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.

The specific protection of only one of many restriction sites in a genome from inactivation by a cognate methyltransferase (MTase) creates a unique cleavage site — an Achilles' heel cleavage (AC) site. In the RecA—AC, or RARE, technique, such specific protection is provided by a synaptic complex composed of RecA protein, a γ-S analog of ATP and a 30–60 nucleotide long oligodeoxynucleotide complementary or identical to the sequence-targeted site in which the protected restriction site is embedded. Upon methylation and the subsequent removal of the protective complex and MTase, the protected site is the only site cut by the cognate restriction enzyme. Two such targeted cuts permit the excision of a unique DNA fragment from the genome. Recent advances include the calibration of DNA clones, the mapping of gaps, and the determination of the sizes of excised fragments by pulsed-field gel electrophoresis, which allows one to measure distances between any two neighboring sequence-targeted sites, in the range of a few kilobases to 10 megabases, with the purpose of physically mapping the genome.

Mapping and manipulation of very large genomes, including the human genome, would be facilitated by the availability of a DNA cleavage method with very high site specificity. Therefore, a general method was devised that extends the effective recognition sequences well beyond the present 8-base pair limit by combining the specificity of the restriction endonuclease with that of another sequence-specific protein that binds tightly to DNA. It was shown that the tightly binding lac or λ repressor protects a restriction site within the operator from specific modification methylases, M⋅Hha I or M⋅Hph I, while all other similar sites are methylated and thus rendered uncleavable. A plasmid containing a symmetric lac operator was specifically cleaved by Hha I, only at the site within the operator, after M⋅Hha I methylation in the presence of the lac repressor, whereas the remaining 31 Hha I sites on this plasmid were methylated and thus not cleaved. Analogous results were obtained with the Hae II site within the lac operator, which was similarly protected by the lac repressor, and with the Hph I site within the phage λ o L operator, which was protected by λ repressor from M⋅Hph I methylation.

Fresh human skin placed in plasma clot culture shows outgrowth of epithelial cells within 3 days followed 3 to 6 days later by fibroblasts. Plasma clot culture of human skin pretreated in medium containing 10% glycerol at 4°C, frozen, and thawed showed outgrowth of fibroblasts only. Using this increased susceptibility of epithelial cells to freeze injury as a criterion, we developed a freezing method in which skin is pretreated with 20 to 30% glycerol at 4°C for 1 to 2 hrs before freezing. Human skin preserved in liquid nitrogen by this method grew out in plasma clot culture very much like fresh skin.