Methods to promote homologous recombination in eukaryotic cells and organisms

• US 5,945,339 A
• Filed: 07/13/1998
• Issued: 08/31/1999
• Est. Priority Date: 01/17/1995
• Status: Expired due to Fees

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University), Thomas Jefferson University (Main Line Health System)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University), Thomas Jefferson University (Main Line Health System)
• Inventors: Kmiec, Eric B., Holloman, William K.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/114,637
• Time in Patent Office: 414 Days
• Field of Search: 435/477, 435/483, 435/484
• US Class Current: 435/477
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00

Abstract

The invention concerns genes encoding recombinases that can be used to promote homologous recombination in eukaryotic cells. The application teaches methods by which a recombinase of one species can be used to isolate a homologous recombinase of a different species and methods to identify the isolated homologs. Recombinases from Ustilago maydis, Saccharomyces cerevisiae and humans are specifically included in the invention.

The invention encompasses the method of producing an isolated recombinase protein in a prokaryotic cell and recovering the product in an active form. The invention also encompasses a genetically engineered gene which encodes a non-naturally occurring recombinase that causes a greater rate of recombination than does the naturally occurring recombinase. The invention further encompasses the use of recombinase proteins and of recombinase genes to promote homologous recombination, including recombination between a host cell genome and a chimeric oligonucleotide, i.e., an oligonucleotide having both RNA and DNA bases.

Claims

1. A method of promoting an alteration at a genetic locus of interest which comprises the steps of:

a) introducing into a eukaryotic cell a nucleic acid which comprises a segment that is homologous with the locus of interest; and

b) introducing into the eukaryotic cell a polydeoxynucleic acid expression vector that comprises a coding portion that encodes a protein that;

(1) is an ATPase;

(2) catalyzes the formation of complementary or identical strand pairings of polydeoxynucleic acids; and

(3) promotes homologous recombination in a eukaryote;

(4) wherein the normalized alignment score of the protein, compared to SEQ ID NO;

2, is at least about 150; and

(5) wherein the coding portion hybridizes to the 2.8 Kb Bam HI REC2 insert of pCM346 when the coding portion is membrane immobilized and a final wash conditions is 40 mM Na₂ PO₄, 1 mM EDTA, 1% SDS at 50°

C.; and

c) expressing the polydeoxynucleic acid expression vector in the cell so as to produce the encoded protein.

2. The method of claim 1, wherein the coding portion encodes a naturally occurring Rec2 recombinase or a naturally occurring recombinase that has been modified to remove a p34^CDC2 kinase consensus phosphorylation site.

3. The method of claim 2, wherein the coding portion encodes a protein comprising a recombinase of an ascomycetes yeast form fungal cell.

4. The method of claim 2, wherein the coding portion encodes a protein comprising a recombinase of an ascomycetes yeast form fungal cell that has been modified by the deletion of a p34^CDC2 kinase consensus phosphorylation site.

5. The method of claim 2, wherein the coding portion encodes a protein comprising a recombinase of a fungus selected from the group of fungal genera consisting of Ustilago, Saccharomyces and Aspergillus.

6. The method of claim 2, wherein the coding portion encodes a protein comprising a recombinase of a fungus selected from the group of fungal genera consisting of Ustilago, Saccharomyces and Aspergillus, which protein has been modified by the deletion of a p34^CDC2 kinase consensus phosphorylation site.