A plasmid model to study genetic recombination in yeast.

Area of Science:

• Molecular Biology
• Genetics
• Yeast Biology

Background:

• Chimeric plasmids with heteroallelic mutant yeast HIS3 genes were constructed.
• These plasmids are designed for studying intramolecular genetic exchange events.

Purpose of the Study:

• To measure the frequency of His+ prototrophs generated by intramolecular exchange during mitotic division in yeast.
• To investigate the role of the rad52 mutation in the genetic exchange process.
• To analyze the nature of recombination events occurring in the constructed plasmids.

Main Methods:

• Construction of chimeric plasmids containing two heteroallelic mutant HIS3 genes in an inverted repeat.
• Transformation of plasmids into his3- yeast strains.
• Measurement of His+ prototroph frequency after mitotic growth.
• Isolation and physical examination of plasmids after genetic exchange.
• Characterization of individual allelic copies within exchanged plasmids.

Main Results:

• 0.1-1% of yeast cells became His+ prototrophs after 20 generations.
• The frequency of His+ prototrophs decreased at least ten-fold in a rad52 mutant strain.
• Less than 10% of exchanged plasmids also showed reciprocal recombination.
• In most cases, only one of the two HIS3 copies became wild-type.

Conclusions:

• The constructed model plasmid undergoes intramolecular genetic exchange events.
• This system is a valuable tool for studying genetic recombination mechanisms.
• The rad52 mutation significantly impacts the frequency of genetic exchange.