High frequency FLP-independent homologous DNA recombination of 2 mu plasmid in the yeast Saccharomyces cerevisiae.
The purpose of this work is to identify and quantitate in vivo 2 mu plasmid FLP-independent recombination in yeast, using a nonselective assay system for rapid detection of phenotypic expression of the recombination events. A tester plasmid was constructed such that in vivo recombination between 2 mu direct repeat sequences produces the resolution of the plasmid into two circular DNA molecules. This recombinational event is detected as a phenotypic shift from red to white colonies, due to the mitotic loss of the plasmid portion containing the yeast ADE8 gene in a recipient ade1 ade2 ade8 genetic background. In the absence of the 2 mu FLP recombinase and/or its target DNA sequence, recombination is not abolished but rather continues at a high frequency of about 17%. This suggests that the FLP-independent events are mediated by the chromosomally-encoded general homologous recombination system. We therefore conclude that the totality of 2 mu DNA recombination events occurring in FLP+ cells is the contribution of both FLP-mediated and FLP-independent events.
Bruschi
C V
CV
Department of Microbiology and Immunology, School of Medicine, East Carolina University, Greenville, NC 27858-4354.
Howe
G A
GA
D000225:Adenine; D015139:Blotting, Southern; D003116:Color; D004274:DNA, Recombinant / Q000032:analysis*; D005800:Genes, Fungal; D010641:Phenotype; D010957:Plasmids; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000032:analysis* / Q000235:genetics; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D000225:Adenine