Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1983 Sep 10;11(17):5921–5940. doi: 10.1093/nar/11.17.5921

Gene expression: chemical synthesis and molecular cloning of a bacteriophage T5 (T5P25) early promoter.

J Rommens, D MacKnight, L Pomeroy-Cloney, E Jay
PMCID: PMC326327  PMID: 6310520

Abstract

A sixty base pair DNA duplex containing the nucleotide sequence of the bacteriophage T5 early (T5P25) promoter has been constructed using a combination of chemical synthesis and enzymatic methods. Subsequent to cloning into pBR322, the promoter has been demonstrated to be biologically active being capable of directing the efficient expression of genes under its control. This serves as a prototype for an approach to the study of the in vivo structure-function relationships and efficiency of promoters.

Full text

PDF
5936

Images in this article

Selected References

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

  1. Caruthers M. H., Khorana H. G. CXI. Total synthesis of the structural gene for an alanine transfer ribonucleic acid from yeast. Synthesis of a dodecadeoxynucleotide and a heptadeoxynucleotide corresponding to the nucleotide sequence 66 to 77. J Mol Biol. 1972 Dec 28;72(2):407–426. doi: 10.1016/0022-2836(72)90154-4. [DOI] [PubMed] [Google Scholar]
  2. Clewell D. B., Helinski D. R. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. doi: 10.1073/pnas.62.4.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dobrynin V. N., Korobko V. G., Severtsova I. V., Bystrov N. S., Chuvpilo S. A., Kolosov M. N. Synthesis of a model promoter for gene expression in Escherichia coli. Nucleic Acids Symp Ser. 1980;(7):365–376. [PubMed] [Google Scholar]
  4. Dugaiczyk A., Boyer H. W., Goodman H. M. Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures. J Mol Biol. 1975 Jul 25;96(1):171–184. doi: 10.1016/0022-2836(75)90189-8. [DOI] [PubMed] [Google Scholar]
  5. Gait M. J., Singh M., Sheppard R. C., Edge M. D., Greene A. R., Heathcliffe G. R., Atkinson T. C., Newton C. R., Markham A. F. Rapid synthesis of oligodeoxyribonucleotides. IV. Improved solid phase synthesis of oligodeoxyribonucleotides through phosphotriester intermediates. Nucleic Acids Res. 1980 Mar 11;8(5):1081–1096. doi: 10.1093/nar/8.5.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jay E., Seth A. K., Rommens J., Sood A., Jay G. Gene expression: chemical synthesis of E. coli ribosome binding sites and their use in directing the expression of mammalian proteins in bacteria. Nucleic Acids Res. 1982 Oct 25;10(20):6319–6329. doi: 10.1093/nar/10.20.6319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jay G., Khoury G., Seth A. K., Jay E. Construction of a general vector for efficient expression of mammalian proteins in bacteria: use of a synthetic ribosome binding site. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5543–5548. doi: 10.1073/pnas.78.9.5543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnsrud L. Contacts between Escherichia coli RNA polymerase and a lac operon promoter. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5314–5318. doi: 10.1073/pnas.75.11.5314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Maniatis T., Jeffrey A., van deSande H. Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry. 1975 Aug 26;14(17):3787–3794. doi: 10.1021/bi00688a010. [DOI] [PubMed] [Google Scholar]
  12. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Miyoshi K., Miyake T., Hozumi T., Itakura K. Solid-phase synthesis of polynucleotides. II. Synthesis of polythymidylic acids by the block coupling phosphotriester method. Nucleic Acids Res. 1980 Nov 25;8(22):5473–5489. doi: 10.1093/nar/8.22.5473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rodriguez R. L., West R. W., Heyneker H. L., Bolivar F., Boyer H. W. Characterizing wild-type and mutant promoters of the tetracycline resistance gene in pBR313. Nucleic Acids Res. 1979 Jul 25;6(10):3267–3287. doi: 10.1093/nar/6.10.3267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  17. Schaller H., Gray C., Herrmann K. Nucleotide sequence of an RNA polymerase binding site from the DNA of bacteriophage fd. Proc Natl Acad Sci U S A. 1975 Feb;72(2):737–741. doi: 10.1073/pnas.72.2.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Seeburg P. H., Nüsslein C., Schaller H. Interaction of RNA polymerase with promoters from bacteriophage fd. Eur J Biochem. 1977 Mar 15;74(1):107–113. doi: 10.1111/j.1432-1033.1977.tb11372.x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Siebenlist U., Simpson R. B., Gilbert W. E. coli RNA polymerase interacts homologously with two different promoters. Cell. 1980 Jun;20(2):269–281. doi: 10.1016/0092-8674(80)90613-3. [DOI] [PubMed] [Google Scholar]
  21. Tu C. P., Cohen S. N. Translocation specificity of the Tn3 element: characterization of sites of multiple insertions. Cell. 1980 Jan;19(1):151–160. doi: 10.1016/0092-8674(80)90396-7. [DOI] [PubMed] [Google Scholar]
  22. Winberg G., Hammarskjöld M. L. Isolation of DNA from agarose gels using DEAE-paper. Application to restriction site mapping of adenovirus type 16 DNA. Nucleic Acids Res. 1980 Jan 25;8(2):253–264. doi: 10.1093/nar/8.2.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. deHaseth P. L., Goldman R. A., Cech C. L., Caruthers M. H. Chemical synthesis and biochemical reactivity of bacteriophage lambda PR promoter. Nucleic Acids Res. 1983 Feb 11;11(3):773–787. doi: 10.1093/nar/11.3.773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. von Gabain A., Bujard H. Interaction of E. coli RNA polymerase with promotors of coliphage T5: the rates of complex formation and decay and their correlation with in vitro and in vivo transcriptional activity. Mol Gen Genet. 1977 Dec 9;157(3):301–311. doi: 10.1007/BF00268667. [DOI] [PubMed] [Google Scholar]
  26. von Gabain A., Bujard H. Interaction of Escherichia coli RNA polymerase with promoters of several coliphage and plasmid DNAs. Proc Natl Acad Sci U S A. 1979 Jan;76(1):189–193. doi: 10.1073/pnas.76.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES