MOLECULAR ANALYSIS OF MUTATION IN MAMMALIAN CELLS

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Molecular analyses of mutation in mammalian cells have been hindered by the complexity of the mammalian genome and the lack of rapid methods for the sequence analysis of induced mutations. Most mammalian loci useful for studying mutagenesis in mammalian cells (hgprt, tk, oua-r, dpt-r, etc.), are not readily amenable to molecular analyses due to the large size of the gene in its normal genomic state, e.g., 35-40 kb in the cast of hgprt, or due to the lack of cloned sequences and/or sequence information concerning the nature of mutations that will lead to the resistant phenotype. There are three notable exceptions, however, which appear promising in their ability to analyze mutations at the molecular level in a genomic target gene. Two of these are the aprt and tk loci in mammalian cells. These loci have been cloned are sequenced and are sufficiently small to allow the application of rapid cloning techniques for analysis of induced mutations. A third system, allowing somewhat greater range of applications, utilizes a single copy of the bacterial gpt gene (456 basepairs) linked to an antibiotic resistance gene, Ampicillin (Apr) or Neomycin (neo-r), integrated into the mammalian genome. Using the SV40 early promotor or a retroviral LTR, the gpt gene expresses the purine salvage pathway enzyme, HGPRT (the bacterial equivalent of HGPRT). In this system, standard techniques used for the isolation of mutations at hgprt can be applied to the isolation of mutations at the integrated gpt sequences.