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. 1981 Sep 25;9(18):4459–4474. doi: 10.1093/nar/9.18.4459

Identification of gene products programmed by restriction endonuclease DNA fragments using an E. coli in vitro system.

J M Pratt, G J Boulnois, V Darby, E Orr, E Wahle, I B Holland
PMCID: PMC327450  PMID: 6272207

Abstract

DNA restriction enzyme fragments have been used to programme the synthesis of polypeptides in an in vitro system without apparent loss in fidelity compared with supercoiled templates. The system is extremely sensitive, less than 1 microgram of DNA can be used to direct the synthesis of 35S-labelled polypeptides of sufficiently high specific activity such that products can be identified by SDS-PAGE after a few hours autoradiography. The ability to analyse fragments can be used to readily assign specific proteins to small regions of the coding template, to identify cloned gene products distinct from those of the vector, and to identify cloned genes expressed from their own promoters. The in vitro system can be used successfully with bacterial DNA from other species and efficient extracts can be prepared from any E. coli K-12 strain, which should greatly facilitate the purification of factors controlling the expression of specific genes by complementation assay.

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Selected References

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

  1. Boyd A., Holland I. B. Regulation of the synthesis of surface protein in the cell cycle of E. coli B/r. Cell. 1979 Oct;18(2):287–296. doi: 10.1016/0092-8674(79)90048-5. [DOI] [PubMed] [Google Scholar]
  2. Collins J. Cell-free synthesis of proteins coding for mobilisation functions of ColE1 and transposition functions of Tn3. Gene. 1979 May;6(1):29–42. doi: 10.1016/0378-1119(79)90083-0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Davis R., Vapnek D. In vivo transcription of R-plasmid deoxyribonucleic acid in Escherichia coli strains with altered antibiotic resistance levels and/or conjugal proficiency. J Bacteriol. 1976 Mar;125(3):1148–1155. doi: 10.1128/jb.125.3.1148-1155.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Depew R. E., Liu L. F., Wang J. C. Interaction between DNA and Escherichia coli protein omega. Formation of a complex between single-stranded DNA and omega protein. J Biol Chem. 1978 Jan 25;253(2):511–518. [PubMed] [Google Scholar]
  6. Dougan G., Sherratt D. The transposon Tn1 as a probe for studying ColE1 structure and function. Mol Gen Genet. 1977 Mar 7;151(2):151–160. doi: 10.1007/BF00338689. [DOI] [PubMed] [Google Scholar]
  7. Fairweather N. F., Orr E., Holland I. B. Inhibition of deoxyribonucleic acid gyrase: effects on nucleic acid synthesis and cell division in Escherichia coli K-12. J Bacteriol. 1980 Apr;142(1):153–161. doi: 10.1128/jb.142.1.153-161.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Konings R. N., Hulsebos T., Van den Hondel C. A. Identification and characterization of the in vitro synthesized gene products of bacteriophage M13. J Virol. 1975 Mar;15(3):570–584. doi: 10.1128/jvi.15.3.570-584.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lindahl L., Post L., Nomura M. DNA-dependent in vitro synthesis of fibosomal proteins, protein elongation factors, and RNA polymerase subunit alpha: inhibition by ppGpp. Cell. 1976 Nov;9(3):439–448. doi: 10.1016/0092-8674(76)90089-1. [DOI] [PubMed] [Google Scholar]
  10. Lutkenhaus J. F., Wu H. C. Determination of transcriptional units and gene products from the ftsA region of Escherichia coli. J Bacteriol. 1980 Sep;143(3):1281–1288. doi: 10.1128/jb.143.3.1281-1288.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Orr E., Fairweather N. F., Holland I. B., Pritchard R. H. Isolation and characterisation of a strain carrying a conditional lethal mutation in the cou gene of Escherichia coli K12. Mol Gen Genet. 1979;177(1):103–112. doi: 10.1007/BF00267259. [DOI] [PubMed] [Google Scholar]
  12. Orr E., Staudenbauer W. L. An Escherichia coli mutant thermosensitive in the B subunit of DNA gyrase: effect on the structure and replication of the colicin E1 plasmid in vitro. Mol Gen Genet. 1981;181(1):52–56. doi: 10.1007/BF00339004. [DOI] [PubMed] [Google Scholar]
  13. Uhlin B. E., Molin S., Gustafsson P., Nordström K. Plasmids with temperature-dependent copy number for amplification of cloned genes and their products. Gene. 1979 Jun;6(2):91–106. doi: 10.1016/0378-1119(79)90065-9. [DOI] [PubMed] [Google Scholar]
  14. Weber L. A., Hickey E. D., Maroney P. A., Baglioni C. Inhibition of protein synthesis by Cl-. J Biol Chem. 1977 Jun 10;252(11):4007–4010. [PubMed] [Google Scholar]
  15. Wilkins B. M., Boulnois G. J., Lanka E. A plasmid DNA primase active in discontinuous bacterial DNA replication. Nature. 1981 Mar 19;290(5803):217–221. doi: 10.1038/290217a0. [DOI] [PubMed] [Google Scholar]
  16. Yang H. L., Heller K., Gellert M., Zubay G. Differential sensitivity of gene expression in vitro to inhibitors of DNA gyrase. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3304–3308. doi: 10.1073/pnas.76.7.3304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Zubay G. In vitro synthesis of protein in microbial systems. Annu Rev Genet. 1973;7:267–287. doi: 10.1146/annurev.ge.07.120173.001411. [DOI] [PubMed] [Google Scholar]

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