Current Microbiology

, Volume 20, Issue 2, pp 133–136 | Cite as

Expression of a DNA methylation (dam) gene inEscherichia coli K-12

  • Judy A. Arraj
  • Te-Hui Wu
  • M. G. Marinus


Plasmid pMQ3, carrying thedam gene ofEscherichia coli on a 6.1 Kb fragment, shows a tenfold increase in relative DNA adenine methylase activity, while plasmid pdam118, with a 1.14 Kbdam insert, shows only a twofold increase, although both plasmids were derived from plasmid pLC13–42. Since a copy number effect did not seem to be the cause of this difference, we have subcloned pMQ3 in order to determine whether the additional chromosomal DNA present in this plasmid is responsible for the enhancement of methylase activity. We show that the 346 base pairs upstream ofdam contain sequences necessary for expression. DNA sequence analysis has revealed that in pdam118 only the 118 bases 5-prime to thedam gene are present in other constructs and that the additional upstream material is pBR322 DNA. This shows that pdam118 carries a DNA duplication.


Methylation Base Pair Adenine Twofold Increase Number Effect 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature Cited

  1. 1.
    Alton NK, Vapnek D (1979) Nucleotide sequence analysis of the chloramphenical Resistance transposon Tn9. Nature 282:864–869Google Scholar
  2. 2.
    Arraj JA, Marinus MG (1983) Phenotypic reversal indam mutants ofEscherichia coli K-12 by a recombinant plasmid containing thedam + gene. J Bacteriol 152:562–565Google Scholar
  3. 3.
    Bale A, d'Alarcao M, Marinus MG (1979) Characterization of DNA adenine methylase (dam) mutants ofEscherichia coli K-12. Mutat Res 59:157–165Google Scholar
  4. 4.
    Brooks JE, Blumenthal RM, Gingeras TR (1983) The isolation and characterization of theEscherichia coli DNA adenine methylasedam gene. Nucleic Acids Res 11:837–851Google Scholar
  5. 5.
    Carraway M, Youderian P, Marinus MG (1987) Spontaneous mutations occur near Dam recognition sites in adam Escherichia coli host. Genetics 116:343–347Google Scholar
  6. 6.
    Casadaban JJ, Chou J, Cohen SN (1980)In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins:Escherichia coli plasmid vectors for the detection and cloning of translation initiation signals. J Bacteriol 143:971–980Google Scholar
  7. 7.
    Clarke L, Carbon J (1976) A colony bank containing syntheticColE1 hybrid plasmids representative of the entireE. coli genome Cell 9:91–99Google Scholar
  8. 8.
    Dente L, Cesareni G, Cortese R (1983) pEMBL: a new family of single stranded plasmids. Nucleic Acids Res 11:1645–1655Google Scholar
  9. 9.
    Herman GE, Modrich P (1982)Escherichia coli dam methylase. Physical and catalytic properties of the homogeneous enzyme. J Biol Chem 257:2605–2612Google Scholar
  10. 10.
    Hughes P, Squali-Houssaini FZ, Forterre P, Kohiyama M (1984)In vitro replication of adam methylated and nonmethylatedoriC plasmid. J Mol Biol 176:155–159Google Scholar
  11. 11.
    Jonczyk P, Hines R, Smith DW (1989) TheEscherichia coli dam gene is expressed as a distal gene of a new operon. Mol Gen Genet 217:85–96Google Scholar
  12. 12.
    Lu AL, Clark S, Modrich P (1983) Methyl-directed repair of DNA base pair mismatchesin vitro. Proc Natl Acad Sci USA 80:4639–4643Google Scholar
  13. 13.
    Marinus MG, Carraway M, Frey AZ, Brown L, Arraj JA (1983) Insertion mutations in thedam gene ofEscherichia coli K-12. Mol Gen Genet 192:288–289Google Scholar
  14. 14.
    Marinus MG (1987) DNA methylation. In: Neidhardt FC et al. (eds)Escherichia coli andSalmonella typhimurium: cellular and molecular biology. Washington DC: American Society of Microbiology, pp 697–702Google Scholar
  15. 15.
    Marinus MG (1987) DNA methylation inEscherichia coli. Annu Rev Genet 231:113–131Google Scholar
  16. 16.
    Messer W, Bellekes U, Lother H (1985) Effect ofdam methylation on the activity of theE. coli replication origin,oriC. EMBO J 4:1327–1332Google Scholar
  17. 17.
    Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor, NY: Cold Spring Harbor LaboratoryGoogle Scholar
  18. 18.
    O'Callaghan CH, Morris A, Kirby SM, Shingler AH (1972) Novel method for detection of beta-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chem 1:283–288Google Scholar
  19. 19.
    Parker B, Marinus MG (1988) A simple and rapid method to obtain substitution mutations inEscherichia coli: Isolation of adam deletion/insertion mutation. Gene 73:531–535Google Scholar
  20. 20.
    Pukkila P, Peterson J, Herman G, Modrich P, Meselson M (1983) Effects of high levels of DNA adenine methylation on methyl-directed mismatch repair inE. coli. Genetics 104:571–582Google Scholar
  21. 21.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  22. 22.
    Smith DW, Garland A, Herman G, Enns RE, Baker TA, Zyskind JW (1985) Importance of state of methylation oforiC GATC sites in initiation of DNA replication inEscherichia coli. EMBO J 4:1319–1327Google Scholar
  23. 23.
    Zagursky RJ, Berman ML (1984) Cloning vectors that yield high levels of single-stranded DNA for rapid sequencing. Gene 27:183–191Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1990

Authors and Affiliations

  • Judy A. Arraj
    • 1
  • Te-Hui Wu
    • 1
  • M. G. Marinus
    • 1
  1. 1.Department of PharmacologyUniversity of Massachusetts Medical SchoolWorcesterUSA

Personalised recommendations