MOLECULAR CHARACTERIZATION OF A SACCHAROMYCES PLASMID CONTAINING THE HIS4 GENE

In a previous study, genetic transformation in yeast was carried out with a mixture of the yeast plasmid 2-micron DNA and total yeast DNA, which was treated with restriction endonuclease PstI and DNA ligase. Strains were derived that contain a plasmid which carries the HIS4 gene. In the present study new plasmids have been constructed by combining the HIS4 carrying yeast plasmid, as well as parts of it, with bacterial plasmids. The hybrid plasmids were propagated in bacteria and analysed. From the data obtained the original plasmid was inferred to be a 2-micron DNA circle with a 9.4 kb insertion carrying the HIS4 gene. This structure was confirmed by restriction endonuclease analysis of plasmid DNA from the original transformant, using molecular hybridization to detect fragments which contain sequences of the 9.4 kb insert. The insert, which is bordered by two PstI sites, was mapped with restriction endonucleases EcoRI, HindIII, and SaII. No BamHI site is present in the insert. © 1979 Carlsberg Laboratory.