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. 1987 Mar;169(3):1017–1023. doi: 10.1128/jb.169.3.1017-1023.1987

Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis.

K M McLean, H R Whiteley
PMCID: PMC211895  PMID: 3546262

Abstract

A ca. 10-kilobase (kb) HindIII fragment of plasmid DNA from Bacillus thuringiensis subsp. israelensis was cloned into plasmid pUC9 and transformed into Escherichia coli. Extracts of the recombinant strain contained a 27-kilodalton (kDa) peptide that reacted with antibodies to a 27-kDa peptide isolated from crystals produced by B. thuringiensis subsp. israelensis. Extracts of the recombinant strain were hemolytic and toxic to Aedes aegypti larvae. Full expression of the 27-kDa peptide required the presence of a ca. 0.8-kb region of DNA located 4 kb upstream from the structural gene; the 0.8-kb region could be present in cis or trans relative to the gene and apparently acted post-transcriptionally. Analysis of maxicells showed that the 10-kb insert also coded for peptides of 67, 20, and 16 kDa; data obtained with different subclones suggest that the 20-kDa peptide is encoded in the 0.8-kb DNA region.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Armstrong J. L., Rohrmann G. F., Beaudreau G. S. Delta endotoxin of Bacillus thuringiensis subsp. israelensis. J Bacteriol. 1985 Jan;161(1):39–46. doi: 10.1128/jb.161.1.39-46.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aronson A. I., Angelo N., Holt S. C. Regulation of extracellular protease production in Bacillus cereus T: characterization of mutants producing altered amounts of protease. J Bacteriol. 1971 Jun;106(3):1016–1025. doi: 10.1128/jb.106.3.1016-1025.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Bulla L. A., Jr, Kramer K. J., Davidson L. I. Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis. J Bacteriol. 1977 Apr;130(1):375–383. doi: 10.1128/jb.130.1.375-383.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Charles J. F., de Barjac H. Sporulation et cristallogenèse de Bacillus thuringiensis var. Israelensis en microscopie électronique. Ann Microbiol (Paris) 1982 May-Jun;133(3):425–442. [PubMed] [Google Scholar]
  7. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  9. Henning U., Schwarz H., Chen R. Radioimmunological screening method for specific membrane proteins. Anal Biochem. 1979 Aug;97(1):153–157. doi: 10.1016/0003-2697(79)90339-7. [DOI] [PubMed] [Google Scholar]
  10. Hurley J. M., Lee S. G., Andrews R. E., Jr, Klowden M. J., Bulla L. A., Jr Separation of the cytolytic and mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis. Biochem Biophys Res Commun. 1985 Jan 31;126(2):961–965. doi: 10.1016/0006-291x(85)90279-7. [DOI] [PubMed] [Google Scholar]
  11. Ibarra J. E., Federici B. A. Isolation of a relatively nontoxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis. J Bacteriol. 1986 Feb;165(2):527–533. doi: 10.1128/jb.165.2.527-533.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Insell J. P., Fitz-James P. C. Composition and Toxicity of the Inclusion of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol. 1985 Jul;50(1):56–62. doi: 10.1128/aem.50.1.56-62.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kronstad J. W., Schnepf H. E., Whiteley H. R. Diversity of locations for Bacillus thuringiensis crystal protein genes. J Bacteriol. 1983 Apr;154(1):419–428. doi: 10.1128/jb.154.1.419-428.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee S. G., Eckblad W., Bulla L. A., Jr Diversity of protein inclusion bodies and identification of mosquitocidal protein in Bacillus thuringiensis subsp. israelensis. Biochem Biophys Res Commun. 1985 Jan 31;126(2):953–960. doi: 10.1016/0006-291x(85)90278-5. [DOI] [PubMed] [Google Scholar]
  15. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  16. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  17. Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Schnell D. J., Pfannenstiel M. A., Nickerson K. W. Bioassay of solubilized Bacillus thuringiensis var. israelensis crystals by attachment to latex beads. Science. 1984 Mar 16;223(4641):1191–1193. doi: 10.1126/science.6701520. [DOI] [PubMed] [Google Scholar]
  19. Thomas W. E., Ellar D. J. Bacillus thuringiensis var israelensis crystal delta-endotoxin: effects on insect and mammalian cells in vitro and in vivo. J Cell Sci. 1983 Mar;60:181–197. doi: 10.1242/jcs.60.1.181. [DOI] [PubMed] [Google Scholar]
  20. Thorne L., Garduno F., Thompson T., Decker D., Zounes M., Wild M., Walfield A. M., Pollock T. J. Structural similarity between the lepidoptera- and diptera-specific insecticidal endotoxin genes of Bacillus thuringiensis subsp. "kurstaki" and "israelensis". J Bacteriol. 1986 Jun;166(3):801–811. doi: 10.1128/jb.166.3.801-811.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tyrell D. J., Bulla L. A., Jr, Andrews R. E., Jr, Kramer K. J., Davidson L. I., Nordin P. Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains. J Bacteriol. 1981 Feb;145(2):1052–1062. doi: 10.1128/jb.145.2.1052-1062.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  24. Waalwijk C., Dullemans A. M., van Workum M. E., Visser B. Molecular cloning and the nucleotide sequence of the Mr 28 000 crystal protein gene of Bacillus thuringiensis subsp. israelensis. Nucleic Acids Res. 1985 Nov 25;13(22):8207–8217. doi: 10.1093/nar/13.22.8207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ward E. S., Ellar D. J. Bacillus thuringiensis var. israelensis delta-endotoxin. Nucleotide sequence and characterization of the transcripts in Bacillus thuringiensis and Escherichia coli. J Mol Biol. 1986 Sep 5;191(1):1–11. doi: 10.1016/0022-2836(86)90417-1. [DOI] [PubMed] [Google Scholar]
  26. Ward E. S., Ellar D. J., Todd J. A. Cloning and expression in Escherichia coli of the insecticidal delta-endotoxin gene of Bacillus thuringiensis var. israelensis. FEBS Lett. 1984 Oct 1;175(2):377–382. doi: 10.1016/0014-5793(84)80772-3. [DOI] [PubMed] [Google Scholar]
  27. Ward E. S., Ridley A. R., Ellar D. J., Todd J. A. Bacillus thuringiensis var. israelensis delta-endotoxin. Cloning and expression of the toxin in sporogenic and asporogenic strains of Bacillus subtilis. J Mol Biol. 1986 Sep 5;191(1):13–22. doi: 10.1016/0022-2836(86)90418-3. [DOI] [PubMed] [Google Scholar]
  28. de Barjac H. Une nouvelle variété de Bacillus thuringiensis très toxique pour les moustiques: B. thuringiensis var. israelensis sérotype 14. C R Acad Sci Hebd Seances Acad Sci D. 1978 Mar 13;286(10):797–800. [PubMed] [Google Scholar]

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