Abstract
The expression of the genes encoding ribonucleotide reductase in Escherichia coli was investigated in cultures synchronized by obtaining the smallest cells in a population after sucrose gradient centrifugation. Specific activity of ribonucleotide reductase and DNA initiation were found to increase in parallel, periodically as a function of the cell cycle. The expression of nrd was also determined in cells synchronized by periodic repeated doubling in a phosphate limited medium. Antibodies directed against the B2 subunit of ribonucleotide reductase were raised in a rabbit and purified. Immunoprecipitation of the B2 subunit and RNA-DNA dot blot hybridization assays were developed and employed to determine the expression of ribonucleotide reductase translational and transcriptional products during the cell cycle. Both of nrd-mRNA and B2 subunit expression were found to increase each generation at approximately the same time DNA synthesis was initiated and then to decrease back to the basal level shortly after DNA initiation. These results provided evidence of cell cycle dependent regulation of ribonucleotide reductase in E. coli. When the upstream regulatory region of nrd was fused to a promoterless lacZ gene on a single copy plasmid, lac-mRNA and beta-galactosidase were found to be synthesized in parallel to nrd expression from the chromosomal operon. When nrd sequences surrounding the promoter were removed from this construct, lac-mRNA and beta-galactosidase synthesis were no longer cell cycle regulated.
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Selected References
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- Aiba H., Adhya S., de Crombrugghe B. Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem. 1981 Nov 25;256(22):11905–11910. [PubMed] [Google Scholar]
- Andrews B. J., Herskowitz I. Regulation of cell cycle-dependent gene expression in yeast. J Biol Chem. 1990 Aug 25;265(24):14057–14060. [PubMed] [Google Scholar]
- Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
- Brosius J., Ullrich A., Raker M. A., Gray A., Dull T. J., Gutell R. R., Noller H. F. Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli. Plasmid. 1981 Jul;6(1):112–118. doi: 10.1016/0147-619x(81)90058-5. [DOI] [PubMed] [Google Scholar]
- Carlson J., Fuchs J. A., Messing J. Primary structure of the Escherichia coli ribonucleoside diphosphate reductase operon. Proc Natl Acad Sci U S A. 1984 Jul;81(14):4294–4297. doi: 10.1073/pnas.81.14.4294. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DAVIS B. D., MINGIOLI E. S. Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950 Jul;60(1):17–28. doi: 10.1128/jb.60.1.17-28.1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Elledge S. J., Davis R. W. Two genes differentially regulated in the cell cycle and by DNA-damaging agents encode alternative regulatory subunits of ribonucleotide reductase. Genes Dev. 1990 May;4(5):740–751. doi: 10.1101/gad.4.5.740. [DOI] [PubMed] [Google Scholar]
- Engström Y., Eriksson S., Jildevik I., Skog S., Thelander L., Tribukait B. Cell cycle-dependent expression of mammalian ribonucleotide reductase. Differential regulation of the two subunits. J Biol Chem. 1985 Aug 5;260(16):9114–9116. [PubMed] [Google Scholar]
- Engström Y., Rozell B. Immunocytochemical evidence for the cytoplasmic localization and differential expression during the cell cycle of the M1 and M2 subunits of mammalian ribonucleotide reductase. EMBO J. 1988 Jun;7(6):1615–1620. doi: 10.1002/j.1460-2075.1988.tb02987.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Engvall E., Perlmann P. Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry. 1971 Sep;8(9):871–874. doi: 10.1016/0019-2791(71)90454-x. [DOI] [PubMed] [Google Scholar]
- Eriksson S., Sjöberg B. M., Hahne S. Ribonucleoside diphosphate reductase from Escherichia coli. An immunological assay and a novel purification from an overproducing strain lysogenic for phage lambdadnrd. J Biol Chem. 1977 Sep 10;252(17):6132–6138. [PubMed] [Google Scholar]
- Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
- Fernandez-Sarabia M. J., Fantes P. A. Ribonucleotide reductase and its regulation during the cell cycle. Trends Genet. 1990 Sep;6(9):275–276. doi: 10.1016/0168-9525(90)90214-q. [DOI] [PubMed] [Google Scholar]
- Filpula D., Fuchs J. A. Regulation of ribonucleoside diphosphate reductase synthesis in Escherichia coli: increased enzyme synthesis as a result of inhibition of deoxyribonucleic acid synthesis. J Bacteriol. 1977 Apr;130(1):107–113. doi: 10.1128/jb.130.1.107-113.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Filpula D., Fuchs J. A. Regulation of the synthesis of ribonucleoside diphosphate reductase in Escherichia coli: specific activity of the enzyme in relationship to perturbations of DNA replication. J Bacteriol. 1978 Aug;135(2):429–435. doi: 10.1128/jb.135.2.429-435.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fuchs J. A. Coordinate control of the synthesis of ribonucleoside diphosphate reductase components in Escherichia coli. J Bacteriol. 1977 May;130(2):957–959. doi: 10.1128/jb.130.2.957-959.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fuchs J. A., Warner H. R. Isolation of an Escherichia coli mutant deficient in glutathione synthesis. J Bacteriol. 1975 Oct;124(1):140–148. doi: 10.1128/jb.124.1.140-148.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gober J. W., Champer R., Reuter S., Shapiro L. Expression of positional information during cell differentiation of Caulobacter. Cell. 1991 Jan 25;64(2):381–391. doi: 10.1016/0092-8674(91)90646-g. [DOI] [PubMed] [Google Scholar]
- Gudas L. J., James R., Paradee A. B. Evidence of the involvement of an outer membrane protein in DNA initiation. J Biol Chem. 1976 Jun 10;251(11):3470–3479. [PubMed] [Google Scholar]
- Hanke P. D., Fuchs J. A. Characterization of the mRNA coding for ribonucleoside diphosphate reductase in Escherichia coli. J Bacteriol. 1983 Dec;156(3):1192–1197. doi: 10.1128/jb.156.3.1192-1197.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanke P. D., Fuchs J. A. Regulation of ribonucleoside diphosphate reductase mRNA synthesis in Escherichia coli. J Bacteriol. 1983 Jun;154(3):1040–1045. doi: 10.1128/jb.154.3.1040-1045.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanke P. D., Fuchs J. A. Requirement of protein synthesis for the induction of ribonucleoside diphosphate reductase mRNA in Escherichia coli. Mol Gen Genet. 1984;193(2):327–331. doi: 10.1007/BF00330689. [DOI] [PubMed] [Google Scholar]
- Hartree E. F. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972 Aug;48(2):422–427. doi: 10.1016/0003-2697(72)90094-2. [DOI] [PubMed] [Google Scholar]
- Hassouna N., Michot B., Bachellerie J. P. The complete nucleotide sequence of mouse 28S rRNA gene. Implications for the process of size increase of the large subunit rRNA in higher eukaryotes. Nucleic Acids Res. 1984 Apr 25;12(8):3563–3583. doi: 10.1093/nar/12.8.3563. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnson R. C., Ball C. A., Pfeffer D., Simon M. I. Isolation of the gene encoding the Hin recombinational enhancer binding protein. Proc Natl Acad Sci U S A. 1988 May;85(10):3484–3488. doi: 10.1073/pnas.85.10.3484. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Joseleau-Petit D., Kepes F., Peutat L., D'Ari R., Rothfield L. I. Biosynthesis of a membrane adhesion zone fraction throughout the cell cycle of Escherichia coli. J Bacteriol. 1990 Nov;172(11):6573–6575. doi: 10.1128/jb.172.11.6573-6575.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Joseleau-Petit D., Képès F., Peutat L., D'Ari R., Képès A. DNA replication initiation, doubling of rate of phospholipid synthesis, and cell division in Escherichia coli. J Bacteriol. 1987 Aug;169(8):3701–3706. doi: 10.1128/jb.169.8.3701-3706.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kepes F., Kepes A. Long - lasting synchrony of the division of enteric bacteria. Biochem Biophys Res Commun. 1981 Apr 15;99(3):761–767. doi: 10.1016/0006-291x(81)91230-4. [DOI] [PubMed] [Google Scholar]
- Kepes F., Kepes A. Synchronisation automatique de la croissance de Escherichia coli. Ann Microbiol (Paris) 1980 Jan-Feb;131(1):3–16. [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Larsen J. E., Albrechtsen B., Valentin-Hansen P. Analysis of the terminator region after the deoCABD operon of Escherichia coli K-12 using a new class of single copy number operon-fusion vectors. Nucleic Acids Res. 1987 Jul 10;15(13):5125–5140. doi: 10.1093/nar/15.13.5125. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Larsson A., Sjöberg B. M. Identification of the stable free radical tyrosine residue in ribonucleotide reductase. EMBO J. 1986 Aug;5(8):2037–2040. doi: 10.1002/j.1460-2075.1986.tb04461.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lutkenhaus J. F., Moore B. A., Masters M., Donachie W. D. Individual proteins are synthesized continuously throughout the Escherichia coli cell cycle. J Bacteriol. 1979 May;138(2):352–360. doi: 10.1128/jb.138.2.352-360.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Milhausen M., Agabian N. Regulation of polypeptide synthesis during Caulobacter development: two-dimensional gel analysis. J Bacteriol. 1981 Oct;148(1):163–173. doi: 10.1128/jb.148.1.163-173.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ohta N., Masurekar M., Newton A. Cloning and cell cycle-dependent expression of DNA replication gene dnaC from Caulobacter crescentus. J Bacteriol. 1990 Dec;172(12):7027–7034. doi: 10.1128/jb.172.12.7027-7034.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- PRITCHARD R. H., LARK K. G. INDUCTION OF REPLICATION BY THYMINE STARVATION AT THE CHROMOSOME ORIGIN IN ESCHERICHIA COLI. J Mol Biol. 1964 Aug;9:288–307. doi: 10.1016/s0022-2836(64)80208-4. [DOI] [PubMed] [Google Scholar]
- Robin A., Joseleau-Petit D., D'Ari R. Transcription of the ftsZ gene and cell division in Escherichia coli. J Bacteriol. 1990 Mar;172(3):1392–1399. doi: 10.1128/jb.172.3.1392-1399.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shapiro L., Gober J. W. Positioning of gene products during Caulobacter cell differentiation. J Cell Sci Suppl. 1989;11:85–97. doi: 10.1242/jcs.1989.supplement_11.7. [DOI] [PubMed] [Google Scholar]
- Thelander L., Reichard P. Reduction of ribonucleotides. Annu Rev Biochem. 1979;48:133–158. doi: 10.1146/annurev.bi.48.070179.001025. [DOI] [PubMed] [Google Scholar]
- Tuggle C. K., Fuchs J. A. Regulation of the operon encoding ribonucleotide reductase in Escherichia coli: evidence for both positive and negative control. EMBO J. 1986 May;5(5):1077–1085. doi: 10.1002/j.1460-2075.1986.tb04325.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tuggle C. K., Fuchs J. A. Regulation of the operon encoding ribonucleotide reductase: role of the negative sites in nrd repression. J Bacteriol. 1990 Apr;172(4):1711–1718. doi: 10.1128/jb.172.4.1711-1718.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]