Construction and Mapping of Recombinant Plasmids Used for the Preparation of DNA Fragments Containing the Escherichia coli Lactose Operator and Promoter*

(Received for publication, September 19, 1978)

Stephen C. Hardies,$ Roger K. Patient&j Ronald D. Klein, Fred Ho, William S. Reznikoff,f and Robert D. Wells11

From the University of Wisconsin, Department of Biochemistry, College of Agricultural and Life Sciences, Madison, Wisconsin 53706

Three DNA restriction fragments of established se- quence containing the Escherichia coli lac genetic con- trolling regions were cloned. In each case a recombi- nant plasmid was constructed which was suitable for the subsequent large scale purification of the lac frag- ment. A 789-base pair Hind11 fragment, containing the lac operator, promoter, and cyclic AMP receptor pro- tein binding site, was ligated into the single Hind11 site of the amplifiable plasmid minicolicin El DNA (pVH51). A 203-base pair Hue III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been “filled in” by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then con- structed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hue III ends. Mapping studies on pVH51 DNA with a number of DNA restriction en- donucleases, including Alu I, Tag I, and Hpa II, are described.

This laboratory is interested in the role of DNA conforma- tion in gene regulation. Prior studies were recently reviewed (1, 2) on conformations in (a) biosynthetic DNA polymers of defined sequence, (b) at specific sites in bacteriophage DNAs, and (c) at sites in mixtures of restriction fragments from viral DNAs. To extend this work efforts were undertaken to purify large quantities of DNA restriction fragments which contain characterized regulatory sites, such as operators and pro- moters. Prior studies (3, 4), using single strand-specific nu-

* This work was supported by grants from the National Institutes of Health (CA 20279 and GM 19670) and the National Science Foundation (PCM 77-15033). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

$ Supported by a predoctoral fellowship from the National Science Foundation. Present address, University of North Carolina, School of Medicine, Department of Bacteriology and Immunology, Chapel Hill, N. C. 27514.

5 Present address, Imperial Cancer Research Fund, Mill Hill Lab- oratories, Burtonhole Lane, London, NW7 lAD, England.

1 Recipient of National Institutes of Health Career Development Award (GM30970) and a Steenbock Career Develonment Award.

11 Partially supported during a portion of this work by the John Simon Guggenheim Foundation.

cleases, revealed the structural uniqueness of a DNA region near the Escherichia coli lac operator. For that reason, and since the lac region is sequenced (5, 6)’ along with regulatory mutants (7-ll), we chose to prepare quantities of several fragments containing the lac regulatory elements. A combi- nation of recombinant DNA technology and high capacity fractionation procedures including RPC-5 column chromatog- raphy was used.

This paper describes the construction of plasmids designed to facilitate the purification of milligram amounts of four restriction fragments containing the lactose operon control elements. The large scale purification and biochemical char- acterization of those fragments is described elsewhere.’ The following paper in this series (12) describes a systematic study of the parameters which influence the resolution of DNA restriction fragments on RPC-5 column chromatography. The last two papers (13, 14) describe the biochemical characteri- zation of fragments purified on RPC-5 and demonstrate that fragments with high AT content bind to this resin more tightly than expected on the basis of size; also, a segment of high AT content within a larger fragment of average AT content can

cause retention on the column. Preliminary accounts of results from all of these papers were reported (15-18).

A number of criteria were considered for the plasmid con- structions described herein. The Zac fragments purified from these plasmids must be sufficiently small for spectroscopic methods to detect small regions within the fragments. How- ever, repressor-binding properties of the lac operator are known to be influenced by the length of the flanking DNA segments (4). This may reflect size-dependent changes in the structural properties of these DNAs. In order to take this possibility into account, we prepared three Zac fragments of different lengths. Thus, the three fragments (789,203, and 95 bp” in length) shown in Fig. 1 were cloned. All three fragments contain the lac operator and promoter, and the 789- and 203- bp DNAs also contain the CRP binding site, a positive regu- latory site.

The methods of construction were chosen to generate plas- mids from which the inserted sequences could be cleaved out leaving only two products, namely the vector and insert frag- ments. The vector chosen was pVH51 (19) which is easily amplified to a high copy number and has a single Hind11 site which was used for cloning the 789-bp Hind11 Zac fragment.

I W. Gilbert, personal communication. ’ S. C. Hardies and R. D. Wells, submitted. ’ The abbreviations used are: CRP, CAMP receptor protein (alter-

nate abbreviations are CAP and CGA); CM, chloramphenicol; bp, base pairs; IPTG, isopropyl thiogalactoside; Lac’, Lac constitutive; RPC, reversed phase chromatography; 0, operator.

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5528 Cloning of lac Operator-Promoter Fragments

Its small size and high copy number under normal growth conditions allowed the direct assay by the method of Barnes (20) of colonies containing plasmids with insertions.

The single Eco RI site of pVH51 was used to clone the 203- bp Hoe III and the 95-bp Ah I fragments by a method adapted from Backman et al. (21). This method results in recovery of Eco RI sites surrounding the Zac insertion. The 203-bp Hue III fragment was also cloned using a 300-bp linker, containing a Hue III half-site at one end and an Eco RI half- site at the other. The linker was isolated from Xplac 5 DNA on RPC-5.

The miniprint supplement of this paper describes the re- striction enzyme map of pVH5I DNA with: HindII, Kpn I (both single cleavage sites), Tag I, AZu I, and Hpa II. Our data were carefully correlated with the previously published (22) partial Hpa II and AZu I maps and with the Hha I, HinfI (23), Hue II, Hue III, and Eco RI maps (23, 24). This mapping information was important for the biochemical characteriza- tions of fragments described in adjoining papers (12-14).

MATERIALS AND METHODS

E. coli Strains-MO is an F- str” strain isogenic with Hfr Hayes. C600 SF8 (rK- mK- recBC lop 11 Iig+) (25) was obtained from J. Abelson (University of California-San Diego). E. coli strain JF290 (ColEI) (26) was the source of the bacteriocin colicin El.

Enzymes and Proteins-The lac repressor was the generous gift of A.D. Riggs (City of Hope Medical Center); the properties of this preparation were described (3, 4). Restriction enzymes and their methods of isolation were as follows: Hind11 and Hind111 (27); Eco RI (28); Hue II and Hue III (29; an additional hydroxylapatite column was run to remove residual exonuclease activity, gift of R. W. Blak- e&y); Hpa II (30); Tag 14; Hha I (31; an additional hydroxylapatite column step was included, gift of S. K. Neuendorf of this laboratory); Alu I (32; DEAE-chromatography step was omitted); and Hinff“ (hydroxylapatite was used as the final purification step). Both Ala I and Hi&I were gifts from U. Muller also of this laboratory. All enzymes used produced sharp bands when digested duplex DNA was displayed on gels, even if a 20-fold excess of enzyme activity was used. Commercial sources of Hpu II (New England Biolabs), and Eco RI1 (Bethesda Research Laboratories) were also used.

Reaction conditions for the following restriction enzymes were as described: Eco RI (33), Hind11 + Hind111 (33), and Hpa II (30). Hae III, Hue II, and Hha I were used under the conditions described for Hae III (33). Tuq I was used in Hue III salts plus 100 fig/ml of gelatin at 50°C. Conditions for other enzymes were: Ala I, 6 mM Tris-HCl (pH 7.4), 6 mM MgC12, 6 mM P-mercaptoethanol, 50 mM NaCl, 100 pg/ml of gelatin at 20°C; Hi&, as for Alu I plus 50 mM ammonium sulfate and at 37°C; and Eco RII, 100 mM Tris-HCl (pH 8.0), 5 mM MgCla at 37°C.

