Cloning and Expression of 130-kd Mosquito-larvicidal Delta-endotoxin Gene of Bacillus Thuringiensis Var. Israelensis in Escherichia Coli

Overview

Journal Mol Gen Genet

Specialties Genetics
Molecular Biology

Date 1987 Jul 1

PMID 2890080

Citations 14

Authors

C Angsuthanasombat

W Chungjatupornchai

S Kertbundit

P Luxananil

C Settasatian

P Wilairat

S Panyim

Affiliations

Soon will be listed here.

Abstract

Five recombinant E. coli clones exhibiting toxicity to Aedes aegypti larvae were obtained from a library of 800 clones containing XbaI DNA fragments of 110 kb plasmid from B. thuringiensis var. israelensis. All the five clones (pMU 14/258/303/388/679) had the same 3.8-kb insert and encoded a major protein of 130 kDa which was highly toxic to A. aegypti larvae. Three clones (pMU 258/303/388) transcribed the 130 kD a gene in the same direction as that of lac Z promoter of pUC12 vector whereas the transcription of the other two (pMU 14/679) was in the opposite direction. A 1.9-kb fragment of the 3.8 kb insert coded for a protein of 65 kDa. Partial DNA sequence of the 3.8 kb insert, corresponding to the 5'-terminal of the 130 kDa gene, revealed a continuous reading frame, a Shine-Dalgarno sequence and a tentative 5'-regulatory region. These results demonstrated that the 3.8 kb insert is a minimal DNA fragment containing a regulatory region plus the coding sequence of the 130 kDa protein that is highly toxic to mosquito larvae.

Citing Articles

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Thammasittirong A, Dechklar M, Leetachewa S, Pootanakit K, Angsuthanasombat C Appl Environ Microbiol. 2011; 77(19):6836-40.

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Crystallization and preliminary X-ray crystallographic analysis of a full-length active form of the Cry4Ba toxin from Bacillus thuringiensis.

Thamwiriyasati N, Sakdee S, Chuankhayan P, Katzenmeier G, Chen C, Angsuthanasombat C Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010; 66(Pt 6):721-4.

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Two conformational states of the membrane-associated Bacillus thuringiensis Cry4Ba delta-endotoxin complex revealed by electron crystallography: implications for toxin-pore formation.

Ounjai P, Unger V, Sigworth F, Angsuthanasombat C Biochem Biophys Res Commun. 2007; 361(4):890-5.

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Specific mutations within the alpha4-alpha5 loop of the Bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170.

Kanintronkul Y, Sramala I, Katzenmeier G, Panyim S, Angsuthanasombat C Mol Biotechnol. 2003; 24(1):11-20.

PMID: 12721492 DOI: 10.1385/MB:24:1:11.

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PMID: 7905597 PMC: 372941. DOI: 10.1128/mr.57.4.838-861.1993.

References

Thorne L, Garduno F, Thompson T, Decker D, Zounes M, Wild M . Structural similarity between the lepidoptera- and diptera-specific insecticidal endotoxin genes of Bacillus thuringiensis subsp. "kurstaki" and "israelensis". J Bacteriol. 1986; 166(3):801-11. PMC: 215197. DOI: 10.1128/jb.166.3.801-811.1986. View

Hanahan D . Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983; 166(4):557-80. DOI: 10.1016/s0022-2836(83)80284-8. View

FAUST R, Abe K, Held G, Iizuka T, Bulla L, Meyers C . Evidence for plasmid-associated crystal toxin production in Bacillus thuringiensis subsp. israelensis. Plasmid. 1983; 9(1):98-103. DOI: 10.1016/0147-619x(83)90034-3. View

Ward E, Ellar D, Todd J . Cloning and expression in Escherichia coli of the insecticidal delta-endotoxin gene of Bacillus thuringiensis var. israelensis. FEBS Lett. 1984; 175(2):377-82. DOI: 10.1016/0014-5793(84)80772-3. View

TYRELL D, Davidson L, Bulla Jr L, Ramoska W . Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes. Appl Environ Microbiol. 1979; 38(4):656-8. PMC: 243556. DOI: 10.1128/aem.38.4.656-658.1979. View

Wabiko H, Raymond K, Bulla Jr L . Bacillus thuringiensis entomocidal protoxin gene sequence and gene product analysis. DNA. 1986; 5(4):305-14. DOI: 10.1089/dna.1986.5.305. View

Donis-Keller H, Maxam A, Gilbert W . Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977; 4(8):2527-38. PMC: 342589. DOI: 10.1093/nar/4.8.2527. View

Kamdar H, Jayaraman K . Spontaneous loss of a high molecular weight plasmid and the biocide of Bacillus thuringiensis var. israeliensis. Biochem Biophys Res Commun. 1983; 110(2):477-82. DOI: 10.1016/0006-291x(83)91174-9. View

Wong H, Schnepf H, WHITELEY H . Transcriptional and translational start sites for the Bacillus thuringiensis crystal protein gene. J Biol Chem. 1983; 258(3):1960-7. View

10.

Messing J . New M13 vectors for cloning. Methods Enzymol. 1983; 101:20-78. DOI: 10.1016/0076-6879(83)01005-8. View

11.

Shibano Y, Yamagata A, Nakamura N, Iizuka T, Sugisaki H, TAKANAMI M . Nucleotide sequence coding for the insecticidal fragment of the Bacillus thuringiensis crystal protein. Gene. 1985; 34(2-3):243-51. DOI: 10.1016/0378-1119(85)90133-7. View

12.

Hurley J, Lee S, Andrews Jr R, Klowden M, Bulla Jr L . Separation of the cytolytic and mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis. Biochem Biophys Res Commun. 1985; 126(2):961-5. DOI: 10.1016/0006-291x(85)90279-7. View

13.

SANGER F, Nicklen S, Coulson A . DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977; 74(12):5463-7. PMC: 431765. DOI: 10.1073/pnas.74.12.5463. View

14.

Gonzalez Jr J, Carlton B . A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis. Plasmid. 1984; 11(1):28-38. DOI: 10.1016/0147-619x(84)90004-0. View

15.

Armstrong J, Rohrmann G, Beaudreau G . Delta endotoxin of Bacillus thuringiensis subsp. israelensis. J Bacteriol. 1985; 161(1):39-46. PMC: 214832. DOI: 10.1128/jb.161.1.39-46.1985. View

16.

TYRELL D, Bulla Jr L, Andrews Jr R, Kramer K, Davidson L, Nordin P . Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains. J Bacteriol. 1981; 145(2):1052-62. PMC: 217217. DOI: 10.1128/jb.145.2.1052-1062.1981. View

17.

Meagher R, Tait R, Betlach M, Boyer H . Protein expression in E. coli minicells by recombinant plasmids. Cell. 1977; 10(3):521-36. DOI: 10.1016/0092-8674(77)90039-3. View

18.

Sekar V, Carlton B . Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis. Gene. 1985; 33(2):151-8. DOI: 10.1016/0378-1119(85)90089-7. View

19.

Lereclus D, Lecadet M, Ribier J, DEDONDER R . Molecular relationships among plasmids of Bacillus thuringiensis: conserved sequences through 11 crystalliferous strains. Mol Gen Genet. 1982; 186(3):391-8. DOI: 10.1007/BF00729459. View

20.

Vieira J, Messing J . Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985; 33(1):103-19. DOI: 10.1016/0378-1119(85)90120-9. View