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. 1980 Apr;77(4):2124–2128. doi: 10.1073/pnas.77.4.2124

Bacteriophage lambda vehicle for the direct cloning of Escherichia coli promoter DNA sequences: feedback regulation of the rplJL-rpoBC operon.

E W Holowachuk, J D Friesen, N P Fiil
PMCID: PMC348664  PMID: 6445564

Abstract

A derivative of bacteriophage lambda, lambda 21, has been constructed and used for the cloning of Escherichia coli DNA fragments carrying promoters. Phage lambda 21 lacks the lac promoter operator and can accept DNA fragments up to 9.8 kilobases in size at a unique HindIII restriction endonuclease site adjacent to lacZ. Recombinant phage that carry promoters are readily identified by their expression of lacZ. Lysogens of these phages in strains harboring a deletion of the chromosomal lac operon are capable of growth on lactose as sole carbon source and can be used to study some of the regulatory signals that act upon the cloned promoter. In principle, lambda 21 can be used to clone any promoter DNA sequence with HindIII termini. PJ, the primary promoter for the rplJL-rpoBC operon, and P beta, a weak promoter for rpoBC, have been cloned in lambda 21. Transcription of lacZ from PJ was found to be subjected to feedback control by ribosomal protein L10 and to a lesser extent by ribosomal protein L7/L12. This suggests a possible L10-binding site near PJ that regulates transcription from that promoter. Lysogens of the phage that carries P beta responded to two regulatory signals: a rho-sensitive termination site preceding rpoBC and induction of beta-galactosidase synthesis by rifampicin. This suggests that P beta is a bona fide promoter for rpoBC.

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Selected References

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

  1. Adhya S., Gottesman M. Control of transcription termination. Annu Rev Biochem. 1978;47:967–996. doi: 10.1146/annurev.bi.47.070178.004535. [DOI] [PubMed] [Google Scholar]
  2. An G., Friesen J. D. Plasmid vehicles for direct cloning of Escherichia coli promoters. J Bacteriol. 1979 Nov;140(2):400–407. doi: 10.1128/jb.140.2.400-407.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. An G., Justesen J., Watson R. J., Friesen J. D. Cloning the spoT gene of Escherichia coli: identification of the spoT gene product. J Bacteriol. 1979 Mar;137(3):1100–1110. doi: 10.1128/jb.137.3.1100-1110.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson E. H. Growth Requirements of Virus-Resistant Mutants of Escherichia Coli Strain "B". Proc Natl Acad Sci U S A. 1946 May;32(5):120–128. doi: 10.1073/pnas.32.5.120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Becker A., Gold M. Isolation of the bacteriophage lambda A-gene protein. Proc Natl Acad Sci U S A. 1975 Feb;72(2):581–585. doi: 10.1073/pnas.72.2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berman M. L., Beckwith J. Fusions of the lac operon to the transfer RNA gene tyrT of Escherichia coli. J Mol Biol. 1979 May 25;130(3):285–301. doi: 10.1016/0022-2836(79)90542-4. [DOI] [PubMed] [Google Scholar]
  7. Berman M. L., Beckwith J. Use of gene fusions to isolate promoter mutants in the transfer RNA gene tyrT of Escherichia coli. J Mol Biol. 1979 May 25;130(3):303–315. doi: 10.1016/0022-2836(79)90543-6. [DOI] [PubMed] [Google Scholar]
  8. Blattner F. R., Williams B. G., Blechl A. E., Denniston-Thompson K., Faber H. E., Furlong L., Grunwald D. J., Kiefer D. O., Moore D. D., Schumm J. W. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science. 1977 Apr 8;196(4286):161–169. doi: 10.1126/science.847462. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cohen S. N., Chang A. C. Recircularization and autonomous replication of a sheared R-factor DNA segment in Escherichia coli transformants. Proc Natl Acad Sci U S A. 1973 May;70(5):1293–1297. doi: 10.1073/pnas.70.5.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dennis P. P., Nomura M. Stringent control of ribosomal protein gene expression in Escherichia coli. Proc Natl Acad Sci U S A. 1974 Oct;71(10):3819–3823. doi: 10.1073/pnas.71.10.3819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fiil N. P., Bendiak D., Collins J., Friesen J. D. Expression of Escherichia coli ribosomal protein and RNA polymerase genes cloned on plasmids. Mol Gen Genet. 1979 May 23;173(1):39–50. doi: 10.1007/BF00267689. [DOI] [PubMed] [Google Scholar]
  16. Franklin N. C. Genetic fusions for operon analysis. Annu Rev Genet. 1978;12:193–221. doi: 10.1146/annurev.ge.12.120178.001205. [DOI] [PubMed] [Google Scholar]
  17. Hayward R. S., Fyfe S. K. Non-coordinate expression of the neighbouring genes rplL and rpoB,C of Escherichia coli. Mol Gen Genet. 1978 Mar 20;160(1):77–80. doi: 10.1007/BF00275121. [DOI] [PubMed] [Google Scholar]
  18. Hayward R. S., Tittawella I. P., Scaife J. G. Evidence for specific control of RNA polymerase synthesis in Escherichia coli. Nat New Biol. 1973 May 2;243(122):6–9. [PubMed] [Google Scholar]
  19. 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]
  20. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Lindahl L., Zengel J. M. Operon-specific regulation of ribosomal protein synthesis in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6542–6546. doi: 10.1073/pnas.76.12.6542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Linn T., Scaife J. Identification of a single promoter in E. coli for rplJ, rplL and rpoBC. Nature. 1978 Nov 2;276(5683):33–37. doi: 10.1038/276033a0. [DOI] [PubMed] [Google Scholar]
  24. Maher D. L., Dennis P. P. In vivo transcription of E. coli genes coding for rRNA, ribosomal proteins and subunits of RNA polymerase: influence of the stringent control system. Mol Gen Genet. 1977 Oct 20;155(2):203–211. doi: 10.1007/BF00393161. [DOI] [PubMed] [Google Scholar]
  25. Miller J. H., Ippen K., Scaife J. G., Beckwith J. R. The promoter-operator region of the lac operon of Escherichia coli. J Mol Biol. 1968 Dec;38(3):413–420. doi: 10.1016/0022-2836(68)90395-1. [DOI] [PubMed] [Google Scholar]
  26. Morse D. E., Guertin M. Amber suA mutations which relieve polarity. J Mol Biol. 1972 Feb 14;63(3):605–608. doi: 10.1016/0022-2836(72)90453-6. [DOI] [PubMed] [Google Scholar]
  27. Ota Y., Kikuchi A., Cashel M. Gene expression of an Escherichia coli ribosomal RNA promoter fused to structural genes of the galactose operon. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5799–5803. doi: 10.1073/pnas.76.11.5799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Platt T., Yanofsky C. An intercistronic region and ribosome-binding site in bacterial messenger RNA. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2399–2403. doi: 10.1073/pnas.72.6.2399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Pribnow D. Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. Proc Natl Acad Sci U S A. 1975 Mar;72(3):784–788. doi: 10.1073/pnas.72.3.784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reznikoff W. S., Michels C. A., Cooper T. G., Silverstone A. E., Magasanik B. Inhibition of lacZ gene translation initiation in trp-lac fusion strains. J Bacteriol. 1974 Mar;117(3):1231–1239. doi: 10.1128/jb.117.3.1231-1239.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sharp P. A., Sugden B., Sambrook J. Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose--ethidium bromide electrophoresis. Biochemistry. 1973 Jul 31;12(16):3055–3063. doi: 10.1021/bi00740a018. [DOI] [PubMed] [Google Scholar]
  33. Subramanian A. R. Copies of proteins L7 and L12 and heterogeneity of the large subunit of Escherichia coli ribosome. J Mol Biol. 1975 Jun 15;95(1):1–8. doi: 10.1016/0022-2836(75)90330-7. [DOI] [PubMed] [Google Scholar]
  34. Watson R. J., Parker J., Fiil N. P., Flaks J. G., Friesen J. D. New chromosomal location for structural genes of ribosomal proteins. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2765–2769. doi: 10.1073/pnas.72.7.2765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Widera G., Gautier F., Lindenmaier W., Collins J. The expression of tetracycline resistance after insertion of foreign DNA fragments between the EcoRI and HindIII sites of the plasmid cloning vector pBR 322. Mol Gen Genet. 1978 Jul 25;163(3):301–305. doi: 10.1007/BF00271959. [DOI] [PubMed] [Google Scholar]
  36. Yamamoto M., Nomura M. Contranscription of genes for RNA polymerase subunits beta and beta' with genes for ribosomal proteins in Escherichia coli. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3891–3895. doi: 10.1073/pnas.75.8.3891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zurawski G., Brown K., Killingly D., Yanofsky C. Nucleotide sequence of the leader region of the phenylalanine operon of Escherichia coli. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4271–4275. doi: 10.1073/pnas.75.9.4271. [DOI] [PMC free article] [PubMed] [Google Scholar]

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