The RAD3 gene of Saccharomyces cerevisiae: Isolation and characterization of a temperature-sensitive mutant in the essential function and of extragenic suppressors of this mutant

Louie Naumovski, Errol C. Friedberg

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Mutations in the RAD3 gene of Saccharomyces cerevisiae were generated by integration of a mutagenized incomplete copy of the cloned gene into wild-type cells. Integrants were mass screened for colonies with abnormal growth characteristics at 37°C. A single temperature-sensitive mutant (rad3ts-1) was isolated and was shown to result from a missense mutation at codon 73 of the RAD3 gene. When shifted from 30° C to 37° C the strain undergoes only 2-4 cell doublings. This phenotype can be rescued by plasmids in which the essential function of the cloned RAD3 gene is intact, but not plasmids in which this function is inactivated. The mutant strain is weakly sensitive to ultraviolet (UV) radiation at restrictive temperatures. Measurement of RNA, DNA and protein synthesis at various times after shifting to restrictive temperatures does not show preferential inactivation of any one of these parameters and the temperature-sensitive mutation does not cause arrest at any specific phase of the cell cycle. The rad3ts-1 strain was transformed with multicopy plasmids from a normal yeast genomic library and two plasmids that partially suppress the temperature-sensitive phenotype were isolated. These suppressor genes (designated SRE1 and SRE2) are distinct from RAD3 and do not suppress the phenotype of several other temperature-sensitive mutants tested. Mutant strains carrying disruptions of the SRE1 gene are viable and are not sensitive to UV or γ radiation.

Original languageEnglish (US)
Pages (from-to)458-466
Number of pages9
JournalMGG Molecular & General Genetics
Volume209
Issue number3
DOIs
StatePublished - Oct 1987

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Saccharomyces cerevisiae
Temperature
Plasmids
Genes
Phenotype
Radiation
Suppressor Genes
Mutation
Genomic Library
Missense Mutation
Codon
Cell Cycle
Yeasts
RNA
DNA
Growth
Proteins

Keywords

  • DNA repair
  • RAD genes
  • UV radiation
  • Yeast

ASJC Scopus subject areas

  • Genetics

Cite this

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abstract = "Mutations in the RAD3 gene of Saccharomyces cerevisiae were generated by integration of a mutagenized incomplete copy of the cloned gene into wild-type cells. Integrants were mass screened for colonies with abnormal growth characteristics at 37°C. A single temperature-sensitive mutant (rad3ts-1) was isolated and was shown to result from a missense mutation at codon 73 of the RAD3 gene. When shifted from 30° C to 37° C the strain undergoes only 2-4 cell doublings. This phenotype can be rescued by plasmids in which the essential function of the cloned RAD3 gene is intact, but not plasmids in which this function is inactivated. The mutant strain is weakly sensitive to ultraviolet (UV) radiation at restrictive temperatures. Measurement of RNA, DNA and protein synthesis at various times after shifting to restrictive temperatures does not show preferential inactivation of any one of these parameters and the temperature-sensitive mutation does not cause arrest at any specific phase of the cell cycle. The rad3ts-1 strain was transformed with multicopy plasmids from a normal yeast genomic library and two plasmids that partially suppress the temperature-sensitive phenotype were isolated. These suppressor genes (designated SRE1 and SRE2) are distinct from RAD3 and do not suppress the phenotype of several other temperature-sensitive mutants tested. Mutant strains carrying disruptions of the SRE1 gene are viable and are not sensitive to UV or γ radiation.",
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AB - Mutations in the RAD3 gene of Saccharomyces cerevisiae were generated by integration of a mutagenized incomplete copy of the cloned gene into wild-type cells. Integrants were mass screened for colonies with abnormal growth characteristics at 37°C. A single temperature-sensitive mutant (rad3ts-1) was isolated and was shown to result from a missense mutation at codon 73 of the RAD3 gene. When shifted from 30° C to 37° C the strain undergoes only 2-4 cell doublings. This phenotype can be rescued by plasmids in which the essential function of the cloned RAD3 gene is intact, but not plasmids in which this function is inactivated. The mutant strain is weakly sensitive to ultraviolet (UV) radiation at restrictive temperatures. Measurement of RNA, DNA and protein synthesis at various times after shifting to restrictive temperatures does not show preferential inactivation of any one of these parameters and the temperature-sensitive mutation does not cause arrest at any specific phase of the cell cycle. The rad3ts-1 strain was transformed with multicopy plasmids from a normal yeast genomic library and two plasmids that partially suppress the temperature-sensitive phenotype were isolated. These suppressor genes (designated SRE1 and SRE2) are distinct from RAD3 and do not suppress the phenotype of several other temperature-sensitive mutants tested. Mutant strains carrying disruptions of the SRE1 gene are viable and are not sensitive to UV or γ radiation.

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