Gene, 2(1977) 75-93
`© Elsevier/North-Holland Biomedical Press, Amsterdam — Printed in The Netherlands
`
`75
`
`CONSTRUCTION AND CHARACTERIZATION OF NEW CLONING
`VEHICLES
`I. AMPICILLIN-RESISTANT DERIVATIVES OF THE PLASMID pMB9
`
`(Plasmid vectors; molecular cloning; tetracycline resistance transposon; res-
`triction enzyme mapping)
`
`FRANCISCO BoL1vAR*, RAYMOND1L,RODRIGUEZ" , MARY c. BETLACH and
`HERBERT w. BOYER
`
`Department of Biochemistry and Biophysics, University of California, San Francisco, Califi
`94143 (U.S.A.)
`
`(Received January 31st, 1977)
`(Accepted June 23rd, 1977)
`
`
`
`SUMMARY
`
`In vitro recombination via restriction endonucleases and the in vivo genetic
`translocation of the Ap resistance (Apr) gene resulted in the construction of
`a new cloning vehicle, the plasmid pBR313. This vector was derived from a
`ColE1-like plasmid and, while it does not produce colicin E1, it still retains
`colicin E1 immunity. The Apr and tetracycline resistance (Tc') markers carried
`in pBR313 were derived from the ampicillin transposon (TnA) of pRSF2124
`and pSC101 respectively. During the construction of pBR313, the TnA com-
`ponent was altered and the Ap’ gene in pBR313 can no longer be translocated.
`This plasmid has a molecular weight of 5.8 Mdalton and has been charac-
`terized using thirteen restriction enzymes, six of which (EcoRI, Smal, Hpal,
`Hirzdlll, BamHI and Sail) cleave the plasmid at unique restriction sites. This
`allows the molecular cloning of DNA fragments generated by these six
`enzymes. The restriction sites for the latter three enzymes, Hindlll, BamHI
`and Sell, are located in the Tc’ gene(s). Cloning DNA fragments into these
`sites alters the expression of the To’ mechanisms thus providing a selection~
`for cells carrying recombinant plasmid molecules. An enrichment method
`for Ap'Tc5 cells carrying recombinant plasmid molecules is described.
`
`
`
`*Present addresses: (F.B.)Departamento Biologia Molecular, Instituto de Investigaciones
`Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico 20, D.F.; (R.L.R.)
`Department of Genetics, Briggs Hall, University of California, Davis, CA 95616 (U.S.A.).
`
`Abbreviations: Ap, ampicillin; Cyc, cycloserine; Tc, tetracycline.
`
`MERCK v. GENENTECH
`MERCK V. GENENTECH
`IPR2016-01373
`IPR2016-01373
`GENENTECH 2034
`— 2°34
`
`

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`76
`
`INTRODUCTION
`
`There are several critical components which facilitate recombinant DNA
`research. The ease with which recombinant DNA research can proceed will in
`part depend on the improvement of one of these components, the cloning
`vehicle. It is now clear that bacterial plasmids, bacteriophages and animal
`viruses can serve as vectors for cloning DNA fragments. Two bacterial plasmids
`used in most of the initial cloning experiments as cloning vectors are pSC101
`(Cohen et al., 1973) and Co1E1 (Hershfield et al., 1974). Other cloning
`vectors have been derived from these two plasmids (Hamer and Thomas,
`1976; Hershfield et al., 1976; So et al., 1976) which improve on their utility.
`We have undertaken the development of a series of plasmids with the goal
`of obtaining a set of multipurpose cloning vehicles. In this paper, we describe
`the construction of plasmid pBR313 with the ColE1 mode of replication
`and which contains the genetic potential for resistance to the antibiotics
`ampicillin (Ap) and tetracycline (Tc). A tentative restriction map has been
`determined for this plasmid and its cloning properties have been characterized.
`pBR313 permits molecular cloning with DNA fragments derived by endo-
`nucleolytic action of the following restriction endonucleases: EcoRI, HindIII,
`BamHI, Sall, Hpal and Smal. A procedure for selecting transformants which
`contain recombinant plasmids has been developed.
