Plasmid Genomes (DNA/Restriction Endonuclease/Incompatibility/R-Plasmids/Heteroduplex Analysis)

Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2242-2246, June 1975

Cloning, Isolation, and Characterization of Replication Regions of Complex Plasmid Genomes (DNA/restriction endonuclease/incompatibility/R-plasmids/heteroduplex analysis)

KENNETH TIMMIS, FELIPE CABELLO, AND STANLEY N. COHEN Department of Medicine, Stanford University School of Medicine, Stanford, California 94305 Communicated by Allan Campbell, April 4, 1975

ABSTRACT EcoRI endonuclease-generated DNA frag- to chloramphenicol (Cm), kanamycin (Km), streptomycin ments carrying replication regions of the F'lac and R6-5 (Sm), and sulfonamide (Su). The pSC102 plasmid was con- plasmids have been cloned and isolated, using as a selection vehicle a nonreplicating ampicillin-resistance DNA structed by in vivo ligation of EcoRI-treated R6-5 DNA, con- fragment derived from a Staphylococcus aureus plasmid. sists of three EcoRI-generated fragments of R6-5, and ex- Heteroduplex analysis of the constructed plasmid chimeras presses Km and Su resistance (11). Plasmid pSC101 codes for and the parent replicons has localized the cloned R6-5 resistance to tetracycline (Tc) (9). Plasmid pSC113 was replication region to a DNA segment between kilobase pair coordinates 1.0 and 88.0 on the R6-5 map. Physical constructed in vitro (6) and contains the entire pSC101 plas- proximity between the plasmid replication functions and mid plus two EcoRI-endonuclease-generated fragments of the locus governing plasmid incompatibility has been the S. aureus penicillinase plasmid pI258 (12); it codes for shown for both parent replicons. The cloning method resistance to penicillin-ampicillin and Tc. Plasmids R100-1 reported appears to be generally applicable for the identi- R192-F7 13 and 14, kindly provided by K. Hardy) fication and isolation of replication regions of a variety of and (refs. complex genomes. are derepressed fertility mutants of R100 and R192, and express resistance to Tc, Cm, Sm, and Su. F'lac is the classic Rapid progress has taken place recently in the study of DNA Paris F' plasmid and was obtained in strain DF109 (= bromo- synthesis involving small replicons such as simian virus 40 (1), deoxyuridine-resistant isolate of DF87, ref. 15) from D. Frei- the bacteriophages M13 and 4X174 (1, 2), and the colicin El felder via R. P. Silver. plasmid (Col El) of Escherichia coli (3, 4). In contrast, bio- The procedures used for conjugal transfer (16) and trans- chemical investigations of the replication of large replicons formation of plasmid DNA (17), radioactive labeling and iso- such as the E. coli chromosome, the sex plasmid F, and some lation of plasmids (18), sucrose and CsCl gradient centrifuga- antibiotic resistance (R) plasmids have been hindered by the tion (5), construction of hybrid plasmid DNA molecules by genetic complexity and structural fragility of these DNA means of the EcoRI restriction endonuclease (11), and agarose molecules. The recent demonstration that recombinant plas- gel electrophoresis (5) have been described. Plasmid hetero- mids are capable of containing and utilizing at least two dis- duplex analysis procedures have been described by Sharp et al. tinct sets of replication functions (5) has made evident still (19). The EcoRI restriction endonuclease was purified from other potential difficulties in investigating the replication of E. coli strain RY-13 according to Greene et al. (20) through large genomes. the phosphocellulose chromatography step. E. coli DNA ligase In this report, we describe the isolation and characteriza- was generously provided by S. Panasenko, P. Modrich, and tion of replication regions of the E. coli plasmids R6-5 and I. R. Lehman. F'lac using an EcoRI-generated fragment of a Staphylococcus aureus plasmid as a selection vehicle. The staphylococcal RESULTS plasmid DNA fragment, which carries genetic information for Isolation of the Selection Vehicle. Chang and Cohen re- penicillin-ampicillin (Ap) resistance, and which apparently cently described the in vitro construction of a Tc and Ap lacks an origin of replication in its original host (R. P. Novick, resistance plasmid chimera, pSC113, that contains the entire personal communication), is capable of propagation in E. coli pSC101 plasmid replicon plus two EcoRI-generated frag- only when linked to another EcoRI DNA fragment carrying ments of the penicillin-ampicillin resistance staphylococcal functions required for replication in this bacterial host (ref. plasmid pI258 (6). Cleavage of the pSC113 plasmid chimera 6 and A. C. Y. Chang and S. N. Cohen, unpublished data). with the EcoRI endonuclease, ligation of the resulting frag- Genetic and molecular investigations of the cloned E. coli ments, and transformation of E. coli with the ligated mixture plasmid replication region fragments demonstrate a physical yielded another plasmid (pSC122) that also expressed resis- proximity between plasmid replication origins, replication tance to both Tc and Ap, but which contained only one of the genes, and incompatibility determinants. two EcoRI-generated fragments of Staphylococcus plasmid MATERIALS AND METHODS DNA originally present in pSC113 (Fig. 1A). This Ap frag- two EcoRI K-12 C600 (7) and nalidixic-acid re- ment, which is the larger of the staphylococcal Escherichia coli strain fragments contained in pSC113, has a buoyant density in CsCl sistant (nalr) mutants of strains CRT46 (8) and CR34 (9) Because of the substantial difference expresses resistance of 1.692 g/cm3 (Table 1). have been described. Plasmid R6-5 (10) in the buoyant density of the Ap fragment and the buoyant = two Abbreviations: Ap, ampicillin-penicillin; Cm, chloramphenicol; density of the pSC101 plasmid (p 1.710 g/cm3) the Km, kanamycin; Sm, streptomycin; Su, sulfonamide; Tc, tetra- DNA species can be separated easily by preparative cen- cycline; CCC, covalently closed circular; kb, kilobase; M.W., trifugation of EcoRI-cleaved pSC122 plasmid DNA in CsCl molecular weight. gradients (Fig. 1B). 2242 Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Cloning of Replication Regions 2243

