Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis

The insecticidal crystal protein gene of the coleopteran-toxic Bacillus thuringiensis var. tenebrionis has been isolated, and the nucleotide sequence has been determined. A total DNA library from var. tenebrionis was made in the plasmid vector pUC12. By using a synthetic 27-base oligonucleotide corr...

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Bibliographic Details
Published in:	Proceedings of the National Academy of Sciences - PNAS Vol. 84; no. 20; pp. 7036 - 7040
Main Authors:	Sekar, V, Thompson, D.V, Maroney, M.J, Bookland, R.G, Adang, M.J
Format:	Journal Article
Language:	English
Published:	Washington, DC National Academy of Sciences of the United States of America 01-10-1987 National Acad Sciences
Subjects:	Amino acids ANTIGENE ANTIGENOS ANTIGENS BACILLUS THURINGIENSIS Biological and medical sciences Biological Sciences: Biochemistry Biotechnology CLONACION CLONAGE CLONING Crystals DNA DNA probes Fundamental and applied biological sciences. Psychology Genes Genetic engineering Genetic technics GENETICA GENETICS GENETIQUE Insect larvae LEPIDOPTERA Methods. Procedures. Technologies Molecular cloning Open reading frames Plasmids PROTEINAS PROTEINE PROTEINS Toxins Characterization Proteins Entomopathogen Bacillales Gene Bacillus thuringiensis Bacteria Primary structure Bacillaceae Molecular cloning Endotoxin Microbial insecticide
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Summary:	The insecticidal crystal protein gene of the coleopteran-toxic Bacillus thuringiensis var. tenebrionis has been isolated, and the nucleotide sequence has been determined. A total DNA library from var. tenebrionis was made in the plasmid vector pUC12. By using a synthetic 27-base oligonucleotide corresponding to a stretch of nine N-terminal amino acids of a tryptic fragment of purified crystal protein of var. tenebrionis as a probe, recombinant colonies were screened by in situ hybridization for the presence of the crystal protein gene. Positive clones obtained from this screening were further tested for toxicity. One recombinant, NSBP544 (which contained a 5.9-kilobase BamHI insert), was toxic to larvae of Colorado potato beetle. Immunoblot analysis revealed that this clone produces two crystal-specific antigens of 65 and 73 kDa as do sporulating var. tenebrionis cells. However, purified crystal inclusions from var. tenebrionis contain a primary peptide component of 65 kDa. A 1932-base-pair open reading frame with a coding capacity of 73,119 Da has been identified by nucleotide sequencing analysis of the cloned crystal protein. In addition, mung bean nuclease mapping indicates that transcription of the crystal protein of var. tenebrionis initiates 130 base pairs upstream from the translational start site. Southern blot analysis using an internal 0.7-kilobase EcoRI fragment of pNSBP544 as a probe revealed that the crystal protein gene is located on a 90-MDa plasmid.
Bibliography:	H01 880522988 To whom reprint requests should be addressed.
ISSN:	0027-8424 1091-6490
DOI:	10.1073/pnas.84.20.7036