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Purification of Proteins Determined by Two Nonessential Genes in Lambda

Frederick Ausubel, Paul Voynow, Ethan Signer, Jette Mistry


The “nonessential” genes in the center of the λ map are involved in prophage integration and excision and in recombination. These genes have been identified and characterized genetically, but, with the exception of exonuclease (Korn and Weissbach, 1963) and beta protein (Radding and Shreffler, 1966) and possibly also int protein (Jordan and Echols, personal communication), specific assays for their products have not yet been devised. Therefore, some other means must be found to guide the purification of these proteins.

A special method was used by Ptashne (1967) to isolate the λ repressor. In principle this method entails establishing conditions under which the product of the gene in question constitutes a relatively large fraction of the proteins being examined, and then identifying the protein as the species eliminated by a nonsense (chain-terminating) mutation in that gene. To enrich for the repressor, Ptashne heavily irradiated λ-immune cells with ultraviolet light to suppress protein synthesis; he then infected with λ and fed the culture radioactive leucine. Under these conditions a significant proportion of label was incorporated into a protein identified as repressor by its dependence on cI gene function. By these means the repressor could be purified radiochemically. It has since been freed of unlabeled proteins as well (Chadwick et al., 1970). Recently, Schwartz (1970) and Hendrix (this volume) utilized nonimmune cells in a procedure analogous to Ptashne’s. They showed that infection of heavily irradiated cells with λ leads to the preferential synthesis of nonessential proteins determined by genes occupying the region extending...

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