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FIG. 2. Analysis of R6-5-Ap and F'lac-Ap hybrid plasmids by 0 10 20 30 agarose gel electrophoresis. CCC plasmid DNA preparations FRACTION NUMBER were cleaved to completion with EcoRI endonuclease and sub- jected to electrophoresis through 0.8% agarose slab gels as FIG. 1. Isolation of staphylococcal Ap-resistance DNA. (A) indicated in Fig. 1. Fragments of all plasmids are numbered Construction of pSC122 plasmid. One microgram of pSC113 from top to bottom in the gels, as was described previously (11). DNA in 50 ,u of a solution of 20 mM Tris.HCl pH 8.0, 1 mM (A) 1, R6-5 + pSC135; 2, pSC135; 3, R6-5; 4, PSC136; 5, Ap Na2EDTA, 10 mM MgCl2 was digested for 30 min with 1 uA of fragment; 6, pSC139; 7, pSC102; 8, R6-5 + pSC102. (B) 1, EcoRI endonuclease prepared as previously described (5). The pSC138; 2, pSC137; 3, F'lac; 4, pSC140; 5, Ap fragment; 6, nuclease was then inactivated by incubation for 5 min at 600 and pSC141. The pSC140 and pSC141 plasmids contain one or more the DNA mixture was ligated by addition of (NH4)2SO4 (to 10 other fragments of F'lac in addition to the replication region mM), NAD (to 100 uAM), bovine serum albumin (to 100 ug/ml), fragments. and 5 units of DNA ligase. After incubation at 140 overnight the mixture was used to transform cells of E. coli K12 C600. Cova- lently closed circular (CCC) plasmid DNAs prepared from transformant clones that expressed Ap-resistance were analyzed pSC122 plasmid were separately mixed with equal amounts of by electrophoresis of their EcoRI digestion products through an EcoRI-cleaved R6-5 or F'lac plasmid DNA and ligation and 0.8% agarose gel in Tris-borate buffer (5). One Ap-resistance transformation were carried out as described in Materials and plasmid, designation pSC122, contained only one of the two Methods and Fig. 1. Covalently closed circular (CCC) plas- original staphylococcal EcoRI endonuclease cleavage products mid DNA samples isolated from 10 separate Ap-resistant of the parent pSC113 plasmid. Fig. 1A is a photograph of an clones obtained by transformation with the R6-5 ligation ethidium bromide-stained agarose gel containing the following mixture were treated with EcoRI endonuclease and examined EcoRI endonuclease-cleaved plasmids: 1, pSC101; 2, pSC113; by agarose gel electrophoresis; several representative plas- 3, pSC122; and 4, Ap DNA fragment isolated as described in 1B below. mids are shown in Fig. 2A. All plasmid DNA preparations (B) Isolation of Ap fragment DNA by buoyant density cen- had in common a single EcoRI fragment of the R6-5 plasmid trifugation. Thirty-three micrograms of [3H]thymidine-labeled in addition to the Ap fragment; one of the clones (pSC136) pSC122 CCC-DNA (specific activity = 2.5 X 103 cpm/pg) were contained a second R6-5 DNA fragment (fragment XI, ref. cleaved with EcoRI enzyme as described above, dialyzed 4 hr 11) which was not essential for replication of the chimera. against a solution of 10 mM Tris HCl, pH 8.0, 1 mM EDTA Because fragments II and III of R6-5 have almost identical (TE buffer) to remove NP40 detergent present in the enzyme mobilities in agarose gels, electrophoresis of a mixture of preparation, and then mixed with TE buffer to give a final volume EcoRI-cleaved pSC135 and R6-5 DNA (Fig. 2A-1) was car- of 8 ml. Solid CsCl was added (refractive index = 1.3985) and ried out to identify the R6-5 DNA fragment that enables the solution was centrifuged in a bovine serum albumin-coated replication of the Ap fragment in E. coli. Mobility measure- centrifuge tube in a 50 Ti rotor at 36,000 rpm for 60 hr at 200. 1 2A Fractions were collected from a hole pierced in the bottom of the ments, using the other R6-5 bands present in gel of Fig. tube and the radioactivity (0) in an aliquot of each fraction was as internal standards, identified the band having increased measured and the refractive index was determined. Fractions fluorescence intensity as EcoRI fragment II of R6-5. A similar indicated by the shaded area were pooled and dialyzed ex- co-electrophoresis experiment indicated that R6-5 fragment haustively against TE buffer. This material was analyzed by II, and not the slightly smaller fragment III as was previously agarose gel electrophoresis (Fig. 1A). About 10 jg of Ap fragment believed (11), is contained also in the pSC102 plasmid (Fig. DNA was recovered. 2A-8). Parallel experiments indicated that a single EcoRI fragment of F'lac (fragment VI of the parent plasmid) was Isolation of Replication Regions of the R6-5 and F'lac Plas- common to all of the F'lac-Ap plasmid chimeras isolated (Fig. mids. Aliquots (1.5,ug) of Ap fragment DNA purified from the 2B); several of the chimeras contained various other F'lac Downloaded by guest on September 26, 2021 2244 Biochemistry: Timmis et al. Proc. Nat. Acad. Sci. USA 72 (1975) TABLE 1. Properties of plasmids

