Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1987 Jan 12;15(1):313–332. doi: 10.1093/nar/15.1.313

Nucleotide sequence and expression of the gene encoding the EcoRII modification enzyme.

S Som, A S Bhagwat, S Friedman
PMCID: PMC340412  PMID: 3029675

Abstract

The gene coding for the EcoRII modification enzyme has been cloned and the nucleotide sequence of 1933 base pairs containing the gene has been determined. The gene codes for a protein of 477 amino acids. Two transcriptional start sites have been mapped by S1 mapping. One deletion that removes 34 N-terminal amino acids was found to have partial enzyme activity. Comparison of the EcoRII methylase sequence with other cytosine methylases revealed several domains of partial homology among all cytosine methylases. Cloning the gene in multicopy pUC vectors increased the expression by 6-18 fold. A 40 fold overproduction of the EcoRII methylase was obtained by cloning the gene in the expression vector carrying the lambda PL promoter.

Full text

PDF
313

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  3. Bougueleret L., Schwarzstein M., Tsugita A., Zabeau M. Characterization of the genes coding for the Eco RV restriction and modification system of Escherichia coli. Nucleic Acids Res. 1984 Apr 25;12(8):3659–3676. doi: 10.1093/nar/12.8.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  5. Brooks J. E., Blumenthal R. M., Gingeras T. R. The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene. Nucleic Acids Res. 1983 Feb 11;11(3):837–851. doi: 10.1093/nar/11.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brosius J., Cate R. L., Perlmutter A. P. Precise location of two promoters for the beta-lactamase gene of pBR322. S1 mapping of ribonucleic acid isolated from Escherichia coli or synthesized in vitro. J Biol Chem. 1982 Aug 10;257(15):9205–9210. [PubMed] [Google Scholar]
  7. Buhk H. J., Behrens B., Tailor R., Wilke K., Prada J. J., Günthert U., Noyer-Weidner M., Jentsch S., Trautner T. A. Restriction and modification in Bacillus subtilis: nucleotide sequence, functional organization and product of the DNA methyltransferase gene of bacteriophage SPR. Gene. 1984 Jul-Aug;29(1-2):51–61. doi: 10.1016/0378-1119(84)90165-3. [DOI] [PubMed] [Google Scholar]
  8. Bur'ianov Ia I., Nesterenko V. F., Kosykh V. G., Baev A. A. Razlichnaia molekuliarnaia struktura DNK-metilaz EcoRII i Eco MRE600 dcmII. Dokl Akad Nauk SSSR. 1981;257(2):495–497. [PubMed] [Google Scholar]
  9. Delaney A. D. A DNA sequence handling program. Nucleic Acids Res. 1982 Jan 11;10(1):61–67. doi: 10.1093/nar/10.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Friedman S. Bactericidal effect of 5-azacytidine on Escherichia coli carrying EcoRII restriction-modification enzymes. J Bacteriol. 1982 Jul;151(1):262–268. doi: 10.1128/jb.151.1.262-268.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Friedman S. Binding of the EcoRII methylase to azacytosine-containing DNA. Nucleic Acids Res. 1986 Jun 11;14(11):4543–4556. doi: 10.1093/nar/14.11.4543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friedman S. The inhibition of DNA(cytosine-5)methylases by 5-azacytidine. The effect of azacytosine-containing DNA. Mol Pharmacol. 1981 Mar;19(2):314–320. [PubMed] [Google Scholar]
  13. Friedman S. The irreversible binding of azacytosine-containing DNA fragments to bacterial DNA(cytosine-5)methyltransferases. J Biol Chem. 1985 May 10;260(9):5698–5705. [PubMed] [Google Scholar]
  14. Goad W. B., Kanehisa M. I. Pattern recognition in nucleic acid sequences. I. A general method for finding local homologies and symmetries. Nucleic Acids Res. 1982 Jan 11;10(1):247–263. doi: 10.1093/nar/10.1.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gottesman M. E., Adhya S., Das A. Transcription antitermination by bacteriophage lambda N gene product. J Mol Biol. 1980 Jun 15;140(1):57–75. doi: 10.1016/0022-2836(80)90356-3. [DOI] [PubMed] [Google Scholar]
  16. Gouy M., Gautier C. Codon usage in bacteria: correlation with gene expressivity. Nucleic Acids Res. 1982 Nov 25;10(22):7055–7074. doi: 10.1093/nar/10.22.7055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grantham R., Gautier C., Gouy M., Jacobzone M., Mercier R. Codon catalog usage is a genome strategy modulated for gene expressivity. Nucleic Acids Res. 1981 Jan 10;9(1):r43–r74. doi: 10.1093/nar/9.1.213-b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Greene P. J., Gupta M., Boyer H. W., Brown W. E., Rosenberg J. M. Sequence analysis of the DNA encoding the Eco RI endonuclease and methylase. J Biol Chem. 1981 Mar 10;256(5):2143–2153. [PubMed] [Google Scholar]
