The overproduction of DNA terminase of coliphage lambda.
An artificial operon containing the genes coding for the two subunits of lambda DNA terminase, Nul and A, has been constructed. Derivatives of plasmid pBR322 served as the cloning vehicles. The transcription is driven by the pL promoter of phage lambda, and translation of the terminase genes was made efficient by the replacement of the wild-type ribosome-binding sites for those of lambda genes cII and/or D. The operon also carries the oL operator, and this enables regulation of its expression by a thermosensitive repressor. The synthesis of genes Nul and A products is extremely efficient upon derepression. Within 40 min after induction of the operon, the two subunits comprise about 20% of the total cellular protein mass. Crude extracts prepared from these overproducing strains are at least 100 times more active than extracts prepared from induced lambda lysogens in both promotion of lambda DNA packaging and cosmid cleaving. The ability to produce highly concentrated terminase would enormously facilitate the study of its structure and mechanism of action. These extracts are also extremely useful in techniques such as lambda DNA packaging, cosmid mapping and cosmid linearization to improve efficiency of integration into mouse eggs.
Chow
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Department of Medical Genetics, University of Toronto, Ontario, Canada.
Daub
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Murialdo
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D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D003360:Cosmids; D004279:DNA, Viral / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D005814:Genes, Viral; D009876:Operon; D010957:Plasmids
D004279:DNA, Viral; D004706:Endodeoxyribonucleases; C030894:terminase