Molecular Buoyant density (g/em3) Molecular weight" Copy Plasmid complete EcoRI fragments 8e(S) lengthb (X 106) no.d Phenotype" pSC122 1.703 1.710, 1.692 32 15.1 i 0.5 10.0 6-8 Tcr, Apr, Tra-, SUPd..A-, AOr F'lac 1.711 85 143.3 95 0.3-0.8 Lac+, Tra+(drd)SupdA+, AO", cllr pSC138 1.702 1.709, 1.692 31 J4.2 :1: 0.5 9.4 0.6-1.0 Apr, Tra-, SUpdRZA+, AOS, 410 R6-5 1.711 75 98.5 65 0.7-3.0 Cmr, Smr, Sur, Kmr, Tra+, SUPdnA +, AOr pSC135 1.705 1.712,1.692 35 17.8 i 1.2 11.8 2-5 Apr, Tra , SUPd..A+, AOr

a Sedimentation coefficient, calculated by sucrose gradient-velocity centrifugation as previously described (5). b Molecular contour length in kilobase (kb) pairs. Double-stranded DNA of phage PM2 (8.92 kb) (27) was used as an internal reference. F'lac and R6-5 values are from refs. 19 and 28. The length of the Ap DNA fragment was determined to be 6.4 kb. ¢ F'lac and R6-5 from refs. 19 and 21. d Expressed per genome equivalent, assuming a molecular weight of 2.5 X 109 for the E. coli chromosome (30). Values obtained by centrifugation of Sarkosyl "whole" lysates to equilibrium in CsCl density gradients in the presence of ethidium bromide (5) and by sedimentation of Brij 58 "cleared" lysates through neutral sucrose gradients (18) were in close agreement. e Tra, ability of the plasmid to transfer itself to recipient bacteria by conjugation; drd, derepressed transfer; SUPdnaA, ability to suppress the dnaA mutation in bacteria grown under non-permissive conditions, presumably by integrative suppression (31); AOs/r, sensitivity/ resistance of plasmid replication to sub-lethal amounts of acridine orange (32), 4JIIr sensitivity resistance to the female-specific bacteriophage 4II.

