IN-VIVO CLONING BY HOMOLOGOUS RECOMBINATION IN YEAST USING A 2-PLASMID-BASED SYSTEM

In order to reduce the number of classical DNA manipulation and ligati on steps in the generation of yeast expression plasmids, a series of v ectors is described which facilitate the assembly of such plasmids by the more efficient 'recombination in vivo' technique. Two sets of vect ors were developed. The first set, called 'expression vectors', contai ns an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon. Subcloni ng in these vectors of a DNA fragment generates a 'transfer vector' wh ich is compatible with the second set of E. coli-yeast shuttle vectors . This set of 'recombination vectors' contains a cassette for a functi onal copy of a gene complementing a host strain auxotrophy or a bacter ial gene conferring an antibiotic resistance to the plasmid-bearing ho st. Plasmid copy numbers can be modulated through the use of URA3 or U RA3-d as the selective marker together with an ARS/CEN and the 2 mu m replicon. Integration of the cloned DNAs into the yeast linearized rep licative vectors occurs by recombination between homologous flanking s equences during transformation in yeast or E. coli. All the vectors co ntain the origin of replication of phage f1 and allow the generation o f single-stranded DNA in E. coli for sequencing or site-directed mutag enesis. The sequence presented (Figure 1a) has been entered in the EMB L data library under Accession Number Z48747.