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.
|Keywords||0 (Histones), 0 (Plasmids), Bacteriophage lambda/genetics, Cloning, Molecular/*methods, Histones/genetics, Plasmids, Saccharomyces cerevisiae/genetics, Support, Non-U.S. Gov't, Transformation, Genetic|
Lund, T., Grosveld, F.G., & Flavell, R.A.. (1982). Isolation of transforming DNA by cosmid rescue.. Proceedings of the National Academy of Sciences of the United States of America, 27, 520–524. Retrieved from http://hdl.handle.net/1765/2349