T4 DNA ligase was prepared as published (34) (gift of E. Selsing of this laboratory) or was purchased from Miles, New England Biolabs, or P-L Biochemicals. All three commercial sources were satisfactory for blunt end ligation reactions; however, the T4 DNA ligase prepared in our laboratory was efficient only for sticky end ligation reactions. The Micrococcus luteus DNA polymerase (gift of J. E. Larson of this laboratory) was prepared and characterized as described (35, 36). Pancreatic RNase A was obtained from Worthington Biochemicals and freed of DNase by boiling as a 2 mg/ml solution for 2 min. Colicin E, was prepared as described (26). The amount used for selections was the minimum required for complete killing in control (no DNA) transformation experiments.

DNA--Xplac 5 DNA was prepared as described (33). Purified Hae III fragments of this DNA were obtained by RPC-5 column chroma- tography (33) for the purification of a 300-bp linker fragment (which had an Eco RI site on one end and a Hae III site on the other) (described under “Results”).

Preparation of Plasmid DNAs-Cultures (100 to 600 ml) of plas- mid-containing cells were grown in M9 medium supplemented with glucose and casamino acids and were amplified (37) for 20 h with 150 pg/ml of CM. Cleared lysates were prepared as described (38). Either 5% Triton X-100 or 0.6% Brij 58 + 0.3% deoxycholate was used to effect lysis. The DNA prepared with the latter mixture of detergents

4 R. J. Roberts, personal communication.

FIG. 1. Genetic and partial restriction endonuclease map of the E. coli luc regulatory region. The three fragments (789, 203, and 95 bp) which were cloned are shown. 0, operator; P, promoter; CRP, CAMP receptor protein binding site. Lac 2 is the coding region for p- galactosidase; luc 1 is the coding region for the Zac repressor, Start refers to the beginning of the lac 2 message which proceeds to the left. 2nd site refers to the secondary lac repressor binding site. The DNA sequence was reported (5, 6).

gave faster reactions with some restriction endonucleases (HindII). The cleared lysates were made 10% in polyethylene glycol 6000 (Union Carbide Corp.) and 0.5 M in NaCl and then were pelleted immediately at 4°C (39). The pellet was resuspended in 0.1 M Tris- HCl (pH 8.0), 10 InM EDTA, and made 1:l by weight with solid CsCl. Two successive preparative buoyant density equilibrium centrifuga- tions in the presence of ethidium bromide were then performed as described (38) except the runs were for 48-h periods. The purified plasmid was then extracted five times with isopropyl alcohol, dialyzed versus 0.5 M NaCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, and then versus 10 mM Tris-HCl (pH 7.4), 0.1 mu EDTA. The DNA solution was extracted three times with phenol, four times with ether, and then dialyzed into storage buffer (10 mrvr Tris-HCl (pH 7.4), 0.1 mM EDTA)

In some cases an alternative method was used to prepare plasmid directly from phenol-extracted lysates. This method is more rapid than that described above and provides DNA of suitable quality for DNA restriction mapping and transformation. Three to six milliliters of CM-amplified cell culture was pelleted. The pellet was suspended in 0.5 ml of 25% sucrose, 50 mu Tris-HCl (pH 8.0), 1 mM EDTA. One- tenth milliliter of lysozyme (10 mg/ml in 50 IIIM Tris-HCl, pH 8.0) was added and the mixture was incubated at 37°C for 15 min. Cells were lysed by blending on a Vortex mixer with 0.4 ml of buffer- saturated distilled phenol. After centrifugation at maximum speed in a table top centrifuge for 15 min at room temperature, the aqueous phase was removed and extracted once with phenol, and then with ether until clear. The nucleic acids were then precipitated with ethanol. After one ethanol wash, the pellet was resuspended in a buffer which was suitable for the subsequent restriction digestions. Pancreatic RNase A (2 pg) was added during the restriction reactions to remove RNA.

Gel Electrophoresis-Seven-tenths per cent agarose gel electro- phoresis was run as described (40). Cylindrical polyacrylamide gel electrophoresis was done as published (41). Polyacrylamide slab gels were prepared and run under similar conditions except that only 5% glycerol (not 25% as described) was included for 8% polyacrylamide slabs.

