JOURNAL OF BACTERIOLOGY, Aug. 1976, p. 982-987Copyright © 1976 American Society for Microbiology

Vol. 127, No. 2Printed in U.S.A.

Method for the Isolation of the Replication Region of aBacterial Replicon: Construction of a Mini-F'km Plasmid

MICHAEL A. LOVETT AND DONALD R. HELINSKI*Department ofBiology, University of California, San Diego, La Jolla, California 92093

Received for publication 3 February 1976

A purified fragment of deoxyribonucleic acid (DNA) that determines resist-ance to kanamycin and is incapable of self-replication was used to select a self-replicating fragment from an EcoRI endonuclease digest of the sex factor F'lac.This F'lac fragment, exhibiting a molecular weight of 6 x 106, carries the genesessential for maintenance of the F replicon in Escherichia coli cells. The con-structed mini-F'km plasmid also retains the incompatibility properties of theparent F'lac plasmid. Large amounts of the kanamycin resistance fragment canbe obtained as a selective agent for this procedure from a hybrid plasmid thatwas constructed by combining the EcoRI-generated kanamycin resistance frag-ment of a molecular weight of 4.5 x 106 with an EcoRI-cleaved, self-replicatingderivative of colicinogenic plasmid El that has a molecular weight of 2.2 x 10".The recombinant plasmid is able to replicate extensively in E. coli in mediumcontaining chloramphenicol, and, therefore, large quantities of this plasmidDNA can be obtained. The substantial difference in size between the twofragments in the recombinant plasmidpreparative agarose gel electrophoresis.

greatly facilitates their separation by

Recently, the finding that cleavage of deoxy-ribonucleic acid (DNA) by restriction endonu-clease EcoRI generates cohesive termini (7, 13)has greatly facilitated the formation in vitro ofrecombinant plasmid DNA molecules. Theserecombinant plasmids have been established(1, 3, 9, 14) in Escherichia coli cells by transfor-mation (4). This report describes the construc-tion of two new plasmids. One plasmid, desig-nated pML21, consists of the 4.5 x 106-daltonfragment of the plasmid pSC105 (3), which de-termines resistance to kanamycin (Km) and isincapable of self-replication, and a mini-ColEl,a self-replicating derivative of colicinogenicplasmid El (ColEl) that exhibits a molecularweight of 2.2 x 106 (8). The recombinant plas-mid possesses the property of the ColEl plas-mid of replicating extensively in a medium con-taining chloramphenicol, making possible theisolation of large amounts of this plasmid DNA.The kanamycin resistance fragment of pML21was separated from the ColEl fragment by pre-parative agarose gel electrophoresis and used toselect a single EcoRI fragment containing aself-replicating region of the sex factor F'lac.The use of the kanamycin resistance fragmentof pML21 to select the replication segment of aplasmid element found in a gram-negative bac-terial species has two advantages over the useby the same procedure of an antibiotic resist-

ance fragment from a Staphylococcus aureusplasmid that has been reported previously (17).It avoids extending the range of antibiotic re-sistance genes in gram-positive bacteria to agram-negative species, and the kanamycin re-sistance fragment can be obtained pure and invery high amounts from the amplifiable mini-ColE1 hybrid plasmid. The mini-F'km plasmid,designated pML31, constructed by this methodis used in an accompanying paper (6) to localizethe F replication region on an F'gal sex factor.

MATERIALS AND METHODSBacterial strains are described in Table 1. ¢ll

bacteriophage was provided by M. Guyer. MS2 bac-teriophage was provided by V. Hershfield. The F'lacplasmid used in this study was obtained initiallyfrom 2310S from the collection of F. Jacob and trans-ferred to the C600 strain by conjugation by J. Col-lins. Preparation of supercoiled plasmid DNA bydye-buoyant density centrifugation was as describedpreviously (10, 11). A final concentration ofTriton of0.2% was used to prepare the cleared lysate. Trans-formation ofE. coli cells was performed as describedelsewhere (4). Cleavage of DNA with the restrictionendonuclease EcoRI was carried out as describedpreviously (9). The reaction was terminated by heatinactivation of the enzyme at 65°C for 10 min. DNAligase from T4 bacteriophage-infected E. coli cellswas purchased from Miles Laboratories, Elkhart,Ind. The buffer used for ligation contained 66 mMtris(hydroxymethyl)aminomethane(Tris), pH 7.5, 50

