SHORT COMMUNICATION Restriction FRANC•
Map of Tn7
VICTOR NORRIS, AND JEAN BREVET
Institut de Microbiologic. Brit. 409 Universiti de Paris-&d. 91405 Orsay Cidex, France Received December 16, 1982; revised March 8, 1983 Tn7, a transposon of 14 kb, encodesresistanceto trimethoprim (Tp) and streptomycin (Sm). A cleavagesite map of this transposon for twenty-two different restriction enzymes as determined by comparison of restriction enzyme cleavage patterns of the plasmids ColE I and ColE I : :Tn7 is presented. The precise localization of these sites was facilitated by the use of two deletion derivatives of ColEl::Tn7: pGB2 and ColEl::Tn7A6, and by the use of pOB14 and pOBlS which contain a part of Tn7 cloned into the plasmid pBR322. This map should aid in the study of the structural and genetic organization of this transposon.
Escherichia coli strain C600 (thr, leu, thi) was generally used unless mentioned otherwise. GM33 (8) is a dam (DNA adenine methylation) mutant of C600. The plasmid ColEI came from S. N. Cohen. ColEl : :Tn7 was a gift from P. Courvalin, but originally came from N. Datta’s laboratory. In vitro deletions of ColE 1: :Tn 7 produced ColE 1: :Tn 7A6 when Hind111was used, and pGB2 when EcoRI was used (Fig. 1). The Hind111 fragments of Tn7, HI and H2, were inserted in the Hind111 site of pBR322 to give the recombinant plasmids pOB14 and pOB15, respectively (9) (Fig. 1). The growing of strains, the purification of plasmids, the transformation procedure, and the extraction of colicine E 1 were previously described (10). Restriction enzymes, obtained from the Boehringer Corporation Ltd. (London) or from New England Bio-Labs, were used in accordance with the suppliers’ recommendations. Digestion patterns of DNA by restriction enzymes were analyzed by electrophoresis on either agarose gel (0.7 or 1%) or polyacrylamide gel (5 to 10%) (II), depending on the size of the fragments being studied. Electrophoreses were performed in Tris-borate buffer (12). Molecular weight markers were the restriction fragments of bacteriophage X (13) or the restriction fragments of pBR322 (14). When necessarythe photographic negatives of gels were analyzed using
Tn7 is a transposon conferring resistance to trimethoprim (Tp)’ and streptomycin (Sm). Initially known as TnC, it was first discovered in the plasmid R483 (1) and its length was estimated at 9.6 * IO6 Da. The use of Tn7 has facilitated the construction of a genetic map of the RP4 plasmid (2,J) and has contributed to the study of the functional organization of the Ti plasmid of Agrobacterium tumefaciens (4-6). Tn7 insertions in the receiver plasmids have been localized by restriction enzyme fragmentations using a map of Tn7 in which only a few sites were positioned relative to one another: the EcoRI site, the two BamHI sites, and the three Hind111sites. The location of the EcaRI site permits the division of Tn7 into two different segments.The shorter one, designatedTn7-L, corresponds to the distance betweencoordinates 0 and 2.7 MDa in Barth’s map (2), and contains the genes conferring resistanceto Tp and Sm; the longer one, Tn 7R, carries the functions required for transposition of Tn7 (( 7) and unpublished results). This paper reports the construction of a cleavage map of Tn7, with a variety of restriction enzymes, as a first step in the study of the genetic organization of this transposon and in the sequencing of its termini. ’ Abbreviations used: Tp. trimethoprim; Sm, streptomycin. 96
0147-619X/83 $3.00 Copyright All
1983 by Academic
in any form
b Hind III C
FIG. I. Partial physical map of the various plasmids. -, Tn7 DNA; -, ColEl DNA; -, pBR322 DNA; I, part of Tn7 DNA removed in ColEl: :Tn7A6. (a) ColEl::Tn7. Map assignement on ColEI for colicine production (ceu), colicine immunity (imm), the mob genes,and the origin of replication (ori) are according to Bhagwat and Person (20). (b) pGB2; (c) pOB14; (d) pOB15. The relative orientation of HI and H2 in pBR322 can be deduced from the position of the C/n1 and BarnHI sites, respectively.
a Kipp and Zonnen D2 microdensitometer to determine the number of fragments of different origins in a single band. The insertion site and orientation of Tn7 in ColE 1 were determined by simple and double digestions with ClaI, HindIIi, EcoRI, and PstI. The asymmetric position of the unique EcoRI site in Tn7 was already known. These results made it possible to locate the insertion of Tn7 next to the unique C/a1 site, and at 2.3 kb from the unique EcoRI site of ColEl. Tn7 is oriented in ColEl in such a way that Tn7-L is closer to the origin of replication. The number of cleavage sites in Tn7 for each of a variety of restriction enzymes was determined by a comparison of the cleavage products of ColEl and ColE 1: :Tn7 DNA. Fifty-eight cleavage sites for 22 different restriction enzymes were located in Tn7. Four enzymes (SacII, SalI, SmaI, and XhoI) have no cleavagesite in Tn7. For the following enzymes, the number of sites in Tn7 are given in brackets; AvaI , AvaII , Bali , BamHI , BcA , [email protected]
, BgflI , BstEII , ClaI , EcoRI [l], &Zinc11, Hind111
, HpaI , KpnI [ 11,PstI [ 11,PvuI , Z’vuII , and Xba I . The precise relative locations of restriction enzyme cleavagesiteswere determined by a series of double or triple digestions.This study wasmade easierby using pGB2, pOB 14,and pOB 15, since each of them contains a different part of Tn7. Since the parts of Tn7 cloned in pOB 14 and pOB 15 have been deleted in ColEl : :Tn7A6, this plasmid was also useful. The cleavage sites for BcA were identified using plasmid DNA extracted from the damstrain GM33 since the Bcii recognition sequence 5’-TLGATCA-3’ includes the sequence GATC in which adenine residues are methylated by the dam gene product (15). This last sequence GATC partially overlaps in particular the ClaI recognition sequence SATICGAT-3’ and the XbaI recognition sequence 5’-TICTAGA-3’, depending on which base pairs follow the cleavage site; hence, a potential C/a1 or XbaI site can be masked in a dam+ strain (16,Z7). While the ColE 1: :Tn7 restriction pattern for XbaI is the same in a dam+ or a dam- strain, examination of the
SHORT COMMUNICATION Eglll
Xbal Hint 0
agl I Ava
I I I
2. The scale extracted HpaI site
Final cleavage map of Tn7 inserted in the plasmid ColEI. The heavy line represents Tn7 DNA. is given in kb. f, the AvuII site present only in ColEI ::Tn7. *, the C/al site only cut in the DNA from the dam- strain. One HpaI site has not been positioned, it is 70 bp away from another in Tn7-L.
