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. 1990 Feb;172(2):1077–1084. doi: 10.1128/jb.172.2.1077-1084.1990

Improved vector system for constructing transcriptional fusions that ensures independent translation of lacZ.

T Linn 1, R St Pierre 1
PMCID: PMC208539  PMID: 2137119

Abstract

An improved vector system has been developed for the in vitro construction of transcriptional fusions to lacZ. The principal feature is an RNaseIII cleavage site inserted between the polylinker cloning site and the promoterless lacZ gene. When these vectors are used to construct transcriptional fusions, the subsequent cleavage of the hybrid mRNA at the RNaseIII site generates an unchanging 5' end for the lacZ mRNA. In contrast to earlier vectors, this feature helps to ensure independent translation of the lacZ mRNA and, thus, the level of beta-galactosidase produced should accurately reflect the frequency of transcription of the upstream DNA sequences. Additional modifications of the vectors include removal of a weak transcriptional terminator between the cloning site and lacZ, insertion of a terminator downstream of lac, and alteration of restriction endonuclease cleavage sites to facilitate the in vitro construction of fusions. Both multicopy plasmid (pTL61T) and single-copy lambda (lambda TL61) vectors have been assembled. These vectors should be generally useful in scanning for transcriptional regulatory signals.

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Selected References

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

  1. Adams C. W., Hatfield G. W. Effects of promoter strengths and growth conditions on copy number of transcription-fusion vectors. J Biol Chem. 1984 Jun 25;259(12):7399–7403. [PubMed] [Google Scholar]
  2. Amann E., Brosius J. "ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli. Gene. 1985;40(2-3):183–190. doi: 10.1016/0378-1119(85)90041-1. [DOI] [PubMed] [Google Scholar]
  3. Appleyard R K. Segregation of New Lysogenic Types during Growth of a Doubly Lysogenic Strain Derived from Escherichia Coli K12. Genetics. 1954 Jul;39(4):440–452. doi: 10.1093/genetics/39.4.440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barry G., Squires C., Squires C. L. Attenuation and processing of RNA from the rplJL--rpoBC transcription unit of Escherichia coli. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3331–3335. doi: 10.1073/pnas.77.6.3331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Belasco J. G., Nilsson G., von Gabain A., Cohen S. N. The stability of E. coli gene transcripts is dependent on determinants localized to specific mRNA segments. Cell. 1986 Jul 18;46(2):245–251. doi: 10.1016/0092-8674(86)90741-5. [DOI] [PubMed] [Google Scholar]
  6. Bertrand K. P., Postle K., Wray L. V., Jr, Reznikoff W. S. Construction of a single-copy promoter vector and its use in analysis of regulation of the transposon Tn10 tetracycline resistance determinant. J Bacteriol. 1984 Jun;158(3):910–919. doi: 10.1128/jb.158.3.910-919.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Borck K., Beggs J. D., Brammar W. J., Hopkins A. S., Murray N. E. The construction in vitro of transducing derivatives of phage lambda. Mol Gen Genet. 1976 Jul 23;146(2):199–207. doi: 10.1007/BF00268089. [DOI] [PubMed] [Google Scholar]
  8. Brosius J., Dull T. J., Sleeter D. D., Noller H. F. Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol. 1981 May 15;148(2):107–127. doi: 10.1016/0022-2836(81)90508-8. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Downing W. L., Dennis P. P. Transcription products from the rplKAJL-rpoBC gene cluster. J Mol Biol. 1987 Apr 20;194(4):609–620. doi: 10.1016/0022-2836(87)90238-5. [DOI] [PubMed] [Google Scholar]
  12. Dunn J. J., Studier F. W. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol. 1983 Jun 5;166(4):477–535. doi: 10.1016/s0022-2836(83)80282-4. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Guyer M. S., Reed R. R., Steitz J. A., Low K. B. Identification of a sex-factor-affinity site in E. coli as gamma delta. Cold Spring Harb Symp Quant Biol. 1981;45(Pt 1):135–140. doi: 10.1101/sqb.1981.045.01.022. [DOI] [PubMed] [Google Scholar]
  15. Hediger M. A., Johnson D. F., Nierlich D. P., Zabin I. DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6414–6418. doi: 10.1073/pnas.82.19.6414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  17. Hoopes B. C., McClure W. R. Studies on the selectivity of DNA precipitation by spermine. Nucleic Acids Res. 1981 Oct 24;9(20):5493–5504. doi: 10.1093/nar/9.20.5493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kalnins A., Otto K., Rüther U., Müller-Hill B. Sequence of the lacZ gene of Escherichia coli. EMBO J. 1983;2(4):593–597. doi: 10.1002/j.1460-2075.1983.tb01468.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. King T. C., Sirdeskmukh R., Schlessinger D. Nucleolytic processing of ribonucleic acid transcripts in procaryotes. Microbiol Rev. 1986 Dec;50(4):428–451. doi: 10.1128/mr.50.4.428-451.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kirschbaum J. B., Konrad E. B. Isolation of a specialized lambda transducing bacteriophage carrying the beta subunit gene for Escherichia coli ribonucleic acid polymerase. J Bacteriol. 1973 Nov;116(2):517–526. doi: 10.1128/jb.116.2.517-526.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Liedke-Kulke M., Kaiser A. D. The c-region of coliphage 21. Virology. 1967 Jul;32(3):475–481. doi: 10.1016/0042-6822(67)90299-1. [DOI] [PubMed] [Google Scholar]
