In vivo cloning by homologous recombination in yeast using a two-plasmid-based system.

Abstract

In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient 'recombination in vivo' technique. Two sets of vectors were developed. The first set, called 'expression vectors', contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon. Subcloning in these vectors of a DNA fragment generates a 'transfer vector' which is compatible with the second set of E. coli-yeast shuttle vectors. This set of 'recombination vectors' contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid-bearing host. Plasmid copy numbers can be modulated through the use of URA3 or URA3-d as the selective marker together with an ARS/CEN and the 2 microns replicon. Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E. coli. All the vectors contain the origin of replication of phage f1 and allow the generation of single-stranded DNA in E. coli for sequencing or site-directed mutagenesis.