Bacillus thuringiensis Chromosomal Genome Sequences and Uses Thereof

Abstract

The present invention relates to nucleic acid sequences from Bacillus thuringiensis and, in particular, to genomic DNA sequences. The invention encompasses nucleic acid molecules present in non-coding regions as well as nucleic acid molecules that encode proteins, fragments of proteins, tRNA's, fragments of tRNA's, rRNA's and fragments of rRNA's. In addition, proteins and fragments of proteins so encoded and antibodies capable of binding the proteins are encompassed by the present invention. The invention also relates to methods of using the disclosed nucleic acid molecules, proteins, fragments of proteins, RNA's, and antibodies, for example, for gene identification and analysis, and preparation of constructs.

1 - 40 . (canceled) 
     
     
         41 . A method for identifying plasmid DNA sequences of a  Bacillus  species, the method comprising the steps of:
 a) identifying a first  Bacillus  species strain which does not contain plasmid DNA;   b) generating a library of chromosomal genomic DNA from said first  Bacillus  species strain;   c) obtaining the nucleotide sequences of said chromosomal genomic DNA from step b);   d) identifying a second  Bacillus  species strain which contains plasmid DNA;   e) generating a library of genomic DNA from said second  Bacillus  species strain, wherein said genomic DNA contains chromosomal and plasmid DNA;   f) obtaining the nucleotide sequences of said genomic DNA from step e);   g) subtracting any common sequences identified in the genomic DNA from step f) which are also identified in the chromosomal genomic DNA from step c) from the nucleotide sequences of step f) to obtain a subtracted set of plasmid DNA sequences; and   h) constructing contigs and sequences of said subtracted set of plasmid DNA sequences;
 wherein said contigs and sequences comprise the plasmid DNA sequences of said second  Bacillus  species. 
   
     
     
         42 . The method according to  claim 41 , wherein said first and second  Bacillus  species is selected from the group consisting of  Bacillus thuringiensis, Bacillus subtilis, Bacillus cereus , and  Bacillus anthracis.    
     
     
         43 . The method according to  claim 42 , wherein said  Bacillus  species is  Bacillus thuringiensis.    
     
     
         44 . The method of  claim 41 , wherein the nucleotide sequences of said chromosomal genomic DNA obtained in step c) comprise the sequences selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:8283. 
     
     
         45 . The method of  claim 41 , wherein said second  Bacillus  species strain contains less than two naturally occurring plasmids. 
     
     
         46 . The method according to  claim 45 , wherein said  Bacillus  species strain is EG 10650. 
     
     
         47 . The method of  claim 45 , wherein said less than two naturally occurring plasmids is selectively tagged with an identifiable marker gene. 
     
     
         48 . The method according to  claim 47 , wherein said marker gene is selected from the group consisting of an antibiotic resistance gene, a gene encoding an essential metabolic or catabolic protein or functional homologue thereof, a gene conferring bioluminescence properties, and a gene encoding an enzyme which catalyzes the metabolism of a substrate which imparts a colored product deposited on or within the  Bacillus  species strain. 
     
     
         49 - 50 . (canceled) 
     
     
         51 . A method for screening one or more bacterial plasmid DNA sequences, the method comprising:
 a) isolating and purifying bacterial plasmid DNA;   b) generating a library comprising one or more bacterial plasmid DNA sequences from the isolated and purified bacterial plasmid DNA;   c) sequencing the library to obtain a set of bacterial plasmid DNA sequences; and   d) determining if the plasmid DNA sequence is a novel sequence by comparing the set of bacterial plasmid DNA sequences to a database comprising a plurality of nucleotide sequences.   
     
     
         52 . The method of  claim 51 , wherein said bacterial plasmid DNA is plasmid DNA of  Bacillus thuringiensis  and said bacterial plasmid DNA sequence is plasmid DNA sequence of  Bacillus thuringiensis.    
     
     
         53 . The method of  claim 52 , wherein said database comprises a set of chromosomal DNA sequences of  Bacillus thuringiensis , wherein said set of chromosomal DNA sequences comprises the sequences selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:8283. 
     
     
         54 . The method of  claim 53 , wherein said determining step d) further comprises:
 1) identifying common sequences in both the set of plasmid DNA sequences and the set of chromosomal DNA sequences;   2) subtracting the common sequences from the set of plasmid DNA sequences to obtain a subtracted set of plasmid DNA sequences;   3) assembling the substracted set of plasmid DNA sequences to contigs and sequences; and   4) determining open reading frames in the contigs and sequences.   
     
     
         55 . A method for identifying one or more novel genes encoding insect inhibitory proteins in one or more plasmid DNA sequences of a  Bacillus  species, the method comprising:
 1) screening one or more plasmid DNA sequences of the  Bacillus  species according to any one of  claims 51  to  54 ; and   2) identifying one or more novel genes encoding insect inhibitory proteins in said one or more plasmid DNA sequences of the  Bacillus  species.