Plasmid Cloning of DNA Polymerase I in Escherichia Coli and Saccharomyces Cerevisiae
The Escherichia coli polA+ gene, which encodes DNA polymerase I, was first cloned on bacteriophage λ (1). More recently a mutant polAI gene, which makes a negligible amount of active protein, was cloned on λ (2) and then transferred to a multicopy plasmid for determination of the polAl nucleotide sequence (3). Also, a sequence encoding the Klenow fragment polymerase I has been cloned on a plasmid expression vector (4). However, attempts by ourselves and others (1) to construct multicopy plasmids with wild-type polA+ genes were uniformally unsuccessful, presumably because high levels of DNA polymerase are lethal. Thus, despite the advantages to biochemical studies gained from these cloned sequences, it remains difficult to study the biological effects of a cloned polymerase gene. The products from the above cloning procedures are either inactive, incomplete, produced in lethal amounts, or are carried by λ vectors which lack the practical versatility of plasmids, and which are unsuitable for introduction into organism other than Escherichia coli.
KeywordsKlenow Fragment polA Mutation Multicopy Plasmid Complement Deficiency Sephadex G200 Column Chromatography
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- 5.Timmis, K.N. (1981) in: Genetics as a Tool in Microbiology, eds. Glover, S.W. and Hopwood, D.A., Cambridge University Press, pp. 49.Google Scholar
- 12.Zissler, J., Signer, E.R. and Schaeffer, F. (1971) in: The Bacteriophage Lambda, ed. Hershey, A.D., Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, pp. 455.Google Scholar
- 22.Sugino, A., Kojo, H., Greenberg, B.D. and Brown, P.O. (1981) The Initiation of DNA Replication, Academic Press Inc.Google Scholar