Achilles' heel cleavage: creation of rare restriction sites in λ phage genomes and evaluation of additional operators, repressors and restriction/modification systems

Eric Grimes, Michael Koob, Waclaw Szybalski

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14 Scopus citations

Abstract

A novel technique for the creation of rare restriction sites was described by Koob et al. [Science 241 (1988) 1084-1086]. This technique, Achilles' heel cleavage (AC), relies on the use of a bound repressor molecule to protect only one of many identical restriction sites from a modification methyltransferase that inactivates all other restriction sites. The technique was applied to a small plasmid and shown to work efficiently with two repressor/operator systems: lac repressor/lacO operator and λ repressor/λoL 1 operator. Here, we have extended these results to a lac operator carried by a much larger vector, namely a 44-kb phage λ construct. In addition, we have evaluated the effect of altering the stability of the lac repressor/lac operator complex by varying both the operator and the repressor. We have also evaluated several more restriction/modification systems (MboI, Dam, MspI and AluI) in addition to HhaII and HaeII used earlier. Finally, we extended the AC technique to a third system, that of the phage 434 repressor and a synthetic 434 operator. From our results we conclude that the AC method should be applicable to the mapping of large genomes and to measuring the strength of operator-repressor interactions. AC could also be applied to identifying and evaluating many different DNA-binding proteins and their sites of action.

Original languageEnglish (US)
Pages (from-to)1-7
Number of pages7
JournalGene
Volume90
Issue number1
DOIs
StatePublished - May 31 1990

Keywords

  • DNA-binding proteins
  • Recombinant DNA
  • bacteriophages λ and 434
  • lacI gene mutants
  • methyltransferase
  • plasmids
  • restriction/modification system
  • synthetic operator

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