NEW IN-VIVO CLONING METHODS BY HOMOLOGOUS RECOMBINATION IN YEAST

We have devised a new strategy to clone DNA sequences from an yeast au tonomously-propagating plasmid into a non-autonomous integrative vecto r by in-vivo recombination. The method consists of a first step in whi ch the replicative plasmid carrying the DNA fragment of interest forms a co-integrate with the nonreplicative plasmid by an induced in-vivo reciprocal exchange accompanied by gene conversion. The dimeric plasmi d obtained is then purified and cut with an appropriate restriction en zyme and ligated independently to obtain the two intact monomeric plas mids, the original autonomous plasmid plus the new non-autonomous plas mid carrying the subcloned DNA fragment. The dimeric co-integrate can also serve as substrate for a second in-vivo reciprocal exchange that produces new autonomous plasmids carrying the desired DNA fragment. Th e technique considerably expands the applications of in-vivo cloning i n yeast by complementing three important characteristics of previously published methods: (1) it can be used to clone into non-propagating v ectors; (2) co-transformation experiments are not required; and (3) th e intermediate co-integrate can be used to generate new types of auton omously-propagating plasmids directly. These characteristics are indep endent of whether the DNA insert is flanked by appropriate restriction sites or whether it does, or does not, express a detectable phenotype in yeast. The method is particularly useful for the cloning of large DNA fragments and can be used for plasmids from organisms other than y easts.