Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Nov;170(11):5368–5370. doi: 10.1128/jb.170.11.5368-5370.1988

Synthesis and overproduction of the 5A protein of insertion sequence IS5.

J M Chernak 1, E J Schlaffer 1, H O Smith 1
PMCID: PMC211615  PMID: 2846516

Abstract

We have demonstrated both the synthesis and overproduction of the 5A protein encoded by the longest open reading frame of the bacterial insertion sequence IS5. Expression was obtained in vitro and in Escherichia coli maxicells from plasmids containing IS5 in either orientation, as well as in vitro from a restriction fragment containing exclusively IS5 DNA. When IS5 was cloned in the appropriate orientation downstream of a strong tac promoter, production of the 5A protein was increased to 10 to 20% of the total protein synthesized in vitro.

Full text

PDF
5370

Images in this article

5369Image
on p.5369
5369Image
on p.5369

Selected References

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

  1. Amann E., Brosius J., Ptashne M. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned genes in Escherichia coli. Gene. 1983 Nov;25(2-3):167–178. doi: 10.1016/0378-1119(83)90222-6. [DOI] [PubMed] [Google Scholar]
  2. Betermier M., Alazard R., Ragueh F., Roulet E., Toussaint A., Chandler M. Phage Mu transposase: deletion of the carboxy-terminal end does not abolish DNA-binding activity. Mol Gen Genet. 1987 Nov;210(1):77–85. doi: 10.1007/BF00337761. [DOI] [PubMed] [Google Scholar]
  3. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  4. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  6. Chen H. Z., Zubay G. Prokaryotic coupled transcription-translation. Methods Enzymol. 1983;101:674–690. doi: 10.1016/0076-6879(83)01047-2. [DOI] [PubMed] [Google Scholar]
  7. DeVries J. K., Zubay G. DNA-directed peptide synthesis. II. The synthesis of the alpha-fragment of the enzyme beta-galactosidase. Proc Natl Acad Sci U S A. 1967 Apr;57(4):1010–1012. doi: 10.1073/pnas.57.4.1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Engler J. A., van Bree M. P. The nucleotide sequence and protein-coding capability of the transposable element IS5. Gene. 1981 Aug;14(3):155–163. doi: 10.1016/0378-1119(81)90111-6. [DOI] [PubMed] [Google Scholar]
  9. Kröger M., Hobom G. Structural analysis of insertion sequence IS5. Nature. 1982 May 13;297(5862):159–162. doi: 10.1038/297159a0. [DOI] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Laskey R. A. The use of intensifying screens or organic scintillators for visualizing radioactive molecules resolved by gel electrophoresis. Methods Enzymol. 1980;65(1):363–371. doi: 10.1016/s0076-6879(80)65047-2. [DOI] [PubMed] [Google Scholar]
  12. Rak B., Lusky M., Hable M. Expression of two proteins from overlapping and oppositely oriented genes on transposable DNA insertion element IS5. Nature. 1982 May 13;297(5862):124–128. doi: 10.1038/297124a0. [DOI] [PubMed] [Google Scholar]
  13. Rak B., von Reutern M. Insertion element IS5 contains a third gene. EMBO J. 1984 Apr;3(4):807–811. doi: 10.1002/j.1460-2075.1984.tb01889.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Raleigh E. A., Kleckner N. Quantitation of insertion sequence IS10 transposase gene expression by a method generally applicable to any rarely expressed gene. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1787–1791. doi: 10.1073/pnas.83.6.1787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rothstein S. J., Jorgensen R. A., Postle K., Reznikoff W. S. The inverted repeats of Tn5 are functionally different. Cell. 1980 Mar;19(3):795–805. doi: 10.1016/s0092-8674(80)80055-9. [DOI] [PubMed] [Google Scholar]
  16. Sancar A., Wharton R. P., Seltzer S., Kacinski B. M., Clarke N. D., Rupp W. D. Identification of the uvrA gene product. J Mol Biol. 1981 May 5;148(1):45–62. doi: 10.1016/0022-2836(81)90234-5. [DOI] [PubMed] [Google Scholar]
  17. Schoner B., Kahn M. The nucleotide sequence of IS5 from Escherichia coli. Gene. 1981 Aug;14(3):165–174. doi: 10.1016/0378-1119(81)90112-8. [DOI] [PubMed] [Google Scholar]
  18. Trinks K., Habermann P., Beyreuther K., Starlinger P., Ehring R. An IS4-encoded protein is synthesized in minicells. Mol Gen Genet. 1981;182(2):183–188. doi: 10.1007/BF00269656. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES