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Synthesis of the Escherichia coli K12 isoenzymes of ornithine transcarbamylase, performed in vitro

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The in vitro synthesis of enzymaticallyactive ornithine transcarbamylase (OTCase) directed by each of the E. coli K-12 OTCase genes (argF and argI) is described. The E. coli OTCase isoenzyme subunits are not identical, whether synthesized in vivo or in vitro, the argF-coded product being about 5% smaller. The OTCase protomers are enzymatically inactive but associate in vitro to an enzymatically active multimer. The rates of subunit association of argF and argI isoenzymes are considerably different. Utilizing the facile assay protocol presented, the regulation of in vitro OTCase synthesis by the specific holorepressor of the arginine regulon is demonstrated. Calculations based upon data presented indicate that there are about 65 molecules of argR gene product per bacterium, a substantially lower estimate than previously reported.

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  1. Bachman, B.J., Low, B.K., Raylor, A.L.: Recalibrated linkage map of Escherichia coli K12. Bacteriol. Rev. 40, 116–167 (1976)

  2. Baumberg, S., Bacon, D.F., Vogel, H.J.: Individually repressible enzymes specified by clustered genes of arginine synthesis. Proc. Nat. Acad. Sci. U.S.A. 53, 1029–1032 (1965)

  3. Crawford, L.V., Gesteland, R.F.: Synthesis of polyoma proteins in vitro. J. Mol. Biol. 74, 627–634 (1973)

  4. Cunin, R., Elseviers, D., Sand, G., Freundlich, G., Glansdorff, N.: On the functional organization of the argECBH cluster of genes in Escherichia coli K12. Molec. gen. Genet. 106, 32–47 (1969)

  5. Cunin, R., Kelker, N., Boyen, A., Yang, H., Zubay, G., Glansdorff, N., Maas, W.K.: Involvement of arginine in in vitro repression of transcription of arginine genes, C. B, and H in Escherichia coli K12. Biochem. Biophys. Res. Commun. 69, 377–382 (1976)

  6. Davis, B.N.: Dise electrophoresis-II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404–427 (1964)

  7. Elseviers, D., Cunin, R., Glansdorff, N.: Control regions within the argECBH gene cluster of Escherichia coli K12. Molec. gen. Genet. 117, 349–366 (1972)

  8. Eshenbaugh, D.L., Sens, D.A., James, E.: A solid phase radioimmune assay for ornithine transcarbamylase. p. 61–73. In: Advances in experimental medicine and biology. Vol. 42 (R.B. Dunlap, ed.) New York: Plenum Press 1974

  9. Eshenbaugh, D.L., Sens, D., James, E.: Rapid sedimentation of Escherichia coli in the presence of polyethylene glycol at lg. Anal. Biochem. 58, 390–394 (1974)

  10. Glansdorff, N., Sand, G., Verhoef, C.: The dual genetic control of ornithine transcarbamylase synthesis in Escherichia coli K12. Mutation Res. 4, 743–751 (1967)

  11. Gorini, L.: Feedback control of the synthesis of arginine in Escherichia coli. Bull. Soc. Chim. Biol. 40, 1939–1952 (1958)

  12. Gorini, L., Gunderson, W., Burger, M.: Genetics of regulation of enzyme synthesis in the arginine biosynthetic pathway of Escherichia coli. Cold Spr. harb. Symp. Quant. Biol. 26, 173–182 (1961)

  13. Gornall, A.G., Bardawill, C.J., David, M.M.: Determination of serum proteins by means of the biuret reaction. JBC 177, 751–766 (1949)

  14. Jacoby, G.A.: Control of the argECBH cluster Escherichia coli. Molec. gen. Genet. 117, 337–348 (1972)

  15. Jacoby, G.A., Gorini, L.: A unitary account of the repression mechanism of arginine biosynthesis in Escherichia coli. J. Mol. Biol. 39, 73–87 (1969)

  16. James, E., Gorini, L: Directed trasposition of argF 6 + gene and isolation of specialized transducing phage ϕ80dargF 6 + . Fed. Proc. 31, No. 2 Abstract 3710 (1972)

  17. James, P.M., Sens, D., Natter, W., Moore, S.K., James, E.: Isolation and characterization of the specialized transducing bacteriophages ϕ80dargF and λh80cI857dargF: Specific cleavage of arginine transducing deoxyribonucleic acid by the endonucleases EcoRI and SmaR. J. Bacteriol. 126, 487–500 (1976)

  18. Kelker, N.E., Maas, W.K., Yang, H., Zubay, G.: In vitro synthesis and repression of arginosuccinase in Escherichia coli K12; partial purification of the arginine repressor. Molec. gen. Genet. 144, 17–20 (1976)

  19. Kikuchi, A., Elseviers, D., Gorini, L.: Isolation and characterization of lambda transducing bacteriophages for argF. argI and adjacent genes. J. Bacteriol. 122, 727–742 (1975)

  20. Laemmli, U.K.: Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970)

  21. Legrain, D., Halleux, P., Stalon, V., Glansdorff, N.: The dual genetic control of ornithine carbamoyltransferase in Escherichia coli. A case of bacterial hybrid enzyme. Eur. J. Biochem. 27, 93–102 (1972)

  22. Maas, W.K.: Studies on repression of arginine biosynthesis in Escherichia coli. Cold Spr. Harb. Symp. Quant. Biol. 26 183–191 (1961)

  23. Miller, J.H.: Cell free synthesis of β-galactosidase. p. 419–424. In: Experiments in molecular genetics (J.H. Miller, ed.). New York: Cold Spring Harbor Laboratory 1972

  24. Ornstein, L.: Disc electrophoresis-I. Background and theory. Ann. N.Y. Sci. 121, 321–347 (1964)

  25. Rosen, J.M., Wood, S.L.C., Comstock, J.P.: Regulation of casein messenger RNA during the development of the rat mammary gland. Biochemistry 14, 2895–2903 (1975)

  26. Sens, D., Eshenbaugh, D., James, E.: Resolution of the DNA strands of the specialized transducing bacteriophage λh80cI857dargF. J. Virol. 16, 85–93 (1975)

  27. Sens, D., James, E.: Regulation of argF RNA synthesis, performed in vitro. Biochem. Biophys. Res. Commun. 64, 169–173 (1975)

  28. Sens, D., Natter, W., James, E.: Evolutionary drift of the argF and argI genes coding for isoenzyme forms of ornithine transcarbamylase in Escherichia coli K12. Cell 10, 275–285 (1977)

  29. Sens, D., Natter, W., James, E.: Transcription of the argF and argI genes of the Arginine Biosynthetic Regulon of Escherichia coli K12, performed in vitro. Molec. gen. Genet. (in press) (1977)

  30. Taylor, A.L., Adelberg, E.A.: Linkage analysis with very high frequency males of Escherichia coli. Genetics 45, 1233–1243 (1960)

  31. Umbarger, H.E.: Regulation of amino acid biosynthesis in microorganisms, p. 13–18. In: Synthesis of amino acids and proteins (H.R.V. Arnstein, ed.) International review of science and biochemistry, Series 1, Vol. 7 Baltimore: University Park Press 1975

  32. Urm, E., Yang, H., Zubay, G., Kelker, N., Maas, W.K.: In vitro repression of N-α-acetyl-L-ornithinase synthesis in Escherichia coli. Molec. gen. Genet. 121, 1–7 (1973)

  33. Weber, K.: New structural model of E. coli aspartate transcarbamylase and the amino-acid sequence of the regulatory polypeptide chain. Nature 218, 1116–1119 (1968)

  34. Wiley, D.C., Lipscomb, W.N.: Crystallographic determination of symmetry of aspartate transcarbamylase. Nature 218, 1119–1121 (1968)

  35. Yang, H., Zubay, G., Urm, E., Reiness, G., Cashel, M.: Effects of guanosine tetraphosphate, guanosine pentaphosphate, and β-γ methylenyl-guanosine pentaphosphate on gene expression of Escherichia coli in vitro. Proc. Nat. Acad. Sci. USA 71, 63–67 (1974)

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Correspondence to Eric James.

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This work is dedicated to Luigi Gorini without whom none of this would have been possible. His unbounded love of science and freedom will be remembered by so many, for so long.

Communicated by F. Gros

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Cleary, M.L., Garvin, R.T. & James, E. Synthesis of the Escherichia coli K12 isoenzymes of ornithine transcarbamylase, performed in vitro. Molec. Gen. Genet. 157, 155–165 (1977).

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  • Arginine
  • Gene Product
  • Ornithine
  • Lower Estimate
  • Regulon