Nucleotide sequence and analysis of the N‐terminal coding region of the Spodoptera‐activeδ‐endotoxin gene of Bacillus thuringiensis aizawai 7.29

Sanchis, Vincent; Lereclus, Didier; Menou, Ghislaine; Chaufaux, Josette; Guo, Suxia; Lecadet, Marguerite-M.

doi:10.1111/j.1365-2958.1989.tb01812.x

Cited by 54 publications

(35 citation statements)

References 37 publications

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“…In a previous study [16] (c) toxins to S. littoralis BBMV preparations (50 pug total protein) were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes. The blots were then soaked overnight in Tris-buffered saline (TBS) to renature the proteins.…”

Section: Introductionmentioning

confidence: 99%

Identification and partial purification of a Bacillus thuringiensis CryIC δ‐endotoxin binding protein from spodoptera littoralis gut membranes

Sanchis

Ellar

1993

FEBS Letters

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Immunoblotting experiments were performed using CryIC and CryIA(c) Bacillus thuringiensis S-endotoxins to detect the presence of specific toxin binding proteins on Spodopteru littoralis brush border membrane vesicles. The CryIC toxin binds two proteins of 40 and 65 kDa and the CryIA(c) binds a protein of 40 kDa. The CryIA(c) toxin also binds faintly to a 120 kDa protein on S littoralis brush border membrane vesicles as does a polyclonal antiserum raised against a putative CryIA(c) 120 kDa binding protein from Manduca sexta. The 40 kDa CryIC binding protein was partially purified by affinity chromatography and is therefore a strong candidate for in vivo S. littornlis CryIC toxin receptor.

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Section: Introductionmentioning

confidence: 99%

Identification and partial purification of a Bacillus thuringiensis CryIC δ‐endotoxin binding protein from spodoptera littoralis gut membranes

Sanchis

Ellar

1993

FEBS Letters

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“…The region upstream of the promoters of the cry1C gene was isolated as a 656-bp BglII/HindIII fragment from a 7-kbp EcoRI fragment containing this gene, including 2.5 kbp upstream of the promoters (13). This fragment was isolated from low melting agarose with GELase (Epicentre Technologies) and cloned into the HindIII and BamHI sites of pUC18.…”

Section: Methodsmentioning

confidence: 99%

Specific Binding of the E2 Subunit of Pyruvate Dehydrogenase to the Upstream Region of Bacillus thuringiensis Protoxin Genes

Walter

Aronson

1999

Journal of Biological Chemistry

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During sporulation, Bacillus thuringiensis produces inclusions comprised of different amounts of several related protoxins, each with a unique specificity profile for insect larvae. A major class of these genes designated cry1 have virtually identical dual overlapping promoters, but the upstream sequences differ. A gel retardation assay was used to purify a potential regulatory protein which bound with different affinities to these sequences in three cry1 genes. It was identified as the E2 subunit of pyruvate dehydrogenase. There was specific competition for binding by homologous gene sequences but not by pUC nor Bacillus subtilis DNA; calf thymus DNA competed at higher concentrations. The B. thuringiensis gene encoding E2 was cloned, and the purified glutathione S-transferase-E2 fusion protein footprinted to a consensus binding sequence within an inverted repeat and to a potential bend region, both sites 200 -300 base pairs upstream of the promoters. Mutations of these sites in the cry1A gene resulted in decreased binding of the E2 protein and altered kinetics of expression of a fusion of this regulatory region with the lacZ gene. Recruitment of the E2 subunit as a transcription factor could couple the change in post exponential catabolism to the initiation of protoxin synthesis.