EcoRI fragments in addition to the one carrying the replica- Heteroduplex Analysis of the pSC13S5, pSC138, and pSC102 tion functions for the plasmid. Plasmids. The location of the R6-5 DNA fragment containing The R6-5-Ap plasmid, pSC135 [molecular weight (M.W.) the cloned replication region of this plasmid was identified by 11.8 X .106] and the F'lac-Ap plasmid pSC138 (M.W. 9.4 X examination of the R6-5/pSC135 heteroduplex (Fig. 3). Two 106), containing the replication region fragments of the re- previously reported inverted repeats on the R6-5 plasmid (21) spective parent plasmids were selected for further study. enabled precise localization of the region of homology. The Table 1 lists some molecular and biological properties of these segment of R6-5 contained in the R6-5-Ap constructed plas- plasmids. The chimeric molecules, unlike the parent replicons, mid chimera is 11.5 4 0.7 kilobase (kb) pairs in length, and is

are nonconjugative plasmids and do not express either the located between coordinates 1.0 kb and 88.0 kb on the R6-5 antibiotic resistances of R6-5 or the lactose utilization of F'lac. map (Fig. 3B). It thus is contained in the resistance transfer However, both types of chimeras have replication properties unit (RTF) region of the plasmid. The pSC135/pSC102 that are characteristic of the parent plasmids: (a) they sup- heteroduplex (Fig. 3C) confirms agarose gel electrophoresis press the dnaA mutation in bacteria grown under nonpermis- data (Fig. 2) indicating that these two plasmids contain the sive conditions; (b) replication of F'lac and pSC138 is sensi- same replication region fragment of R6-5; the entire 11.5 kb tive to acridine orange, whereas that of R6-5 and pSC135 is segment of pSC135 derived from the R6-5 parent is present not; (c) although the chimeric plasmids are much smaller also in pSC102. Furthermore, the pSC135/pSC102 hetero- than the parent replicons from which they were derived, duplex demonstrates that the three EcoRI fragments com- they are maintained by host bacteria at the same copy num- prising pSC102 are not contained contiguously in the R6-5 ber as the parent plasmids. plasmid, since the reverse repeat IR2 is located 20 kb and

TABLE 2. Compatibility of hybrid plasmids with related and unrelated replicons

C600 C600 C600 C600 C600 CR34 CR34 DNA Selection C600 (pSC101) (F'lac) (pSC135) (pSC102) (R6-5) (R100-1) (R192-F7) pSC122 I 2.9 X 105 - 1.4 X 106 - 2.2 X 105 3.1 X 106 1.2 X 105 2.1 X 10O I + R - 1.4 X 105 2.6 X 105 3.0 X 105 1.0 X 105 1.7 X 106 incompatibility index - - 1.0 0.85 1.0 1.2 1.2 pSC138 I 1.2 X 105 1.9 X 105 7.6 X 10- 2.5 X 103 8.4 X 103 - - I + R 1.9 X 105 1.9 X 101 2.5 X 103 8.3 X 10- - incompatibility index - 1.0 42 - 1.0 1.0 pSC135 I 1.9 X 106 2.4 X 105 1.1 X 106 - 2.4 X 104 5.3 X 104 2.4 X 102 1.5 X 103 I + R - 2.0 X 10 1.1 X 106 - 3.0 X 101 1.4 X 103 6 4.8 X 101 incompatibility index 1.2 1.0 - 667 38 40 31 pSC102 I 2.6 X 106 2.0 X 10 - 9.8 X 103 - 3.4 X 103 1.3 X 103 I + R - 2.4 X 105 1.2 X 101 - - 9.6 X 101 3.6 X 101 incompatibility index 0.8 - 817 35 36

