Point mutations have been implicated as important etiological factors in a number of human genetic disorders including several of the haemopathies, osteogenesis imperfecta, as well as in the activation of some oncogenes. Little is known, however, about the mechanisms by which such mutations occur in mammalian cells. Determination of mechanistic pathways by generating mutational spectra using DNA sequence analysis has proven extremely powerful in bacterial systems. Similar studies in mammalian cells have been limited due to technical problems associated with the rapid isolation of target gene sequences from mammalian cells. We are in the process of analyzing both spontaneous and induced point mutations in a Chinese hamster ovary (CHO) cell line, AS52, that carries a single copy of the bacterial gpt gene transfected and functionally integrated into the CHO genome. The gpt gene is analogous to the mammalian hgprt gene and mutations at either locus can be isolated by selecting for resistance to the purine analog, 6-thioguanine (6TG). The small size of the gpt gene (456 base pairs) provides for the convenient rescue of mutant gpt sequences from the CHO genome for DNA sequence analyses. Our data suggest that genomic sequences at the site of the gpt integration may influence sequence stability in cloning experiments. We are in the process of performing cloning experiments utilizing alternative E. coli hosts known to stabilize genomic inserts. In addition, we have begun to amplify the gpt gene sequences using the polymerase chain reaction (PCR) technique. This approach bypasses the requirement of cloning each mutant gene and provides enough DNA to allow direct sequence analysis without further subcloning. Initial studies with the PCR technique indicate the entire 456 base pair gpt structural gene can be amplified from 1 g of genomic DNA. Using these amplified DNA sequences we will perform DNA sequence analysis and determine the utility of the PCR technique in generating point mutational spectra from mammalian cells.
