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.
KeywordsGlycerol EDTA Electrophoresis Polyacrylamide Tryptophan
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