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. 1990 Oct 25;18(20):5961–5967. doi: 10.1093/nar/18.20.5961

Selection for mutations in the PR promoter of bacteriophage lambda.

S Brown 1, J Ferm 1, S Woody 1, G Gussin 1
PMCID: PMC332391  PMID: 2146590

Abstract

Insertion of DNA containing PR, the early rightward promoter of bacteriophage lambda, is lethal to M13-derived vectors when the promoter directs transcription (using the '+' strand as template) toward the M13 origin of replication (ori). Lethality can be relieved by mutation of PR, repression of the promoter by the lambda cl repressor, or by insertion of a strong transcription terminator between PR and ori. We have used selection for plaque formation in the absence of repressor to isolate 14 different mutations at 8 sites in PR. This method of isolating promoter mutants in vivo is applicable generally to strong promoters whose activity is regulated either positively or negatively.

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Selected References

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  1. Backman K., Ptashne M. Maximizing gene expression on a plasmid using recombination in vitro. Cell. 1978 Jan;13(1):65–71. doi: 10.1016/0092-8674(78)90138-1. [DOI] [PubMed] [Google Scholar]
  2. Berget P. B., Poteete A. R., Sauer R. T. Control of phage P22 tail protein expression by transcription termination. J Mol Biol. 1983 Mar 15;164(4):561–572. doi: 10.1016/0022-2836(83)90050-5. [DOI] [PubMed] [Google Scholar]
  3. Carty M., Menzel R. The unexpected antitermination of gyrA-directed transcripts is enhanced by DNA relaxation. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8882–8886. doi: 10.1073/pnas.86.22.8882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Deuschle U., Kammerer W., Gentz R., Bujard H. Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J. 1986 Nov;5(11):2987–2994. doi: 10.1002/j.1460-2075.1986.tb04596.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Goliger J. A., Yang X. J., Guo H. C., Roberts J. W. Early transcribed sequences affect termination efficiency of Escherichia coli RNA polymerase. J Mol Biol. 1989 Jan 20;205(2):331–341. doi: 10.1016/0022-2836(89)90344-6. [DOI] [PubMed] [Google Scholar]
  6. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hwang J. J., Brown S., Gussin G. N. Characterization of a doubly mutant derivative of the lambda PRM promoter. Effects of mutations on activation of PRM. J Mol Biol. 1988 Apr 20;200(4):695–708. doi: 10.1016/0022-2836(88)90481-0. [DOI] [PubMed] [Google Scholar]
  8. Kornberg A. DNA replication. J Biol Chem. 1988 Jan 5;263(1):1–4. [PubMed] [Google Scholar]
  9. Maurer R., Meyer B., Ptashne M. Gene regulation at the right operator (OR) bacteriophage lambda. I. OR3 and autogenous negative control by repressor. J Mol Biol. 1980 May 15;139(2):147–161. doi: 10.1016/0022-2836(80)90302-2. [DOI] [PubMed] [Google Scholar]
  10. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Meyer B. J., Maurer R., Ptashne M. Gene regulation at the right operator (OR) of bacteriophage lambda. II. OR1, OR2, and OR3: their roles in mediating the effects of repressor and cro. J Mol Biol. 1980 May 15;139(2):163–194. doi: 10.1016/0022-2836(80)90303-4. [DOI] [PubMed] [Google Scholar]
  12. Panasenko S. M., Cameron J. R., Davis R. W., Lehman I. R. Five hundredfold overproduction of DNA ligase after induction of a hybrid lambda lysogen constructed in vitro. Science. 1977 Apr 8;196(4286):188–189. doi: 10.1126/science.322281. [DOI] [PubMed] [Google Scholar]
  13. Pruss G. J., Drlica K. DNA supercoiling and prokaryotic transcription. Cell. 1989 Feb 24;56(4):521–523. doi: 10.1016/0092-8674(89)90574-6. [DOI] [PubMed] [Google Scholar]
  14. Reichardt L., Kaiser A. D. Control of lambda repressor synthesis. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2185–2189. doi: 10.1073/pnas.68.9.2185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rosenberg M., Ho Y. S., Shatzman A. The use of pKc30 and its derivatives for controlled expression of genes. Methods Enzymol. 1983;101:123–138. doi: 10.1016/0076-6879(83)01009-5. [DOI] [PubMed] [Google Scholar]
  16. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Telesnitsky A. P., Chamberlin M. J. Sequences linked to prokaryotic promoters can affect the efficiency of downstream termination sites. J Mol Biol. 1989 Jan 20;205(2):315–330. doi: 10.1016/0022-2836(89)90343-4. [DOI] [PubMed] [Google Scholar]
  18. Tsao Y. P., Wu H. Y., Liu L. F. Transcription-driven supercoiling of DNA: direct biochemical evidence from in vitro studies. Cell. 1989 Jan 13;56(1):111–118. doi: 10.1016/0092-8674(89)90989-6. [DOI] [PubMed] [Google Scholar]
  19. Wu T. H., Liao S. M., McClure W. R., Susskind M. M. Control of gene expression in bacteriophage P22 by a small antisense RNA. II. Characterization of mutants defective in repression. Genes Dev. 1987 Apr;1(2):204–212. doi: 10.1101/gad.1.2.204. [DOI] [PubMed] [Google Scholar]
  20. Yang X. J., Hart C. M., Grayhack E. J., Roberts J. W. Transcription antitermination by phage lambda gene Q protein requires a DNA segment spanning the RNA start site. Genes Dev. 1987 May;1(3):217–226. doi: 10.1101/gad.1.3.217. [DOI] [PubMed] [Google Scholar]
  21. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  22. Youderian P., Bouvier S., Susskind M. M. Sequence determinants of promoter activity. Cell. 1982 Oct;30(3):843–853. doi: 10.1016/0092-8674(82)90289-6. [DOI] [PubMed] [Google Scholar]
  23. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucleic Acids Res. 1982 Oct 25;10(20):6487–6500. doi: 10.1093/nar/10.20.6487. [DOI] [PMC free article] [PubMed] [Google Scholar]

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