Search tips
Search criteria 


Logo of narLink to Publisher's site
Nucleic Acids Res. 1987 December 10; 15(23): 9781–9796.
PMCID: PMC306531

Cloning, sequencing and expression of the Taq I restriction-modification system.


The Taq I modification and restriction genes (recognition sequence TCGA) have been cloned in E. coli and their DNA sequences have been determined. Both proteins were characterized and the N-terminal sequence of the endonuclease was determined. The genes have the same transcriptional orientation with the methylase gene 5' to the endonuclease gene. The methylase gene is 1089 bp in length (363 amino acids, 40,576 daltons); the endonuclease gene is 702 bp in length (234 amino acids, 27,523 daltons); they are separated by 132 bp. Both methylase and endonuclease activity can be detected in cell extracts. The clones fully modify the vector and chromosomal DNA but they fail to restrict infecting phage. Clones carrying only the restriction gene are viable even in the absence of modification. The restriction gene contains 7 Taq I sites; the modification gene contains none. This asymmetric distribution of sites could be important in the regulation of the expression of the endonuclease gene.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.7M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Roberts RJ. Restriction enzymes and their isoschizomers. Nucleic Acids Res. 1987;15 (Suppl):r189–r217. [PMC free article] [PubMed]
  • Xia Y, Burbank DE, Van Etten JL. Restriction endonuclease activity induced by NC-1A virus infection of a Chlorella-like green alga. Nucleic Acids Res. 1986 Aug 11;14(15):6017–6030. [PMC free article] [PubMed]
  • Sato S, Hutchinson CA, 3rd, Harris JI. A thermostable sequence-specific endonuclease from Thermus aquaticus. Proc Natl Acad Sci U S A. 1977 Feb;74(2):542–546. [PubMed]
  • Barker D, Hoff M, Oliphant A, White R. A second type II restriction endonuclease from Thermus aquaticus with an unusual sequence specificity. Nucleic Acids Res. 1984 Jul 25;12(14):5567–5581. [PMC free article] [PubMed]
  • Sato S, Nakazawa K, Shinomiya T. A DNA methylase from Thermus thermophilus HB8. J Biochem. 1980 Sep;88(3):737–747. [PubMed]
  • Mann MB, Rao RN, Smith HO. Cloning of restriction and modification genes in E. coli: the HbaII system from Haemophilus haemolyticus. Gene. 1978 Apr;3(2):97–112. [PubMed]
  • Newman AK, Rubin RA, Kim SH, 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]
  • Greene PJ, Gupta M, Boyer HW, Brown WE, Rosenberg JM. Sequence analysis of the DNA encoding the Eco RI endonuclease and methylase. J Biol Chem. 1981 Mar 10;256(5):2143–2153. [PubMed]
  • Walder RY, Hartley JL, Donelson JE, Walder JA. Cloning and expression of the Pst I restriction-modification system in Escherichia coli. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1503–1507. [PubMed]
  • Gingeras TR, Brooks JE. Cloned restriction/modification system from Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 1983 Jan;80(2):402–406. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Kiss A, Posfai G, Keller CC, Venetianer P, Roberts RJ. Nucleotide sequence of the BsuRI restriction-modification system. Nucleic Acids Res. 1985 Sep 25;13(18):6403–6421. [PMC free article] [PubMed]
  • Blumenthal RM, Gregory SA, Cooperider JS. Cloning of a restriction-modification system from Proteus vulgaris and its use in analyzing a methylase-sensitive phenotype in Escherichia coli. J Bacteriol. 1985 Nov;164(2):501–509. [PMC free article] [PubMed]
  • Howard KA, Card C, Benner JS, Callahan HL, Maunus R, Silber K, Wilson G, Brooks JE. Cloning the DdeI restriction-modification system using a two-step method. Nucleic Acids Res. 1986 Oct 24;14(20):7939–7951. [PMC free article] [PubMed]
  • Lacks SA, Mannarelli BM, Springhorn SS, Greenberg B. Genetic basis of the complementary DpnI and DpnII restriction systems of S. pneumoniae: an intercellular cassette mechanism. Cell. 1986 Sep 26;46(7):993–1000. [PubMed]
  • Kosykh VG, Buryanov YI, Bayev AA. Molecular cloning of EcoRII endonuclease and methylase genes. Mol Gen Genet. 1980;178(3):717–718. [PubMed]
  • Caserta M, Zacharias W, Nwankwo D, Wilson GG, Wells RD. Cloning, sequencing, in vivo promoter mapping, and expression in Escherichia coli of the gene for the HhaI methyltransferase. J Biol Chem. 1987 Apr 5;262(10):4770–4777. [PubMed]
  • Clewell DB, Helinski DR. Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an opern circular DNA form. Proc Natl Acad Sci U S A. 1969 Apr;62(4):1159–1166. [PubMed]
  • Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. [PMC free article] [PubMed]
  • Mandel M, Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. [PubMed]
  • Lederberg EM, Cohen SN. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Chen EY, Seeburg PH. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. [PubMed]
  • Haltiner M, Kempe T, Tjian R. A novel strategy for constructing clustered point mutations. Nucleic Acids Res. 1985 Feb 11;13(3):1015–1025. [PMC free article] [PubMed]
  • Hattori M, Sakaki Y. Dideoxy sequencing method using denatured plasmid templates. Anal Biochem. 1986 Feb 1;152(2):232–238. [PubMed]
  • Biggin MD, Gibson TJ, Hong GF. 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. [PubMed]
