Saccharomyces cerevisiae RAD2 gene: isolation, subcloning, and partial characterization

Area of Science:

• Molecular Biology
• Yeast Genetics
• DNA Repair

Background:

• The RAD2 gene in Saccharomyces cerevisiae plays a role in DNA repair, specifically in complementing UV sensitivity.
• Understanding the RAD2 gene is crucial for comprehending DNA repair mechanisms and genetic stability in yeast.

Purpose of the Study:

• To isolate and characterize the RAD2 gene from Saccharomyces cerevisiae.
• To determine the essentiality of the RAD2 gene for cell viability.
• To analyze the RNA transcript of the RAD2 gene.

Main Methods:

• Isolation of a complementing plasmid (pNF2000) from a yeast genomic library.
• Genetic analysis of yeast strains transformed with integrating plasmids containing RAD2 gene fragments.
• Gene disruption via integration of internal RAD2 gene fragments.
• RNA-DNA hybridization to measure RNA transcript size.

Main Results:

• A 9.7-kilobase pair DNA insert in plasmid pNF2000 was identified as containing the RAD2 gene.
• The smallest functional subclone of the RAD2 gene was determined to be 4.5 kilobase pairs.
• Disruption of the RAD2 gene did not affect haploid cell viability under normal growth conditions.
• The RNA transcript of the RAD2 gene was measured to be approximately 3.2 kilobases.

Conclusions:

• The isolated plasmid contains the functional RAD2 gene of Saccharomyces cerevisiae.
• The RAD2 gene is not essential for haploid cell viability.
• The study provides insights into the molecular characteristics and functional role of the RAD2 gene in DNA repair.