Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro

Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642-6. doi: 10.1073/pnas.74.9.3642.

Abstract

A HindII restriction fragment comprising the Escherichia coli lac regulatory region and the genetic information for the alpha peptide of beta-galactosidase (beta-D-galactosidegalactohydrolase, EC. 3.2.1.23) has been inserted into 1 of the 10 Bsu I cleavage sites of M13 by blunt end ligation. A stable hybrid phage was isolated and identified by its ability to complement the lac alpha function. Further characterization of the hybrid phage includes retransformation studies, agarose gel electrophoresis, DNA-DNA hybridization, and heteroduplex mapping. The insertion point has been localized at 0.083 map unit on thewild-type circular map-i.e., within the intergenic region. The results prove that part of the intergenic region is nonessential and that the phage can be used as a cloning vehicle.

MeSH terms

  • Coliphages / metabolism
  • DNA Restriction Enzymes
  • DNA, Recombinant / metabolism*
  • DNA, Single-Stranded / metabolism*
  • DNA, Viral / metabolism*
  • Escherichia coli / metabolism
  • Genes, Viral
  • Lactose Factors*
  • Nucleic Acid Hybridization
  • Plasmids*

Substances

  • DNA, Recombinant
  • DNA, Single-Stranded
  • DNA, Viral
  • DNA Restriction Enzymes