Deletion mutagenesis and inactivation of tumor suppressor genes is an important step leading to cellular immortality, carcinogenesis, and metastatic tumor progression. Active repair of DNA lesions helps to avoid the formation of mutations and cancer. Recent evidence indicates that DNA sequence, chromosomal structure, and genomic location all contribute to the specificity and efficiency of DNA repair and mutagenesis. Until now, it was not possible to separate these variables and study the repair and mutagenesis of one gene at more than one genomic site. Several stable gpt+ transgenic V79 cell lines were recently developed each containing one integrated copy of the gpt gene located at a unique site within the genome and exhibiting spontaneous mutagenesis that is not the result of gene loss of inactivation. The g12 cell line, when treated with clastogens, shows a small colony mutant phenotype which may indicate the formation of multilocus deletions extending into an adjacent essential gene and thus provide a convenient way to score, select, and analyze this type of mutation. The g12 cells plus one or two additional cell lines (chosen based on their bleomycin- and EMS-induced mutation frequencies) will be studied further. The gpt gene in these cells will be mapped and its location within the V79 genome determined by in situ hybridization. The small colony mutant phenotype of the g12 cell line will be studied to determine if it is the result of large deletions or rearrangements extending beyond the gpt gene. Deletion mutations that include a portion of the gpt gene will be cloned and sequenced. The gpt sequences in nonrevertable mutants will be amplified by polymerase chain reaction and intragenic deletions and mutations analyzed. UV- and EMS-induced repair of the gpt gene will also be analyzed using a novel technique based on Hanawalt's gene-specific repair assay. The modified assay will measure the incorporation of BrdUrd into repair patches within the gpt gene by UV photolysis of the BrdUrd-containing sequences prior to quantitation of the DNA by the Southern technique. The sequence dependence of induced mutagenesis within the gene will be analyzed and compared with the strand specificity of repair. The results of these studies will provide new insights into the role of chromosomal location in DNA mutagenesis and help to explain the genetic variability of clastogens.
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| Frenkel, K; Klein, C B (1993) Methods used for analyses of ""environmentally"" damaged nucleic acids. J Chromatogr 618:289-314 |