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CONSTRUCTION OF A MINI-F'Km PLASMID 983

TABLE 1. Bacterial strains

Strain PropertiesaHfrH Hfr (Hayes) thi-1C600 thr leu Thi- Lac-C600 recA trpR - thr leu Thi- Lac- trpR- trpEtrpE 10220 recA

CR34 Nalr thr leu Thi- Lac- Thy- NalrHB 101 thi- leu pro lac gal his hsr

hsm recA StrrJC 411 argG his leu metB lacY malA

xyl strA

a Genetic symbols are as given by Taylor andTrotter (16).

mM NaCl, 10 mM MgCl2, 10 mM dithiothreitol, and6 mM adenosine 5'-triphosphate. The ligation of theEcoRI digest of the F'lac plasmid and the purifiedKm fragment was carried out at 15°C for 3 h in atotal volume of 2 ml, containing equal amounts (8gg of each) of F'lac and Km DNA and 0.5 units ofT4DNA ligase. The ligation was terminated by theaddition of ethylenediaminetetraacetic acid (EDTA)to 25 mM. An appropriate amount of 0.1 M CaCl2was added to adjust the concentration of CaCl2 to0.03 M prior to transformation with this DNA sam-ple.

Agarose slab gel electrophoresis was performedbasically as described elsewhere (P. J. Greene, M.C. Betlach, H. M. Goodman, and H. W. Boyer, In R.B. Wickner [ed. ], Methods in Molecular Biology, vol.9). The Km fragment was separated from the mini-ColEl fragment by preparative agarose gel electro-phoresis. The electrophoresis apparatus consisted ofa plexiglass cylinder with an internal diameter of 10cm and a height of 15 cm. The cylinder contained acolumn of 0.8% agarose gel that was 8 cm in heightand was held in the apparatus by placement of di-alysis tubing across the bottom of the cylinder. Asample well of 8 cm in diameter was formed in thegel by positioning a plastic beaker approximately1.5 cm into the molten gel. The top 7 cm of theapparatus was covered with 500 ml of Tris-boratebuffer, and the apparatus was positioned on asponge in a lower buffer chamber that also con-tained 500 ml of Tris-borate buffer. The DNA sam-ple, containing up to 4.5 mg ofEcoRI-cleaved pML21DNA with 6% glycerol and 0.0016% bromophenylblue, was underlayered into the sample well, and acircular electrode was placed in the buffer above thesample. Electrophoresis was carried out at roomtemperature at 100 mA until the bromophenyl blueran off the gel (about 10 h). The gel was then stainedfor 5 min with ethidium bromide (0.5 gg/ml), andthe separated bands of DNA were sliced out of thegel. To recover the DNA, the gel slice was finelyminced with a scalpel and covered with 40 to 50 ml ofa buffer containing 100 mM Tris, pH 7.5, 50 mMNaCl, and 25 mM EDTA in a 250-ml plastic bottle. Amagnetic stirring bar was added, and the DNA waseluted from the gel by mixing at 37°C overnight. Theagarose was then pelleted by centrifugation for 5min at 5,000 rpm at 10°C in a Sorvall GSA rotor. The

recovery of the Km fragment from the gel was ap-proximately 40%. Solid cesium chloride (3.6 g) and0.3 ml of a 5-mg/ml solution of ethidium bromidewas added to each 3.8 ml of the supernatant, and theDNA solution was centrifuged to equilibrium in aSpinco Ti6O rotor at 38,000 rpm for 36 h at 15°C. Thisequilibrium centrifugation concentrated the KmDNA and removed small pieces of agarose remain-ing in the supernatant after the low-speed centrifu-gation step. The presence ofethidium bromide in thegradient allowed the visualization of the DNA bandwith shortwave ultraviolet light.

