Molecular engineering with the FRT sequence of the yeast 2 microm plasmid: [cir0] segregant enrichment by counterselection for 2 microm site-specific recombination.
Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for manipulating DNA both in vitro and in living organisms. In this work we describe the isolation of yeast Saccharomyces cerevisiae segregants which have lost the highly stable 2 microm DNA plasmid, exploiting the site-specific recombination system of 2 microm itself. We efficiently isolated [cir0] segregants from two haploid yeast strains and also a diploid. Moreover, the effect of mutations in the core region of the FRT (Flp Recognition Target) sequence was investigated in vivo, studying the result of the recombination event between several mutated and wild-type FRT sequences. From our result it seems that the identity between the core regions of two FRT sites is necessary but not sufficient, indicating that the core sequence itself has a relevant function in the recombination mechanism in vivo.
Storici
F
F
Microbiology Group, ICGEB, AREA Science Park, Trieste, Italy.
Bruschi
C V
CV
D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA
D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase