Functional expression of cloned yeast DNA in Escherichia coli.

Abstract

A collection of hybrid circular DNA was constructed in vitro using the poly(dA.cntdot.Dt) connector method; each hybrid circle contained 1 molecule of poly(dT)-tailed DNA of plasmid ColE1 (made linear by digestion with EcoRI endonuclease) annealed to a poly(dA)-tailed fragment of yeast (Saccharomyces cerevisiae) DNA, produced originally by shearing total yeast DNA to an average size of 8 .times. 106 daltons. This DNA preparation was used to transform E. coli, selecting colicin-E1-resistant clones that contain hybrid ColE1-yeast DNA plasmids. Sufficient numbers of transformant clones were obtained to ensure that the hybrid plasmid population was representative of the entire yeast genome. Various hybrid ColE1-yeast DNA plasmids capable of complementing E. coli auxotrophic mutations were selected from this population. Plasmid pYeleu10 complements several different point or deletion mutations in the E. coli or [Salmonella] typhimurium leuB gene (.beta.-isopropylmalate dehydrogenase); plasmids pYeleu11, pYeleu12 and pYeleu17 are specific suppressors of the leuB6 mutation in E. coli C600. Plasmid pYehis2 complements a deletion in the E. coli hisB gene (imidazole glycerol phosphate dehydratase). Complementation of bacterial mutations by yeast DNA segments does not appear to be a rare phenomenon.