RESULTSConstruction of a mini-ColE1-Km recombi-

nant plasmid. Supercoiled DNA of plasmidpSC105, consisting of the 5.8 x 106-dalton plas-mid pSC101 and a 4.5 x 106;-dalton Km frag-ment derived from EcoRI-cleaved R6-5, was di-gested with EcoRI endonuclease and centri-fuged on a preparative, neutral 5 to 20% sucrosedensity gradient. The slowest sedimenting,one-third portion of the broad DNA peak ob-tained was precipitated with ethanol. After re-suspension in a buffer containing 20 mM Tris,pH 7.5, 20 mM NaCl, and 1 mM EDTA (TEN),an equal amount of EcoRI endonuclease-treated mini-ColEl DNA in TEN buffer wasadded. About 2 ug of this mixture was used totransform strain C600 trpR - trpE recA for kan-amycin resistance. Kanamycin-resistant colo-nies obtained from this transformation weretested for immunity to colicin El. Several ofthekanamycin-resistant and colicin El-trans-formed clones were examined further and foundto possess supercoiled DNA that sedimented asa 30S molecule. This supercoiled DNA yielded,upon treatment with EcoRI endonuclease, the4.5 x 106-dalton Km fragment and the 2.2 x 106-dalton mini-ColEl DNA (Fig. 10). This recom-binant plasmid was designated pML21. At leastseveral hundred copies of this plasmid accumu-late per chromosome per cell after incubation ofthe cells in the presence of chloramphenicol (8),as has been described for the ColE1-containingrecombinant plasmids PVH5 and pML2 (9).Construction of a recombinant plasmid

consisting of the replication region of F'lacand the Km fragment. Under no condition,including attempts to transform several E. colistrains to kanamycin resistance with the puri-fied Km fragment, has the Km fragment beenshown to be capable of self-replication. There-fore, this fragment, purified from pML21 asdescribed in Materials and Methods, was usedto select for the plasmid replication region pres-ent on one or more EcoRI-generated fragmentsderived from a plasmid element. The procedureused for the purification of the Km fragment

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A D Li

_-a

FIG. 1. Agarose slab gel electrophoresis ofEcoRI endonuclease-digested DNA samples. DNA samples (0.1to 1.0 Ag) contained 6% glycerol and 0.0016% bromophenyl blue in a volume of100 X. Electrophoresis was

carried out at room temperature for 100 min at 160 V. The agarose concentration was 0.8% and the Tris-borate buffer contained 10.8 g ofTris, 0.93 g ofEDTA, and 5.5 g ofboric acid per liter. The gel was stained inTris-borate buffer with 1.0 pg of ethidium bromide per ml and photographed with a shortwave ultravioletlight source. (A) Fragments of F'lac; molecular weights are approximately 13 (doublet), 7.7, 6.8, 6.2(doublet), 6.0, 5.4, 5.0, 3.4, 3.2 (doublet), 2.9, 1.7, 1.4, and 1.3 x 106, and 4.1, 3.1, and 2.9 x 105. (B)Fragments ofpML31; 6.0 x 106 and 4.5 x 106 molecular weight. (C) Fragments ofpML21; 4.5 x 106 and 2.2X 106 molecular weight. (D) Purified kanamycin fragment; 4.5 x 106 molecular weight. (E) X Fragments:molecular weights of 13.70, 4.68, 3.70, 3.56, 3.03, and 2.09 x 106 (9). The 3.70 X 106 and 3.56 x 106fragments appear as a single band under these conditions.

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CONSTRUCTION OF A MINI-F'Km PLASMID 985

gives little or no detectable contamination withthe DNA of the EcoRI-derived mini-ColEl frag-ment of pML21 (Fig. 1D). It was expected,therefore, that any kanamycin-resistant clonesobtained after transformation with the ligatedmixture of the Km fragment and EcoRI-gener-ated fragments derived from a replicon shouldhave resulted from the selection of an EcoRIfragment(s) possessing the essential propertiesof the replicon. Approximately 5 ,ug of the li-gated mixture of the EcoRI endonuclease-gen-erated fragments of the DNA of the sex factorF'lac, isolated from C600 recA trpR - trpE(F'lac), and the purified Km fragment ofpML21 was used to transform E. coli C600.Supercoiled plasmid DNA isolated from threekanamycin-resistant clones was found in eachcase to sediment as a 36S molecule in a neutralsucrose gradient and yield on EcoRI cleavagethe Km fragment and a 6 x 106-dalton frag-ment that is also generated by EcoRI digestionof the F'lac (Fig. lb). This recombinant plas-mid, containing the Km fragment and the seg-ment of the F'lac plasmid responsible for itsreplication, was designated pML31 and is alsoreferred to as mini-F'km.

Properties of pML31. The mini-F'km plas-mid does not possess properties associated withthe fertility of the sex factor F'lac. The conjugalmating of C600 (pML31) with JC411 did notresult in the transfer of pML31 under condi-tions where a frequency of transfer of 10-9 or

greater would have been detected, indicatingthat pML31 is non-self-transmissible. C600(pML31) also was found to be resistant to themale-specific MS2 phage and sensitive to thefemale-specific k11 phage (Table 2). The anti-biotic resistance plasmid R100-1 was able topromote the transfer of pML31 at low fre-quency. Under conjugal mating conditionswhere the R100-1 plasmid was transferred fromC600 (R100-1) (pML31) to a CR34 Nalr recipientat a frequency of 5 x 10-4, the pML31 plasmidwas transferred at a frequency of 1.5 x 10-l.The mini-F'km plasmid does retain the prop-

erty of incompatibility with F-prime plasmidsexhibited by the parent F'lac plasmid. WhenC600 recA trpR - trpE (F'lac) was mated withboth HB101 (pML31) and HB101, the number ofLac+ colonies appearing on MacConkey lactoseplates containing streptomycin as the counter-selective agent was approximately 110 of 210total colonies and 93 of 155 colonies, respec-tively, indicating the absence of surface exclu-sion of the parent F'lac in pML31. However,when the recipient clones of HB101 (pML31) onMacConkey lactose plates containing strepto-mycin were replica plated to MacConkey lac-

TABLE 2. Bacteriophage typing ofpML31lNo. of plaques

Strain o11 MS2

5 x lo-5 b 10 62.5x 10-8

HfrH 38 3 1 > 1,000C600 >1,000 >500 136 0C600 (pML31) >1,000 >500 115 0C600 recA trpR- 170 30 5 >1,000trpE (Flac)

C600 recA trpR- > 1,000 253 69 0trpEa A 0.1-ml amount ofan overnight L-broth culture

of each strain tested was added to 2.5 ml of melted0.7% L agar containing 2 x 10-3 M CaCl2. A 0.1-mlportion of each dilution of the bacteriophage prepa-ration was added, and the mixtures were pouredonto L-agar plates containing 2 x 10-3 M CaCl2,0.1% glucose, and 0.001% thymidine. Plaques werecounted after overnight incubation at 37°C.

b Dilution.

tose plates containing kanamycin and strepto-mycin, 58 of 61 Lac- colonies were replicated,but only 39 of 93 original Lac+ colonies werereplicated and each of these colonies was Lac-upon replica plating to MacConkey plates con-taining kanamycin. Fifteen Lac+ colonies fromthe mating were also transferred to MacConkeylactose-streptomycin plates and then replicaplated to minimal lactose plates and to Mac-Conkey lactose plates containing streptomycinand kanamycin. Fourteen of the 15 Lac+ colo-nies replicated to minimal lactose plates, and 8of the 15 replicated to the kanamycin plates,but each of these 8 colonies was found to beLac-. These data indicate that the mini-F'kmplasmid cannot stably coexist with F'lac.pML31 also retained the stringent control of

replication of F plasmids that limits their copynumber to one to two per bacterial chromo-some. The data presented in Table 3 show thatapproximately 0.59% of the DNA in C600(pML31) is in the form of 36S plasmid DNA.Assuming that the size of the E. coli chromo-some is 2.5 x 109 daltons, there are approxi-mately 1.4 copies of the 10.5 x 106-daltonpML31 per chromosome.