Transformation was carried out as described (17). Recipient bacteria were selected for the expression of antibiotic resistance determinants carried by either incoming (I) plasmid DNA, or by both incoming and resident (I + R) plasmid DNAs. The numbers of trans- formants per yg of DNA are given in the table. The incompatibility index (given in italics) is the ratio I/(I + R); it is about 1.0 for compatible replicons and greater than 1.0 for incompatible plasmids. Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Cloning of Replication Regions 2245

63.9 kb from the ends of EcoRI fragment II in the complete R6-5 plasmid and is situated 1.7 kb and 7 kb from the ends of this fragment in pSC102. Thus, formation of the pSC102 plasmid necessarily occurred by intracellular ligation of separate DNA fragments, and not simply by in vivo recir- culation of a single DNA segment containing the three EcoRI fragments. Direct examination of the pSC102/R6-5 heteroduplex (data not shown) confirmed this interpretation. The only region of homology seen in fifteen pSC135/pSC138 heteroduplexes (e.g., Fig. 3D) is the 6.4 kb Ap fragment, indicating that the EcoRI replication region fragments of R6-5 and F'lac contain dissimilar base sequences. This is consistent with the observed differences in replication-associ- ated properties shown by F'lac and R6-5 (copy number, acridine orange sensitivity, compatibility; Table 1 and ref. 22). The F'lac/pSC138 heteroduplex (Fig. 3E) shows a region of homology 7.8 kb in length, which corresponds to the size estimated by gel electrophoresis for the EcoRI replication region fragment cloned from the F'lac plasmid (M.W. 5 X 106). Compatibility Studies. Although incompatibility between bacterial plasmids has been considered to be a property of ('Sl1b REP jIR1 (ISl)a IIR2{Sl)b I= I /MEE \ /\/\ I plasmid replication (23), direct evidence for a structural 2.2 1.3 1.0 88.0 55.0 44.1 28.0 27.2 2.2 1.3 interrelationship between these separate functions is lacking. 0/98.5 24.1 21.0 __ RTF - r-DETERMINANT Using constructed Ap resistance plasmid chimeras containing the replication regions of R6-5 or F'lac, we have investigated FIG. 3. Heteroduplex analysis of R6-5-Ap and F'lac-Ap compatibility of the plasmid chimeras with those plasmids hybrid plasmids. Standard procedures for plasmid DNA hetero- from which their replication functions have been derived duplex analysis and electron microscopy were followed (19). (Table 2). Incompatibility was studied by transformation OX174 single-stranded and PM2 duplex DNA were used as of the R6-5-Ap plasmid chimera pSC135, or the F'lac-Ap internal standards for molecular length measurements (19, 27). plasmid chimera pSC138 into bacteria containing the parent The bar in each part of the figure represents 1 kb. Arrows indicate or a plasmid related to it, and was measured by the frequency the junctions of single-strand (SS) and double-stranded (D) of expression of genes carried by the incoming plasmid in the regions of the heteroduplexes. (A) Heteroduplex between pSC135 and R6-5 plasmids. The region of homology is 11.5 kb in length presence or in the absence of selection for determinants car- and is located between 1.0 and 88.0 kb on the standard R6-5 ried by the resident plasmid. physical map [shown in (B); see refs. 28 and 29]. A large single- The pSC122 plasmid, which provided the Ap fragment for stranded substitution loop (SS2) containing the two inverted these plasmid chimeras, is seen to be compatible with F'lac, repeats IR1 and IR2 represents the R6-5 segment absent in the R6-5, R100-1, R192-F7, and pSC102 plasmids, and hence the pSC135 plasmid. The smaller substitution loop (SS1) represents Ap fragment does not contribute to incompatibility. In con- the Ap-DNA fragment contribution to pSC135. (B) Map of trast, the R6-5-Ap plasmid pSC135 is incompatible with R6-5 showing the location of the cloned replication region. (C) pSC102, R6-5, and other large plasmids (R100-1, R192-F7) The pSC135/pSC102 heteroduplex. The nonhomologous regions (Table 2) of the same incompatibility group as R6-5 (FII, ref. comprising the Ap-DNA