Genomic rearrangements are among the most serious mutations arising in mammalian cells, implicated in the process of carcinogenesis and in the etiology of a variety of human genetic disorders. While many mutagens and carcinogens induce genomic rearrangements, most mammalian mutagenesis assays are limited in their ability to detect large scale genomic mutations. Often genomic rearrangements are lethal to the cell, however, the mechanisms by which such rearrangements result in viable cells deserve further study. To study the mechanisms by which genomic rearrangements arise, we use the transgenic Chinese hamster ovary cell line, AS52, which carries a single functional copy of the bacterial gpt gene stably integrated into the CHO genome. AS52 cells are well defined as being capable of detecting mutations induced by a variety of clastogens and radio-mimetic agents that are not detected as mutagens in other mammalian mutational assay systems, apparently due to the location of the genomic integration of the bacterial gpt transgene. We are characterizing the genomic site of gpt integration and have defined a nonfunctional gpt/plasmid related sequence that is closely linked to the functional gpt gene. In order to rapidly distinguish deletions and rearrangements from putative point mutations, we have developed primers and reaction conditions useful for multiplex PCR of AS52 genomic DNA. Our multiplex PCR yields sequences from the dhfr gene (which is not linked to gpt) as well as from both the functional and the nonfunctional gpt sequences. The nonfunctional gpt sequence can serve as a marker for rapidly distinguishing smaller deletions/rearrangements while the dhfr sequences serve as an internal PCR control. If the PCR conditions are appropriate then we should always observe the dhfr PCR fragment, while loss of both the functional and the nonfunctional gpt related PCR fragments allows the distinction of larger scale deletion/rearrangement events. Thus, we can now rapidly distinguish complete deletions (i.e., large scale genomic rearrangements) from partial deletions (i.e., smaller scale genomic rearrangements), from point mutations simply using PCR. We are presently characterizing the molecular fine structure of deletions induced by the base analog, 5-azacytidine, and the chemotherapeutic agent, U73,975.