`
`MATERIALS AND METHODS
`
`(a) Bacterial strains
`The following derivatives of E. coli K12 were used as recipient cells in
`transformation experiments: HB101 F‘ pro Ieu thi lacY Str' rfimf; Endol‘,
`recA’ (Boyer and Roulland-Dussoix, 1969); RR1 F“ pro leu thi lacY Str'
`rfgmk; EndoI'. The E. coli B strain HB50 (pro leu try his arg met thr gal lac-Y
`Sir’ rf;mf,;) was used to prepare unmethylated plasmid DNA for EcoRII
`digestion (Yoshimori et al., 1972). The bacterial plasmids pRSF2124 and
`pSC101 in the E. coli K12 C600 background (F‘ leu thi thr lacY) were kind-
`ly provided by S. Falkow and S. Cohen, respectively. The plasmid Co1E1
`(JC411 Ff leu his arg met gal mat xyl thy lacY Str') was obtained from
`D. Helinski. The plasmids pMB8 and pMB9 were maintained in and prepared
`from HB101.
`
`(b) Preparation of plasmid DNA
`Preparation of plasmid DNA by amplification in the presence of chlor-
`amphenicol (170 pg/ml) was performed according to Clewell (1972). The
`DNA was purified by a modification of the cleared lysate technique of
`Gueny et al., 1973. The cleared lysate was extracted with an equal volume
`of phenol and the aqueous phase precipitated with two volumes of cold
`ethanol. The resuspended DNA, in a 5 ml volume of A50 agarose buffer
`(500 mM NaCl, 50 mM Tris pH 8.0, 1 mM EDTA), was applied to a 25 cm X
`
`

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`77
`
`50 cm Bio-Gel A50 agarose column (Bio-Rad), and the first peak fractions as
`determined by Amnm were pooled and ethanol-precipitated. The plasmid
`DNA was further purified by dye-buoyant centrifugation in a CsCl-propidium
`diiodide gradient, equilibrated for 18 h at 36 000 rpm, 20° C, in a Spinco
`SW 50.1 rotor. Small molecular weight RNA contaminates the plasmid DNA
`at this stage.
`‘
`The band of supercoiled DNA as visualized with UV light was collected
`and the propidium diiodide extracted by passing the DNA over a 1 cm X 4 cm
`Dowex (AG 50W-X8 Bio-Rad) column. The eluent was dialyzed against 10 mM
`Tris-HCI, 1 mM EDTA, pH 7.4, ethanol-precipitated and resuspended in
`50 mM Tris-HC1, 10 mM NaCl, 1 mM EDTA pH 7.4. DNA concentrations
`were determined spectrophotometrically in the above buffer; Ammm of 1.0 =
`50 pg DNA/ml (Padmanabhan and Wu, 1972).
`Preparation of plasmid DNA for the rapid analysis of the restriction endo-
`nuclease digestion pattern was performed according to the procedure described
`by Meagher et al. (1977).
`
`(c) Enzymes
`EcoRI restriction endonuclease was purified according to the procedure
`of Greene et al. (1974). The restriction endonucleases, Alul (Roberts et al.,
`1976), HaeII (Roberts et al., unpublished observations), HaeIII (Roberts et
`al., unpublished observations), Bgll (Wilson and Young, unpublished observa-
`tions), BamHI (Wilson and Young, 1975), EcoRII (Yoshimori et a1., 1975),
`HincII (Landy et al., 1974), HindIII (Danna et al., 1973), Pstl (Smith et a1.,
`1976), and Sail (Arrand et a.l., 1976), were purified according to the proce-
`dure described by Heyneker et al. (1976). Hpal (Gromkova and Goodgal,
`1972) was obtained from BRL laboratories. T4 polynucleotide ligase, a gift
`from H. Heyneker, was purified according to the procedure of Panet et al.
`(1973). Colicin E1 was prepared from the strain JC411 as described by
`Schwartz and Helinski (1971). All restriction enzymes were stored at —20°C
`in 50% glycerol, 20 mM KH,PO..-~K2HPO4 pH 7.0, 1 mM EDTA, 1 mM NaN3,
`and 100 mM NaCl.
`
`(d) Preparation of various restriction endonuclease DNA fragments
`DNA fragments were generated with various restriction endonucfiases by
`digesting from 0.1 to 5.0 pg of DNA in a 10 to 30 ul volume according to the
`following conditions for each endonuclease: Bgll, Alul, Haell, and HaeIII,
`6 mM Tris-HCI pH 7.9, 6 mM (3—mercaptoethanol, 6 mM MgCl,; Hpal, 50 mM
`Tris"-HCI pH 7.5, 5 mM B-mercaptoethanol, 5 mM MgCl,, 50 mM NaCl; Smal,
`15 mM Tris-HCI pH 9, 6 mM MgCl,, 15 mM KCI; BamHI, 100 mM Tris-HCI,
`5 mM MgCl; pH 7.6; EcoRI, 100 mM Tris-HCI, 5 mM MgC1,, 100 mM NaCl.
