Achilles' heel cleavage: creation of rare restriction sites in lambda phage genomes and evaluation of additional operators, repressors and restriction/modification systems.
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 lambda repressor/lambda oL1 operator. Here, we have extended these results to a lac operator carried by a much larger vector, namely a 44-kb phage lambda 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 HhaI 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.
Grimes
E
E
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Koob
M
M
Szybalski
W
W
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D007763:Lac Operon; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism*; D015183:Restriction Mapping; D014157:Transcription Factors / Q000378:metabolism*
D012097:Repressor Proteins; D014157:Transcription Factors; D004262:DNA Restriction Enzymes