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. 1982;1(7):791–799. doi: 10.1002/j.1460-2075.1982.tb01249.x

Cloning and expression of the crystal protein genes from Bacillus thuringiensis strain berliner 1715.

A Klier, F Fargette, J Ribier, G Rapoport
PMCID: PMC553111  PMID: 6329704

Abstract

From a clone bank of the entire genome of Bacillus thuringiensis, one clone that contains a plasmid ( pBT 15-88) harboring a sporulation gene was identified by molecular hybridization. This gene, identified as the crystal protein gene, occurs both on a large host plasmid DNA and in the chromosomal DNA in B. thuringiensis strain berliner 1715. The inserted sequence of pBT 15-88, which corresponds to the chromosomal sequence, was not expressed in Escherichia coli. In B. thuringiensis (kurstaki), the crystal gene was found only on a large host plasmid while in B. thuringiensis ( dendrolimus ), it is only on the chromosomal DNA. The plasmid crystal gene was cloned by ligation of a 14-kb BamHI fragment of a host plasmid DNA of 42 megadaltons from strain berliner 1715 into the BamHI site of the bifunctional vector pHV33 . In E. coli and in sporulating B. subtilis the plasmid pBT 42-1 coded for a polypeptide, detected by antibodies against the crystal protein, with the same electrophoretic mobility as the crystal protein of B. thuringiensis. The crystal gene was not expressed in vegetative cells of B. subtilis, suggesting that the control at the transcriptional level is the same in B. subtilis and in B. thuringiensis. Protein extracts from the clones harboring the hybrid plasmid are toxic for the larvae of Pierris brassicae and the protein antigen forms cytoplasmic inclusion bodies in E. coli and B. subtilis, which are visible under the light microscope.

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Selected References

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

  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bowen B., Steinberg J., Laemmli U. K., Weintraub H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 1980 Jan 11;8(1):1–20. doi: 10.1093/nar/8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Colman A., Byers M. J., Primrose S. B., Lyons A. Rapid purification of plasmid DNAs by hydroxyapatite chromatography. Eur J Biochem. 1978 Nov 2;91(1):303–310. doi: 10.1111/j.1432-1033.1978.tb20966.x. [DOI] [PubMed] [Google Scholar]
  6. Denhardt D. T. A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun. 1966 Jun 13;23(5):641–646. doi: 10.1016/0006-291x(66)90447-5. [DOI] [PubMed] [Google Scholar]
  7. DiCioccio R. A., Strauss N. Patterns of transcription in Bacillus subtilis during sporulation. J Mol Biol. 1973 Jun 25;77(2):325–336. doi: 10.1016/0022-2836(73)90338-0. [DOI] [PubMed] [Google Scholar]
  8. François M., Glatron P., Rapoport G. Translation of a stable mRNA fraction from sporulating cells of Bacillus thuringiensis in a cell-free system from Escherichia coli. Structural homologies between the native crystal protein and the products synthesized in vitro. Biochimie. 1976;58(1-2):119–129. doi: 10.1016/s0300-9084(76)80362-8. [DOI] [PubMed] [Google Scholar]
  9. Glatron M. F., Rapoport G. Biosynthesis of the parasporal inclusion of Bacillus thuringiensis: half-life of its corresponding messenger RNA. Biochimie. 1972;54(10):1291–1301. doi: 10.1016/s0300-9084(72)80070-1. [DOI] [PubMed] [Google Scholar]
  10. González J. M., Jr, Dulmage H. T., Carlton B. C. Correlation between specific plasmids and delta-endotoxin production in Bacillus thuringiensis. Plasmid. 1981 May;5(3):352–365. doi: 10.1016/0147-619x(81)90010-x. [DOI] [PubMed] [Google Scholar]
  11. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haldenwang W. G., Lang N., Losick R. A sporulation-induced sigma-like regulatory protein from B. subtilis. Cell. 1981 Feb;23(2):615–624. doi: 10.1016/0092-8674(81)90157-4. [DOI] [PubMed] [Google Scholar]
  13. Klier A. F., Kunst F., Rapoport G. Structure of cloned ribosomal DNA cistrons from Bacillus thuringiensis. Nucleic Acids Res. 1979 Oct 25;7(4):997–1010. doi: 10.1093/nar/7.4.997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Klier A. F., Lecadet M. M. Arguments based on hybridization-competition experiments in favor of the in vitro synthesis of sporulation-specific mRNAs by the RNA polymerase of B. thuringiensis. Biochem Biophys Res Commun. 1976 Nov 22;73(2):263–270. doi: 10.1016/0006-291x(76)90702-6. [DOI] [PubMed] [Google Scholar]
  15. Klier A. F., Lecadet M. M., Dedonder R. Sequential modifications of DNA-dependent RNA polymerase during sporogenesis in Bacillus thuringiensis. Eur J Biochem. 1973 Jul 16;36(2):317–327. doi: 10.1111/j.1432-1033.1973.tb02916.x. [DOI] [PubMed] [Google Scholar]
  16. Klier A., Lecadet M. M. Evidence in favour of the modification in vivo of RNA polymerase subunits during sporogenesis in Bacillus thuringiensis. Eur J Biochem. 1974 Aug 15;47(1):111–119. doi: 10.1111/j.1432-1033.1974.tb03674.x. [DOI] [PubMed] [Google Scholar]
  17. Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maizels N. Dictyostelium 17S, 25S, and 5S rDNAs lie within a 38,000 base pair repeated unit. Cell. 1976 Nov;9(3):431–438. doi: 10.1016/0092-8674(76)90088-x. [DOI] [PubMed] [Google Scholar]
  19. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  20. McMaster G. K., Carmichael G. G. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci U S A. 1977 Nov;74(11):4835–4838. doi: 10.1073/pnas.74.11.4835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Niaudet B., Ehrlich S. D. In vitro genetic labeling of Bacillus subtilis cryptic plasmid pHV400. Plasmid. 1979 Jan;2(1):48–58. doi: 10.1016/0147-619x(79)90005-2. [DOI] [PubMed] [Google Scholar]
  22. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  23. Rain-Guion M. C., Petit-Glatron M. F., Klier A., Lecadet M. M., Rapoport G. Coding capacity of the transcript products synthesized in vitro by the RNA polymerases from Bacillus thuringiensis. Biochem Biophys Res Commun. 1976 Jun 7;70(3):709–716. doi: 10.1016/0006-291x(76)90650-1. [DOI] [PubMed] [Google Scholar]
  24. Rapoport G., Klier A., Billault A., Fargette F., Dedonder R. Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis. Mol Gen Genet. 1979 Oct 3;176(2):239–245. doi: 10.1007/BF00273218. [DOI] [PubMed] [Google Scholar]
  25. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  26. Rogolsky M. Nonenteric toxins of Staphylococcus aureus. Microbiol Rev. 1979 Sep;43(3):320–360. doi: 10.1128/mr.43.3.320-360.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schnepf H. E., Whiteley H. R. Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli. Proc Natl Acad Sci U S A. 1981 May;78(5):2893–2897. doi: 10.1073/pnas.78.5.2893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]

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