`0.02% NP40 (Particle Data Laboratories) pH 7.6; EcoRI*, 2 mM Tris--H01,
`2 mM MgC1,, 20% glycerol pH 8.8; EcoRII, 100 mM Tris-HCl, 5 mM MgCl;
`pH 7.6; Hincli, 100 mM Tris-~HCl, 66 mM MgCl,, 500 mM NaCl, 60 mM 3-
`mercaptoethanol pH 8.0; HindIII, 6.6 mM Tris-HCI, 6.6 mM MgCl,, 50 mM
`
`

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`78
`
`NaCl, 7 mM 3-mercaptoethanol pH 7.5; Pstl, 90 mM Tris-HCl, 10 mM MgSO4
`pH 7.5; Sall, 8 mM Tris-HCl, 6 mM MgCl;, 150 mM NaCl, 0.2 mM EDTA,
`50 pg/ml bovine serum albumin. Endonuclease reactions were incubated at
`37°C and stopped by heating to 63°C for 5 min or by the addition of 10 pl
`of 5% sodium dodecyl sulfate, 25% glycerol, 0.025% bromophenol blue (stop
`mixture).
`
`(e) Molecular weight standards
`For agarose gel electrophoresis, the following markers were used: the six
`EcoRI-generated fragments of the Pi bacteriophage genome (13.7, 4.68, 3.7,
`3.56, 3.03, and 2.09 md.); the EcoRI-generated linear forms of the plasmids
`pVH51 (2.2 md.) (Hershfield et al., 1976) and pMB8 (1.7 md.) and the six
`HindII_I-generated fragments of the SV40 genome (1.13, 0.75, 0.68, 0.35,
`0.28, 0.13 md.). For acrylamide gel electrophoresis the seven HindIII-generated
`fragments of the bacteriophage PM2 (3.5, 1.34, 0.6, 0.29, 0.26, 0.155, 0.07
`md.) were used as molecular weight standards (Wes Brown, personal commu-
`nication).
`
`(f) Ligation of DNA fragments
`The ligation reactions were incubated at 12°C in volumes ranging from 50
`to 1000 pl containing 66 mM Tris—HCl, 6.6 mM MgCl,, 10 mM dithiothreitol
`(Sigma), 0.2 mM ATP pH 7.6 and varying amounts of DNA. The reaction was
`started by the addition of 0.5 to 5.0 ul of T4 polynucleotide ligase.-DNA
`ligation was monitored by the electrophoresis of small aliquots of the reaction
`taken at intervals.
`
`(g) Agarose and acrylamide electrophoresis
`Slab gels containing 0.8 to 1.0% agarose (Seakem) were prepared by auto-
`claving the agarose for 10 min in Tris--EDTA--borate buffer (90 mM Trizma
`Base (Sigma), 2.5 mM Na, EDTA, 90 mM H3803 pH 8.2). After the gels
`were poured and allowed to solidify, samples containing from 0.2 to 1 ug of
`DNA were loaded onto the gels in a 20 to 40 pl volume per slot. Electro-
`phoresis was performed at 130 to 150 V at room temperature for 2 h. Slab
`gels of 7.5% acrylamide (1 mm thickness) were prepared by mixing 2.4 ml
`of 10X Tris-EDTA—borate buffer with 15.6 ml of water, 6 ml of 28% acryl-
`amide (Bio-Rad), 0.6% bisacrylamide and 0.12 ml of 10% NH4S03. After de-
`gassing, TEMED (12 ,uml) (Bio-Rad) was added and the mixture poured im-
`mediately. Acrylamide electrophoresis was performed at 120 V for 2 h. After
`electrophoresis, all gels were soaked for 5 min in 4 pg/ml ethidium bromide
`solution and illuminated with a short-wave UV transilluminator (Ultraviolet
`Products, San Gabriel, Calif.). A yellow no. 9 Kodak Wratten gelatin filter
`and NP Type 55 Polaroid film were used with a MP-3 Polaroid camera to
`photograph the gels.
`
`

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`79
`
`(h) Transformation of E. coli
`Chloroform-sterilized DNA in a 100 1.11 Volume was adjusted to a final con-
`centration of 30 mM CaCl2 and added to 0.2 ml of cells prepared for trans-
`formation by the procedure of Cohen et al. (1972). The transformation mix-
`ture was allowed to stand on ice for 1 h after which it received a 60 sec, 42°C
`heat pulse. The heat pulse was terminated by the addition of 5 ml of Luria
`broth. Transformed cells were plated immediately or allowed to proceed into
`the logarithmic phase of cell growth before plating.
`
`RESULTS
`
`(a) Construction and characterization of pMB9
`The construction and characterization of a series of Co1E1-like plasmid
`derivatives have been reported (Betlach et al., 1976). From one of these
`plasmids we derived a small plasmid which is similar to pVH51 (Hershfield et
`al., 1976). pMB8 has a molecular weight of 1.72 X 10‘ daltons, is immune to
`colicin E1, exhibits a ColE1 mode of DNA replication in terms of copy num-
`ber and replication in the presence of chloramphenicol but does not form a
`detectable relaxation complex (Clewell and Helinski, 1969). Although pMB8
`offers some advantages as a cloning vector (e.g., small size, good yields of
`DNA, low background of protein synthesis in minicell systems), it does not
`have a good selective marker or substrate sites for several of the commonly
`used restriction endonucleases other than EcoRI. We then constructed a
`composite plasmid which affords cloning with different restriction enzymes
`by incorporating into pMB8 some components of the pSC101 plasmid which
`can confer Tc’ to the cell. This plasmid, pMB9, was constructed by ligating
`the products of an Eco RI* endonuclease (Polisky et al., 1975) digest of
`pSC101 DNA with an EcoRI endonuclease digest of pMB8 (Rodriguez et al.,
`1976).
`The plasmid pMB9 has a molecular weight of 3.5 -10‘ daltons and one sub-
`strate site for each of the following restriction endonucleases: EcoRI, Hindlll,
`Sail and BamHI. The relative positions of these sites (Fig. 1) were determined
`by acrylamide gel electrophoresis of double and triple digestions of plasmid
`DNA with various restriction enzymes. Since pMB8 has no Hindlll, BamHI
`or Sall sites, these sites should be associated with that pSC101 DNA frag-
`ment introduced in pMB9. This assumption is supported by the fact that
`these three sites are present in pSC101 in the same relative positions (data
`not shown) and that the molecular cloning of DNA into the Hindlll, BamHI
`and Sall sites alters the Te’ mechanism (Hamer and Thomas, 1976; Rodriguez
`et al., 1976). Since DNA inserted into the EcoRI site of pMB9 does not alter
`the expression of the Te‘ mechanism, we believe that this site lies outside the
`Tc’ gene. However, cloning into the EcoRI site of pSC101 has been found to
`affect the level and inducibility of Tc‘ (Tait et al., 1977).
`Although EcoRI-generated recombinant plasmids of pMB9 can be selected
`by virtue of their resistance to tetracycline, transformants with recombinant
`
`

`
`80
`
`plasmids constructed by insertion of DNA fragments generated by HindIII,
`BamHI and Sall digestion and incorporated into the respective restriction
`sites can only be selected by colicin E1 immunity. Since the action of colicin
`E1 is extremely dependent on the physiological state of the cell and is
`accompanied by a high frequency of spontaneous mutations to colicin
`tolerance (Nagel De Zwaig and Luria, 1967), we chose to introduce another
`selective marker into pMB9. This was accomplished by the genetic transloca-
`tion of an ampicillin resistance marker (Ap') from pRSF2124 (So et al., 1976)
`to pMB9.
`
`(b) Construction of Ap’-Tc’ derivatives of pMB9
`The Ap' marker has been shown to be translocated on a 3.2 -10‘ daltons
`sequence of DNA which has been termed TnA (Heffron et al., 1975). Trans-
`location of the TnA from pRSF2124 to pMB9 was accomplished by cotrans-
`forming E. coli strain RRI with a total of 5 ug of supercoiled pMB9 and
`pRSF2124 DNA at a respective molecular ratio of 2 : 1. Ap‘-Tc’ transformants
`which occurred at a frequency of 6 '10" after 5 generations were screened for
`plasmid DNA which gave a linear 6.7 mdalton plasmid molecule upon di-
`gestions with EcoRI. While the majority of Apr-Tc‘ transformants contained
`varying ratios of pMB9 and pRSF2124, approximately 20% of the total Ap"-
`Tc’ transformants give one linear plasmid DNA molecule upon EcoRI di-
`gestion. These EcoRI-generated linear molecules had molecular weights of
`6.7 -10‘ daltons which corresponds to the sum of the molecular weights of
`the TnA (3.2 -10‘ daltons) and pMB9 (3.5 -10‘ daltons). These plasmid
`molecules were shown to confer resistance to Ap and Tc when transformed
`back into E. coli RR1.
`The presence of a single asymmetrically located BamHI site about 1 -10‘
`daltons from the end of the TnA (Heffron et al., 1977) made it possible to
`localize the various TnA insertion sites in pMB9 (Fig. 1). In the case of two
`Ap'-Tc’ derivatives of pMB9, designated pBR312 and pBR26, mapping the
`relative positions of the restriction and TnA insertion sites was carried out
`by digestion with combinations of various restriction enzymes as shown in
`Plate 1(a). In relation to the molecular weight standards (slots 4, 7 and 10)
`linear molecules of pBR312 and pBR26, having molecular weights of 6.7 -10‘
`daltons, were also generated by digestion with Hindlll (data not shown).
`BamHl digests of pBR312 and pBR26 gave two fragments in each case
`with molecular weights of 5.1 and 1.6 -10‘ daltons and 5.06 and 1.65 -10‘
`daltons, respectively. By following a BamHI digestion of these plasmids with
`an EcoRI digest, the smallest band of pBR312 and the largest band of pBR26
`were each cleaved by EcoRI to give a third band of 0.4 -10‘ daltons. These
`results enable us to localize the TnA insertion counterclockwise to the EcoRI
`
`site in pBR312 and clockwise to the EcoRI site in pBR26. Since the smallest
`BamHI fragments of pBR312 and pBR26 are less than 2.2 -10‘ daltons, this
`indicates that the orientation of the TnA is such that the one—third portion
`which is known to carry the Ap’ gene (Heffron et al., 1977) is proximal to
`the BamHI site in the Tc’ gene (Fig. 1).
`
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`81
`
`Fig. 1. Schematic representation of pMB9 DNA showing the Tc’ region with the EcoRI,
`HindIII, BamHI and Sall sites. The arrows represent the position of the IR from TnA in
`two Ap’ derivatives of pMB9: pBR312 and pBR26.
`
`(c) Construction and characterization of pBR313
`Although pBR312 and pBR26 now possess a second strong selective marker
`in the form of Apr, their potential usefulness as molecular cloning vectors is
`diminished by the presence of an extra BamHI site and their increased molec-
`ular weights. Consequently, we decided to remove the BamHI site contributed
`by the TnA and simultaneously reduce the size of pBR312 by partial EcoRI*
`digestion. After digestion, the DNA was ligated in a 500 111 volume and then
`transformed into RR1. Ap’-Tc’ transformants, which occurred at a frequency
`of 3 -10"’, were screened for plasmid DNA giving linear molecules upon
`Baml-II digestion. Out of 16 Apr-Tc’ clones, 6 gave linear molecules of varying
`molecular weights after treatment with BamHI. The three smallest plasmids
`(pBR313, pBR315 and pMB316) were selected for further study.
`The molecular weight and relative position of restriction sites of pBR313
`were determined as described above and the results of the acrylamide gel
`electrophoresis are shown in Plate I(b). The data from Plate I(b) have been
`summarized in Fig. 2a. As can be seen in Fig. 2b, the EcoRI* digestion has
`removed one of the two BamHI sites from pBR313, pBR315 and pBR316
`and reduced their molecul weights by 0.8, 1.5 and 1.4 -10‘ daltons respec-
`tively. One useful feature o EcoRI* digestions is the ability to lose and
`
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`83
`
`1966) and the Tc’-associated proteins detected in the minicell system (Levy
`and McMurry, 1974; Tait et al., 1977). Positioning the left-hand boundary of
`the Te’ gene was based on our knowledge that cloning into the EcoRI site of
`pBR313 did not affect Tc’ while cloning into the HindIII site did affect the
`expression of the Tc’ mechanism. The position and size of the Tc’ region is
`also consistent with the orientation of the TnA in pBR26. This follows from
`the consideration of the known position of the inverted repeats to the BamHI
`site (Heffron et al., 1977 ). The 1.65 -10‘ daltons BamHI-generated fragment
`of pBR26 allows for 0.65 -10‘ daltons of DNA between the pMB9 specified
`BamHI site and the end of the To’ gene(s) after accounting for the 1.0 -10‘
`dalton segment of the TnA.
`The restriction endonuclease Pstl was used to further characterize pBR313.
`As shown in Plate II(a) (slot 5), pBR313 has three Pstl sites which give frag-
`ments of 0.4, 1.25 and 4.15 -10‘ daltons. Since pMB9 was found to have no
`Pstl sites (Plate II(a), slot 2), it was concluded that the Pstl sites were associ-
`ated with the TnA. This conclusion is further supported by the presence of
`five Pstl sites in pRSF2124 (Plate II(a), slot 3). Two of these five sites are
`known to be carried on the ColE1 portion of pRSF2124 (data not shown). It
`can also be seen that two of the three Pstl fragments present in pBR312
`(Plate II(a), slot 4) 1.7 and 0.4 -10‘ daltons, are present in pRSF2124, while
`only the smallest fragment is present in pBR313. From these results, we
`concluded that the 1.7 -10‘ dalton fragment of pBR312 was reduced to
`1.25 -10‘ daltons in pBR313 by the EcoRI* digestion. The Pstl-EcoRI com-
`bination digest shown in slots 7, 8, and 9 of Plate II(a), corroborate the
`placement of the TnA counterclockwise to the EcoRI site in pBR312 (Fig. 1)
`
`Plate I. (a) Agarose slab gel electrophoresis of plasmids pBR312 and pBR26 cleaved by
`EcoRI, BamHI and Sail endonucleases. Digested DNA (0.3 to 0.5 #8) was applied to the
`sample slots in 40 ul volumes. Agarose gel electrophoresis was carried out as described in
`MATERIALS AND METHODS. Molecular weight estimates are based on the 6 A fragments
`generated by EcoRI, the linear forms of the plasmids pMB8 and pVH51 and the six HindIII-
`generated fragments of the SV40 genome (slots 4, 7, 10) (see MATERIALS AND
`METHODS). Slots 1, 2, and 5 show the EcoRI, SalI and BamHI digestions respectively of
`pBR26 plasmid DNA. Slot 3 shows the double digestion EcoRI-SaII and slot 6 shows the
`EcoRI-BamHI double digestions both in pBR26 DNA. Slots 9, 12, and 13 show the BamHI,
`SalI and EcoRI digestions respectively of pBR312 plasmid DNA. Slot 8 shows the EcoRI-
`Bafi1HI and slot 11 the EcoRI-Sall double digestions of pBR312 DNA. For explanation
`see RESULTS (construction of Ap'~Tc' derivatives of pMB9).
`(b) Acrylamide slab gel electrophoresis of plasmid pBR313 DNA fragments obtained by
`double and triple digestions using EcoRI, Hindlll, Bamlil and Sall restriction endonucleases.
`Gel electrophoresis was carried out as described in MATERIALS AND METHODS.
`Molecular weight estimates are based on the (seven) Hindlll-(generated) fragments of the
`PM2 phage genome. The restriction endonuclease digestion combinations for Fig. 3 are as
`follows: (Slot 1) EcoRI-BamHI; (Slot 2) EcoRI-BamHI-Sell; (Slot 3) Bamlil-Sail; (Slot 4)
`EcoRI-Sall; (Slot 5) EcoRI-Hindlll-Sail; (Slot 6) HindIII-Sail; (Slot 7) Hindlll-BamHI;
`(Slot 8) Hindlll-Bamfll-Sail; (Slot 9) EcoRI-HindIII. Hindlll-digested PM2 markers are
`also present in slots 2 (bands 2, 3, 4, 5, 6, 8 and 10), slot 5 (bands 2, 3, 4, 6, 7, 8 and 9),
`slot 8 (bands 2, 3, 4, 5, 6, 8 and 10).
`
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`87
`
`that there is one EcoRII site between HindIII and BamHI sites and no EcoRII
`
`site between BamHI and Sall sites. Combination digests with additional
`restriction endonucleases have enabled us to localize other EcoRII sites on
`
`pBR313 (Fig. 2a).
`
`(e) Mapping the substrate sites of the restriction endonucleases HincII, Hpal,
`Smal, Bgll, Alu, HaeII and HaeIII
`The restriction enzyme Hincll recognizes the sequence
`G T Py‘Pu A c
`(Landy et al., 1974)
`
`C APuTPy T G
`
`As shown in Fig. 2a, there are four Hincll sites in pBR313, one of which, is
`present in the 0.4 -10‘ dalton Pstl fragment missing in pBR317. It should be
`noted at this point that the Hpal-HincII and SalI-HincII double digestion
`patterns are identical to the Hincll pattern (data not shown). This is due to
`the purine-pyrimidine ambiguity present in the HincII substrate site which
`enables this particular enzyme to recognize both the Hpal
`
`t
`GTTAAC
`
`C A A?T T G
`
`and Sail
`
`l
`GTCGAC
`
`C A G C TTG
`
`substrate
`
`sites (Danna et al., 1973; Bolivar and Shine, 1976, unpublished observation).
