Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1982;1(10):1217–1224. doi: 10.1002/j.1460-2075.1982.tb00016.x

Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda.

M Zabeau, K K Stanley
PMCID: PMC553192  PMID: 6327257

Abstract

Hybrid plasmids carrying cro-lacZ gene fusions have been constructed by joining DNA segments carrying the PR promoter and the start of the cro gene of bacteriophage lambda to the lacZ gene fragment carried by plasmid pLG400 . Plasmids in which the translational reading frames of the cro and lacZ genes are joined in-register (type I) direct the synthesis of elevated levels of cro-beta-galactosidase fusion protein amounting to 30% of the total cellular protein, while plasmids in which the genes are fused out-of-register (type II) produce a low level of beta-galactosidase protein. Sequence rearrangements downstream of the cro initiator AUG were found to influence the efficiency of translation, and have been correlated with alterations in the RNA secondary structure of the ribosome-binding site. Plasmids which direct the synthesis of high levels of beta-galactosidase are conditionally lethal and can only be propagated when the PR promoter is repressed. Deletion of sequences downstream of the lacZ gene restored viability, indicating that this region of the plasmid encodes a function which inhibits the growth of the cells. The different applications of these plasmids for expression of cloned genes are discussed.

Full text

PDF
1217

Images in this article

1222Fig. 6.
on p.1222

Selected References

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

  1. Bernard H. U., Remaut E., Hershfield M. V., Das H. K., Helinski D. R., Yanofsky C., Franklin N. Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter. Gene. 1979 Jan;5(1):59–76. doi: 10.1016/0378-1119(79)90092-1. [DOI] [PubMed] [Google Scholar]
  2. Büchel D. E., Gronenborn B., Müller-Hill B. Sequence of the lactose permease gene. Nature. 1980 Feb 7;283(5747):541–545. doi: 10.1038/283541a0. [DOI] [PubMed] [Google Scholar]
  3. Casadaban M. J., Chou J., Cohen S. N. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. doi: 10.1128/jb.143.2.971-980.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casadaban M. J. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. J Mol Biol. 1976 Jul 5;104(3):541–555. doi: 10.1016/0022-2836(76)90119-4. [DOI] [PubMed] [Google Scholar]
  5. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Davis A. R., Nayak D. P., Ueda M., Hiti A. L., Dowbenko D., Kleid D. G. Expression of antigenic determinants of the hemagglutinin gene of a human influenza virus in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5376–5380. doi: 10.1073/pnas.78.9.5376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Furlong D. Direct evidence for 6-fold symmetry of the herpesvirus hexon capsomere. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2764–2766. doi: 10.1073/pnas.75.6.2764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Goeddel D. V., Kleid D. G., Bolivar F., Heyneker H. L., Yansura D. G., Crea R., Hirose T., Kraszewski A., Itakura K., Riggs A. D. Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci U S A. 1979 Jan;76(1):106–110. doi: 10.1073/pnas.76.1.106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gough N. M., Webb E. A., Cory S., Adams J. M. Molecular cloning of seven mouse immunoglobulin kappa chain messenger ribonucleic acids. Biochemistry. 1980 Jun 10;19(12):2702–2710. doi: 10.1021/bi00553a026. [DOI] [PubMed] [Google Scholar]
  11. Gray H. B., Jr, Ostrander D. A., Hodnett J. L., Legerski R. J., Robberson D. L. Extracellular nucleases of Pseudomonas BAL 31. I. Characterization of single strand-specific deoxyriboendonuclease and double-strand deoxyriboexonuclease activities. Nucleic Acids Res. 1975 Sep;2(9):1459–1492. doi: 10.1093/nar/2.9.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guarente L., Lauer G., Roberts T. M., Ptashne M. Improved methods for maximizing expression of a cloned gene: a bacterium that synthesizes rabbit beta-globin. Cell. 1980 Jun;20(2):543–553. doi: 10.1016/0092-8674(80)90640-6. [DOI] [PubMed] [Google Scholar]
  13. Iserentant D., Fiers W. Secondary structure of mRNA and efficiency of translation initiation. Gene. 1980 Apr;9(1-2):1–12. doi: 10.1016/0378-1119(80)90163-8. [DOI] [PubMed] [Google Scholar]
  14. Koenen M., Rüther U., Müller-Hill B. Immunoenzymatic detection of expressed gene fragments cloned in the lac Z gene of E. coli. EMBO J. 1982;1(4):509–512. doi: 10.1002/j.1460-2075.1982.tb01199.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Müller-Hill B., Kania J. Lac repressor can be fused to beta-galactosidase. Nature. 1974 Jun 7;249(457):561–563. doi: 10.1038/249561a0. [DOI] [PubMed] [Google Scholar]
  19. Pirrotta V., Ineichen K., Walz A. An unusual RNA polymerase binding site in the immunity region of phage lambda. Mol Gen Genet. 1980;180(2):369–376. doi: 10.1007/BF00425850. [DOI] [PubMed] [Google Scholar]
  20. Remaut E., Stanssens P., Fiers W. Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda. Gene. 1981 Oct;15(1):81–93. doi: 10.1016/0378-1119(81)90106-2. [DOI] [PubMed] [Google Scholar]
  21. Roberts T. M., Kacich R., Ptashne M. A general method for maximizing the expression of a cloned gene. Proc Natl Acad Sci U S A. 1979 Feb;76(2):760–764. doi: 10.1073/pnas.76.2.760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Salser W. Globin mRNA sequences: analysis of base pairing and evolutionary implications. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):985–1002. doi: 10.1101/sqb.1978.042.01.099. [DOI] [PubMed] [Google Scholar]
  23. Schwartz M., Roa M., Débarbouillé M. Mutations that affect lamB gene expression at a posttranscriptional level. Proc Natl Acad Sci U S A. 1981 May;78(5):2937–2941. doi: 10.1073/pnas.78.5.2937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schwarz E., Scherer G., Hobom G., Kössel H. Nucleotide sequence of cro, cII and part of the O gene in phage lambda DNA. Nature. 1978 Mar 30;272(5652):410–414. doi: 10.1038/272410a0. [DOI] [PubMed] [Google Scholar]
  25. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shine J., Fettes I., Lan N. C., Roberts J. L., Baxter J. D. Expression of cloned beta-endorphin gene sequences by Escherichia coli. Nature. 1980 Jun 12;285(5765):456–463. doi: 10.1038/285456a0. [DOI] [PubMed] [Google Scholar]
  27. Silhavy T. J., Casadaban M. J., Shuman H. A., Beckwith J. R. Conversion of beta-galactosidase to a membrane-bound state by gene fusion. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3423–3427. doi: 10.1073/pnas.73.10.3423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Smith A. J. The use of exonuclease III for preparing single stranded DNA for use as a template in the chain terminator sequencing method. Nucleic Acids Res. 1979 Mar;6(3):831–848. doi: 10.1093/nar/6.3.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Steege D. A. A ribosome binding site from the pR RNA of bacteriophage lambda. J Mol Biol. 1977 Aug 25;114(4):559–568. doi: 10.1016/0022-2836(77)90178-4. [DOI] [PubMed] [Google Scholar]
  30. Sugden B., De Troy B., Roberts R. J., Sambrook J. Agarose slab-gel electrophoresis equipment. Anal Biochem. 1975 Sep;68(1):36–46. doi: 10.1016/0003-2697(75)90676-4. [DOI] [PubMed] [Google Scholar]
  31. Sugimoto K., Sugisaki H., Okamoto T., Takanami M. Studies on bacteriophage fd DNA. IV. The sequence of messenger RNA for the major coat protein gene. J Mol Biol. 1977 Apr 25;111(4):487–507. doi: 10.1016/s0022-2836(77)80065-x. [DOI] [PubMed] [Google Scholar]
  32. Teather R. M., Müller-Hill B., Abrutsch U., Aichele G., Overath P. Amplification of the lactose carrier protein in Escherichia coli using a plasmid vector. Mol Gen Genet. 1978 Feb 27;159(3):239–248. doi: 10.1007/BF00268260. [DOI] [PubMed] [Google Scholar]
  33. Wood W. B. Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA. J Mol Biol. 1966 Mar;16(1):118–133. doi: 10.1016/s0022-2836(66)80267-x. [DOI] [PubMed] [Google Scholar]
  34. Zuker M., Stiegler P. Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res. 1981 Jan 10;9(1):133–148. doi: 10.1093/nar/9.1.133. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES