Summary
Escherichia coli infected with bacteriophage lambda-arabinose transducing phage were tested as sources of araC protein. Infection of cells with such phage produces an intracellular concentration of araC protein up to 100 times that present in wild-type E. coli, apparently resulting from fusion of the araC gene to bacteriophage lambda promoters. Lysates from these phage-infected cells may be fractionated to yield another 100-fold enrichment in araC activity so that the total enrichment is 10,000-fold. A nonsense mutation in araC provided proof of the identification on gel electrophoresis of a band in the purified material. Biologically active araC protein is a dimer with 28,000 M.W. subunits.
The araC gene in these phage replaces the int-xis genes but is oriented in the opposite direction. Nonetheless, it appears to be transcribed in this position by the phage promoter pr via transcription the long way around. Furthermore, because araC gene is in this position, we were able to isolate phage on which the araC gene was under phage late gene control by deletion of the late gene transcription stop signals in the b2 region.
Similar content being viewed by others
References
Adhya, S., Gottesman, M., deCrombrugghe, B.: Release of polarity in Escherichia coli Gene N of phage λ: Termination and antitermination of Transcription. Proc. nat. Acad. Sci. (Wash.) 71, 2534–2538 (1974)
Albertsson, P.O.: Partition of cell particles and macromolecules, 2nd ed., p. 30. New York: Wiley Interscience 1971
Carter, B.J., Smith, M.G.: Intracellular pools of Bacteriophage λ Deoxyribonucleic acid. J. molec. Biol. 50, 713–718 (1970)
Casadaban, M.J.: Regulation of the regulatory gene for the arabinose pathway, araC. J. molec. Biol. 104, 557–566 (1976)
Franklin, N.C.: Altered reading of genetic signals fused to the N operon of bacteriophage λ: Genetic evidence for modification of polymerase by the protein product of the N gene. J. molec. Biol. 89, 33–48 (1974)
Hirsh, J., Schleif, R.: Electron microscopy of gene regulation: The L-arabinose operon. Proc. nat. Acad. Sci (Wash.) 73, 1518–1522 (1976)
Inoko, H., Imai, M.: Involvement of some host function of Escherichia coli in the turn off control of phage ϕ80 and λ. Molec. gene. Genet. 129, 49–59 (1974)
Krell, K., Gottesman, M.E., Parks, J.S.: Escape synthesis of the bioitin operon in induced λb2 lysogens. J. molec. Biol. 68, 69–82 (1972)
Lis, J.T., Schleif, R.: Different cyclic AMP requirements for induction of the arabinose and lactose operons of Escherichia coli. J. molec. Biol. 79, 149–162 (1973)
Lis, J.T., Schleif, R.: The isolation and characterization of plaqueforming arabinose transducing bacteriophage λ. J. molec. Biol. 95, 395–407 (1975a)
Lis, J.T., Schleif, R.: The regulatory region of the L-arabinose operon: A physical, genetic and physiological study. J. molec. Biol. 95, 417–431 (1975b)
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)
Miller, J.H., Platt, T., Weber, K.: In the lactose operon (Beckwith, J. and Zipster, D. eds.), p. 343. New York: Cold Sring Harbor Press 1970
Pero, J.: Location of the phage λ gene responsible for turning off λ-exonuclease synthesis. Virology 40, 65–71 (1970)
Schleif, R.: An L-arabinose binding protein and arabinose permeation in Escherichia coli. J. molec. Biol. 46, 185–196 (1969)
Steffen, D.L.: Purification of araC protein. Doctoral Dissertation, Brandeis University (1977)
Weber, K., Osborn, M.: The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406–4412 (1969)
Wilcox, G., Boulter, J., Lee, N.: Direction of transcription of the regulatory gene araC in Escherichia coli B/r. Proc. nat. Acad. Sci. (Wash.) 71, 3635–3639 (1974)
Zubay, G., Chambers, D., Cheong, L.: In the lactose operon (Beckwith, J. and Zipster, E., eds.), p. 375. New York: Cold Spring Harbor Press 1970
Author information
Authors and Affiliations
Additional information
Communicated by W. Arber
Rights and permissions
About this article
Cite this article
Steffen, D., Schleif, R. Overproducing araC protein with lambda-arabinose transducing phage. Molec. Gen. Genet. 157, 333–339 (1977). https://doi.org/10.1007/BF00268671
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00268671