Characterization of the I-SpomI endonuclease from fission yeast

Abstract

Isolated DNAs encoding the enzyme I-SpomI and its recognition and cutting site are provided. The DNA sequences can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

1 . An isolated DNA sequence comprising the nucleotide sequence of SEQ ID NO:8 or a fragment thereof, wherein said DNA sequence encodes a polypeptide having SpomI endonuclease activity.  
     
     
         2 . An isolated, DNA sequence comprising the nucleotide sequence of SEQ ID NO:9 or a fragment thereof, wherein said DNA sequence encodes a polypeptide having I-SpomI endonuclease activity.  
     
     
         3 . An isolated RNA complementary to the nucleotide sequence of  claim 1  or  2 .  
     
     
         4 . An expression vector that directs expression of I-SpomI enzyme.  
     
     
         5 . The vector of  claim 4 , wherein said vector expresses said enzyme in mammalian cells.  
     
     
         6 . The vector of  claim 5 , wherein said vector is an adenovirus vector.  
     
     
         7 . The vector of  claim 4 , wherein said vector expresses said enzyme in insect cells.  
     
     
         8 . A vector comprising an I-SpomI restriction site.  
     
     
         9 . A recombinant chromosome comprising an I-SpomI site.  
     
     
         10 . A recombinant cell comprising an I-SpomI site.  
     
     
         11 . The recombinant chromosome of  claim 9 , wherein said chromosome is from a eukaryotic or prokaryotic organism.  
     
     
         12 . The recombinant cell of  claim 10 , wherein said cell is from a eukaryotic or prokaryotic organism.  
     
     
         13 . The cell of  claim 10 , farther comprising a Group I intron encoded endonuclease site.  
     
     
         14 . The cell of  claim 13 , wherein said Group I intron encoded endonuclease site is selected from I-CeuI, I-Crel, I-DmoI, and I-SceI sites.  
     
     
         15 . A recombinant chromosome comprising a nucleic acid encoding I-SpomI enzyme.  
     
     
         16 . A recombinant cell comprising a nucleic acid encoding I-SpomI enzyme.  
     
     
         17 . The recombinant chromosome of  claim 15 , wherein said chromosome is from a eukaryotic or prokaryotic organism.  
     
     
         18 . The recombinant cell of  claim 16 , wherein said cell is from a eukaryotic or prokaryotic organism.  
     
     
         19 . The recombinant chromosome of  claim 11  or  17 , wherein said chromosome is from a mammalian, insect, fungal, plant, yeast, bacterial, or nematode organism.  
     
     
         20 . The recombinant cell of  claim 12  or  18 , wherein said cell is from a mammalian, insect, flmgal, plant, yeast, bacterial, or nematode organism.  
     
     
         21 . A transgenic organism comprising a recombinant chromosome comprising an I-SpomI site.  
     
     
         22 . A transgenic organism comprising a nucleic acid encoding I-SpomI enzyme.  
     
     
         23 . The transgenic organism of  claim 21  or  22 , wherein said organism is a mammalian, insect, fungal, plant, yeast, bacterial, or nematode organism.  
     
     
         24 . A method of inducing at least one site-directed double-strand break in DNA of a cell comprising: 
 (a) providing cells containing double-stranded DNA, wherein said DNA comprises at least one I-SpomI restriction site;    (b) providing the cells with I-SpomI endonuclease; and    (c) selecting cells in which at least one double-strand break has been induced.    
     
     
         25 . The method of  claim 24 , wherein said cell is a mammalian, insect, fungal, plant, yeast, bacterial, or nematode cell.  
     
     
         26 . A method of inducing homologous recombination between chromosomal DNA of a cell and exogenous DNA added to said cell, said method comprising 
 (a) providing cells containing chromosomal DNA, wherein said DNA comprises at least one I-SpomI restriction site;    (b) providing I-SpomI enzyme and exogenous DNA to said cells; and    (c) selecting cells in which said exogenous DNA is inserted into said chromosomal DNA.    
     
     
         27 . The method of  claim 26 , wherein said cell is a mammalian, insect, fungal plant, yeast, bacterial, or nematode cell.  
     
     
         28 . A method of inducing at least one site-directed double-strand break in DNA of a cell comprising: 
 (a) providing cells containing DNA, wherein said DNA comprises at least one I-SpomI restriction site and at least one additional Group I intron encoded endonuclease site;    (b) providing the cells with I-SpomI endonuclease and Group I intron encoded endonuclease; and    (c) selecting cells in which at least two double-strand break have been induced.    
     
     
         29 . The method of  claim 28 , wherein said Group I intron encoded endonuclease is selected from I-CeuI, I-CreI, I-DmoI, and I-SceI sites.  
     
     
         30 . The method of  claim 28 , wherein said cell is a mammalian, insect, fungal, plant, yeast, bacterial, or nematode cell.  
     
     
         31 . A vector comprising a DNA sequence encoding a polypeptide having I-SpomI endonuclease activity.  
     
     
         32 . The vector of  claim 32 , wherein said vector is a plasmid deposited at CNCM under accession number I-2643.  
     
     
         33 . An isolated polypeptide comprising the amino sequence of SEQ ID NO:11 or a fragment thereof, wherein said polypeptide has I-SpomI endonuclease activity.  
     
     
         34 . An isolated polypeptide comprising the amino sequence of SEQ ID NO:12 or a fragment thereof, wherein said polypeptide has I-SpomI endonuclease activity.