Method for identifying suppressor mutations for common p53 cancer mutations

• US 6,183,964 B1
• Filed: 11/02/1998
• Issued: 02/06/2001
• Est. Priority Date: 05/01/1996
• Status: Expired due to Term

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Boeke, Jef D., Brachmann, Rainer K.
• Primary Examiner(s): Arthur, Lisa B.
• Assistant Examiner(s): GOLDBERG, JEANINE ANNE
• Application Number:
  US09/184,073
• Time in Patent Office: 827 Days
• Field of Search: 536/23.1, 435/325, 435/254.2, 435/320.1, 435/69.1, 435/6, 435/255.1
• US Class Current: 435/6.12
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/4746 p53

Abstract

Intragenic suppressor mutations of common p53 mutations are able to function in cis and/or trans. These mutations are useful for identifying small molecule drugs which function in a similar fashion. In addition, the mutations themselves may be useful therapeutically, especially if they function in trans. Methods for rapidly obtaining this type of mutant employ a yeast selection system. Cells having both the negative mutation and intragenic suppressor are useful for studying the interactions of the two, in particular in determining the structure of the homotetramers and heterotetramers.

Claims

1. A method for identifying yeast cells which carry mutations in a human p53 gene, wherein said mutations suppress the phenotype of dominant-negative p53 mutations, comprising the step of:

determining the phenotype of a cell comprising a nucleic acid encoding human p53 which comprises a dominant negative mutation which has been further mutagenized to induce a suppressor mutation, if the phenotype of the cell is that of a cell carrying a wild-type p53 then the cell is a candidate cell for carrying a human p53 suppressor mutation which suppresses the phenotype of the dominant-negative mutation.

2. The method of claim 1 wherein the phenotype is determined by observing expression of a gene which is dependent on p53 for expression.

3. The method of claim 2 wherein the pS3dependent gene is selected from the group consisting of p₂₁^WAF1, GADD45, IGF-BP3, and bax.

4. The method of claim 2 wherein the p53dependent gene is a reporter gene under the control of a p53 responsive transcription element.

5. The method of claim 1 wherein he dominant-negative mutation is selected from the group consisting of:

V143A, R175H, G245D, G245S, R248W, R249S, R248Q, R273H, R273C, R282W, and R273P.

6. The method of claim 1 wherein the nucleic acid encoding human p53 was mutagenized using PCR-generated fragments to recombine with a gapped p53-encoding molecule.

7. The method of claim 1 wherein the mutagenized nucleic acid encoding human p53 comprises an oligomerization domain of human p53.

8. A method for identifying yeast cells which carry mutations in a human p53 gene, wherein said mutations suppress the phenotype of dominant negative p53 mutations, comprising the steps of:

mutagenizing a nucleic acid comprising a dominant-negative allele of human p53;

supplying the nucleic acid which has been mutagenized to a cell;

determining the phenotype of the cell;

if the phenotype is that of a cell carrying a wild-type p53, then the cell is a candidate cell for carrying a human p53 suppressor mutation which suppresses the phenotype of the dominant-negative mutation.

12. The method of claim 8 wherein the nucleic acid which has been mutagenized comprises an oligomerization domain of human p53.

9. A method for identifying yeast cells which carry mutations in a human p53 gene, wherein said mutations suppress the phenotype of dominant-negative p53 mutations, comprising the steps of:

introducing a mutagenized nucleic acid encoding human p53 into a cell comprising a dominant-negative p53 expression construct;

determining the phenotype of the cell;

if the phenotype is that of a cell carrying a wild-type p53, then the cell is a candidate cell for carrying a human p53 suppressor mutation which suppresses the phenotype of the dominant-negative mutation.

10. The method of claim 9 wherein the nucleic acid comprises an oligomerization domain of human p53.

11. A method for identifying yeast cells which carry mutations in a human p53 gene, wherein said mutations suppress the phenotype of dominant-negative p53 mutations, comprising the steps of:

introducing a mutegenized nucleic acid encoding human p53 into cells comprising (a) a dominant-negative p53 expression construct and (b) a selectable reporter gene operably linked to a DNA sequence to which human p53 specifically binds;

selecting a cell expressing the reporter gene, wherein said cell is a candidate cell for carrying a human p53 suppressor mutation which suppresses the phenotype of the dominant-negative p53.

13. The method of claim 11 wherein the mutagenized nucleic acid comprises an oligomerization domain of human p53.