppress the HS-sensitive phenotype by reducing Ras and/or Cyr activity. One of these, pPSI3, contains a cDNA that encodes the C-terminal region (aa 166-550) of the Sz. pombe Dbp2 protein, a homologue of the human p68 RNA helicase. We have amplified cDNAs encoding the full-length Sz. pombe Dbp2 protein by the polymerase chain reaction method and have cloned them into a S. cerevisiae expression vector. The iral- cells harboring these plasmids retained their HS-sensitive phenotype. These results suggest that the truncated Dbp2, but not the full-length protein, is capable of interfering with Ras and/or Cyr activity.

Original languageEnglish (US)
Pages (from-to)147-152
Number of pages6
JournalGene
Volume129
Issue number1
DOIs
StatePublished - Jul 15 1993

Fingerprint

Schizosaccharomyces
Saccharomyces cerevisiae
Complementary DNA
Phenotype
Cyclic AMP
Clone Cells
Proteins
DEAD-box RNA Helicases
ras Proteins
Mutation
Phosphoric Diester Hydrolases
Adenylyl Cyclases
Plasmids
Polymerase Chain Reaction
Genes

Keywords

  • heat shock
  • Ras
  • Recombinant DNA
  • RNA helicase
  • yeast

ASJC Scopus subject areas

  • Genetics

Cite this

Identification and genetic analysis of Schizosaccharomyces pombe cDNAs that suppress deletion of IRA1 in Saccharomyces cerevisiae. / Matviw, Heather; Yu, Gang; Young, Dallan.

In: Gene, Vol. 129, No. 1, 15.07.1993, p. 147-152.

Research output: Contribution to journalArticle

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abstract = "Ira1 is a negative regulator of Ras proteins in Saccharomyces cerevisiae. Deletion of IRA1 leads to constitutive activation of the Ras/cyclic AMP (cAMP) pathway, which results in several phenotypes including sensitivity to heatshock (HS) treatment. We have identified eight Schizosaccharomyces pombe cDNAs that, when overexpressed, suppress the HS-sensitive phenotype associated with the deletion of IRA1 in S. cerevisiae. To determine where these cDNAs act, we tested their ability to suppress other mutations that activate the Ras/cAMP pathway in S. cerevisiae. Two of the cDNA clones, pPSI1 and pPSI2, failed to suppress the HS-sensitive phenotype induced by the activating RAS2Val19 mutation. Clone pPSI2 encodes Gap1/Sar1, a Sz. pombe homologue of Ira1, which has been previously identified. Three of the six RAS2Val19 suppressors could suppress the deletion of PDE1 and PDE2, the cAMP phosphodiesterase (Pde)-encoding genes, suggesting that they act downstream from adenylyl cyclase (Cyr). The remaining three clones, pPSI3, pPSI6 and pPSI7, encode proteins that may suppress the HS-sensitive phenotype by reducing Ras and/or Cyr activity. One of these, pPSI3, contains a cDNA that encodes the C-terminal region (aa 166-550) of the Sz. pombe Dbp2 protein, a homologue of the human p68 RNA helicase. We have amplified cDNAs encoding the full-length Sz. pombe Dbp2 protein by the polymerase chain reaction method and have cloned them into a S. cerevisiae expression vector. The iral- cells harboring these plasmids retained their HS-sensitive phenotype. These results suggest that the truncated Dbp2, but not the full-length protein, is capable of interfering with Ras and/or Cyr activity.",
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N2 - Ira1 is a negative regulator of Ras proteins in Saccharomyces cerevisiae. Deletion of IRA1 leads to constitutive activation of the Ras/cyclic AMP (cAMP) pathway, which results in several phenotypes including sensitivity to heatshock (HS) treatment. We have identified eight Schizosaccharomyces pombe cDNAs that, when overexpressed, suppress the HS-sensitive phenotype associated with the deletion of IRA1 in S. cerevisiae. To determine where these cDNAs act, we tested their ability to suppress other mutations that activate the Ras/cAMP pathway in S. cerevisiae. Two of the cDNA clones, pPSI1 and pPSI2, failed to suppress the HS-sensitive phenotype induced by the activating RAS2Val19 mutation. Clone pPSI2 encodes Gap1/Sar1, a Sz. pombe homologue of Ira1, which has been previously identified. Three of the six RAS2Val19 suppressors could suppress the deletion of PDE1 and PDE2, the cAMP phosphodiesterase (Pde)-encoding genes, suggesting that they act downstream from adenylyl cyclase (Cyr). The remaining three clones, pPSI3, pPSI6 and pPSI7, encode proteins that may suppress the HS-sensitive phenotype by reducing Ras and/or Cyr activity. One of these, pPSI3, contains a cDNA that encodes the C-terminal region (aa 166-550) of the Sz. pombe Dbp2 protein, a homologue of the human p68 RNA helicase. We have amplified cDNAs encoding the full-length Sz. pombe Dbp2 protein by the polymerase chain reaction method and have cloned them into a S. cerevisiae expression vector. The iral- cells harboring these plasmids retained their HS-sensitive phenotype. These results suggest that the truncated Dbp2, but not the full-length protein, is capable of interfering with Ras and/or Cyr activity.

AB - Ira1 is a negative regulator of Ras proteins in Saccharomyces cerevisiae. Deletion of IRA1 leads to constitutive activation of the Ras/cyclic AMP (cAMP) pathway, which results in several phenotypes including sensitivity to heatshock (HS) treatment. We have identified eight Schizosaccharomyces pombe cDNAs that, when overexpressed, suppress the HS-sensitive phenotype associated with the deletion of IRA1 in S. cerevisiae. To determine where these cDNAs act, we tested their ability to suppress other mutations that activate the Ras/cAMP pathway in S. cerevisiae. Two of the cDNA clones, pPSI1 and pPSI2, failed to suppress the HS-sensitive phenotype induced by the activating RAS2Val19 mutation. Clone pPSI2 encodes Gap1/Sar1, a Sz. pombe homologue of Ira1, which has been previously identified. Three of the six RAS2Val19 suppressors could suppress the deletion of PDE1 and PDE2, the cAMP phosphodiesterase (Pde)-encoding genes, suggesting that they act downstream from adenylyl cyclase (Cyr). The remaining three clones, pPSI3, pPSI6 and pPSI7, encode proteins that may suppress the HS-sensitive phenotype by reducing Ras and/or Cyr activity. One of these, pPSI3, contains a cDNA that encodes the C-terminal region (aa 166-550) of the Sz. pombe Dbp2 protein, a homologue of the human p68 RNA helicase. We have amplified cDNAs encoding the full-length Sz. pombe Dbp2 protein by the polymerase chain reaction method and have cloned them into a S. cerevisiae expression vector. The iral- cells harboring these plasmids retained their HS-sensitive phenotype. These results suggest that the truncated Dbp2, but not the full-length protein, is capable of interfering with Ras and/or Cyr activity.

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