Isolation of the centromere-linked CDC10 gene by complementation in yeast

Area of Science:

• Molecular Biology
• Yeast Genetics
• Recombinant DNA Technology

Background:

• The CDC10 gene in Saccharomyces cerevisiae is essential for cell cycle progression.
• Previous studies had identified a 40 kilobase pair region around the LEU2 locus on chromosome III.

Purpose of the Study:

• To clone and physically map the CDC10 locus on yeast chromosome III.
• To establish the physical and genetic map relationship in the LEU2-CDC10 interval.

Main Methods:

• Construction of a hybrid plasmid colony bank in Escherichia coli using the E. coli-Saccharomyces cerevisiae shuttle vector pLC544.
• Isolation of a plasmid containing the CDC10 locus by complementation of a temperature-sensitive cdc10 mutation in yeast.
• Characterization of the cloned DNA segment and its overlap with the previously identified LEU2 locus region.

Main Results:

• An 8 kilobase pair DNA segment containing the CDC10 locus was isolated.
• The cloned DNA physically maps the CDC10 locus on chromosome III, establishing directionality relative to the genetic map and centromere.
• The relationship between physical and genetic distance in the leu2-cdc10 interval was determined to be approximately 3 kilobase pairs per centimorgan.

Conclusions:

• The study successfully cloned and physically mapped the yeast CDC10 gene.
• This provides a physical anchor for the genetic map of chromosome III and aids in understanding gene organization.
• The established physical to genetic distance ratio is valuable for future yeast genome mapping efforts.