`As shown in Fig. 2a, the EcoRI site is located 0.29 -10‘ daltons from the
`Hincll situated in the Ap’ gene (S. Falkow, personal communication) and
`0.35 -10‘ daltons from the Hincll-Sall site. Since a Sail-Hpal double digestion
`generates a 1.3 -10‘ dalton fragment which is also present in a Hincll digest,
`this places the Hpal site clockwise of the Sail site as shown in Fig. 2a. When
`the 2.29 -10‘ dalton fragment produced by a Hincll digest of pBR313 is
`cleaved by Smal, this fragment is reduced by 0.12 -10" daltons. The fact that
`
`Plate II. (:1) Analysis of PstI and Pstl-EcoRI single and double digestions of pMB9,
`pRSF2124, pBR312 and pBR313 using agarose gel electrophoresis. Molecular weights
`estimates are based on the 6 A fragments generated by EcoRI, the linear forms of the
`plasmids pMB8 and pVH51 and the HindIII generated fragments of the SV40 genome
`(slots 1, 6 and 11). The Pstl digestion patterns of the various plasmids are as follows:
`(slot 2) pMB9; (slot 3) pSF2124; (slot 4) pBR312 and (slot 5) pBR313. Pstl-EcoRI double
`digestions of these plasmids are as follows: (slot 7) pMB9; (slot 8) pSF2124; (slot 9)
`pBR312 and (slot 10) pBR313.
`(b) Acrylarnide slab gel electrophoresis of EcoRII cleaved pMB9, pBR312, pBR313 and
`pBR316 DNAs. Purified plasmids DNA were cleaved with EcoRII as described in
`MATERIALS AND METHODS and the fragments were dialyzed and subjected to acryl-
`amide gel electrophoresis. The seven ‘PM2 Hindlll-generated fragments were used as
`molecular weight markers (slots 4, 7 and 10). Slots 5, 6, 8 and 9 show the EcoRII pattern
`of plasmids pBR313, pBR316, pBR312 and pMB9 respectively. Double digestions EcoRII-
`Hindlll, EcoRII-Sail and EcoRlI-BamHI of pBR313 DNA are shown in slots 1, 2 and 3
`respectively. For explanation see the text (RESULTS Section (d)).
`
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`88
`
`the 1.42 -10‘ dalton fragment produced by a Sall-Smal double digest is also
`reduced by 0.12 -10‘ daltons when digested with Hpal, places the Smal site
`0.12 -10‘ daltons clockwise from the Hpal site (Fig. 2a).
`Combination digest of pBR313 and derivative pBR plasmids (Bolivar et al.,
`1977) have enabled us to map the five Bgll restriction sites shown in Fig. 2a.
`The Alul restriction enzyme cleaves pBR313 into more than eighteen frag-
`ments, some of which have been mapped by analysis of double digestion
`patterns. At present, we have been able to localize seven Alul sites on pBR313.
`The Alul site between the EcoRI and HindIII sites (Fig. 2a) was localized by
`the determination of the nucleotide sequence in this region of the DNA
`(J. Shine, unpublished observation). Using the strategy of combination digests,
`we were also able to map eleven Haell sites and 4 HaeIII sites present in
`pBR313 (Fig. 2a).
`
`(f) Molecular cloning of various restriction endonuclease-generated fragments
`in pBR313
`DNA fragments from various sources were produced by digestion with
`EcoRI, Hindlll, BamHI, Sall and HindIII-BamHI restriction enzymes and
`cloned in their respective sites in pBR313 (Table I). EcoRI-recombinant
`plasmids of pBR313 gave Ap'-Tc’ phenotypes while BamHI, Sall and HindIII-
`BamHI-recombinant plasmids were Ap’-Tc‘. While some transformant-carrying
`Hindlll-recombinant plasmids were Ap'Tc“, others were found to have a low-
`1evelTc' which was observed when recombinant transfonnants wereincubated
`for more than 24 h on Luria agar plates containing 10 pg/ml Tc. As in the
`case of the EcoRI recombinant plasmid, cloning of DNA fragments into the
`Hpal or Smal sites of pBR313 does not affect the expression of To’ (data
`not shown).
