Gain of function mutations in ATP-dependent transposition proteins

Abstract

The invention is specifically directed to efficient, random, simple insertion of a transposon or derivative transposable element into DNA in vivo or in vitro. The invention is particularly directed to mutations in ATP-utilizing regulatory transposition proteins that permit insertion with less target-site specificity than wild-type. The invention encompasses gain-of-function mutations in TnsC, an ATP-utilizing regulatory transposition protein that activates the bacterial transposon Tn7. Such mutations enable the insertion of a Tn7 transposon or derivative transposable element in a non-specific manner into a given DNA segment. Insertion can be effected in plasmid and cosmid libraries, cDNA libraries, PCR products, bacterial artificial chromosomes, yeast artificial chromosomes, mammalian artificial chromosomes, genomic DNAs, and the like. Such insertion is useful in DNA sequencing methods, for genetic analysis by insertional mutagenesis, and alteration of gene expression by insertion of a given genetic sequence.

That what is claimed:  
     
         1 . An isolated ATP-utilizing regulatory protein encoded by a transposon, said protein containing a mutation that allows efficient and simple insertion of and reduced target site specificity on a transposable element derived from said transposon.  
     
     
         2 . The protein of  claim 1  wherein said protein is TnsC.  
     
     
         3 . The protein of  claim 2  wherein said mutation is valine at amino acid number 225.  
     
     
         4 . A transposon encoding an ATP-utilizing regulatory protein, the protein containing a mutation that allows efficient and simple insertion of and reduced target site specificity on said transposon.  
     
     
         5 . The transposon of  claim 4 , wherein said transposon is Tn7 or a derivative thereof.  
     
     
         6 . The transposon of  claim 5  wherein said mutation is valine at amino acid number 225.  
     
     
         7 . A composition containing an isolated ATP-utilizing regulatory protein encoded by a transposon, said protein containing a mutation that allows efficient and simple insertion of and reduced target site specificity on said transposon and a transposable element derived from said transposon.  
     
     
         8 . The composition of  claim 7  wherein said protein is TnsC.  
     
     
         9 . The composition of  claim 8  wherein said mutation is valine at amino acid number 225.  
     
     
         10 . A composition containing a transposon encoding an ATP-utilizing regulatory protein, the protein containing a mutation that allows efficient and simple insertion of and reduced target site specificity on said transposon.  
     
     
         11 . The composition of  claim 10  wherein said transposon is Tn7 or a derivative thereof.  
     
     
         12 . The composition of  claim 11  wherein said mutation is valine at amino acid number 225.  
     
     
         13 . The composition according to  claim 7  further containing a transposon or transposable element derived therefrom capable of being activated by said protein.  
     
     
         14 . The composition of  claim 13  further comprising target DNA into which said transposon or derivative is capable of inserting.  
     
     
         15 . The composition of  claim 13  in which said transposon or derivative contains at least one primer binding site that is native to said transposon or heterologous.  
     
     
         16 . The composition of  claim 15  further comprising primers that hybridize to said primer binding site on the transposon or derivative.  
     
     
         17 . The composition of  claim 13  in which said transposon or derivative contains a heterologous DNA sequence.  
     
     
         18 . The composition according to  claim 10  further comprising a target sequence into which said transposon is capable of insertion.  
     
     
         19 . The composition according to  claim 11  which said transposon contains at least one primer binding site that is native to said transposon or heterologous.  
     
     
         20 . The composition of  claim 19  further comprising primers that hybridize to the primer binding site on said transposon.  
     
     
         21 . The composition according to  claim 11  in which said transposon contains a heterologous DNA sequence.  
     
     
         22 . A composition containing an isolated ATP-utilizing regulatory protein encoded by a transposon, said protein containing a mutation that allows efficient and simple insertion of and reduced target site specificity on a transposable element derived from said transposon, further comprising a transposon or transposable derived therefrom capable of being activated by said protein.  
     
     
         23 . The composition of  claim 22  wherein said transposon is a Tn7 or a derivative thereof and said protein is TnsC.  
     
     
         24 . The composition of  claim 23  wherein said mutation is valine at amino acid number 225.  
     
     
         25 . A kit containing the composition according to claim  22 .