DISCUSSIONThe selection of a single EcoRI-generated

fragment from F'lac that serves as a molecularvehicle for the stable maintenance of the Kmfragment derived from pML21 indicates thatthe purified Km fragment can be used to selectEcoRI-generated fragments containing the es-sential components of a replicon. pML21 offers

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986 LOVETT AND HELINSKI

TABLE 3. Number of copies ofpML31 DNA perbacterial chromosomea% DNA sedimenting No. of cop-

Clearedly-36S DNA ~ies ofExpt sate (cpm); 36S DNA 36S DNA pML31/bac-

total lysate cleared ly-(cpm)/total terial(cpm) sate (cpm) lysate (cpm) chromosome

1 1.18 49 0.58 1.42 1.52 39 0.59 1.4

a C600 (pML31) spheroplasts labeled with[3H]thymine were lysed with Triton X-100 as de-scribed elsewhere (10, 11). The crude lysate wascentrifuged at 48,000 x g for 20 min at 0°C. Thecleared lysate was analyzed by centrifugation on 5 to20% neutral sucrose density gradients containing 50mM Tris, pH 7.5, 0.5 M NaCl, and 5 mM EDTA at50,000 rpm for 100 min at 15°C in a Spinco SW50.1rotor. Differentially labeled supercoiled ColEl DNA(23S) was included as a sedimentation reference.The percentage of labeled DNA in the gradient sedi-menting at the 36S position was calculated, and thenumber of copies of pML31 per bacterial chromo-some was determined as described in the text.

two advantages in the preparation of the Kmfragment; the 2.2 x 106-dalton mini-ColEl vehi-cle is easily separated from the 4.5 x 106 Kmfragment by preparative agarose gel electro-phoresis, and the ability of the plasmid to repli-cate extensively after the addition of chloram-phenicol permits the isolation of large quanti-ties of the supercoiled plasmid DNA. Recently,an antibiotic-resistant fragment from an S. au-reus plasmid has been used to clone the replica-tion segments of plasmid R6-5 and F'lac (17).The properties of the replication segment ofF'lac isolated by this procedure are similar tothose reported here.

In an accompanying paper (6) the analysis ofheteroduplexes between pML31 and two F-prime plasmids by electron microscopy has lo-calized the replication fragment of pML31 tothe region of the F-prime plasmid deduced tocontain the genes involved in replication of theplasmid. The fact that pML31 retains the strin-gent control of replication of F elements proba-bly means that the 6 x 106-dalton fragmentcontains those genes of F'lac responsible forstringent replication control. The retention ofthe incompatibility property of F'lac by pML31does not distinguish whether this phenomenonis due to competition of a similar or identicalreplicon for the same essential cellular site, orif some other gene product or DNA site on the6.0 x 106 fragment is responsible for this prop-erty. It is clear, however, that both replicationand incompatibility functions of F'lac are pres-ent on this 6.0 x 106 segment of F'lac plasmid.A similar observation has been made for the

isolated replication regions of R6-5 and F'lacdescribed by Timmis et al. (17). The high fre-quency with which F'lac entered C600 (pML31)suggests that pML31 does not carry the gene forsurface exclusion. This function has been foundto map within a region of F containing thegenes required for the expression of fertility(18).The 6.0 x 106 fragment represents 6.3% ofthe

size of F'lac (94.9 ± 2.1 x 106 daltons [15]).Apparent clustering of genes required for DNAreplication and existence as a plasmid has alsobeen noted in the cases ofpSC101, which repre-sents less than 10% of the size of the R6-5plasmid from which it was derived (2), X dv,which represents less than 15% of the size ofbacteriophage X (12), the mini-ColEl plasmid(8), and, recently, for R6-5 and F'lac (17), wherea conceptually similar procedure was used forthe isolation of the replication region. The rea-son for this clustering of replication genes andits implications with respect to the control ofplasmid replication and the evolution of plas-mid elements remain to be determined.

ACKNOWLEDGMENTSWe thank William A. Moller for his excellent technical

assistance, and we thank Herbert Boyer and John Collinsfor helpful discussions.

This investigation was supported by U.S. Public HealthService research grant AI-07194 from the National Instituteof Allergy and Infectious Diseases and National ScienceFoundation grant GB-29492.

LITERATURE CITED1. Chang, A. C. Y., and S. N. Cohen. 1974. Genome con-

struction between bacterial species in vitro: replica-tion and expression of Staphylococcus plasmid genesin Escherichia coli. Proc. Natl. Acad. Sci. U.S.A.71:1030-1034.