Plusmid Constructions-These experiments were done in compli- ance with the National Institutes of Health Guidelines on Recombi- nant DNAs under categories Pl, EKl. The general method is as follows: Ligations were carried out on 0.8 pg of total DNA in a 20.~1 reaction volume with lo-’ units (units defined in Ref. 42) of T4 DNA ligase at 15°C for 16 h. The ratio of insert to vector DNA is discussed under “Results.” The reaction conditions were similar to those re- ported by Sgaramella (43) and were 20 mM Tris-HCl (pH 7.4), 10 mM

MgCL, 1 mM diethiothreitol, 1 mM EDTA, 50 mrvr ATP. Where indicated, “sticky ends” were filed in (21) before addition of ligase by incubation of the DNA in ligase buffer with 0.1 unit of M. luteus DNA polymerase and 2 PM of each of the four deoxytriphosphates at 15°C for 60 min. Two-tenths microgram DNA was used per transformation of E. coli C600 SF8 or MO.

CaCl:, treatment of the cells and transformation were as described (44) except for the following modifications. The mixture of DNA- and CaCla-treated cells was heated to 37’C for 30 s, chilled on ice for 90 min, and then inoculated into 4 ml of L-broth (45) and shaken 90 min at 37°C. Two hundred microliters of this culture was then treated with colicin E, for 30 min at 37°C and plated on TYE-XG plates (46)

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Cloning of lac Operator-Promoter Fragments 5529

containing 0.5% deoxycholate. Cells transformed by Zac operator- containing plasmids are rendered lac constitutive and turn blue as described (47). Only clones which did not segregate white colonies on continuous repurification were further characterized.

Screening for Insertions by Plasmid Size-The size of plasmids was determined as described (20) with the following modifications. Either 2 to 3 mg of cells scraped from a plate or the pellet from 5 ml of CM-amplified cultures was used as starting material. Forty micro- grams of RNase was added to each sample during lysis.

Purification of Linker-A Hue III digest of hplac 5 DNA, when fractionated by RPC-5, yielded a pool containing several (three or four) h fragments, one of which had a single Eco RI site (see Fig. 1 in Ref. 33, Fractions 156 to 159). This 1300-bp fragment was cleaved by Eco RI into lOOO- and 300-bp pieces. After treatment of the pooled fragments with Eco RI, this mixture (90 ag of total DNA) was refractionated on an RPC-5 column (900 x 1.5 mm) with a sodium acetate gradient (12, 33, 50) to purify the 300-bp Eco RI-Hue III linker fragment. This linker was pure after chromatography and dialysis (see Fig. 5, second gel from top).

Other Methods-Recovery of fragments from gels was as follows. Gel slices were pulverized and soaked overnight at 37°C in 10 mM Tris-HCl (pH 7.9), 1 mu EDTA, 100 mM NaCl. Polyacrylamide debris were removed by centrifugation. The DNA was chromatographed on DEAE-cellulose as described (48) except that the sample was loaded in the above buffer and the DNA was eluted with 1 M NaCl, 10 mM Tris-HCl (pH 7.9), 1 mM EDTA.

Purification of Zac operator fragments by repressor binding was described (4). One hundred micrograms of lac repressor was used to bind out -50% of the 203-bp Hue III Zac 0 fragment from 1 mg of Hue III-digested hplac 5 DNA. The material which eluted with IPTG was predominantly 203 bp in length although the 169-bp fragment carrying the secondary repressor binding site (7,49) was also present (see top gel in Fig. 5). A slight background of X fragments originating from nonspecific binding to repressor was observed also.

