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Plasmid Cloning of DNA Polymerase I in Escherichia Coli and Saccharomyces Cerevisiae

  • Ad Spanos
  • Steven Sedgwick
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 179)

Abstract

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.

Keywords

Klenow Fragment polA Mutation Multicopy Plasmid Complement Deficiency Sephadex G200 Column Chromatography 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1984

Authors and Affiliations

  • Ad Spanos
    • 1
  • Steven Sedgwick
    • 1
  1. 1.Genetics DivisionNational Institute for Medical ResearchLondonGreat Britain

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