MOLECULAR ANALYSIS OF DELETION MUTATIONS IN CHINESE HAMSTER OVARY CELLS

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We study large scale genomic rearrangements at the gpt locus in the Chinese hamster ovary (CH0) AS52 cell line. The gpt locus in AS52 cells is more responsive to genomic rearrangements induced by clastogenic or radiomimetic agents allowing for the recovery and characterization of mutants not recovered at other selectable loci, like hprt. We detect such rearrangements as the result of either an altered PCR profile or an altered Southern blot. Those mutants that yield a PCR product with rearranged sequences can be sequenced directly. The antitumor agent, Adozelesin (ADZ), induces approximately 90% large scale deletions or rearrangements as detected by PCR. However, a substantial number of smaller deletions, insertions or rearrangements are observed as well. These rearrangements include a 202-bp insert with a 3-base flanking repeat, several small deletions (9-13 bp) all with flanking sequence repeats, deletions of repeated adjacent 3-base direct repeats and a few short deletions (1-5 bp) without flanking homologous sequences. In one mutant, a rearrangement that includes a duplication of about 100 bp appears to be mediated at a 5-base region of sequence homology in which the last 3 bases include an ADZ consensus binding sequence. Such a mutant may arise as a result of ADZ-DNA adduct blocking the polymerase resulting in the breathing and aberrant reannealing of the synthesized strand, either on the same template strand or on a replicating sister chromatid. These data suggest that in most cases short stretches of sequence homology are required for deletions, insertions and duplications. In addition, we are cloning the site of the gpt integration in AS52 cells to more accurately define the types of large scale genomic rearrangements observed. Presently, we cannot distinguish between intrachromosomal deletions, mitotic recombination, gene/chromosome conversion or aneuploidy and reduplication. By characterizing the site of gpt integration and defining polymorphic flanking genomic sequences, we will be able to assess the type and frequency of specific rearrangements induced by environmental mutagens and carcinogens. We are presently characterizing several lambda clones derived from an AS52 genomic library.