Claims for Patent: 5,310,664

Summary for Patent: 5,310,664

Title:	Site specific double stranded DNA endonuclease
Abstract:	An endonuclease which cleaves double stranded DNA at a specific site, producing staggered ends with 4 base pair, 3\' overhangs, has been isolated, purified and characterized. A method of producing sufficient quantities of the endonuclease for purification and characterization is disclosed. A method to use the endonuclease to cleave DNA, producing fragments useful for gene mapping is also disclosed.
Inventor(s):	Butow; Ronald A. (Dallas, TX), Perlman; Philip S. (Worthington, OH)
Assignee:	Board of Regents, The University of Texas System (Austin, TX)
Application Number:	07/997,653
Patent Claims:	1. An endonuclease protein encoded in the fourth intron of yeast cytochrome oxidase having an apparent molecular weight of about 29,000 daltons as determined by SDS-PAGE, and which is capable of cleaving double-stranded DNA at a site identified as follows: ##STR2## 2. The endonuclease protein of claim 1, further defined as a mitochondrial-derived enzyme. 3. The endonuclease protein of claim 2, further defined as a mitochondrial enzyme derived from yeast. 4. The endonuclease protein of claim 3, further defined as a Saccharomyces cerevisiae-derived enzyme. 5. The endonuclease of claim 1, further defined as having a biological activity of up to about 100 units/mg protein. 6. The endonuclease of claim 5 further defined as having a biological activity of up to about 34,000 units/mg after phosphocellulose chromatography. 7. The endonuclease of claim 6 further defined as having a biological activity of up to about 50,000 units/mg after heparin chromatography. 8. The endonuclease of claim 7 further defined as having a biological activity of up to about 200,000 units/mg after gel filtration. 9. The endonuclease of claim 8 further defined as having a biological activity of up to about 500,000 units/mg after DNA affinity chromatography. 10. The endonuclease of claim 1 wherein the endonuclease is translated from a fusion between the upstream exons of the mitochondrial cytochrome oxidase subunit I gene (cox1) of yeast and the open reading frame (ORF) within the 4th intron (aI4.alpha.) of the cox1 gene. 11. The endonuclease of claim 1 wherein the enzyme is capable of cleaving recipient DNA molecules near the site of yeast mitochondrial cox1 intron (aI4.alpha.) insertion. 12. The endonuclease of claim 1 which is also capable of acting as a maturase under certain conditions, said conditions comprising the coincidence of a point mutation in the intron reading frame, the mim-2 mutation or the presence of the nuclear NAM2 gene. 13. A method for preparing an endonuclease having the capability of cleaving double stranded DNA, comprising the steps: (a) culturing yeast that are incapable of splicing the aI4.alpha. intron of the cox1 gene; (b) preparing a mitochondrial extract from the yeast; (c) fractionating the extract; and (d) selecting a fraction or fractions which comprises an endonuolease as defined by claim 1. 14. The method in claim 13 wherein the yeast comprises the WA12/PZ27 strain of Saccharomyces cerevisiae. 15. The method in claim 13 wherein the yeast comprises a strain incapable of expressing the bI4 maturase activity of the cytochrome b gene (cob) in an appropriate culture media. 16. A method of cleaving DNA comprising the steps of: (a) preparing an enzyme as defined by claim 1; and (b) incubating the enzyme with DNA so as to effectuate the endonucleolytic cleaving of the DNA.

Details for Patent 5,310,664

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2011-05-10
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2011-05-10
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2011-05-10