fragment (SS3) and the segment of 22, and N. Datta, personal communication). The lowest incom- pSC102 (SS4) containing IR2 are indicated. (D) pSC135/pSC138 heteroduplex. The duplex region represents the Ap DNA frag- patibility ratios (about 40) were observed between pSC135 (or ment common to both plasmids. The replication region frag- pSC102) and the R6-5, R100-1, and R192-F7 plasmids, which ments of R6-5 (SS5) and F/lac(SS6) are indicated. (E) pSC138/ appear to contain more than one set of replication functions F'lac heteroduplex. The region of homology is 7.8 kb, which (11, 24, 25). The greatest incompatibility ratio (about 800) corresponds to the length of EcoRI fragment VI of F'lac. The was observed between pSC135 and pSC102. The pSC138 single-strand substitution loops SS7 and SS8 are Ap DNA plasmid, which contains a replication region from F'lac, is fragments and the nonhomologous segment of F'lac, respec- entirely compatible with plasmid R6-5 and its derivative tively. pSC102, consistent with the absence of nucleotide sequence homology in the replication regions of the R6-5-Ap and F'lac- plasmids, as determined by examination of CCC-DNA, did Ap chimeras (Fig. 3D). As expected, pSC138 is incompatible not occur in cells carrying compatible plasmid replicons. with the F'lac plasmid. Measurements of incompatibility, which used a standard DISCUSSION conjugation method (16) and which employed nonconjugative The Ap-resistance EcoRI staphylococcal plasmid DNA frag- plasmids as resident replicons, yielded data which supported ment used in these experiments has particular advantages the interpretations derived from Table 2. In the absence of as a probe and vehicle for selection and cloning of replication continued selection for antibiotic resistance determinants regions in E. coli. Because its buoyant density is substan- carried by both plasmids, rapid segregation occurred in in- tially different from the buoyant density of pSC101 DNA, the stances where a high incompatibility index was observed. fragment can be prepared in large quantities by CsCl den- However, in both transformation and conjugation experi- sity gradient equilibrium centrifugation of the EcoRI-cleaved ments, detectable segregation of or recombination between composite plasmid pSC122. The fragment can be separated Downloaded by guest on September 26, 2021 2246 Biochemistry: Timmis et al. Proc. Nat. Acad. Sci. USA 72 (1975) from cloned E. coli plasmid replication region fragments by 1. Kasamatsu, H. & Vinograd, J. (1974) Annu. Rev. Biochem. the same procedure, thus permitting the isolation of large 43, 695-719. amounts of replication 2. Schekman, R., Weiner, A. & Kornberg, A. (1974) Science region DNA for physical character- 186, 987-993. ization and in vitro studies. While the level of ampicillin 3. Sakakibara, Y. & Tomizawa, J.-I. (1974) Proc. Nat. Acad. resistance expressed by the staphylococcal DNA fragment in Sci. USA 71, 802-806. E. coli (minimum inhibitory concentration, 200 ,g/ml) is 4. Lovett, M. A., Katz, L. & Helinski, D. R. (1974) Nature substantially lower than the levels achieved by common 251, 337-340. 5. Timmis, K., Cabello, F. & Cohen, S. N. (1974) Proc. Nat. penicillin-ampicillin resistance plasmids indigenous to and Acad. Sci. USA 71, 4556-4560. widely distributed among E. coli (Cabello and Cohen, in 6. Chang, A. C. Y. & Cohen, S. N. (1974) Proc. Nat. Acad. Sci. preparation), it is sufficient for the selection of replication re- USA 71, 1030-1034. gions as described here. Although earlier results, and those 7. Bachman, B. J. (1972) Bacteriol. Rev. 36, 525-557. presented in Table 2, suggest that at least two separate 8. Hirota, Y., Mordoh, J. & Jacob, F. (1970) J. Mol. Biol. 53, replication regions are located on R6-5 and related plasmids 369-387. 9. Cohen, S. N. & Chang, A. C. Y. (1973) Proc. Nat. Acad. (11, 24, 25), all of the plasmid chimeras cloned in these studies Sci. USA 70, 1293-1297. by the use of the Ap fragment selection vehicle contain a 10. Silver, R. P. & Cohen, S. N. (1972) J. Bacteriol. 