FLP-FRT mediated intrachromosomal recombination on a tandemly duplicated YEp integrant at the ILV2 locus of chromosome XIII in Saccharomyces cerevisiae.
A YEp chimaeric plasmid carrying SMR1 and URA3 genetic markers was integrated into chromosome XIII at the ilv2-delta 1 locus in a [cir (o)] background. The 1.5 kb BglII deletion of ilv2-delta 1 allowed the clear identification of an integrant structure which consisted of a direct tandem duplication (TD) of the chimaeric plasmid. Within the integrant structure, a single copy of the plasmid sequence was flanked by a direct duplication of the 2 microns site-specific recombinase (FLP) recognition target (FRT). Isogenic [cir (o)] and [cir+] diploids formed by crossing the [cir (o)] TD strain to complementary haploids were analyzed for plasmid marker loss and chromosomal DNA alterations in the presence and absence of selection pressure for the URA3 and SMR1 plasmid borne markers. [cir (o)] diploids showed no plasmid marker loss and maintained the TD structure. In the absence of selection pressure, the [cir+] diploid underwent FLP-FRT mediated unequal interchromatid recombination, resulting in the breakage-fusion-bridge cycle and homozygotization of chromosome XIII (Rank et al. 1988). Maintenance of selection pressure for the centromere distal plasmid URA3 marker selected against FLP-FRT interchromatid recombinants so that the effects of site specific recombinase on intrachromatid recombination could be evaluated. Intrachromatid recombination at the directly duplicated FRT sites of the TD structure resulted in the loss of a diagnostic internal fragment. These results show that in the presence of FLP, FRT sites separated by up to 13.3 kb of chromosomal DNA function as substrates for intra and interchromatid recombination.
Rank
G H
GH
Department of Biology, University of Saskatchewan, Saskatoon, Canada.
Arndt
G M
GM
Xiao
W
W
D015139:Blotting, Southern; D002678:Chimera; D002874:Chromosome Mapping; D002875:Chromosomes / Q000502:physiology*; D005838:Genotype; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*