`
`(g) Tetracycline-cycloserine enrichment for recombinant transforman ts
`Since transfonnation of E. coli K12 with in vitro ligated recombinant DNA
`may yield recombinant transformants at a frequency as low as 10“ to 10"’/
`ml/,u g of DNA, a procedure was needed for enriching the number of re-
`combinant transformants in the total cell population. Such a technique was
`developed by taking advantage of the bacteriostatic nature of To and the
`bactericidal effect of Ap and Cyc. The rationale behind this procedure is the
`temporary inhibition of the growth of Tc‘-recombinant transformants by the
`addition of To to the growth medium. After allowing the Tc’-transformants
`a 45 min interval of exposure to Tc, Cyc was added at a concentration which
`promoted the exponential lysis of growing cells (Curtiss et al., 1965). The
`Tc‘-recombinant cells can be recovered after the removal of the To and Cyc.
`Nontransforrned cells can be eliminated from the culture by the addition of
`Ap either before or after the Tc-Cyc lytic step. A mixed-culture reconstruc-
`tion experiment was conducted to demonstrate the practicality of this
`rationale. As shown in Table II, recombinant transformants containing
`N. crassa DNA (pBR313-NCS8) initially present at a frequency of 1-10"
`were enriched to 3 -10" by this procedure.
`
`

`
`

`
`

`
`91
`
`cultures were centrifuged and resuspended in 100 ml of growth medium
`without antibiotics and incubated for 10 h. The percentages of cells that
`were Ap'Tc" (i.e., cells carrying recombinant plasmids) in the Sail and BamHI
`experiments were enriched to 92% and 88% respectively.
`
`DISCUSSION
`
`By means of the EcoRI and EcoRI* reactions and the genetic translocation
`of ampicillin resistance translocon (TnA), we have constructed a series of
`bacterial plasmid cloning vectors with a ColE1 replication mode. These
`plasmids have been characterized with thirteen restriction enzymes. One of
`these plasmids, pBR313, has some features which make it a more efficient
`cloning vehicle than the currently used vectors, for example pSC101 (Cohen
`et al., 1973), ColE1 (Hershfield et al., 1974), pMB9, and phage lambda
`(Cameron et al., 1975). The advantages of using pBR313 as a cloning vector
`are summarized as follows. (1) The molecular cloning of EcoRI, Hindlll,
`BamHI, Sall, Hpal and Smal can now be achieved in a single low molecular
`weight, amplifiable plasmid. (2) The substrate sequences for HindIII, BamHI
`and Sall restriction endonuclease are located in Tc’ region thus facilitating
`the recovery of cells harboring recombinant DNA by virtue of their Apr-Tc‘
`phenotypes. Although recombinant plasmids generated by EcoRI, Hpal and
`Smal do not inactivate the Tc’ mechanism in pBR313, double-digested DNA
`fragments involving any one of these enzymes and either Hindlll, BamHI or
`Sail will produce Tc‘ recombinants. We believe that the use of these six
`restriction enzymes and the 14 possible combination digests will provide not
`only the opportunity for cloning many interesting DNA fragments but also
`the further dissection of these DNAs into their component parts. This feature
`is of particular importance for the DNA sequencing technique recently
`developed by Maxam and Gilbert (Maxam and Gilbert, 1977). (3) Recom-
`binant transformants with Ap”-Tc“ phenotypes are amenable to enrichment
`over non-recombinant and non-transformant cells by the use of the Ap-Tc-Cyc
`lytic procedure. (4) As a result of the EcoRI* digestion of pBR312, the Ap’
`gene can no longer be translocated from pBR313 (S. Falkow, personal com-
`munication). This eliminates the possibility of translocating cloned DNA
`from the plasmid vector to either the chromosome or other resident episomes.
`While cloning into the BamHI and Sail sites in pBR313 clearly inactivates
`the Tc’ mechanism, cloning in the Eco RI site does not. Many DNA fragments
`inserted into the Hindlll site also inactivate the Tc’ mechanism; however,
`other pieces of DNA only reduce the level of Te resistance. Preliminary data
`suggest that the HindIII site may be localized in a regulatory region, i.e., a
`promoter for E. coli DNA polymerase. This notion is supported by the fact
`that the HindIII site in pBR313 is protected from digestion in the presence
`of RNA polymerase (Rodriguez et al., 1977).
`At present, we have mapped more than forty restriction sites in pBR313
`using thirteen restriction endonucleases. At least fourteen of these sites are
`
`

`
`

`
`93
`
`Heffron, R., Bedinger, P., Champoux, J.J. and Falkow, S., Proc. Natl. Acad. Sci. USA, 74
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