Methods for obtaining nucleotide sequences coding for polypeptides specifically active for larvae of S. littoralis

Abstract

This invention provides a method for obtaining a nucleotide sequence that codes for the active part of a polypeptide specifically active for larvae of S. littoralis. The method comprises hybridizing at least one nucleotide probe to DNA from a strain of B. thuringiensis active against S. littoralis, wherein at least one nucleotide probe is derived from the 5′ part of a restriction fragment of a gene for δ endotoxin of B. thuringiensis strain aizawa 7-29, isolating the DNA from the strain of B. thuringiensis active against S. littoralis that hybridized to the probe, cloning the isolated DNA into a vector, and purifying the vector to thereby obtain the nucleotide sequence that codes for the active part of a polypeptide specifically active for larvae of S. littoralis, wherein the specifically active polypeptide has (1) a specificity index of less than 1.0 and (2) a higher specific toxic activity towards S. littoralis than the native crystal proteins of the strains aizawai 7-29 or berliner 1715.

1 - 36 . (canceled)  
     
     
         37 . A method for obtaining a nucleotide sequence that codes for the active part of a polypeptide specifically active for larvae of  S. littoralis,  wherein the method comprises: 
 (A) hybridizing at least one nucleotide probe to DNA from a strain of  B. thuringiensis  active against  S. littoralis,  wherein at least one nucleotide probe is derived from the 5′ part of a restriction fragment of a gene for δ endotoxin of  B. thuringiensis  strain aizawa 7-29;    (B) isolating the DNA from the strain of  B. thuringiensis  active against  S. littoralis  that hybridized to the probe;    (C) cloning the isolated DNA into a vector; and    (D) purifying the vector to thereby obtain the nucleotide sequence that codes for the active part of a polypeptide specifically active for larvae of  S. littoralis;      wherein the specifically active polypeptide has (1) a specificity index of less than 1.0 and (2) a higher specific toxic activity towards  S. littoralis  than the native crystal proteins of the strains aizawai 7-29 or berliner 1715.    
     
     
         38 . The method as claimed in  claim 37 , wherein the nucleotide probe comprises the HindIII-PstI restriction fragment of the δ endotoxin of  B. thuringiensis  strain aizawa 7-29.  
     
     
         39 . The method as claimed in  claim 37 , wherein the nucleotide probe comprises the HincII-PstI fragment of the δ endotoxin of  B. thuringiensis  strain aizawa 7-29 and further comprises the HindIII-HincII restriction fragment of  B. thuringiensis  strain entomocidus 6-01.  
     
     
         40 . The method as claimed in  claim 37 , wherein the nucleotide probe encodes amino acids 281-620 of the δ endotoxin of  B. thuringiensis  strain aizawa 7-29.  
     
     
         41 . The method as claimed in  claim 37 , wherein the specificity index is less than 0.5.  
     
     
         42 . The method as claimed in  claim 38 , wherein the specificity index is less than 0.5.  
     
     
         43 . The method as claimed in  claim 39 , wherein the specificity index is less than 0.5.  
     
     
         44 . The method as claimed in  claim 40 , wherein the specificity index is less than 0.5.  
     
     
         45 . The method as claimed in  claim 37 , wherein the specific toxic activity of the specifically active polypeptide towards  S. littoralis  is greater than 2.5 times the specific toxic activity of the native crystal proteins of the strains aizawai 7-29 or berliner 1715 towards  S. littoralis.    
     
     
         46 . The method as claimed in  claim 38 , wherein the specific toxic activity of the specifically active polypeptide towards  S. littoralis  is greater than 2.5 times the specific toxic activity of the native crystal proteins of the strains aizawai 7-29 or berliner 1715 towards  S. littoralis.    
     
     
         47 . The method as claimed in  claim 39 , wherein the specific toxic activity of the specifically active polypeptide towards  S. littoralis  is greater than 2.5 times the specific toxic activity of the native crystal proteins of the strains aizawai 7-29 or berliner 1715 towards  S. littoralis.    
     
     
         48 . The method as claimed in  claim 40 , wherein the specific toxic activity of the specifically active polypeptide towards  S. littoralis  is greater than 2.5 times the specific toxic activity of the native crystal proteins of the strains aizawai 7-29 or berliner 1715 towards  S. littoralis.