RESULTS5

Cloning of 789-bp lac Fragment-A 789-bp Hind11 frag- ment, which was sequenced (5,6), extends from the end of the Zac i gene coding for the COOH terminus of the Zac repressor through the CAMP receptor protein binding site and the lac promoter and operator into the beginning of the .z gene (Fig. 1). It was cloned into the single Hind11 site of pVH51 as follows. A HindII+III digest of hplac 5 DNA was mixed with HindII+III-cleaved pVH51 (Hind111 does not cleave pVH51) and ligated by T4 DNA ligase. E. coli MO was transformed with the reaction products, selected for colicin immunity, and screened for lac constitutivity as described under “Materials and Methods.” As a control, E. coli MO was transformed with uncleaved pVH51. Agarose gel electrophoresis was conducted on sodium dodecyl sulfate-lysed, -amplified cultures of a num- ber of selected candidates which were lac constitutive and on one isolate from the control transformation. DNA from three candidates which were larger than pVH51 were prepared for restriction mapping.

Representative data is shown for the plasmids designated pRZ2 and pRZ3 (Fig. 2). Panel A shows agarose gel electro- phoretograms from which the sizes of intact pRZ2 and pRZ3 can be estimated relative to the positions of monomer- and dimer-supercoiled pVH51 DNA. Both are approximately 800 bp larger than pVH51. Hpa II digestion of the plasmids (Panel B) shows that the insertion is in the largest (940 bp) Hpa II fragment of pVH51. (More extensive mapping studies with several enzymes are presented in the miniprint supplement” to this paper.) Three new fragments are found at 328,254, and 1129 bp. From the known sequence of the 789-bp Zac fragment,

’ Portions of this paper (including Figs. 6 and 7 and Table I) are presented in a miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda. Md. 20014. Reauest Document No. 78M-1661. cite authors, and include a check or money order for $1.20 per set of photocopies.

pVH 51

A pRZ 2 UNCUT pRZ 3

BASE PAIRS 800 400 200 100 1

Hpa II -

C $7”: Hpa II - pRZ3 >- Hin+d 11

I / I /

FIG. 2. Gel electrophoretic characterization of plasmids containing 789-bp Hind11 lac fragment. Panel A, shows 0.7% agarose gel electro- phoretograms of the indicated plasmids. The five bands indicated by arrows are from an Eco RI digest of Xplac 5 DNA added as markers. The bands resulting from each plasmid are (from right to left): supercoiled monomer, nicked circular monomer, supercoiled dimers, respectively. Panels B and C are 5% polyacrylamide gels of the indicated digests. The base pair scale applies to Panels B and C. All gels were run from left to right. In Panel B, for the Hpa II digest of pVH51, products of partial digestion appear at 400 bp and 110 bp. In Panel C, for pVH51, a product of partial Hind11 digestion appears at 951 bp.

it is apparent that the 254-bp fragment is derived from the Zac insertion (see Fig. 1). Since the 328-bp Hpa II fragment is cleaved into 104- and 224-bp fragments by Hind11 (Panel C), the new 328-bp fragment in both pRZ2 and pRZ3 must represent a fusion between the 224-bp end of the 789-bp lac insertion and the 104-bp HindII-Hpa II segment of pVH51. The 104-bp fragment falls in a multiplet with other Hpa II fragments of this size. Similarly the 293-bp end of the lac insertion plus the 836bp HindII-Hpa II pVH51 segment produces the new 1129-bp in pRZ2 and PRZ3 DNAs. Double digestions were done with Eco RI and Hpa II on these plasmids in order to locate the insertions relative to the single Eco RI site in pVH51 (data not shown). In both plasmids the insertion is oriented such that the promoter is directed away from the Eco RI site which is 138 bp away from the Hind11 site.

Panel C also shows that the Hind11 sites were regenerated at both ends of the insertion in pRZ3, but only at one end in pRZ2. Fig. 3 summarizes these maps. Since the fusion frag- ment containing the lost Hind11 site in pRZ2 is not detectably different in size from the comparable fragment in pRZ3, we presume that an alteration (deletion) of only a few bp oc- curred.

Fig. 3 also shows the structure of pRZ1 DNA which has picked up a 360-bp X fragment in addition to the lac insertion.

