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. 1979 Nov;140(2):400–407. doi: 10.1128/jb.140.2.400-407.1979

Plasmid vehicles for direct cloning of Escherichia coli promoters.

G An, J D Friesen
PMCID: PMC216663  PMID: 387734

Abstract

A multicopy plasmid cloning vehicle, pGA22, which carries genes for ampicillin resistance (Apr), tetracycline resistance (Tcr), chloramphenicol resistance (Cmr), and kanamycin resistance (Kmr) has been constructed. This plasmid has five unique sites for restriction endonucleases EcoRI, PstI, XhoI, SmaI, and SalI within antibiotic resistance genes. pGA22, which is 5.1 megadaltons in size, has a low copy number (probably fewer than 10 per genome), is capable of relaxed replication, and is mobilized by F-factor at a frequency of 10(-5). A series of promoter-cloning vehicles, pGA24, pGA39, and pGA46, has been developed from pGA22. In these plasmids the natural promoter for Tcr has been removed and has been replaced by small deoxyribonucleic acid fragments carrying unique sites for several restriction endonucleases. Cells carrying these vectors are sensitive to tetracycline unless insertional activation of the Tcr occurs by cloning a promoter-carrying deoxyribonucleic acid fragment in one of the unique sites adjacent to the 5' end of Tcr. In this way, promoters carried on a HindIII-generated deoxyribonucleic acid fragment can be inserted at the HindIII site of plasmid pGA24, pGA39, or pGA46. A promoter in fragments generated by digestion with restriction endonuclease XmaI or PstI or by any restriction endonucleases which generate flush ends, such as SmaI, PvuII, HpaI, HincII, or HaeIII, can be clones in plasmid pGA39. Plasmid pGA46 can be used to detect a promoter fragment carried on a BglII, BamHI, MboI, or PstI fragment. We also describe a plasmid, pGA44, with a unique KpnI site in the rifampin resistance gene rpoB.

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Selected References

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  1. 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]
  2. Cabello F., Timmis K., Cohen S. N. Replication control in a composite plasmid constructed by in vitro linkage of two distinct replicons. Nature. 1976 Jan 29;259(5541):285–290. doi: 10.1038/259285a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Clewell D. B., Helinski D. R. Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry. 1970 Oct 27;9(22):4428–4440. doi: 10.1021/bi00824a026. [DOI] [PubMed] [Google Scholar]
  5. Collins J. Gene cloning with small plasmids. Curr Top Microbiol Immunol. 1977;78:122–170. [PubMed] [Google Scholar]
  6. Errington L., Glass R. E., Hayward R. S., Scaife J. G. Structure and orientation of an RNA polymerase operon in Escherichia coli. Nature. 1974 Jun 7;249(457):519–522. doi: 10.1038/249519a0. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. Lovett M. A., Helinski D. R. Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'kn plasmid. J Bacteriol. 1976 Aug;127(2):982–987. doi: 10.1128/jb.127.2.982-987.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Neve R. L., West R. W., Rodriguez R. L. Eukaryotic DNA fragments which act as promoters for a plasmid gene. Nature. 1979 Jan 25;277(5694):324–325. doi: 10.1038/277324a0. [DOI] [PubMed] [Google Scholar]
  13. Newman A. J., Linn T. G., Hayward R. S. Evidence for co-transcription of the RNA polymerase genes rpoBC with a ribosomal protein gene of escherichia coli. Mol Gen Genet. 1979 Jan 31;169(2):195–204. doi: 10.1007/BF00271671. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Reeve J. N. Bacteriophage infection of minicells: a general method for identification of "in vivo" bacteriophage directed polypeptide biosynthesis. Mol Gen Genet. 1977 Dec 14;158(1):73–79. doi: 10.1007/BF00455121. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Sutcliffe J. G. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3737–3741. doi: 10.1073/pnas.75.8.3737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Tait R. C., Rodriguez R. L., Boyer H. W. Altered tetracycline resistance in pSC101 recombinant plasmids. Mol Gen Genet. 1977 Mar 16;151(3):327–331. doi: 10.1007/BF00268797. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Yamamoto M., Nomura M. Organization of genes for transcription and translation in the rif region of the Escherichia coli chromosome. J Bacteriol. 1979 Jan;137(1):584–594. doi: 10.1128/jb.137.1.584-594.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]

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