Yeast/E. coli shuttle vectors with multiple unique restriction sites

Abstract

Two yeast/E. coli shuttle vectors have been constructed. The two vectors, YEp351 and YEp352, have the following properties: (1) they can replicate autonomuosly in Saccharomyces cerevisiae and in E. coli; (2) they contain the β‐lactamase gene and confer ampicillin resistance to E. coli; (3) they contain the entire sequence of pUC18; (4) all ten restriction sites of the multiple cloning region of pUC18 including EcoRI, SacI, KpnI, SmaI, BamH1, XbaI, SbaI, SalI, PstI, SphI and HindIII are unique in YEp352; these sites are also unique in YEp351 except for EcoRI and KpnI, which occur twice; (5) recombinant plasmids with DNA inserts in the multiple cloning region of YEp351 and YEp352 can be recognised by loss of β‐galactosidase function in appropriate E. coli hosts; (6) YEp351 and YEp352 contain the yeast LEU2 and URA3 genes, respectively, allowing for selection of these grown under non‐selective conditions indicative of high plasmid copy number. The above properties make the shuttle vectors suitable for constructions of yeast genomic libraries and for cloning of DNA fragments defined by a large number of different restriction sites. The two vectors have been further modified by deletion of the sequences necessary for antunomous replication in yeast. The derivative plasmids YIp651 and YIp352 can therefore be used ti integrate specific sequences into yeast chromosomal DNA.