Cloning of 203 bp lac Fragment-A plasmid was then constructed in which the 203-bp Hue III fragment containing the Zac operator, promoter, and CRP binding site (Fig. 1) was inserted into the Eco RI site of pVH51. This fragment was purified using the lac repressor as described under “Materials and Methods.”

This material was ligated in a 1:1 molar ratio with Eco RI- cleaved pVH51 DNA which had been fiied in at the ends by reaction with deoxynucleoside triphosphates and the M. lu- teus DNA polymerase as described under “Materials and Methods.” This ligation mixture was transformed into E. coli C600 SF8 which had approximately a lo-fold higher transfor- mation frequency than E. coli MO. The resulting plasmids were analyzed in an analogous manner to pRZ2 and pRZ3 (data not shown). Fig. 3 summarizes the resulting maps. pRW2 and pRW3 each have a single 203-bp insertion with opposite

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5530 Cloning of lac Operator-Promoter Fragments

pvti 51

pR.Z I

p RZ 2

p RZ 3

pRW I

p RW 8

p RW9

p RW 2

p RW3

p RW IO

pRWll

p RW 7

p RW12

pRWl3

p RW 6

p RWl4

p RW 5

p RW4

! immunity ori t ‘i’P

-200 bp

orientations and with both Eco RI sites regenerated. pRW10 and pRWl1 are similar but lack one Eco RI site each. pRW7, pRW12, and pRW13 have deletions which were useful for purposes of restriction mapping (miniprint supplement).

Cloning of Multiple 203bp lac Fragments-Since one of the goals of this work was to prepare large amounts of the inserted restriction fragments, we attempted to increase the yields by constructing plasmids containing multiple copies of this Zac fragment.

The 203-bp Zac insertion was isolated as an Eco RI fragment from pRW2 DNA as described elsewhere.2 The 203-bp frag- ment was first ligated to itself to give a homologous series of oligomers ranging up to ll-mer (data not shown, but the gel analysis of a similar reaction is presented in Footnote 2). This mixture was then ligated to Eco RI-cleaved pVH51 at a vector to insert ratio of 1:l on a nucleotide basis and transformed into E. coli C600. Selected colonies which were colicin-im- mune and Lac’ were analyzed for the size of the insertion as described under “Materials and Methods.” The plasmid DNA was prepared from several candidates having a 400-bp or larger insertion by the procedure described under “Materials and Methods” which allowed restriction mapping directly from cell lysates. This procedure substantially reduced the

FIG. 3. Summary of maps of plas- mids. The positions of relevant restric- tion sites and inserted sequences is shown for all plasmids described in this paper. Symbols are: R = Eco RI; K = Kpn I; H = HindII; E = Hae III; I = lac insertion; rz = X insertion; o = de- leted region. Only Hae III sites referred to in the text are shown. Numerous other Hae III sites exist in pVH51 (see mini- print supplement) and in the 7%bp Zac insertion (7). Horizontal arrows show orientation of the Zac promoter. The par- ent plasmid, pVH51, is shown at the top with approximate locations of the origin of replication and colicin immunity gene. The scale is at the lower left.

time required to screen candidates for an appropriate restric- tion map.

Fig. 4 shows representative gel analyses of Hae III, Eco RI, and Hpa II digestions of one of the isolates. Panel A demon- strates that the insert-containing Hae III fragment in pRW6 is -400 bp larger than for pRW2 DNA (which already contains one 203-bp Eco RI fragment, see above). Thus, a total of three 203-bp fragments were inserted. Panel B shows a partial Eco RI digestion of pRW6 DNA on 0.7% agarose gels where the vector plus one, two, or three additional 203-bp segments are visible. Panel C shows a partial Eco RI digest of pRW6 DNA on 5% polyacrylamide gels where monomers, dimers, and trimers of 203 bp are visible. When Panels B and C are considered together, they show that all four Eco RI sites among the three Zac inserted regions are regenerated. Panel D shows that the Hpa II cleavage products of pRW6 DNA are identical with those of pRW2 except for a greater intensity which co-migrates with a 205-bp Hpa II band from the vector.

