Isolation of transforming DNA by cosmid rescue.
A procedure has been developed to allow the recovery of an integrated plasmid genome from a transformed cell, together with large areas of the flanking DNA sequences. DNA from Saccharomyces cerevisiae BAS2, in which the pBR322--ura 3 plasmid (Y1p5) is integrated at the yeast histone H2A and H2B locus, was used to generate a cosmid library, using a new cosmid vector (pTL5) that is ampicillin sensitive and tetracycline resistant. Colonies were selected for ampicillin resistance, which was conferred by the incorporation of the integrated pBR322 beta-lactamase gene into the recombinant cosmid. Restriction enzyme and blot hybridization analyses show that the rescued clones contain the yeast histone genes in addition to the Y1p5 sequences; a total of approximately 50 kilobase pairs of DNA sequences flanking the plasmid was recovered as a series of overlapping cosmids. This approach should allow the recovery of most genes that can be linked to a marker pBR322 sequence and for which a specific phenotype can be selected in a recipient eukaryotic cell.
D010582:Bacteriophage lambda / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D006657:Histones / Q000235:genetics; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
D006657:Histones