Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1984-02-16
1988-12-20
Warden, Robert J.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 4, 435 29, 435 34, 935 55, 935 70, 935 71, 935 84, C12Q 168, C12Q 102, C12Q 104
Patent
active
047925205
ABSTRACT:
A new and improved cell assay has been developed for mutagens and potential carcinogens to precisely identify the predominant type of mutation(s) each such compound induces in the DNA of cells. The test utilizes, for example, a selectable genetic marker such as adenine phosphoribosyltransferase (APRT) which is now extensively characterized on the DNA, protein and cellular phenotype levels. Specific mutations such as transitions, transversions, point insertions or point deletions are engineered at specific known sites in a mouse APRT gene deduced from the determined gene sequence, such that the gene cannot be properly expressed. These mutant genes are then introduced into non-reverting APRT deficient mammalian cells. These hybrid constructs represent the basic test medium for detection of mutagenic activity. The tester cells are treated with mutagens known to preferentially induce specific DNA mutations in mammalian cells. Reversion within the appropriate tester cell culture detected by growth in selection medium or other detection systems, will confirm or refute the mode of action of these mutagens. As an additional approach for the identification of mutagens that produce frameshifts, the amino acid sequences of major frameshift peptides have been determined from the nucleotide sequence of the mouse APRT gene. These frameshift peptides are synthesized and individually used to elicit antisera. Mutant colonies, arising as a consequence of frameshift mutation, are identified in situ by virtue of their binding one of the specific antisera which are coupled to a color-development assay. Additionally, methods for identifying mutagens which produce mutations at the APRT locus, and which exert their effect by inducing DNA rearrangements, transpositions or excision are employed. The tester lines and reagents enable the exact nature of mutation that any mutagen produces in cells to be rapidly established. Further, portions of the nucleotide sequence of a mouse APRT DNA strand as well as its encoded amino acid sequence are disclosed.
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Stambrook Peter J.
Tischfield Jay A.
Medical College of Georgia Research Institute
Spiegel Jack
University of Cincinnati
Warden Robert J.
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