Therefore, all three lac insertions in pRW6 are oriented the same as in pRW2 DNA. Of the two dimeric and one trimeric insertions which were examined, all had this feature (Fig. 3). These plasmids were stable in both rec.- (CSOO SF8) and ret+ (MO) cells as judged by their failure to segregate white

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A

6

C

D

pRW2 pRW6

Cloning of lac Operator-Promoter Fragments

t

Hae III

PRW6(

PRW

pRW2 pRW6

609 406 303

COMPLETE Eco

I- PARTIAL RI

COMPLETE Eco

>- FYiRTIAL RI

HpaII

5531

FIG. 4. Gel electrophoretic analysis of restriction enzyme digests of recombinant plasmids containing multiple insertions of the lac 203- bp Hue III fragment. Panels A (5% polyacrylamide gels) and B (0.7% agarose gels) show digests performed on phenol-extracted cell lysates as described under “Materials and Methods.” In Panel A, a 789-bp marker was added to the digests prior to electrophoresis. In pRW2, the 813-bp band containing a single 203-bp insert is just above the 789 marker and is indicated by an arrow. In pRW6 this band has increased in size by -400 bp. In Panel B, the positions of markers (an

colonies and by the homogeneity of plasmid size when ana- lyzed from lysed colonies or in chloramphenicol-amplified cultures. .

Cloning of 203-bp lac Fragment with Retention of Hue III Ends-The 203-bp lac fragment produced by Hue III was converted into an Eco RI fragment when cloned as described above. Alternatively, this Hue III fragment was cloned by a procedure which permitted retention of its Hue III ends. This

construction utilized Eco RI-Hue III linkers, molecules with an Eco RI half-site at one end and a Hue III half-site at the other. The linker was prepared as under “Materials and Methods.”

To prepare the recombinant plasmid, a 1:2:1 molar mixture of Eco RI cut pVH51, linker, and repressor-bound Hue III 203-bp lac fragment was ligated and transformed into E. coli C600 SF8 as described under “Materials and Methods.” The plasmid DNA from six colicin immune LacC candidates was prepared for restriction mapping. Fig. 5 shows a gel analysis on an Eco RI-Hue III double digest of pRW1 DNA which turned out to be the desired product. The 203-bp lac repres- sor-bound fragment and the 300-bp linker as well as a control double digest of pVH51 are shown on parallel gels. The Hue III-Eco RI double digest of pRW1 DNA released, in addition to the bands from the vector, a doublet at the position of the linker and a band at the position of 203 bp.

The maps of pRW1 and two other isolates (pRW8 and pRW9) from this experiment are shown in Fig. 3. For pRW9, a direct joint was made between an Eco RI sticky end and a Hue III blunt end without regeneration of the Eco RI site. In pRW8, one joint of this type occurred and one Eco RI-Hue III joint was made with regeneration of an Eco RI site. In the latter case, the single-stranded 5’-end from the Eco RI site must have been ligated directly to a Hue III blunt end with repair of the four base gap in Go. This is a novel reaction which may have been catalyzed by T4 DNA ligase or may have occurred in uiuo after transformation. These examples show that joining of incompatible ends can occur.

Cloning 95-bp lac Fragment-We also wished to clone

Eco RI digest of hplac 5 DNA) are shown by arrows. The high background on these gels in Panels A and B is due to contaminating chromosomal DNA which is not entirely removed by this rapid isolation procedure. Panels C and D are 5% polyacrylamide gels on the purified plasmid DNAs. The designations of 406 and 609 bp for the dimer and trimer, respectively, in Panel C do not include the four bp per Eco RI site at the junctions. The arrow below Panel D shows the position of the 203-bp fragment. In each case the indicated digests were run from left to right.

