The infeasibiblity of isolating chick DNA photolyase gene by direct transfection of cosmid library
Murley, Jeffrey S.
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Recent work has suggested that the Pyr(6-A)Pyo lesion, a minor component of the DNA lesions generated after exposure to ultraviolet light, may be more mutagenic than the dominant (5-6) cyclobutane dimer. Testing this hypothesis in cells from placental mammals has not been possible because the presence of a photolyase, an enzyme capable of monomerizing the (5-6) dimer but not the (6-A) lesion, has not been clearly demonstrated. Previous experiments have demonstrated photolyase activity in a variety of embryonic chick tissues. The work described herein details the use of direct transfection and the screening of a genomic cosmid library to isolate the gene(s) encoding this photolyase. The results show the difficulties in using transfection and failure of the library to generate a phr+ transfectant. Isolated phr would be used to generate phr+ mammalian cells. Generation of photolyase-containing excision- deficient mammalian cells would allow the mutagenic and lethal effects of minor photoproducts induced by ultraviolet light to be compared to the dominant (5-6) cyclobutane dimer. Isolation of the chick photolyase gene would also enable evolutionary comparisons to be made with the E. coli and yeast phr genes.