Identification of theEcoKI andEcoR124I type I restriction-modification enzyme subunits by non-equilibrium pH gradient two-dimensional gel electrophoresis
Tóm tắt
Effectively optimized and reproducible procedure for monitoring the composition of type I restriction-modification endonucleasesEcoKI andEcoR124I by non-equilibrium pH gradient two-dimensional (2-D) gel electrophoresis is described. Three subunits of the enzyme complex, which widely differ from one another in their isoelectric points and molar mass, were identified in crude cell extracts ofE. coli. For the first time all three subunits of bothEcoKI andEcoR124I were detected as distinct spots on a single 2-D gel. A sensitive immunoblotting procedure was suggested suitable for routine use in determining the identity of individual subunits. Potential application of this method for detailed studies of regulation of the function and stoichiometry of the enzyme complexes is discussed.
Tài liệu tham khảo
Appleyard R.K.: Segregation of new lysogenic types during growth of doubly lysogenic strains derived fromEscherichia coli K-12.Genetics 39, 440–452 (1954).
Barcus V.A., Titheradge A.J., Murray N.E.: The diversity of alleles at thehsd locus in natural populations ofEscherichia coli.Genetics 140, 1187–1197 (1995).
Bradford M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248–254 (1976).
Dybvig K., Huilan Y.: Regulation of a restriction and modification systemvia DNA inversion inMycoplasma pulmonis.Mol. Microbiol. 12, 547–560 (1994).
Firman K., Price C., Glover S.W.: TheEcoR124 andEcoR124/3 restriction and modification systems: cloning the genes.Plasmid 14, 224–234 (1985).
Firman K.D., Dutta C., Weiserová M., Janščák P.: The role of subunit assembly in the functional control of type I restriction-modification enzymes.Mol. Biol. Today 1 34–41 (2000).
Hubáček J., Glover S.W.: Complementation analysis of temperature-sensitive host specificity mutations inEscherichia coli.J. Mol. Biol. 50, 111–127 (1970).
Janščák P., Dryden D.T., Firman K.: Analysis of the subunit assembly of the type IC restriction-modification enzymeEcoR124I.Nucl. Acids Res. 26, 4439–4445 (1998).
Kobayashi I., Nobusato A., Kobayashi-Takahashi N., Uchiyama I.: Shaping the genome-restriction-modification systems as mobile genetic elements.Curr. Opin. Genet. Dev. 9, 649–656 (1999).
Laemmli U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–685 (1970).
Li X.M., Vohradský J., Weiser J.: The use of protein synthesis elongation factor EF-Tu as internal calibration standard in two-dimensional electrophoretic studies of differentiation inStreptomyces.Electrophoresis 15, 1198–1204 (1994).
Lopez M.F.: Nonequilibrium pH gel electrophoresis (NEPHGE).Meth. Mol. Biol. 112, 129–131 (1999).
Murray N.E.: Type, I restriction systems: sophiticated molecular machines (a legacy of Bertani and Weigle).Microbiol. Mol. Biol. Rev. 64, 412–434 (2000).
O’Farrell P.H.: High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 250, 4007–4021 (1975).
O’Farrell P.Z., Goodman H.M., O'Farrell P.H.: High resolution two-dimensional electrophoresis of basic as well as acidic proteins.Cell 12, 1133–1141 (1977).
Patel J., Taylor I., Dutta C.F., Kneale G., Firman K.: High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M.EcoR124.Gene 112, 21–27 (1992).
Prakash-Cheng A., Chung S.S., Ryu J.: The expression and regulation ofhsdK genes after conjugative transfer.Mol. Gen. Genet. 241, 491–496 (1993).
Rabilloud T.: A comparison between low background silver diammine and silver nitrate protein stains.Electrophoresis 13, 429–439 (1992).
Redaschi N., Bickle T.A.: Posttranscriptional regulation ofEcoP1I andEcoPI5I restriction activity.J. Mol. Biol. 257, 790–803 (1996).
Sambrook J.C., Fritsch E.F., Maniatis T.:Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA) 1989.
Saunders N.J., Peden J.F., Hood D.W., Moxon E.R.: Simple sequence repeats in theHelicobacter pylori genome.Mol. Microbiol. 27, 1091–1098 (1998).
Taylor I., Patel J., Firman K., Kneale G.: Purification and biochemical characterization of theEcoR124 type I modification methylase.Nucl. Acids Res. 20, 179–186 (1992).
Titheradge A.J., Ternent D., Murray N.E.: A third family of allelichsd genes inSalmonella enterica: sequence comparisons with related proteins identify conserved regions implicated in restriction of DNA.Mol. Microbiol. 22 437–447 (1996).
Tomb J.F., White O., Kerlavage A.R., Clayton R.A., Sutton G.G., Fleischmann R.D., Ketchum K.A., Klenk H.P., Gill S., Dougherty B.A., Nelson K., Quackenbush J., Zhou L., Kirkness E.f., Peterson S., Loftus B., Richardson D., Dodson R., Khalak H.G., Glodek A., McKenney K., Fitzegerald L.M., Lee N., Adams M.D., Hickey E.K., Berg, D.E., Gocayne J.D., Utterback T.R., Peterson J.D., Kelley J.M., Cotton M.D., Weidman J.M., Fuji C., Bowman C., Watthey L., Wallin E., Hayes W.S., Borodovsky M., Kapp P.D., Smith H.O., Fraser C.M., Venter J.C.: The complete genome sequence of the gastric pathogenHelicobacter pylori.Nature 388, 539–547 (1997).
Weiser J., Mikulík K., Žižka Z., Šťastná J., Janda I., Jiráňová A.: Isolation and characterization ofStreptomyces aureofaciens protein-synthesis elongation factor Tu in an aggregated state.Eur. J. Biochem. 129, 127–132 (1982).
Weiserová M., Janščák, P., Benada O., Hubáček J., Zinkevich V.E., Glover S.W., Firman K.: Cloning, production and characterization of wild-type and mutant forms of the R.EcoK endonucleases.Nucl. Acids Res. 21, 373–379 (1993); publishederratum: Nucl. Acids Res. 21, 1686 (1993).
Yanisch-Perron C., Vieira J., Messing J.: Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.Gene 33, 103–119 (1985).
Zeindl-Eberhart E., Jungblut P.R., Rabes H.M.: A new method to assign immunodetected spots in the complex two-dimensional electrophoresis pattern.Electrophoresis 18, 799–801 (1997).