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“…A 5 kb HindlII-EcoRI DNA fragment from Bacillus thuringiensis subsp, aizawai 7-29 that contains the entire coding sequence, the promoter and putative terminator of the crylC gene ( [19]; and J. MullerCohn and V. Sanchis, unpublished results) was cloned into the HindlII and EcoRI sites of the plasmid vector pHT315 [20]. The resulting plasmid was designated pHTIC.…”

Section: In Vivo J~s-labelling Of the Crylc A-endotoxinmentioning

confidence: 99%

“…The CrylC toxin is highly specific towards S. littoralis and other insects of the Noctuidae family that are poorly susceptible to the other types of a-endotoxins [19]. It was also 7-fold more toxic for B. mori larvae (LC50 of 10 ng/cm2).…”

Section: Insect Bioassaysmentioning

confidence: 99%

A comparison and analysis of the toxicity and receptor binding properties of Bacillus thuringiensis CryIC ∂‐endotoxin on Spodoptera littoralis and Bombyx mori

Sanchis

Chaufaux

Pauron³

1994

FEBS Letters

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The binding of L-[35S]methionine in vivo labelledCrylC toxin to its receptor in brush border membrane vesicle (BBMV's) prepared from Spodoptera littoralis and Bombyx mori was studied. Both insect species were highly susceptible to the CrylC toxin in bioassays, B. mori being 7-fold more sensitive to CrylC than S. littoralis (LC50's of 10 ng/cm 2 and 70 ng/cm 2, respectively). Competition and direct binding experiments revealed saturable high-affinity binding sites on BBMV's from both insects which had similar binding characteristics for the CrylC toxin (Kd = 10 nM, B~ = 8 to 9 pmol/mg BBMV's and IC50 = 37 nM for both insect species). Thus a specific receptor for the CrylC toxin is present in both insect species and the 7-fold greater potency of CrylC towards B. moriis not due to qualitative or quantitative differences in binding affinity or receptor site concentration. Dissociation experiments also indicated that the binding of [35S]CrylC to B. mori BBMV's is partially reversible.Key words." O-Endotoxin; Toxicity; Binding affinity; Bacillus thuringiensis; Spodoptera littoralis; Bombyx mori ~odu~onDuring sporulation Bacillus thuringiensis synthesizes crystalline inclusions composed of one or several insecticidal proteins (known as ~-endotoxins or Cry proteins) that form a large family of related proteins. The target of these toxins is the brush border membrane of the larval midgut epithelial cells [1] and it is now generally accepted that the ~-endotoxins act by opening or by forming new specific ion-selective channels [2][3][4][5] or non selective pores [6,7] in the midgut cells of susceptible insects. Thereby, the toxins destroy the osmotic balance across the cell membrane causing swelling and eventual lysis of midgut epithelial cells.The a-endotoxins have been classified into five major classes according to their insecticidal properties and molecular structures (CryI, II, III, IV and V). They are active against Lepidoptera (CryI), Lepidoptera and Diptera (CrylI), Coleoptera (CrylII), Diptera (CrylV) and Lepidoptera and Coleoptera (CryV) [8,9]. Individual toxins are highly specific for particular insects and the high insect specificity of these toxins has been correlated with the presence of specific toxin receptors in BBMV preparations from the gut of susceptible insect species [10][11][12]. Recently, several authors have indicated that both the affinity and the number of binding sites for the toxin appear to be important factors in the insecticidal specificity and potency of the O-endotoxins [ 1 I, 13,14]. It has also been proposed that there is a general correlation between larvicidal potency and the product of receptor site concentration and affinity [14]. However, Wolfersberger [15] found that the dissociation constants for CrylA(b) and CrylA(c) toxin binding to Lymantria dispar larvae were inversely correlated with their potency towards this insect species. Also, Garczynski et al. [16] (33) (1) 45 68 89 38. ing sites for CryIA(c) but the larvae were not killed by this toxin.Therefore, an import...

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Nucleotide sequence and analysis of the N‐terminal coding region of the Spodoptera‐activeδ‐endotoxin gene of Bacillus thuringiensis aizawai 7.29

Cited by 54 publications

References 37 publications

Identification and partial purification of a Bacillus thuringiensis CryIC δ‐endotoxin binding protein from spodoptera littoralis gut membranes

Identification and partial purification of a Bacillus thuringiensis CryIC δ‐endotoxin binding protein from spodoptera littoralis gut membranes

Specific Binding of the E2 Subunit of Pyruvate Dehydrogenase to the Upstream Region of Bacillus thuringiensis Protoxin Genes

A comparison and analysis of the toxicity and receptor binding properties of Bacillus thuringiensis CryIC ∂‐endotoxin on Spodoptera littoralis and Bombyx mori

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