  • Ansorge W, Labeit S. Field gradients improve resolution on DNA sequencing gels. J Biochem Biophys Methods. 1984 Dec;10(3-4):237–243. [PubMed]
  • Olsson A, Moks T, Uhlén M, Gaal AB. Uniformly spaced banding pattern in DNA sequencing gels by use of field-strength gradient. J Biochem Biophys Methods. 1984 Nov;10(1-2):83–90. [PubMed]
  • 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. [PubMed]
  • Raleigh EA, Wilson G. Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9070–9074. [PubMed]
  • Keller C, Corcoran M, Roberts RJ. Computer programs for handling nucleic acid sequences. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):379–386. [PMC free article] [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Strickler JE, Hunkapiller MW, Wilson KJ. Utility of the gas-phase sequencer for both liquid- and solid-phase degradation of proteins and peptides at low picomole levels. Anal Biochem. 1984 Aug 1;140(2):553–566. [PubMed]
  • Hunkapiller MW, Hood LE. Analysis of phenylthiohydantoins by ultrasensitive gradient high-performance liquid chromatography. Methods Enzymol. 1983;91:486–493. [PubMed]
  • Shine J, Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. [PubMed]
  • Revel HR, Luria SE. DNA-glucosylation in T-even phage: genetic determination and role in phagehost interaction. Annu Rev Genet. 1970;4(0):177–192. [PubMed]
  • Ross TK, Braymer HD. Localization of a genetic region involved in McrB restriction by Escherichia coli K-12. J Bacteriol. 1987 Apr;169(4):1757–1759. [PMC free article] [PubMed]
  • Mise K, Nakajima K. Purification of a new restriction endonuclease, StyI, from Escherichia coli carrying the hsd+ miniplasmid. Gene. 1985;33(3):357–361. [PubMed]
  • Yoshida Y, Mise K. Occurrence of small Hsd plasmids in Salmonella typhi, Shigella boydii, and Escherichia coli. J Bacteriol. 1986 Feb;165(2):357–362. [PMC free article] [PubMed]
  • Som S, Bhagwat AS, Friedman S. Nucleotide sequence and expression of the gene encoding the EcoRII modification enzyme. Nucleic Acids Res. 1987 Jan 12;15(1):313–332. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Walder RY, Walder JA, Donelson JE. The organization and complete nucleotide sequence of the PstI restriction-modification system. J Biol Chem. 1984 Jun 25;259(12):8015–8026. [PubMed]
  • Mannarelli BM, Balganesh TS, Greenberg B, Springhorn SS, Lacks SA. 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. [PubMed]
  • Macdonald PM, Mosig G. Regulation of a new bacteriophage T4 gene, 69, that spans an origin of DNA replication. EMBO J. 1984 Dec 1;3(12):2863–2871. [PubMed]
  • Theriault G, Roy PH, Howard KA, Benner JS, Brooks JE, Waters AF, Gingeras TR. 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. [PMC free article] [PubMed]
  • Schoner B, Kelly S, Smith HO. The nucleotide sequence of the HhaII restriction and modification genes from Haemophilus haemolyticus. Gene. 1983 Oct;24(2-3):227–236. [PubMed]
  • Hattman S, Wilkinson J, Swinton D, Schlagman S, Macdonald PM, Mosig G. Common evolutionary origin of the phage T4 dam and host Escherichia coli dam DNA-adenine methyltransferase genes. J Bacteriol. 1985 Nov;164(2):932–937. [PMC free article] [PubMed]
  • Szomolányi E, Kiss A, Venetianer P. Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli. Gene. 1980 Aug;10(3):219–225. [PubMed]
  • Janulaitis A, Povilionis P, Sasnauskas K. Cloning of the modification methylase gene of Bacillus centrosporus in Escherichia coli. Gene. 1982 Dec;20(2):197–204. [PubMed]
  • Kiss A, Baldauf F. Molecular cloning and expression in Escherichia coli of two modification methylase genes of Bacillus subtilis. Gene. 1983 Jan-Feb;21(1-2):111–119. [PubMed]
  • Behrens B, Noyer-Weidner M, Pawlek B, Lauster R, Balganesh TS, Trautner TA. Organization of multispecific DNA methyltransferases encoded by temperate Bacillus subtilis phages. EMBO J. 1987 Apr;6(4):1137–1142. [PubMed]
  • Günthert U, Reiners L, Lauster R. Cloning and expression of Bacillus subtilis phage DNA methyltransferase genes in Escherichia coli and B. subtilis. Gene. 1986;41(2-3):261–270. [PubMed]
  • Noyer-Weidner M, Jentsch S, Kupsch J, Bergbauer M, Trautner TA. DNA methyltransferase genes of Bacillus subtilis phages: structural relatedness and gene expression. Gene. 1985;35(1-2):143–150. [PubMed]
  • Herman GE, Modrich P. Escherichia coli dam methylase. Physical and catalytic properties of the homogeneous enzyme. J Biol Chem. 1982 Mar 10;257(5):2605–2612. [PubMed]
  • Brooks JE, Blumenthal RM, Gingeras TR. The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene. Nucleic Acids Res. 1983 Feb 11;11(3):837–851. [PMC free article] [PubMed]
  • Schlagman SL, Hattman S. Molecular cloning of a functional dam+ gene coding for phage T4 DNA adenine methylase. Gene. 1983 May-Jun;22(2-3):139–156. [PubMed]
  • Walder RY, Langtimm CJ, Chatterjee R, Walder JA. Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII. J Biol Chem. 1983 Jan 25;258(2):1235–1241. [PubMed]
  • Lacks SA, Springhorn SS. Cloning in Streptococcus pneumoniae of the gene for DpnII DNA methylase. J Bacteriol. 1984 Mar;157(3):934–936. [PMC free article] [PubMed]

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