  22. Linn T., Ralling G. A versatile multiple- and single-copy vector system for the in vitro construction of transcriptional fusions to lacZ. Plasmid. 1985 Sep;14(2):134–142. doi: 10.1016/0147-619x(85)90073-3. [DOI] [PubMed] [Google Scholar]
  23. Loenen W. A., Brammar W. J. A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene. 1980 Aug;10(3):249–259. doi: 10.1016/0378-1119(80)90054-2. [DOI] [PubMed] [Google Scholar]
  24. Mileham A. J., Revel H. R., Murray N. E. Molecular cloning of the T4 genome; organization and expression of the frd--DNA ligase region. Mol Gen Genet. 1980;179(2):227–239. doi: 10.1007/BF00425449. [DOI] [PubMed] [Google Scholar]
  25. Mitchell D. H., Reznikoff W. S., Beckwith J. R. Genetic fusions defining trp and lac operon regulatory elements. J Mol Biol. 1975 Apr 15;93(3):331–350. doi: 10.1016/0022-2836(75)90281-8. [DOI] [PubMed] [Google Scholar]
  26. Mott J. E., Galloway J. L., Platt T. Maturation of Escherichia coli tryptophan operon mRNA: evidence for 3' exonucleolytic processing after rho-dependent termination. EMBO J. 1985 Jul;4(7):1887–1891. doi: 10.1002/j.1460-2075.1985.tb03865.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mousset S., Thomas R. Ter, a function which generates the ends of the mature lambda chromosome. Nature. 1969 Jan 18;221(5177):242–244. doi: 10.1038/221242a0. [DOI] [PubMed] [Google Scholar]
  28. Munson L. M., Stormo G. D., Niece R. L., Reznikoff W. S. lacZ translation initiation mutations. J Mol Biol. 1984 Aug 25;177(4):663–683. doi: 10.1016/0022-2836(84)90043-3. [DOI] [PubMed] [Google Scholar]
  29. Murray N. E., Brammar W. J., Murray K. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet. 1977 Jan 7;150(1):53–61. doi: 10.1007/BF02425325. [DOI] [PubMed] [Google Scholar]
  30. Padan E., Arbel T., Rimon A., Shira A. B., Cohen A. Biosynthesis of the lactose permease in Escherichia coli minicells and effect of carrier amplification on cell physiology. J Biol Chem. 1983 May 10;258(9):5666–5673. [PubMed] [Google Scholar]
  31. Peden K. W. Revised sequence of the tetracycline-resistance gene of pBR322. Gene. 1983 May-Jun;22(2-3):277–280. doi: 10.1016/0378-1119(83)90112-9. [DOI] [PubMed] [Google Scholar]
  32. Post L. E., Strycharz G. D., Nomura M., Lewis H., Dennis P. P. Nucleotide sequence of the ribosomal protein gene cluster adjacent to the gene for RNA polymerase subunit beta in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1697–1701. doi: 10.1073/pnas.76.4.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ralling G., Linn T. Evidence that Rho and NusA are involved in termination in the rplL-rpoB intercistronic region. J Bacteriol. 1987 May;169(5):2277–2280. doi: 10.1128/jb.169.5.2277-2280.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ralling G., Linn T. Relative activities of the transcriptional regulatory sites in the rplKAJLrpoBC gene cluster of Escherichia coli. J Bacteriol. 1984 Apr;158(1):279–285. doi: 10.1128/jb.158.1.279-285.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Robertson H. D. Escherichia coli ribonuclease III cleavage sites. Cell. 1982 Oct;30(3):669–672. doi: 10.1016/0092-8674(82)90270-7. [DOI] [PubMed] [Google Scholar]
  36. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  37. Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene. 1983 Nov;25(1):71–82. doi: 10.1016/0378-1119(83)90169-5. [DOI] [PubMed] [Google Scholar]
  38. Silhavy T. J., Beckwith J. R. Uses of lac fusions for the study of biological problems. Microbiol Rev. 1985 Dec;49(4):398–418. doi: 10.1128/mr.49.4.398-418.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Simons R. W., Houman F., Kleckner N. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene. 1987;53(1):85–96. doi: 10.1016/0378-1119(87)90095-3. [DOI] [PubMed] [Google Scholar]
  40. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  41. Wong E. M., Muesing M. A., Polisky B. Temperature-sensitive copy number mutants of CoIE1 are located in an untranslated region of the plasmid genome. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3570–3574. doi: 10.1073/pnas.79.11.3570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yu X. M., Munson L. M., Reznikoff W. S. Molecular cloning and sequence analysis of trp-lac fusion deletions. J Mol Biol. 1984 Jan 25;172(3):355–362. doi: 10.1016/s0022-2836(84)80032-7. [DOI] [PubMed] [Google Scholar]

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