Summary
A 760 base pair nucleotide sequence of transposon Tn1721 containing the resolvase (tnpR) gene has been determined. A 186 triplet open reading frame was assigned to tnpR, and the allocation of-35 and-10 promoter boxes was supported by mapping transcription initiation at 70 base pairs upstream of tnpR. Expression of tnpR under tac promoter control generated sufficient resolvase protein for enzyme purification and for in vitro studies. Purified Tn1721 resolvase requires supercoiling and two directly oriented resolution (res) sites. The enzyme resolves cointegrate substrates containing repeat copies of Tn1721 res, of Tn21 res, of Tn21 res/Tn1721 res, but not of Tn3 res.
Similar content being viewed by others
References
Abdel-Meguid SS, Grindley NDF, Templeton NS, Steitz TA (1984) Cleavage of the site-specific recombination protein γδ resolvase: The smaller of two fragments binds DNA specifically. Proc Natl Acad Sci USA 81:2001–2005
Aiba H, Adhya S, de Crombrugghe B (1981) Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem 256:11905–11910
Altenbuchner J, Schmitt R (1983) Transposon Tn1721: site-specific recombination generates deletions and inversions. Mol Gen Genet 190:300–308
Altenbuchner J, Choi C-L, Grinsted J, Schmitt R, Richmond MH (1981) The transposons Tn501(Hg) and Tn1721(Tc) are related. Genet Res Camb 37:285–289
Altenbuchner J, Schmid K, Schmitt R (1983) Tn1721-encoded tetracycline resistance: mapping of structural and regulatory genes mediating resistance. J Bacteriol 153:116–123
Amann E, Brosius J, Ptashne M (1983) Vectors bearing a hybrid trp-lac promoter useful for regulated gene expression of cloned genes in Escherichia coli. Gene 25:167–178
Bolivar F, Rodriguez RL, Greene PH, Betlach MC, Heynecker HL, Boyer HW (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:97–113
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
Diver WP, Grinsted J, Fritzinger DC, Brown NL, Altenbuchner J, Rogowsky P, Schmitt R (1983) DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721. Mol Gen Genet 191:189–193
Green MR, Roeder RG (1980) Definition of a novel promoter for the major adenovirus-associated virus mRNA. Cell 22:231–242
Grinsted J, de la Cruz F, Altenbuchner J, Schmitt R (1982) Complementation of transposition of tnpA mutants of Tn3, Tn21, Tn501, and Tn1721. Plasmid 8:276–286
Halford S, Jordan SL, Kirbride EA (1985) The resolvase protein from the transposon Tn21. Mol Gen Genet 200:169–175
Hawley DK, McClure WR (1983) Compilation and analysis of Escherichia coli promoter DNA sequences. Nucl Acids Res 11:2237–2255
Heffron F (1983) Tn3 and its relatives. In: Shapiro JA (ed) Mobile genetic elements. Academic Press, New York, p 223
Heffron F, McCarthy BJ, Ohtsubo H, Ohtsubo E (1979) DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3. Cell 18:1153–1164
Johnson RA, Walseth TF (1979) The enzymatic preparation of [α-32P]ATP, [α-32P]GTP, [32P]cAMP, and [32P]cGMP and their use in the assay of adenylate and guanylate cyclases and cyclic nucleotide phosphodiesterases. Adv Cycl Nucl Res 10:135–167
Kitts P, Symington D, Burke M, Reed R, Sherratt D (1982) Transposon-specified site-specific recombination. Proc Natl Acad Sci USA 79:46–50
Krasnow MA, Cozzarelli NR (1983) Site-specific relaxation and recombination by the Tn3 resolvase: recognition of the DNA path between oriented res sites. Cell 32:1313–1324
Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavage. Methods Enzymol 65:499–559
Novick RP, Clowes RC, Cohen SN, Curtiss R III, Datta N, Falkow S (1976) Uniform nomenclature for bacterial plasmids: A proposal. Bacteriol Rev 40:168–189
Reed RR (1981) Transposon-mediated site-specific recombination: a defined in vitro system. Cell 25:713–719
Rogowsky P, Schmitt R (1984) Resolution of a hybrid cointegrate between transposons Tn501 and Tn1721 defines the recombination site. Mol Gen Genet 193:162–166
Schmitt R, Altenbuchner J, Grinsted J (1982) Complementation of transposition functions encoded by transposons Tn501 (HgR) and Tn1721 (TetR) In: Levy SB, Clowes RC, Koenig EL (eds) Molecular biology, pathogenicity and ecology of bacterial plasmids. Plenum Press, New York, p 359
Smitt R, Mötsch S, Rogowsky P, de la Cruz F, Grinsted J (1985) On the transposition and evolution of Tn1721 and its relatives. In: Helinski D, Cohen S, Clewell D (eds) Plasmids in bacteria. Plenum Press, New York, p 79
Soberon X, Covarrubias L, Bolivar F (1980) Construction and characterization of new cloning vehicles, IV. Deletion derivatives of pBR322 and pBR325. Gene 9:287–305
Zieg J, Simon M (1980) Analysis of the nucleotide sequence of an invertible controlling element. Proc Natl Acad Sci USA 77:4196–4200
Author information
Authors and Affiliations
Additional information
Communicated by A. Böck
Rights and permissions
About this article
Cite this article
Rogowsky, P., Schmitt, R. Tn1721-encoded resolvase: structure of the tnpR gene and its in vitro functions. Molec. Gen. Genet. 200, 176–181 (1985). https://doi.org/10.1007/BF00383332
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00383332