2. Cohen, S. N., and A. C. Y. Chang. 1973. Recirculariza-tion and autonomous replication of a sheared R-factorDNA segment in Escherichia coli transformants.Proc. Natl. Acad. Sci. U.S.A. 70:1293-1297.

3. Cohen, S. N., A. C. Y. Chang, H. W. Boyer, and R. B.Helling. 1973. Construction of biologically functionalbacterial plasmids in vitro. Proc. Natl. Acad. Sci.U.S.A. 70:3240-3244.

4. Cohen, S. N., A. C. Y. Chang, and C. L. Hsu. 1972.Nonchromosomal antibiotic resistance in bacteria:genetic transformation ofEscherichia coli by R-factorDNA. Proc. Natl. Acad. Sci. U.S.A. 69:2110-2114.

5. Davidson, N., R. C. Deonier, S. Hu, and E. Ohtsubo.1975. Electron microscope heteroduplex relationsamong plasmids ofEscherichia coli. X. Deoxyribonu-cleotide acid sequence organization of F and F-primes,and the sequences involved in Hfr formation, p. 56-65. In D. Schlessinger (ed.), Microbiology-1974.American Society for Microbiology, Washington,D.C.

6. Guyer, M. S., D. Figurski, and N. Davidson. 1976.Electron microscope study of a plasmid chimera con-taining the replication region of the Escherichia coliF plasmid. J. Bacteriol. 127:988-997.

7. Hedgpeth, J., H. M. Goodman, and H. W. Boyer. 1972.DNA nucleotide sequence restricted by the RI endo-

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nuclease. Proc. Natl. Acad. Sci. U.S.A. 69:3448-3452.8. Hershfield, V., H. W. Boyer, L. Chow, and D. R. Helin-

ski. 1975. Characterization of a mini-ColEl plasmid.J. Bacteriol. 126:447453.

9. Hershfield, V., H. W. Boyer, C. Yanofsky, M. A. Lov-ett, and D. R. Helinski. 1974. Plasmid ColEl as amolecular vehicle for cloning and amplication ofDNA. Proc. Natl. Acad. Sci. U.S.A. 71:3455-3459.

10. Katz, L., D. T. Kingsbury, and D. R. Helinski. 1973.Stimulation by cyclic adenosine monophosphate ofplasmid deoxyribonucleic acid replication and catabo-lite repression of the plasmid deoxyribonucleic acid-protein relaxation complex. J. Bacteriol. 114:577-591.

11. Lovett, M. A., D. G. Guiney, and D. R. Helinski. 1974.Relaxation complexes of plasmids ColEl and ColE2:unique site of the nick in the open circular DNA ofthe relaxed complexes. Proc. Natl. Acad. Sci. U.S.A.71:3854-3857.

12. Matsubara, K., and A. D. Kaiser. 1968. dv: an autono-mously replicating DNA fragment. Cold Spring Har-bor Symp. Quant. Biol. 33:769-775.

13. Mertz, J. E., and R. W. Davis. 1972. Cleavage of DNAby Rl restriction endonuclease generates cohesiveends. Proc. Natl. Acad. Sci. U.S.A. 69:.3370-3374.

14. Morrow, J. F., S. N. Cohen, A. C. Chang, H. W. Boyer,H. M. Goodman, and R. Helling. 1974. Replicationand transcription of eukaryotic DNA in Escherichiacoli. Proc. Natl. Acad. Sci. U.S.A. 71:1743-1747.

15. Sharp, P. A., M. Hsu, E. Ohtsubo, and N. Davidson.1972. Electron microscope heteroduplex studies of se-

quence relations among plasmids ofEscherichia coli.I. Structure of F-prime factors. J. Mol. Biol. 71:471-497.

16. Taylor, A. L., and C. D. Trotter. 1967. Revised linkagemap ofEscherichia coli. Bacteriol. Rev. 31:332-352.

17. Timmis, K., F. Cabello, and S. N. Cohen. 1975. Clon-ing, isolation and characterization of replication re-gions of complex plasnid genomes. Proc. Natl. Acad.Sci. U.S.A. 72:2242-2246.

18. Willetts, N. 1974. Mapping loci for surface exclusionand incompatibility on the F factor ofEscherichia coliK-12. J. Bacteriol. 118:778-782.

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