Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Nov;61(11):3826–3831. doi: 10.1128/aem.61.11.3826-3831.1995

Specific PCR primers directed to identify cryI and cryIII genes within a Bacillus thuringiensis strain collection.

J Cerón 1, A Ortíz 1, R Quintero 1, L Güereca 1, A Bravo 1
PMCID: PMC167686  PMID: 8526493

Abstract

In this paper we describe a PCR strategy that can be used to rapidly identify Bacillus thuringiensis strains that harbor any of the known cryI or cryIII genes. Four general PCR primers which amplify DNA fragments from the known cryI or cryIII genes were selected from conserved regions. Once a strain was identified as an organism that contains a particular type of cry gene, it could be easily characterized by performing additional PCR with specific cryI and cryIII primers selected from variable regions. The method described in this paper can be used to identify the 10 different cryI genes and the five different cryIII genes. One feature of this screening method is that each cry gene is expected to produce a PCR product having a precise molecular weight. The genes which produce PCR products having different sizes probably represent strains that harbor a potentially novel cry gene. Finally, we present evidence that novel crystal genes can be identified by the method described in this paper.

Full Text

The Full Text of this article is available as a PDF (316.1 KB).

Selected References

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

  1. Bourque S. N., Valéro J. R., Mercier J., Lavoie M. C., Levesque R. C. Multiplex polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thuringiensis. Appl Environ Microbiol. 1993 Feb;59(2):523–527. doi: 10.1128/aem.59.2.523-527.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carozzi N. B., Kramer V. C., Warren G. W., Evola S., Koziel M. G. Prediction of insecticidal activity of Bacillus thuringiensis strains by polymerase chain reaction product profiles. Appl Environ Microbiol. 1991 Nov;57(11):3057–3061. doi: 10.1128/aem.57.11.3057-3061.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ceron J., Covarrubias L., Quintero R., Ortiz A., Ortiz M., Aranda E., Lina L., Bravo A. PCR analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis. Appl Environ Microbiol. 1994 Jan;60(1):353–356. doi: 10.1128/aem.60.1.353-356.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chak K. F., Chao D. C., Tseng M. Y., Kao S. S., Tuan S. J., Feng T. Y. Determination and Distribution of cry-Type Genes of Bacillus thuringiensis Isolates from Taiwan. Appl Environ Microbiol. 1994 Jul;60(7):2415–2420. doi: 10.1128/aem.60.7.2415-2420.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chambers J. A., Jelen A., Gilbert M. P., Jany C. S., Johnson T. B., Gawron-Burke C. Isolation and characterization of a novel insecticidal crystal protein gene from Bacillus thuringiensis subsp. aizawai. J Bacteriol. 1991 Jul;173(13):3966–3976. doi: 10.1128/jb.173.13.3966-3976.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Donovan W. P., Rupar M. J., Slaney A. C., Malvar T., Gawron-Burke M. C., Johnson T. B. Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleoptera species. Appl Environ Microbiol. 1992 Dec;58(12):3921–3927. doi: 10.1128/aem.58.12.3921-3927.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gleave A. P., Williams R., Hedges R. J. Screening by polymerase chain reaction of Bacillus thuringiensis serotypes for the presence of cryV-like insecticidal protein genes and characterization of a cryV gene cloned from B. thuringiensis subsp. kurstaki. Appl Environ Microbiol. 1993 May;59(5):1683–1687. doi: 10.1128/aem.59.5.1683-1687.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hofmann C., Vanderbruggen H., Höfte H., Van Rie J., Jansens S., Van Mellaert H. Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7844–7848. doi: 10.1073/pnas.85.21.7844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Höfte H., Whiteley H. R. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol Rev. 1989 Jun;53(2):242–255. doi: 10.1128/mr.53.2.242-255.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Höfte H., de Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J., Vanderbruggen H., van Montagu M., Zabeau M. Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur J Biochem. 1986 Dec 1;161(2):273–280. doi: 10.1111/j.1432-1033.1986.tb10443.x. [DOI] [PubMed] [Google Scholar]
  11. Kalman S., Kiehne K. L., Libs J. L., Yamamoto T. Cloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp. galleriae. Appl Environ Microbiol. 1993 Apr;59(4):1131–1137. doi: 10.1128/aem.59.4.1131-1137.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kirk-Mason K. E., Turner M. J., Chakraborty P. R. Cloning and sequence of beta tubulin cDNA from Giardia lamblia. Nucleic Acids Res. 1988 Mar 25;16(6):2733–2733. doi: 10.1093/nar/16.6.2733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lambert B., Höfte H., Annys K., Jansens S., Soetaert P., Peferoen M. Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae. Appl Environ Microbiol. 1992 Aug;58(8):2536–2542. doi: 10.1128/aem.58.8.2536-2542.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lambert B., Theunis W., Aguda R., Van Audenhove K., Decock C., Jansens S., Seurinck J., Peferoen M. Nucleotide sequence of gene cryIIID encoding a novel coleopteran-active crystal protein from strain BTI109P of Bacillus thuringiensis subsp. kurstaki. Gene. 1992 Jan 2;110(1):131–132. doi: 10.1016/0378-1119(92)90457-z. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Sanchis V., Lereclus D., Menou G., Chaufaux J., Lecadet M. M. Multiplicity of delta-endotoxin genes with different insecticidal specificities in Bacillus thuringiensis aizawai 7.29. Mol Microbiol. 1988 May;2(3):393–404. doi: 10.1111/j.1365-2958.1988.tb00044.x. [DOI] [PubMed] [Google Scholar]
  17. Sick A., Gaertner F., Wong A. Nucleotide sequence of a coleopteran-active toxin gene from a new isolate of Bacillus thuringiensis subsp. tolworthi. Nucleic Acids Res. 1990 Mar 11;18(5):1305–1305. doi: 10.1093/nar/18.5.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Van Rie J., Jansens S., Höfte H., Degheele D., Van Mellaert H. Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins. Appl Environ Microbiol. 1990 May;56(5):1378–1385. doi: 10.1128/aem.56.5.1378-1385.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Van Rie J., Jansens S., Höfte H., Degheele D., Van Mellaert H. Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects. Eur J Biochem. 1989 Dec 8;186(1-2):239–247. doi: 10.1111/j.1432-1033.1989.tb15201.x. [DOI] [PubMed] [Google Scholar]
  21. Van Rie J., McGaughey W. H., Johnson D. E., Barnett B. D., Van Mellaert H. Mechanism of insect resistance to the microbial insecticide Bacillus thuringiensis. Science. 1990 Jan 5;247(4938):72–74. doi: 10.1126/science.2294593. [DOI] [PubMed] [Google Scholar]
  22. Visser B., Munsterman E., Stoker A., Dirkse W. G. A novel Bacillus thuringiensis gene encoding a Spodoptera exigua-specific crystal protein. J Bacteriol. 1990 Dec;172(12):6783–6788. doi: 10.1128/jb.172.12.6783-6788.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES