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. 1991 Aug;173(16):5105–5109. doi: 10.1128/jb.173.16.5105-5109.1991

Analysis of Streptococcus pneumoniae sequences cloned into Escherichia coli: effect of promoter strength and transcription terminators.

J P Dillard 1, J Yother 1
PMCID: PMC208201  PMID: 1860821

Abstract

Difficulties encountered in the cloning of DNA from Streptococcus pneumoniae and other AT-rich organisms into ColE1-type Escherichia coli vectors have been proposed to be due to the presence of a large number of strong promoter-acting sequences in the donor DNA. The use of transcription terminators has been advocated as a means of reducing instability resulting from disruption of plasmid replication caused by strong promoters. However, neither the existence of promoter-acting sequences of sufficient strength and number to explain the reported cloning difficulties nor their role as a source of instability has been proven. As a direct test of the "strong promoter" hypothesis, we cloned random fragments from S. pneumoniae into an E. coli vector containing transcription terminators, identified strong promoter-acting sequences, and subsequently removed the transcription terminators. We observed that terminator removal resulted in reduced copy numbers for the strongest promoter-acting sequences but not in reduced promoter strengths or altered plasmid stabilities. Our results indicate that promoters strong enough to require transcription terminators for plasmid stability are probably rare in S. pneumoniae DNA.

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Selected References

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

  1. 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]
  2. 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]
  3. Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
  4. Bujard H., Baldari C., Brunner M., Deuschle U., Gentz R., Hughes J., Kammerer W., Stuber D. Integration of efficient promoters of the E. coli system into plasmid vectors. Gene Amplif Anal. 1983;3:65–87. [PubMed] [Google Scholar]
  5. Bujard H., Stueber D., Gentz R., Deuschle U., Peschke U. Insertion of transcriptional elements outside the replication region can interfere with replication, maintenance, and stability of Co1E1-derived plasmids. Basic Life Sci. 1985;30:45–52. doi: 10.1007/978-1-4613-2447-8_6. [DOI] [PubMed] [Google Scholar]
  6. Chan P. T., Ohmori H., Tomizawa J., Lebowitz J. Nucleotide sequence and gene organization of ColE1 DNA. J Biol Chem. 1985 Jul 25;260(15):8925–8935. [PubMed] [Google Scholar]
  7. Chandler M. S., Morrison D. A. Competence for genetic transformation in Streptococcus pneumoniae: molecular cloning of com, a competence control locus. J Bacteriol. 1987 May;169(5):2005–2011. doi: 10.1128/jb.169.5.2005-2011.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen J. D., Morrison D. A. Cloning of Streptococcus pneumoniae DNA fragments in Escherichia coli requires vectors protected by strong transcriptional terminators. Gene. 1987;55(2-3):179–187. doi: 10.1016/0378-1119(87)90278-2. [DOI] [PubMed] [Google Scholar]
  9. Chen J. D., Morrison D. A. Construction and properties of a new insertion vector, pJDC9, that is protected by transcriptional terminators and useful for cloning of DNA from Streptococcus pneumoniae. Gene. 1988 Apr 15;64(1):155–164. doi: 10.1016/0378-1119(88)90489-1. [DOI] [PubMed] [Google Scholar]
  10. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  11. Frost J. W., Bender J. L., Kadonaga J. T., Knowles J. R. Dehydroquinate synthase from Escherichia coli: purification, cloning, and construction of overproducers of the enzyme. Biochemistry. 1984 Sep 11;23(19):4470–4475. doi: 10.1021/bi00314a036. [DOI] [PubMed] [Google Scholar]
  12. Gentz R., Langner A., Chang A. C., Cohen S. N., Bujard H. Cloning and analysis of strong promoters is made possible by the downstream placement of a RNA termination signal. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4936–4940. doi: 10.1073/pnas.78.8.4936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Greaves D. R., Patient R. K. RecBC, sbcB independent, (AT)n-mediated deletion of sequences flanking a Xenopus laevis beta globin gene on propagation in E. coli. Nucleic Acids Res. 1986 May 27;14(10):4147–4158. doi: 10.1093/nar/14.10.4147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  15. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Knaus R., Bujard H. PL of coliphage lambda: an alternative solution for an efficient promoter. EMBO J. 1988 Sep;7(9):2919–2923. doi: 10.1002/j.1460-2075.1988.tb03150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kobayashi T., Kato C., Kudo T., Horikoshi K. Excretion of the penicillinase of an alkalophilic Bacillus sp. through the Escherichia coli outer membrane is caused by insertional activation of the kil gene in plasmid pMB9. J Bacteriol. 1986 Jun;166(3):728–732. doi: 10.1128/jb.166.3.728-732.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kuriki Y. Response to temperature shifts of expression of the amp gene on pBR322 in Escherichia coli K-12. J Bacteriol. 1987 May;169(5):2294–2297. doi: 10.1128/jb.169.5.2294-2297.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. MARMUR J., DOTY P. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J Mol Biol. 1962 Jul;5:109–118. doi: 10.1016/s0022-2836(62)80066-7. [DOI] [PubMed] [Google Scholar]
  20. Macrina F. L., Evans R. P., Tobian J. A., Hartley D. L., Clewell D. B., Jones K. R. Novel shuttle plasmid vehicles for Escherichia-Streptococcus transgeneric cloning. Gene. 1983 Nov;25(1):145–150. doi: 10.1016/0378-1119(83)90176-2. [DOI] [PubMed] [Google Scholar]
  21. Martin B., Alloing G., Boucraut C., Claverys J. P. The difficulty of cloning Streptococcus pneumoniae mal and ami loci in Escherichia coli: toxicity of malX and amiA gene products. Gene. 1989 Aug 15;80(2):227–238. doi: 10.1016/0378-1119(89)90287-4. [DOI] [PubMed] [Google Scholar]
  22. Martin B., Prats H., Claverys J. P. Cloning of the hexA mismatch-repair gene of Streptococcus pneumoniae and identification of the product. Gene. 1985;34(2-3):293–303. doi: 10.1016/0378-1119(85)90138-6. [DOI] [PubMed] [Google Scholar]
  23. Moran C. P., Jr, Lang N., LeGrice S. F., Lee G., Stephens M., Sonenshein A. L., Pero J., Losick R. Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis. Mol Gen Genet. 1982;186(3):339–346. doi: 10.1007/BF00729452. [DOI] [PubMed] [Google Scholar]
  24. Morrison D. A., Jaurin B. Streptococcus pneumoniae possesses canonical Escherichia coli (sigma 70) promoters. Mol Microbiol. 1990 Jul;4(7):1143–1152. doi: 10.1111/j.1365-2958.1990.tb00689.x. [DOI] [PubMed] [Google Scholar]
  25. Mulligan M. E., McClure W. R. Analysis of the occurrence of promoter-sites in DNA. Nucleic Acids Res. 1986 Jan 10;14(1):109–126. doi: 10.1093/nar/14.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Prats H., Martin B., Claverys J. P. The hexB mismatch repair gene of Streptococcus pneumoniae: characterisation, cloning and identification of the product. Mol Gen Genet. 1985;200(3):482–489. doi: 10.1007/BF00425735. [DOI] [PubMed] [Google Scholar]
  27. Radloff R., Bauer W., Vinograd J. A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc Natl Acad Sci U S A. 1967 May;57(5):1514–1521. doi: 10.1073/pnas.57.5.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Reddy P., Peterkofsky A., McKenney K. Hyperexpression and purification of Escherichia coli adenylate cyclase using a vector designed for expression of lethal gene products. Nucleic Acids Res. 1989 Dec 25;17(24):10473–10488. doi: 10.1093/nar/17.24.10473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rhee D. K., Morrison D. A. Genetic transformation in Streptococcus pneumoniae: molecular cloning and characterization of recP, a gene required for genetic recombination. J Bacteriol. 1988 Feb;170(2):630–637. doi: 10.1128/jb.170.2.630-637.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  31. Sheflin L. G., Kowalski D. Altered DNA conformations detected by mung bean nuclease occur in promoter and terminator regions of supercoiled pBR322 DNA. Nucleic Acids Res. 1985 Sep 11;13(17):6137–6154. doi: 10.1093/nar/13.17.6137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Stassi D. L., Lacks S. A. Effect of strong promoters on the cloning in Escherichia coli of DNA fragments from Streptococcus pneumoniae. Gene. 1982 Jun;18(3):319–328. doi: 10.1016/0378-1119(82)90170-6. [DOI] [PubMed] [Google Scholar]
  33. Stueber D., Bujard H. Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes. EMBO J. 1982;1(11):1399–1404. doi: 10.1002/j.1460-2075.1982.tb01329.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Summers D. K., Sherratt D. J. Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability. Cell. 1984 Apr;36(4):1097–1103. doi: 10.1016/0092-8674(84)90060-6. [DOI] [PubMed] [Google Scholar]

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