Identification of Gene Products Programmed by Restriction Endonuclease DNA Fragments Using an E. Coli in Vitro System

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1981 Sep 25

PMID 6272207

Citations 22

Authors

J M PRATT

G J Boulnois

V Darby

E Orr

E Wahle

I B Holland

Affiliations

Soon will be listed here.

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.

Citing Articles

Testing the conservation of the translational machinery over evolution in diverse environments: assaying Thermus thermophilus ribosomes and initiation factors in a coupled transcription-translation system from Escherichia coli.

Thompson J, Dahlberg A Nucleic Acids Res. 2004; 32(19):5954-61.

PMID: 15534366 PMC: 528807. DOI: 10.1093/nar/gkh925.

Characterization of the tpr gene product and isolation of a specific protease-deficient mutant of Porphyromonas gingivalis W83.

Park Y, McBride B Infect Immun. 1993; 61(10):4139-46.

PMID: 8406803 PMC: 281136. DOI: 10.1128/iai.61.10.4139-4146.1993.

Coupled transcription--translation in extracts of Streptomyces lividans.

Thompson J, Rae S, Cundliffe E Mol Gen Genet. 1984; 195(1-2):39-43.

PMID: 6593562 DOI: 10.1007/BF00332721.

Nucleotide sequence encoding the flavoprotein and hydrophobic subunits of the succinate dehydrogenase of Escherichia coli.

Wood D, Darlison M, Wilde R, Guest J Biochem J. 1984; 222(2):519-34.

PMID: 6383359 PMC: 1144207. DOI: 10.1042/bj2220519.

Resistance to fusidic acid in Escherichia coli mediated by the type I variant of chloramphenicol acetyltransferase. A plasmid-encoded mechanism involving antibiotic binding.

Bennett A, Shaw W Biochem J. 1983; 215(1):29-38.

PMID: 6354181 PMC: 1152360. DOI: 10.1042/bj2150029.

References

Zubay G . In vitro synthesis of protein in microbial systems. Annu Rev Genet. 1973; 7:267-87. DOI: 10.1146/annurev.ge.07.120173.001411. View

Orr E, Staudenbauer W . 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):52-6. DOI: 10.1007/BF00339004. View

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; 125(3):1148-55. PMC: 236194. DOI: 10.1128/jb.125.3.1148-1155.1976. View

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; 9(3):439-48. DOI: 10.1016/0092-8674(76)90089-1. View

Weber L, Hickey E, Maroney P, Baglioni C . Inhibition of protein synthesis by Cl-. J Biol Chem. 1977; 252(11):4007-10. View

Dougan G, Sherratt D . The transposon Tn1 as a probe for studying ColE1 structure and function. Mol Gen Genet. 1977; 151(2):151-60. DOI: 10.1007/BF00338689. View

Depew R, Liu L, Wang J . Interaction between DNA and Escherichia coli protein omega. Formation of a complex between single-stranded DNA and omega protein. J Biol Chem. 1978; 253(2):511-8. View

Dagert M, Ehrlich S . Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979; 6(1):23-8. DOI: 10.1016/0378-1119(79)90082-9. View

Collins J . Cell-free synthesis of proteins coding for mobilisation functions of ColE1 and transposition functions of Tn3. Gene. 1979; 6(1):29-42. DOI: 10.1016/0378-1119(79)90083-0. View

10.

Uhlin B, Molin S, Gustafsson P, Nordstrom K . Plasmids with temperature-dependent copy number for amplification of cloned genes and their products. Gene. 1979; 6(2):91-106. DOI: 10.1016/0378-1119(79)90065-9. View

11.

Yang H, 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; 76(7):3304-8. PMC: 383813. DOI: 10.1073/pnas.76.7.3304. View

12.

Boyd A, Holland I . Regulation of the synthesis of surface protein in the cell cycle of E. coli B/r. Cell. 1979; 18(2):287-96. DOI: 10.1016/0092-8674(79)90048-5. View

13.

Orr E, Fairweather N, Holland I, Pritchard R . 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):103-12. DOI: 10.1007/BF00267259. View

14.

Fairweather N, Orr E, Holland I . Inhibition of deoxyribonucleic acid gyrase: effects on nucleic acid synthesis and cell division in Escherichia coli K-12. J Bacteriol. 1980; 142(1):153-61. PMC: 293920. DOI: 10.1128/jb.142.1.153-161.1980. View

15.

Lutkenhaus J, Wu H . Determination of transcriptional units and gene products from the ftsA region of Escherichia coli. J Bacteriol. 1980; 143(3):1281-8. PMC: 294497. DOI: 10.1128/jb.143.3.1281-1288.1980. View

16.

Wilkins B, Boulnois G, LANKA E . A plasmid DNA primase active in discontinuous bacterial DNA replication. Nature. 1981; 290(5803):217-21. DOI: 10.1038/290217a0. View

17.

Konings R, Hulsebos T, van den Hondel C . Identification and characterization of the in vitro synthesized gene products of bacteriophage M13. J Virol. 1975; 15(3):570-84. PMC: 354494. DOI: 10.1128/JVI.15.3.570-584.1975. View