There are about 70,000 synthetic chemicals in current commercial use, and about 1,000 new products are added to this repertoire each year. Since many of these compounds are potentially mutagenic and/or carcinogenic, the need for more effective mutagen screening is imperative. This need has recently been emphasized in a report from the National Academy of Sciences. In this application, we propose to develop a novel mammalian cell assay for mutagens and potential carcinogens that will precisely identify the predominant type of mutations(s) each such compound induces in the DNA of mammalian cells. Our test utilizes the selectable genetic marker adenine phosphoribosyltransferase (APRT), which we have extensively characterized on the DNA, protein and cellular phenotype levels. We will engineer specific mutations (transitions, transversions or point insertions) at sites in a mouse APRT gene such that the gene cannot be expressed, and will introduce these mutant genes into non-reverting, APRT deficient human cell Recipients. These hybrid constructs will represent our basic test medium for detection of mutagenic activity. As a model system, the tester cells will be treated with mutagens known to preferentially induce specific DNA base-pair transitions in prokaryotes and possibly in mammalian cells. Reversion within the appropriate tester cell culture will then confirm or refute the mode of action of these mutagens. As an additiona approach for the identification of mutagens that produce frameshifts, we have deduced, from our knowledge of the nucleotide sequence of the mouse APRT gene, the amino acid sequences of major frameshift peptides. These frameshifts peptides will be synthesized and individually used to elicit antisera. Mutant colonies, arising as a consequence of frameshift mutation, will be identified in situ by virtue of their binding one of the specific antisera which will be coupled to a color-development assay. Once our tester lines have been constructed and our reagents have been produced, we expect to be able to rapidly establish the exact nature of mutation that any mutagen produces in animal cells.
NIH ExPorter
NIH ExPorter