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The influence of homology polymorphism and length on plasmid integration in yeast

Homologous recombination operates on DNA molecules with identical or almost identical sequences of nucleotides. The results obtained in different experimental systems indicated that the frequency of recombination increases with increasing length of homology and decreases with decreasing sequence identity. We tested these general concepts using plasmid integration as a model. The plasmids contained two yeast genes, URA3 and CYC1 so that transfbrmants could arise by integration in either homology present in the yeast genome. Different changes were introduced in the region containing the CYC1 gene, while the URA3 gene was kept intact. By Southern blot analysis of transformants we found that the presence of a single base-pair substitution in the 856 bp fragment was sufficient to decrease the proportion of transformants obtained by integration in the CYC1 gene from 66% to 29%. However, addition of one, five or eight more point mutations did not have cumulative effect: with 1% sequence divergence (nine mutations) 21% of transformation events were still due to plasmid integration in the CYC1 gene. The presence of one, two or six point mutations did not have inhibitory effect on integration when plasmids were used for transformation in the single-stranded form. By increasing the size of homology from 108 to 1681 bps the proportion of integrations in the CYC1 region was raised from 2% to 70%. This increase was not a linear function of the length of homology indicating that factors other than homology size and identity influence the frequency of plasmid integration.

homology; polymorphism; length; plasmid integration; yeast

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