Generation of a chick genomic cosmid library : the first step towards isolation of the chick photolyase gene
Burd, Christopher G.
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Ultraviolet light-induced effects on biological systems are well documented. The most intensely studied effects are lethality, mutagenesis, and inhibition of DNA synthesis. It is generally agreed that the basis of these effects lie in the response to DNA lesions such as the K5-6) cyclobutane dimer, which is the most prominent lesion formed after irradiation with 254 nm UV. There are, however, a variety of minor lesions that, though not formed as efficently as the (5-6) dimer, may actually be responsible for the induced effects. A number of mechanisms have evolved that enable a cell to remove or tolerate the various chemical modifications induced in DNA by UV radiation. Common to prokaryotes and eukaryotes are excision repair and photoreactivation. Postreplication repair is also common to both groups, although the specific repair mechanisms are fundamentally different. E. coli has an inducible damage tolerance system termed the SOS response that has no counterpart in eukaryotic cells. The research project described here details the construction of a cosmid library of the chicken genome. Evidence is given that the library was completed and is representative of the entire genome. Two methods for isolating the chick photolyase gene are discussed, and a method for generating a PR+ mammalian cell line is suggested. Finally, experiments that will delineate (5-6) effects from effects due to minor lesions in mammalian cells via use of the chick photolyase are discussed.