Abstract
An RNA-DNA hybridization method is described for determining the fraction of a radioisotopically labeled RNA preparation isolated from bacteria that is message RNA. Experiments are performed under conditions where over 95% of the input RNA is converted to an RNA-DNA complex. Competitive hybridization methods are described that partition the RNA-DNA hybrids into ribosomal RNA-DNA and nonribosomal RNA-DNA hybrids. Evidence is presented that the competition is specific. Message RNA radioactivity was assumed to be RNA radioactivity not competed by ribosomal RNA and transfer RNA. The fraction of RNA made at a given instant under balanced growth conditions (30 C, in minimal medium), that is, message RNA, was determined to be 65%. The method was also used to measure the size distribution of newly synthesized mRNA in Escherichia coli. That size was found to average 3–8 × 105 daltons.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Adesnik, M., and Levinthal, C. (1969). Synthesis and saturation of ribosomal RNA in Escherichia coli. J. Mol. Biol. 46281.
Bishop, D. H. L., Claybrook, J. R., and Spiegelman, S. (1967). Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Mol. Biol. 26373.
Bishop, J. O., and Irving, M. I. (1971). A method for measuring the content of a specific messenger ribonucleic acid in Escherichia coli. Biochem. J. 121105.
Contesse, G., Crepin M., and Gros, F. (1970). In Beckwith, J., and Zipser, D. (eds.), The Lactose Operon, Cold Spring Harbor Laboratories, New York.
Gillespie, D. (1968). The formation and detection of DNA-RNA hybrids. Meth. Enzymol. 12B641.
Gillespie, D. and Spiegelman, S. (1965). A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12829.
Gillespie, S., and Gillespie D. (1971). RNA-DNA hybridization in aqueous solutions and in solutions containing formamide. Biochem. J. 125481.
Guttman, B., and Novick, A. (1963). A messenger RNA for β-galactosidase in E. coli. Cold Spring Harbor Symp. Quant. Biol. 28873.
Hayashi, M., and Spiegelman, S. (1961). The selective synthesis of informational RNA in bacteria. Proc. Natl. Acad. Sci. 471564.
Hayashi, M., Spiegelman, S., Franklin, N., and Luria, S. (1963). Separation of the RNA message transcribed in response to a specific inducer. Proc. Natl. Acad. Sci. 49 729.
Hecht, N., and Woese, C. (1968). Separation of bacterial ribosomal ribonucleic acid from its macromolecular precursors by polyacrylamide gel electrophoresis. J. Bacteriol. 95986.
Imamoto, F., and Yanofsky, C. (1967). Transcription of the tryptophan operon in polarity mutants of E. coli. I. Characterization of the tryptophan messenger RNA of polar mutants. J. Mol. Biol. 281.
Imamoto, F., Morikawa, N., and Sato, K. (1965). On the transcription of the tryptophan operon in E. coli. III. Multicistronic messenger RNA and polarity for transcription. J. Mol. Biol. 13169.
Ishihama, A., Mizuno, N., Takai, M., Otaka, E., and Osawa, S. (1962). Molecular and metabolic properties of messenger RNA from normal and T2-injected E. coli. J. Mol. Biol. 5251.
Jacobson, A., and Gillespie, D. (1968). Metabolic events occurring during recovery from prolonged glucose starvation in E. coli. J. Bacteriol. 951030.
Kennell, D. (1964). Persistence of messenger RNA activity in Bacillus megaterium treated with actinomycin. J. Mol. Biol. 9789.
Kennell, D. (1968). Titration of gene sites on DNA by DNA-RNA hybridization. J. Mol. Biol. 3485.
Kennell, D. (1971). Principles and practices of nucleic acid hybridization. Prog. Nucleic Acid Res. 11259.
Kurland, C. (1960). Molecular characterization of ribonucleic acid from Escherichia coli ribosomes. I. Isolation and molecular weights. J. Mol. Biol. 283.
Lazzarini, R. A., and Dahlberg, A. E. (1971). The control of RNA synthesis during amino acid deprivation in E. coli. J. Biol. Chem. 246420.
Maaløe, O., and Kjeldgaard, N. O. (1966). Control of Macromolecular Synthesis, W. A. Benjamin, New York.
Marmur, J. (1961). A procedure for the isolation of DNA from microorganisms. J. Mol. Biol. 3208.
Martin, R. (1963). The one operon-one messenger theory of transcription. Cold Spring Harbor Symp. Quant. Biol. 28357.
Meijs, W. H., and Schilperoort, R. A. (1971). Determination of the amount of DNA on nitrocellulose membrane filters. FEBS Letters, 12166.
Monier, R., Naono, S., Hayes, D., Hayes, F., and Gros, F. (1962). Studies on the heterogeneity of messenger RNA from E. coli. J. Mol. Biol. 5311.
Pato, M., and von Meyenburg, K. (1970). Residual RNA synthesis in Escherichia coli after inhibition of initiation of transcription by rifampicin. Cold Spring Harbor Symp. Quant. Biol. 35497.
Pettijohn, D. E., Clarkson, K., Kossman, C. R., and Stonington, O. G. (1970). Synthesis of ribosomal RNA on a protein-DNA complex isolated from bacteria: A comparison of ribosomal RNA synthesis in vitro and in vivo. J. Mol. Biol. 52281.
Rose, J., Mosteller, R., and Yanofsky, C. (1970). Tryptophan messenger ribonucleic acid elongation rates and steady state levels of tryptophan operon enzymes under various growth conditions. J. Mol. Biol. 51541.
Stamato, T. D., and Pettijohn, D. E. (1971). Regulation of ribosomal RNA synthesis in stringent bacteria. Nature New Biol. 23499.
Summers, W. C. (1970). A simple method for extraction of RNA from E. coli utilizing diethylpyrocarbonate. Anal. Biochem. 33459.
Winslow, R. M., and Lazzarini, R. A. (1969). Amino acid regulation of the rates of synthesis and chain elongation of ribonucleic acid in Escherichia coli. J. Biol. Chem. 2443387.
Author information
Authors and Affiliations
Additional information
This research was supported by grant GM-14368 from the Institute of General Medical Sciences, National Institutes of Health. Two of the authors (D. P. and A. J.) were supported by predoctoral fellowships from the National Institute of General Medical Sciences.
Rights and permissions
About this article
Cite this article
Gillespie, D., Jacobson, A., Gillespie, S. et al. Use of exhaustive nucleic acid hybridization for determining the amount and size distribution of newly synthesized bacterial message RNA. Biochem Genet 10, 175–190 (1973). https://doi.org/10.1007/BF00485764
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00485764