Genetic insulator for preventing influence by another gene promoter

Abstract

A 16 bp polynucleotide sequence of Arabidopsis thaliana is a genetic insulator that can effectively isolate a transgene from positional effects of neighboring gene activities in transgenic plant cells.

What is claimed is:  
     
         1 . An isolated polynucleotide, comprising: 
 (a) at least one copy of a polynucleotide having the sequence set forth in SEQ ID NO:9; or    (b) a polynucleotide which is a variant or fragment of the polynucleotide set forth in SEQ ID NO:9, wherein the variant or fragment has a plant genetic insulator activity.    
     
     
         2 . The isolated polynucleotide of  claim 1 , wherein the polynucleotide comprises at least one copy of a polynucleotide having a set forth in the group consisting of SEQ ID NOS: 1, 5, 9, 10, 11, 12, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 34, 35 or 36.  
     
     
         3 . The isolated polynucleotide of  claim 1 , further comprising a replicable vector; into which the polynucleotide is inserted.  
     
     
         4 . The isolated polynucleotide of  claim 3 , wherein the vector is an expression vector.  
     
     
         5 . The isolated polynucleotide of  claim 3 , wherein the vector is a plant vector.  
     
     
         6 . The isolated polynucleotide of  claim 3 , further comprising a host cell, in which the vector is situated.  
     
     
         7 . The isolated polynucleotide of  claim 3 , wherein the host cell is a plant cell.  
     
     
         8 . The isolated polynucleotide of  claim 3 , further comprising a transgenic plant, in which the vector is situated.  
     
     
         9 . The isolated polynucleotide of  claim 8 , wherein the plant is Arabidopsis or tobacco.  
     
     
         10 . The isolated polynucleotide of  claim 3 , further comprising a transgenic seed, in which the vector is situated.  
     
     
         11 . A recombinant polynucleotide, comprising: 
 (a) at least one copy of a polynucleotide having the sequence set forth in SEQ ID NO:9; or    (b) a polynucleotide which is a variant or fragment of the polynucleotide set forth in SEQ ID NO:9, wherein said variant or fragment has a plant genetic insulator activity.    
     
     
         12 . A method for expressing a polypeptide in a plant cell, comprising the steps of: 
 (a) providing a vector comprising: 
 (i) at least one copy of either 
 (A) a polynucleotide having the sequence set forth in SEQ ID NO:9; or  
 (B) a polynucleotide which is a variant or fragment of the polynucleotide set forth in SEQ ID NO:9, wherein the variant or fragment has a plant genetic insulator activity; and  
 
 (ii) a structural polynucleotide coding for a polypeptide;  
   (b) inserting the vector into a plant cell, wherein the genetic insulator polynucleotide is recombined into the genomic DNA of the plant; and    (c) allowing the plant cell to express the polypeptide.    
     
     
         13 . The method according to  claim 12 , wherein the genetic insulator polynucleotide is located immediately upstream of the polynucleotide encoding the polypeptide.  
     
     
         14 . The method according to  claim 13 , wherein the plant is Arabidopsis or tobacco.  
     
     
         15 . A method of making a recombinant plant cell having reduced variability of expression of a transgenic polypeptide therein, said method comprising: 
 (a) providing a plant cell capable of regeneration;    (b) transfecting said plant cell with a polynucleotide construct comprising 
 (i) a genetic insulator polypeptide, comprising: 
 (A) at least one copy of a polynucleotide having the sequence set forth in SEQ ID NO:9; or  
 (B) a polynucleotide which is a variant or fragment of the polynucleotide set forth in SEQ ID NO:9, wherein the variant or fragment has a plant genetic insulator activity;  
 
 (ii) a transcription initiation region; and  
 (iii) a structural polynucleotide encoding a polynucleotide;  
   wherein the genetic insulator polypeptide, the transcription initiation region and the structural polynucleotide are operatively associated;    wherein the polynucleotide expression has a reduced variability as compared with a plant cell transfected with a polynucleotide construct that does not contain the genetic insulator polypeptide.    
     
     
         16 . The method of  claim 15 , wherein expression of the transgenic polypeptide occurs in more of a plurality of the plant cells as compared to a plurality of the plant cells transfected with a polynucleotide construct that does not contain the genetic insulator polypeptide.  
     
     
         17 . A method for insulating the expression of a transgenic polypeptide from cis-acting regulatory elements in the plant chromosome into which the polynucleotide coding for the expressed polypeptide has integrated, comprising: 
 transfecting a plant cell with a polynucleotide construct comprising 
 (a) a genetic insulator polypeptide, comprising: 
 (i) at least one copy of a polynucleotide having the sequence set forth in SEQ ID NO:9; or  
 (ii) a polynucleotide which is a variant or fragment of the polynucleotide set forth in SEQ ID NO:9, wherein the variant or fragment has a plant genetic insulator activity;  
 
 (b) a transcription initiation region; and  
 (c) a structural polynucleotide encoding a polynucleotide;  
 wherein the genetic insulator polypeptide, the transcription initiation region and the structural polynucleotide are operatively associated;  
 wherein the transfected polynucleotide construct integrates into a chromosome of the plant cell; and  
 wherein the expression of the polypeptide from the integrated polynucleotide is insulated from cis-acting regulatory elements in the plant chromosome into which the polynucleotide coding for the expressed polypeptide has integrated.