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Last Updated: September 25, 2020

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Claims for Patent: 4,792,520

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Summary for Patent: 4,792,520
Title: 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.
Inventor(s): Stambrook; Peter J. (Cincinnati, OH), Tischfield; Jay A. (Augusta, GA)
Assignee: University of Cincinnati (Cincinnati, OH) Medical College of Georgia Research Institute (Augusta, GA)
Application Number:06/580,876
Patent Claims:1. 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.

2. A method of claim 1 wherein the mutated clone gene encodes adenine phosphoribosyltransferase (APRT) when in an unmutated form.

3. A method of claim 2 wherein the sequence of the mutated cloned APRA gene is selected from a group of sequences consisting of a human mutated APRT gene sequence and a mouse mutated APRA gene sequence.

4. A method of claim 2 wherein the nonreverting mammalian cells cannot revert to APRT producing cells absent undergoing base substitution mutagenic reversion at the specific site in the mutated cloned APRT gene.

5. A method of claim 4 wherein the nonreverting mammalian cells are selected from a group of nonreverting mammalian cells consisting of human nonreverting mammalian cells and mouse nonreverting mammalian cells.

6. A method of claim 5 wherein the nonreverting human cells are human recessive phenotype adenine phosphoribosyltransferase (APRT.sup.-) line HTD-114 cells.

7. A method of claim 5 wherein the nonreverting mouse cells are mouse recessive phenotype adenine phosphoribosyltransferase (APRT.sup.-) LS-24b or CAK cells.

8. A method of claim 1 wherein said specific site is a Pst 1 restriction site at an intron/exon junction in a mouse gene encoding adenine phosphoribosyltransferase (APRT) when in an unmutated form.

9. A method of claim 1 including the further step of introducing a selectable marker having a DNA sequence encoding a known selectable characteristic into the mammalian cells for conferring to the transfectants of the mammalian cells an additional selectable characteristic when cultured in the cell culture medium following base substitution mutagenic reversion in the cloned gene at the specific site.

10. A method of claim 9 wherein the selectable marker is a plasmid containing a neomycin-resistance gene conferring G418 drug resistance to the transfectants of the mammalian cells as the additional selectable characteristic.

11. A method of claim 9 including the further step of introducing the selectable marker and mutated cloned gene into the mammalian cells by a method selected from a group of methods consisting of cotransfection, transfection with the mutated cloned gene ligated to the selectable marker and microinjection.

12. A method of claim 1 wherein the cell culture medium contains adenine, alanosine, azaserine or any combination thereof.

13. A method of claim 1 wherein the mutagenic agent is selected from a group of mutagenic agents consisting of a compound, radiation, gamma rays and electron beams.

14. A method of claim 1 including the further step of

using in vitro site speciic mutagenesis to construct the mutated gene.

15. 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.

16. A method of claim 15 wherein the mutated clone gene encodes adenine phosphoribosyltransferase (APRT) when in an unmutated form.

17. A method of claim 16 wherein the sequence of the mutated clone APRT gene is selected from a group of sequences consisting of a human mutated APRT gene sequence and a mouse mutated APRT gene sequence.

18. A method of claim 16 wherein the nonreverting mammalian cells cannot revert to APRT producing cells absent undergoing mutagenic frameshift reversion at the specific site in the mutated cloned APRT gene.

19. A method of claim 15 wherein the mutagenic agent is selected from a group of mutagenic agents consisting of a compound, radiation, gamma rays and electron beams.

20. A method of claim 15 wherein the cell culture medium contains adenine, alanosine, azaserine or any combination thereof.

21. A method of claim 15 wherein the mutated cloned gene is introduced into the nonreverting mammalian cells by a method selected from a group of methods consisting of transfection and microinjection.

22. A method of claim 15 wherein the phenotypic reversion induced by the mutagenic agent results from same site or second site frameshift mutation.

23. A method of claim 15 including the further step of

using in vitro site specific mutagenesis to construct the mutated gene.

24. A method of claim 15 including the further step of

introducing a selectable marker having a DNA sequence encoding a known selectable characteristic into the mammalian cells for conferring to the mammalian cells an additional selectable characteristic when cultured in a cell culture medium following frameshift mutagenic reversion at the specific site.

25. A method of claim 24 wherein the selectable marker is a plasmid containing a neomycin-resistance gene conferring G418 drug resistance to the mammalian cells as the additional selectable characteristic.

26. A method of claim 18 wherein the non-reverting mammalian cells are selected from a group of non-reverting mammalian cells consisting of human nonreverting mammalian cells and mouse nonreverting mammalian cells.

27. A method of claim 26 wherein the nonreverting human cells are nonreverting human APRT.sup.- line HTD-114 cells.

28. A method of claim 26 wherein the nonreverting mouse cells are nonreverting mouse APRT.sup.- LS-24b or APRT.sup.- CAK cells.