203 bp & 300 bp linker pRWl pVH5I

HoacIII

&o RI

FIG. 5. Polyacrylamide gel analysis of a Hue III + Eco RI double digest of the recombinant plasmid pRW1. The linker-containing plasmid was constructed and isolated as described under “Materials and Methods” and “Results.” Parallel control gels were also run on the 203.bp lac fragment and on the 300-bp h linker fragment with a Hae III site at one end and an Eco RI site at the other. A parallel double digest of the vector pVH51 DNA is also shown. The gels were run from left to right.

the 95-bp Alu I fragment (Fig. 1) which contains only the lac operator and promotor and lacks a portion of the CRP binding site. The 95-bp fragment was obtained from an Alu I digest of pRZ3 (construction described above) which was fractionated by electrophoresis on a 5% polyacrylamide gel. The 95-bp Zac fragment was eluted from a section of the gel as described (“Materials and Methods”). Since this fragment co-migrates with another band from pRZ3, the recovered material was not pure.

The mixture was cloned into the Eco RI site of pVH51 in an analogous fashion to the construction of pRW2. In this case a large molar excess of the Zac fragment was used result- ing in 50% Lac” colonies. We have found that molar excesses of lo-fold will give rise to insertion frequencies of greater than 50%.

DISCUSSION

The plasmids described in this paper were designed to simplify the purification of fragments containing the lac ge- netic control elements. Four Zac fragments, each contained within the next larger fragment, were cloned. All contain the Zac operator and all or part of the Zac promoter. The purpose of obtaining quantities of these DNAs is to perform biochem- ical and physical studies to evaluate the role of DNA proper- ties in Zac genetic regulation. Other workers have cloned fragments containing portions of the Zac control region (21,

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5532 Cloning of lac Operator-Promoter Fragments

26,51) and containing chemically synthesized oligonucleotides corresponding to a portion of this region (47, 52-54). The novel aspect of the plasmid constructions described herein is their utility for the large scale preparation of fragments.’

The construction of pRW1 demonstrates the utility of DNA linkers isolated from natural sources for gene cloning. Thus, it is not necessary to resort to the tedious steps of chemical polynucleotide synthesis in order to obtain linkers. Moreover, linkers from natural sources can easily be obtained with a variety of sizes which contain different restriction sites. In some cases, such as for pRW1, the relatively large size of the linkers was advantageous since it facilitated the purification from the desired insert. Furthermore, linkers from natural sources can be chosen so that a genetic marker such as the Zac operator is present to facilitate detection of the final plasmid. Also, biological functions, such as promoters or ter- minators, can be carried on the linker if desired.

The construction of pRW1 required the coordinated ligation of 4 different molecules (the 203-bp insert, the vector, and 2 linker molecules in the proper orientation). Its success dem- onstrated the feasibility of such complicated constructions.

pRW6 was designed to increase the yields of the lac frag- ment by producing multiple copies of the fragment per mole- cule of vector. Several other laboratories (21,52-54) have also found multiple inserts. However, when we attempted to con- struct plasmids with an even greater number of Zac inserts (using the purified 6-mer or lo-mer of self-ligated 203-bp Eco RI fragment), only plasmids containing deletions extending into the lac insertion were recovered. Others (53) have dis- cussed problems in obtaining stable plasmids with multiple insertions. It was proposed that only direct repeats are stable because inverted repeats led to abortive replication structures (54). This is consistent with the fact that we only obtained direct repeats.

Some of the plasmids described herein were used in this series of papers for other purposes. Digests of pRZ2 were used to explore column chromatography on RPC-5 further (12-14). Base composition and physical analyses on this molecule and its restriction fragments were performed. The restriction map- ping presented in the miniprint supplement to this paper was used in the interpretation of that work. We have not relied on only a size estimate and the position of a few bands to characterize our insertions. Rather, we have also examined each of them with at least two frequently cutting restriction enzymes. A number of product plasmids with unexpected maps were isolated during the course of this study. This may be due to the diversity of mechanisms by which the recipient bacteria may alter and repair the incoming ligation products. Since unexpected plasmids can arise which are similar to the expected products, we have found that it is generally advisable to verify the organization of new plasmids with several restric- tion digests.

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For additional references, please see below.

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