110, 1082- unique EcoRI fragment. This finding suggests that essential 1088. components of other R6-5 replication region(s) may be dis- 11. Cohen, S. N., Chang, A. C. Y., Boyer, H. W. & Helling, on R. B. (1973) Proc. Nat. Acad. Sci. USA 70, 3240-3244. tributed separate EcoRI fragments of the R6-5 plasmid. 12. Peyru, G., Wexler, L. F. & Novick, R. P. (1969) J. The use of other restriction endonucleases for cloning replica- Bacteriol. 98, 215-221. tion regions of R6-5 should permit investigation of this pos- 13. Nishimura, Y., Ishibashi, M., Meynell, E. & Hirota, Y. sibility. (1967) J. Gen. Microbiol. 49, 89-98. The demonstration that a 5.2 megadalton fragment of F'lac 14. Meynell, E. & Cooke, M. (1969) Genet. Res. 14, 309-313. 15. Freifelder, D. R. & Freifelder, D. (1968) J. Mol. Biol. 32, DNA and a 7.6 megadalton fragment of the R6-5 plasmid 15-23. carry all of the functions required for replication of a DNA 16. Timmis, K. (1972) J. Bacteriol. 109, 12-20. fragment in E. coli indicates that the replication origin and 17. Cohen, S. N., Chang, A. C. Y. & Hsu, L. (1972) Proc. Nat. the replication genes of the F'lac and R6-5 plasmids are clus- Acad. Sci. USA 69, 2110-2114. tered together in a small region of the genomes of these plas- 18. Clewell, D. B. & Helinski, D. R. (1969) Proc. Nat. Acad. Sci. USA 62, 1159-1166. mids; similar clustering of replication origin and replication 19. Sharp, P. A., Hsu, M.-T., Ohtsubo, E. & Davidson, N. genes has been observed in the genome of the bacteriophage X (1972) J. Mol. Biol. 71, 471-497. (26). 20. Greene, P. J., Betlach, M. D., Goodman, H. M. & Boyer, H. The ability to clone specific segments of complex genomes W. (1974) in Methods in Molecular Biology, ed. Wickner, R. B. (Marcel Dekker, Inc. New York), Vol. 9, pp. 87- that carry genetic information for particular biological func- 103. tions such as DNA replication appears to be highly useful for 21. Sharp, P. A., Cohen, S. N. & Davidson, N. (1973) J. Mol. genetic and biochemical investigations of such functions. Biol. 75, 235-255. The current experiments report the selective cloning and 22. Willetts, N. (1972) Anau. Rev. Genet. 6, 257-268. study of EcoRI-generated replication region fragments of 23. Jacob, F., Brenner, S. & Cuzin, F. (1963) Cold Spring two R6-5 and F'lac. The methods Harbor Symp. Quant. Biol. 28, 329-348. large plasmid genomes, 24. Yoshikawa, M. (1974) J. Bacteriol. 118, 1123-1131. described are potentially applicable for the isolation of DNA 25. Rownd, R. H., Perlman, D. & Goto, N. (1974) in Micro- segments containing the replication origin and/or genes of biology-1974, ed. Schlessinger, D. (American Society for any complex replicon capable of functioning in bacteria and Microbiology), pp. 76-94. may be useful in the study of chromosome replication. The 26. Stevens, W. F., Adhya, S. & Szybalski, W. (1970) in The Bacteriophage Lambda, ed. Hershey, A. D. (Cold Spring sequestration of replication functions onto small plasmid DNA Harbor Laboratory, Cold Spring Harbor, N.Y.), pp. 515- molecules should facilitate in vivo and in vitro investiga- 533. tions of gene products involved in replication. With appro- 27. Espejo, R. T., Canelo, E. S. & Sinsheimer, R. L. (1969) priate modification, the procedure describe1 may also permit Proc. Nat. Acad. Sci. USA 63, 1164-1168. the isolation of DNA functions 28. Hu, S., Ohtsubo, E., Davidson, N. & Saedler, H. (1975) J. particular segments specifying Bacteriol., 122, 764-775. involved in the conjugal transfer of plasmids. 29. Ptashne, K. & Cohen, S. N. (1975) J. Bacteriol., 122, 776- These studies were supported by National Institute of Allergy 781. and Infectious Diseases Grant Al 08619, National Science 30. Cooper, S. & Helmstetter, C. (1968) J. Mol. Biol. 31, 519- Foundation Grant GB-30581, and American Cancer Society 540. Grant VC-139. K.T. is the recipient of a postdoctoral fellowship 31. Nishimura, Y., Caro, L., Berg, C. M. & Hirota, Y. (1971) from the Helen Hay Whitney Foundation. We thank J. Zabielski J. Mol. Biol. 55, 441-456. for technical assistance during part of this investigation. 32. Hirota, Y. (1960) Proc. Nat. Acad. Sci. USA 46, 57-60. Downloaded by guest on September 26, 2021