ABSTRACT
Polymerase chain reaction screening using cryV-specific oligonucleotides, designed to amplify the 5' half of cryV-type genes, revealed the presence of such genes in 7 of 21 Bacillus thuringiensis serotypes examined. Restriction analysis and hybridization studies indicated that these putative genes fall into at least three subclasses. The nucleotide sequence of the cryV-type gene cloned from B. thuringiensis subsp. kurstaki DSIR732 revealed an open reading frame coding for a protein of 719 amino acids, and lysates of Escherichia coli cells expressing the 81.2-kDa CryV732 protein were toxic to Epiphyas postvittana (Lepidoptera: Tortricidae).
Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Bacterial Toxins , Endotoxins , Genes, Bacterial , Bacillus thuringiensis/classification , Bacillus thuringiensis Toxins , Base Sequence , Cloning, Molecular , DNA Probes , DNA, Bacterial/genetics , Escherichia coli/genetics , Gene Expression , Hemolysin Proteins , Molecular Sequence Data , Polymerase Chain Reaction , SerotypingABSTRACT
The nucleotide (nt) sequence of a DNA segment containing the majority of a gene cloned from Bacillus thuringiensis DSIR517 encoding a 130 kDa insecticidal crystal protein has been determined. Sequence analysis reveals an open reading frame (ORF) of 3453 nt. The ATG initiation codon, which is preceded by a potential ribosome-binding site sequence, was confirmed by N-terminal amino acid sequencing. The ORF extends beyond the 3' terminus of the cloned fragment; however, the high degree of homology between the deduced amino acid sequence of this ORF and other Cry proteins suggests the clone lacks only five C-terminal amino acids. Making this assumption, the ORF of 3468 nt encodes a protein of 1156 amino acids with an estimated molecular mass of 129700 Da. Analysis of the deduced amino acid sequence reveals a number of features characteristic of Cry proteins. Alignment of the Cry 517 protein sequence with other Cry proteins suggests it is most closely related to the cryIA-E genes but sufficiently different to form a new cryI gene subclass.