ColE 1: :Tn7 digestion pattern with CfuI has revealed a new site positioned in Tn7-L (see Fig. 2). It is also of interest that an A&I site exists on the ColE 1 part of ColE 1: :Tn7 but not on ColE 1 itself (see Fig. 2). This phenomenon may be viewed as a case of microevolution of a plasmid (18). The Tn7 map generated by our studies is presented in Fig. 2. Complete documentation is on file at the editorial office of Plasmid or may be obtained from the authors. By adding up the lengths of the various restriction fragments, we estimate the length of Tn7 to be 14 kb. Sequence data (19) permitted us to locate the insertion of Tn7 at 20 bp from the CfuI site in ColEl and, using the easily calculated pGB2 length of 7.3 kb, a length of 5 kb for Tn7-L is suggested. The functions of Tn7 are now being localized using the map presented here; at least one function has already been revealed by the
fact that Tn7A6 can only transpose when complemented by a wild-type Tn 7 (( 7) and unpublished results). ACKNOWLEDGMENTS We are grateful to P. Schaeffer for critical reading of the manuscript and P. Courvahn for his generous gift of a strain containing ColEl::Tn7. We are indebted to D. Borowsky for her diligent assistance. F.G.-T. was the recipient of Predoctoral Fellowship 81373 from the Del& gation GenCrale a la Recherche Scientifique et Technique (DGRST). This work was supported by the Centre National de la Recherche Scientifique (L.A. 136) (France).
REFERENCES I. BARTH, P. T., DATTA, N., HEDGES, R. W., AND GRINTER,N. J., J. Bucteriol. 125,800-8 10 (1976). 2. BARTH, P. T., AND GRINTER, N. J., J. Mol. Biol. 113,455-474 (1977). 3. BARTH, P. T., GRATER, N. J., AND BRADLEY, D. E., J. Bucteriol. 133, 43-52 (1978).
SHORT COMMUNICATION 4. HERNALSTEENS, J. P., DE GREVE, H., VAN MONTAGU, M., AND SCHELL, J., Plasmid 1, 2 18-225 (1978). 5. HOLSTERS, M., SILVA, B., VAN VLIET, F., GENETELLO, C., DE BLOCK, M., DHAESE, P., DEPICXER, A., INzB, D., ENGLER, G., VILLARROEL, R., VAN MONTAGU, M., AND SCHELL, J., Playmid 3,212-
230 (1980). 6. DEGREVE, H., DECRAEMER, H., SEURINCK, J., VAN MONTAGU, M., AND SCHELL, J., Plasmid 6,235-
248 (1981). 7. SHERRATT, D., ARTHUR, A., AND BURKE, M., Cold Spring Harbor Symp. Quant. Biol. XLV, 275-28 I
(1980). 8. MARINIJS, M. G., AND MORRIS, N. R., J. Bacterial. 114, 1143-I 150 (1973).
9. OUARTSI, A., These de 3eme cycle, No. 2962, UniversiG de Paris-Sud, 198 1.
10. HASSAN, D. M., AND BREVET, J., Plasmid 10, 3144 (1983). 11. MANIATIS, T., JEFFREY, A., AND VAN DE SANDE, H., Biochemistry 14, 3787-3794 (1975).
12. MC DONELL, M. W., SIMON, M. N., AND SIIJDIER, F. W., J. Mol. Biol. 110, 119-146 (1977). 13. DANIELS, D. L., DEWET, J. R., AND BLATTNER, F. R., J. Viral. 33, 390-400 (1980). 14. S~TCLIFFE, J. G., Nucl. Acids. Res. 5, 2721-2728
(1978). 15. GEIER, G. E., AND MODRICH, P., J. Biol. Chem. 254, 1408-1413 (1979). 16. BACKMAN, K., Gene 11, 169-171 (1980). 17. MC CLELLAND, M., Nuci. Acid. Res. 9, 5859-5866
(1981). 18. COHEN, S. N., BREVET, J., CABELLO, F., CHANG, A. C. Y., CHOU, J., KOPECKO, D. J., KRETS~HMER, P. J., NISEN, P., AND TIMMIS, K., In “Microbiology” (D. Schlessinger ed.), pp. 2 17-220. American Society for Microbiology, Washington, D.C., 1978.
19. GOSTI-TESTU, F., AND BREVET, J., C. R. Acad. Sci. Paris Ser. III 294, 193-196 (1982). 20. BHAGWAT, A. S., AND PERSON, S.. Mol. Gen. Genet. 182, 505-507 (1981).