29. A method of identifying a mutagenic agent which induces or facilitates DNA transposition in DNA in mammalian cells comprising the steps of

(a) inserting a retrovirus proviral DNA or other transposable DNA at or near a target locus in a DNA segment of hemizygous or heterozygous mammalian cells which are selectable for a known characteristic when cultured in a cell culture selection medium, said inserting the hemizygous or heterozygous mammalian cells nonselectable for said characteristic when cultured in the cell culture medium,

(b) exposing the mammalian cells to a mutagenic agent for a sufficient amount of time to induce mutagenic reversion at or near the target locus in the DNA segment by DNA transposition, loss or excision of the proviral or other transposable DNA,

(c) culturing the exposed mammalian cells in the cell culture medium which selects for those mammalian cells that are selectable for said characteristic as a result of mutagenic reversion at or near the target locus in the DNA segment, and

(d) identifying the mutagenic agent which has induced the DNA mutagenic reversion at or near the target locus in the DNA segment in the selected mammalian cells.

30. A method of claim 29 wherein the DNA segment corresponds to a gene encoding adenine phosphoribosyltransferase (APRT) and the mammalian cells are hemizygous or heterozygous at or near the target locus of the gene.

31. A method of claim 29 wherein the retrovirus proviral DNA is derived from murine leukemia virus.

32. A method of claim 29 wherein the selective cell culture medium contains adenine, alanosine, azaserine or any combination thereof.

33. A method of claim 29 wherein the mutagenic agent is selected from a group of mutagenic agents consisting of a compound, radiation, gamma rays and electron beams.

34. A method of claim 29 including the further step of introducing a selectable marker having a DNA sequence encoding a known selectable characteristic into the mammalian cells for conferring to the mammalian cells an additional selectable characteristic when cultured in a cell culture medium following mutagenic reversion at the target locus in the DNA segment.

35. A method of claim 34 wherein the selectable marker is a plasmid containing a neomycin-resistant gene conferring G418 drug resistant to the mammalian cells as the additional selectable characteristic.

36. A method of identifying a mutagenic agent which induces mutation in DNA in mammalian cells and the type of mutation induced thereby comprising the step of

(a) exposing mammalian cells, into which a gene having a known mutation at or near a specific site in the gene has been inserted, to a mutagenic agent for a sufficient amount of time to induce specific mutagenic DNA reversion at or near the specific site, the mutated gene having a DNA sequence encoding a known selectable characteristic when in an unmutated form, the mammalian cells being nonselectable for said characteristic when cultured in a cell culture selection medium absent undergoing mutagenic DNA reversion at or near the specific site,

(b) culturing the exposed mammalian cells in the cell culture medium which selects for those exposed mammalian cells that are selectable for said characteristic as a result of specific mutagenic DNA reversion at or near the specific site, and

(c) identifying the mutagenic agent which has induced the specific mutagenic DNA reversion at or near the specific site in the selected mammalian cells.

37. A method of claim 36 including the further step of

using in vitro site specific mutagenesis to construct the mutated gene.

38. A method of claim 36 wherein the specific mutation is selected from a group of mutations consisting of base substitution mutation and frameshift mutation.

39. A method of claim 37 wherein the specific mutation is selected from a group of mutations consisting of base substitution mutation and frameshift mutation.

40. A method of claim 36 wherein the known mutation is due to the introduction of foreign DNA into the endogenous DNA of the mammalian cells at or near the specific site.

41. A method of claim 37 wherein the known mutation is due to the introduction of foreign DNA into the endogenous DNA of the mammalian cells at or near the specific site.

42. A method of claim 36 including the further step of

introducing a selectable marker having a DNA sequence encoding a known selectable characteristic into the mammalian cells for conferring to the mammalian cells an additional selectable characteristic when cultured in the cell culture medium following mutagenic DNA reversion at or near the specific site.

43. A method of claim 42 wherein the mutated gene and selectable marker are introduced simultaneously into the mammalian cells by a method selected from a group of methods consisting of contransfection, transfection with the gene ligated to the selectable marker and microinjection.

44. A method of claim 36 including the further step of

inserting a retrovirus proviral DNA or other transposable DNA at or near the selected site in the gene to generate the mutated gene, the mammalian cells being hemizygous or heterozygous at or near the specific site.

45. A method of claim 44 wherein the specific site is in or near the gene encoding adenine phosphoribosyltransferase (APRT) when in an unmutated form.

46. A method of claim 44 wherein the retrovirus proviral DNA is derived from murine leukemia virus.

47. A method of claim 36 wherei the medium is a cell culture medium containing adenine, alanosine, azaserine or any combination thereof.

48. A method of claim 36 wherein the mutated gene is a constructed mutated cloned gene encoding adenine phosphoribosyltransferase (APRT) when in an unmutated form.

49. A method of claim 48 wherein the mammalian cells are nonreverting mammalian cells that cannot revert to adenine phosphoribosyltransferase (APRT) producing cells absent undergoing specific DNA mutagenic reversion at the specific site in the gene.

50. An assay ensemble for determining whether an agent induces mutation in DNA in mammalian cells and characterizing the type of mutation induced thereby comprising

an effective number of mammalian cells containing a gene which has been introduced into said mammalian cells, the gene having a known mutation introduced at or near a specific site, the gene having a DNA sequence encoding a known selectable characteristic when in an unmutated form and the mammalian cells being nonselectable for said characteristic when cultured in a cell culture selection medium absent undergoing mutagenic DNA reversion at or near the specific site, and

an effective amount of the cell culture medium which selects for the mammalian cells which have undergone specific mutagenic DNA reversion at or near the specific site in the gene when exposed to an agent that has induced the mutation and characterizing the type of mutation induced by the agent.

51. An assay ensemble of claim 50 wherein the mutated gene encodes adenine phosphoribosyltransferase (APRT).

52. An assay ensemble of claim 51 wherein the mammalian cells are nonreverting mammalian cells that cannot revert to APRT producing cells absent undergoing mutagenic DNA reversion at or near the specific site.

53. An assay of claim 50 wherein the medium is cell culture medium containing adenine, alanosine, azaserine or any combination thereof.

54. An assay ensemble of claim 50 wherein the mammalian cells further include a selectable marker having a DN sequence encoding a known selectable characteristic for conferring to the mammalian cells an additional selectable characteristic when cultured in cell culture medium following mutagenic DNA reversion at or near the specific site.

55. An assay ensemble of claim 54 wherein the selectable marker is a plasmid containing a neomycin-resistance gene conferring G418 drug resistance to the mammalian cells as the additional selectable characteristic.

56. An assay ensemble of claim 50 wherein the mutated gene is a recombinant vector adapted for transforming the mammalian cells, the recombinant vector comprising a plasmid or phage into which a DNA segment encoding adenine phosphoribosyltransferase (APRT) has been inserted.

57. An assay ensemble of claim 56 wherein the plasmid or phage is a bacterial plasmid or phage and the DNA segment encodes APRT when in an unmutated form.

58. 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.

59. A kit of claim 58 wherein the introduced mutated cloned gene encodes adenine phosphoribosyltransferase (APRT) when in an unmutated form.

60. A kit of claim 59 wherein the mammalian cells are selected from a group of mammalian cells consisting of human mammalian cells that are nonrevertable for producing APRT when in an untransfected form and mouse mammalian cells that are nonrevertable for producing APRT when in an untransfected form.

61. A kit of claim 60 wherein the human cells are human recessive phenotype adenine phosphoribosyltransferase (APRT.sup.-) line HTD-114 cells.

62. A kit of claim 60 wherein the mouse cells are mouse recessive phenotype adenine phosphoribosyltransferase (APRT.sup.-) LS-24b or CAK cells.

63. A kit of claim 58 wherein the specific site is a Pst 1 restriction site at an intron/exon junction in a mouse gene encoding adenine phosphoribosyltransferase (APRT) when in an unmutated form.

64. A kit of claim 58 wherein the mammalian cells further comprise a selectable marker having a DNA sequence encoding a known selectable characteristic for conferring to the mammalian cells an additional selectable characteristic when cultured in a cell culture medium following DNA mutagenic reversion at or near the specific site.

65. A kit of claim 64 wherein the selectable marker is a plasmid containing a neomycin-resistance gene for conferring G418 drug resistance to the mammalian cells as the additional selectable characteristic.

66. A kit of claim 58 wherein the cell culture medium contains adenine, alanosine, azaserine or any combination thereof.

67. A kit of claim 58 wherein the frameshift reversion induced by the agent is same or second site frameshift reversion.

68. A kit of claim 58 wherein the retrovirus proviral DNA is derived from murine leukemia virus.

69. A kit of claim 58 wherein the introduced mutated cloned gene is a recombinant plasmid adapted for transforming the mammalian cells, the recombinant plasmid comprising a plasmid into which a DNA segment encoding adenine phosphoribosyltranferase (APRT) has been inserted.

70. A kit of claim 69 wherein the recombinant plasmid is pSAM-1 wherein the plasmid is bacterial plasmid pBR328 and the DNA segment encodes mouse APRT.

71. A kit of claim 58 wherein the introduced mutated selectable clone gene is a recombinant vector adapted for transforming the mammalian cells, the recombinant vector comprising a phage into which a DNA segment encoding human adenine phosphoribosyltransferase (APRT) has been inserted.

Details for Patent 4,792,520

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Schering INTRON A interferon alfa-2b VIAL 103132 001 1986-06-04   Start Trial University of Cincinnati (Cincinnati, OH) Medical College of Georgia Research Institute (Augusta, GA) 2039-02-26 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 002 1986-06-04   Start Trial University of Cincinnati (Cincinnati, OH) Medical College of Georgia Research Institute (Augusta, GA) 2039-02-26 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 003 1986-06-04   Start Trial University of Cincinnati (Cincinnati, OH) Medical College of Georgia Research Institute (Augusta, GA) 2039-02-26 RX search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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