  19. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  20. Hattman S., Schlagman S., Cousens L. Isolation of a mutant of Escherichia coli defective in cytosine-specific deoxyribonucleic acid methylase activity and in partial protection of bacteriophage lambda against restriction by cells containing the N-3 drug-resistance factor. J Bacteriol. 1973 Sep;115(3):1103–1107. doi: 10.1128/jb.115.3.1103-1107.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kiss A., Posfai G., Keller C. C., Venetianer P., Roberts R. J. Nucleotide sequence of the BsuRI restriction-modification system. Nucleic Acids Res. 1985 Sep 25;13(18):6403–6421. doi: 10.1093/nar/13.18.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kosykh V. G., Buryanov Y. I., Bayev A. A. Molecular cloning of EcoRII endonuclease and methylase genes. Mol Gen Genet. 1980;178(3):717–718. doi: 10.1007/BF00337884. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Mannarelli B. M., Balganesh T. S., Greenberg B., Springhorn S. S., Lacks S. A. Nucleotide sequence of the Dpn II DNA methylase gene of Streptococcus pneumoniae and its relationship to the dam gene of Escherichia coli. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4468–4472. doi: 10.1073/pnas.82.13.4468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  26. May M. S., Hattaman S. Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes. J Bacteriol. 1975 Apr;122(1):129–138. doi: 10.1128/jb.122.1.129-138.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Newman A. K., Rubin R. A., Kim S. H., Modrich P. DNA sequences of structural genes for Eco RI DNA restriction and modification enzymes. J Biol Chem. 1981 Mar 10;256(5):2131–2139. [PubMed] [Google Scholar]
  28. Pósfai G., Baldauf F., Erdei S., Pósfai J., Venetianer P., Kiss A. Structure of the gene coding for the sequence-specific DNA-methyltransferase of the B. subtilis phage SPR. Nucleic Acids Res. 1984 Dec 11;12(23):9039–9049. doi: 10.1093/nar/12.23.9039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pósfai G., Kiss A., Erdei S., Pósfai J., Venetianer P. Structure of the Bacillus sphaericus R modification methylase gene. J Mol Biol. 1983 Nov 5;170(3):597–610. doi: 10.1016/s0022-2836(83)80123-5. [DOI] [PubMed] [Google Scholar]
  30. Roberts R. J. Restriction and modification enzymes and their recognition sequences. Nucleic Acids Res. 1985;13 (Suppl):r165–r200. doi: 10.1093/nar/13.suppl.r165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  32. Rosenberg M., Ho Y. S., Shatzman A. The use of pKc30 and its derivatives for controlled expression of genes. Methods Enzymol. 1983;101:123–138. doi: 10.1016/0076-6879(83)01009-5. [DOI] [PubMed] [Google Scholar]
  33. Sanger F., Air G. M., Barrell B. G., Brown N. L., Coulson A. R., Fiddes C. A., Hutchison C. A., Slocombe P. M., Smith M. Nucleotide sequence of bacteriophage phi X174 DNA. Nature. 1977 Feb 24;265(5596):687–695. doi: 10.1038/265687a0. [DOI] [PubMed] [Google Scholar]
  34. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Santi D. V., Norment A., Garrett C. E. Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6993–6997. doi: 10.1073/pnas.81.22.6993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schoner B., Kelly S., Smith H. O. The nucleotide sequence of the HhaII restriction and modification genes from Haemophilus haemolyticus. Gene. 1983 Oct;24(2-3):227–236. doi: 10.1016/0378-1119(83)90083-5. [DOI] [PubMed] [Google Scholar]
  37. Staden R. An interactive graphics program for comparing and aligning nucleic acid and amino acid sequences. Nucleic Acids Res. 1982 May 11;10(9):2951–2961. doi: 10.1093/nar/10.9.2951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Takano T., Watanabe T., Fukasawa T. Mechanism of host-controlled restriction of bacteriophage lambda by R factors in Escherichia coli K12. Virology. 1968 Feb;34(2):290–302. doi: 10.1016/0042-6822(68)90239-0. [DOI] [PubMed] [Google Scholar]
  39. Theriault G., Roy P. H., Howard K. A., Benner J. S., Brooks J. E., Waters A. F., Gingeras T. R. Nucleotide sequence of the PaeR7 restriction/modification system and partial characterization of its protein products. Nucleic Acids Res. 1985 Dec 9;13(23):8441–8461. doi: 10.1093/nar/13.23.8441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tran-Betcke A., Behrens B., Noyer-Weidner M., Trautner T. A. DNA methyltransferase genes of Bacillus subtilis phages: comparison of their nucleotide sequences. Gene. 1986;42(1):89–96. doi: 10.1016/0378-1119(86)90153-8. [DOI] [PubMed] [Google Scholar]
  41. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  42. Walder R. Y., Walder J. A., Donelson J. E. The organization and complete nucleotide sequence of the PstI restriction-modification system. J Biol Chem. 1984 Jun 25;259(12):8015–8026. [PubMed] [Google Scholar]
  43. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES