US 4,792,520 A · Methods and kits for identifying mutagenic agents and molecular mutations in DNA in mammalian cells

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.

Claims71

Dependent claims

A method of identifying a mutagenic agent which induces base substitution mutation in DNA in mammalian cells comprising the steps of

(a) introducing a cloned gene having a DNA sequence encoding a known selectable characteristic when in an unmutated form into nonreverting mammalian cells to produce transfectants of the mammalian cells, the untransfected nonreverting mammalian cells being nonselectable for said characteristic when cultured in a cell culture selection medium and the cloned gene having a base substitution at a specific site rendering the gene or its product nonselectable for said characteristic, whereupon said introduction of the cloned gene into the nonreverting mammalian cells renders the transfectants of the mammalian cells selectable for the characteristic when cultured in the cell culture medium following base substitution mutagenic reversion in the cloned gene at the specific site,
(b) exposing the transfected mammalian cells to a mutagenic agent for a sufficient amount of time to induce reversion by base substitution mutagenesis in the cloned gene at the specific site,
(c) culturing the exposed transfected mammalian cells in the cell culture medium which selects for those transfected mammalian cells that are selectable for said characteristic as a result of base substitution mutagenic reversion in the cloned gene at the specific site, and
(d) identifying the mutagenic agent which has induced the base substitution mutation at the specific site in the cloned gene in the selected transfected mammalian cells.

1 more element

A method of identifying a mutagenic agent which induces frameshift mutation in DNA in mammalian cells comprising the steps of

(a) introducing a cloned gene having a DNA sequence encoding a known selectable characteristic when in an unmutated form into nonreverting mammalian cells to produce transfectants of the mammalian cells, the untransfected nonreverting mammalian cels being nonselectable for said characteristic when cultured in a cell culture selection medium and the cloned gene having a frameshift mutation at a specific site rendering the gene or its product nonselectable for said characteristic, whereupon said introduction of the cloned gene into the nonreverting mammalian cells renders the transfectants of the mammalian cells selectable for the characteristic when cultured in the cell culture medium following frameshift mutagenic reversion in the cloned gene at the specific site,
(b) exposing the transfected mammalian cells to a mutagenic agent for a sufficient amount of time to induce reversion by frameshift mutagenesis in the cloned gene at the specific selected site,
(c) culturing the exposed transfected mammalian cells in the cell culture medium which selects for those transfected mammalian cells that are selectable for said characteristic as a result of frameshift mutagenic reversion in the cloned gene at the specific site, and
(d) identifying the mutagenic agent which has induced the frameshift mutation at the specific site in the cloned gene in the selected transfected mammalian cells.

1 more element

A kit for determining whether an agent induces base substitution, frameshift or DNA transposition mutation in DNA in mammalian cells comprising

(a) an effective number of at least one of the following types of mammalian cells selected from a group of mammalian cells consisting of
(i) mammalian cells containing an introduced mutated cloned gene having a base substitution at a specific site, the mutated cloned gene having a DNA sequence encoding a known selectable characteristic when the gene is in an unmutated form, the transfected mammalian cells being nonrevertable for the characteristic when in an untransfected form and nonselectable for said characteristic when cultured in a cell culture selection medium absent undergoing base substitution mutagenic reversion at the specific site,
(ii) mammalian cells containing an introduced mutated cloned gene having a frameshift mutation at a specific site, the mutated cloned selectable gene having a DNA sequence encoding a known selectable characteristic when the gene is in an unmutated form, the transfected mammalian cells being nonrevertable for the characteristic when in an untransfected form and nonselectable for said characteristic when cultured in a cell culture selection medium absent undergoing frameshift mutagenic reversion at the specific site, and
(iii) hemizygous or heterozygous mammalian cells containing a retrovirus proviral DNA or other transposable DNA at or near a specific site in a DNA segment of the hemizygous or heterozygous mammalian cells, the DNA segment having a DNA sequence encoding a known selectable characteristic when in an unmutated form and the retrovirus proviral DNA or other transposable DNA render the hemizygous or heterozygous mammalian cells nonselectable for said characteristic when cultured in a cell culture selection medium absent undergoing DNA mutagenic reversion at or near the specific site, and
(b) an effective amount of cell culture medium which selects for those mammalian cells that are selectable for said characteristic when cultured in said cell culture medium as a result of DNA mutagenic reversion at or near the specific site when exposed to an agent for identifying the agent as a mutagen which induces base substitution, frameshift or DNA transposition mutation in DNA in mammalian cells.

2 more elements

Dependencies are inferred from the claim text.

Methods and kits for identifying mutagenic agents and molecular mutations in DNA in mammalian cells

Life of this patent

Methods and kits for identifying mutagenic agents and molecular mutations in DNA in mammalian cells

Life of this patent

Abstract

Claims71