Gene. ~ (1976) 81--92 81 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

A GENERAL METHOD FOR INSERTING SPECIFIC DNA SEQUENCES ONmG vF.mCLES (Synthetic decadeoxyribonucleotides; linker sequences; lac DNA; plasmids; restriction endonucleases; cloned DNA) CHANDER P. BAHL, KENNETH J. MARiANS and RAY WU* Section o f Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, N Y 14853 (U.S.A.) and

JACEK STAWINSKYand SARAN A. NARANG Division o f Biolo&ical Sciences, National Research Council of Canada, Ottawa, Ontario (Canada)

(Received September 30th, 1976) (Revision received and accepted October 5th, 1976)

SUMMARY

A general method has been developed to introduce any double,stranded DNA molecule into cloningHehicles at different restriction endonuclease sites. In this method a chemically synthesized decsdeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joined by DNA tigase to both ends of the DNA to be cloned. The resulting new duplex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic/ac operator DNA at the BamI and H/ndIII cleavage sites of the plasmid pMB9 DNA. INTRODUCTION

Molecular cloning has become a powerful tool for the amplification of *To whom correspondence should be addressed. is paper ~ X ~ 8 series on "Nueleotide Sequence Analysis of DNA". P a ~ XXVIII is by Jay and Wu, 1976. This work was supported by Grants GM 18887 and CA 14989 ~ m the National Institutes of Health, and No. 15576 from the National Research Coun

orC,m

Abbreviations: ll~(;J, isopropyl thiofalactoside; ONPG, o-n|trophenyl-~-D.galactoside;X-gal, 6-bromo-5-ehloro-8,indoyl~-b~de.

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specific DNA fragments and their subsequent isolation in high yields. Two basic steps are involved in molecular cloning. First the DNA fragment~ to be cloned are joined in vitro ~ an autonomously replicating vehicle molecule, e.g. plasmid DNA (Cohen et aL, 1973; Tanaka and Weisblum, 1975) or ), phage DNA (Thomas et al., I974; Murray and Murray, 1974). The hyht~d D N A formed is then introduced into £. coli by ~ f o r m a t i o n and then c l o n e d by single colony isolation or plaque formation. .... .... In one cloning method, two different DNA molecules are cut by the same restriction endonuclease ~ produce identical cohesive ends. The DNA molecules are annealed to one another and then covalent!y joined by DNA ligase. This method limits the size and kind of DNA fragments that can be cloned since it often requires cloning of a much larger DNA fragment than one is intereste~ in. For example, if one wants to clone a small DNA fragment such as a promoter (e.g. an RNA polymerase p n ) ~ fragment), the nearest restriction endonuclease recognition sites may be relatively distant, and thus extraneous DNA sequences must be included in the cloned DNA. Furthermore,

many DNA fragments cannot be cloned because of the lack o f a suitable restriction enzyme to produce mole,.des with cohesive ends. In this communication we report a general procedure to overcome these limitations. The procedure utilizes a chemically synthesized decadeoxynucleotide such as d(C-C-G-G-AT-C-C-G-G) or d(A-C-A-A-G-C-T-T-G-T) to generate restriction enzyme recognition sites at the ends of any natural or synthetic DNA molecule to be cloned, thereby making the cloning procedure much more selective and versatile. By this method, we have inserted the synthetic/ac operator DNA at the BamI and HindlII sites, respectively, of pMB9 plasmid. The insertion of the lac operator into the EcoRI site of pMB9 has recently been accomplished for the large scale production of lac operator (Marians et al. and Heyneker et al., personal commun.). MATERIALS AND METHODS

The lactose operator duplex DNA was chemically synthesized as described by Bahl et al. (1976). Lactose repressor (Plattet al., 1973) and T4 DNA ligase (Weiss et al., 1968 and Planet et al., 1973) were puttied by published methods. All the procedures for labeling and purifying DNA have been described (Wu et al., 1976).

Chemical synthesis of the decadeoxyribonucleotides The two decadeoxyribonucleotides d(C-C-G-G-A-T-C.C.G.G) (BamI linker sequmce) and d(A-C-A-A-G~-T-T,G-T)(HindIII linker sequence)were syn. thesiz~ b y t h e ~ p r o v e d phosphotriester method d e v e | o p ~ m o ~ labora~ry ( I t ~ ~ et i ~ u m et ~., 1975~ K et et~,, 1975b, and Stawinsky et al,, 1976). conditions are summarized in Fig. 1 and Table I, ~pectively. ~ 0 new steps were introduced in these syntheses: the dimethoxytn'tyl group was ~removed

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Fig. 1. The modified phosphotriester approach for the synthesis of the decadeoxyribonucleotide d(A-C-A-A-G4~-T-T-G-T).

by a 2% solu~on of benzene-sulfonic acid in chloroform and the chlorophenyl phosphate protecting group was removed by treatment with concentrated ammonium hydroxide for 4--6 h (Stawinsky et al., 1976). The oligonucleotides were characterized by 2-D electrophoresis-homochromatography of their partial venom phosphodiesterase digestion products (Jay et al., 1974 and Tu et et al., 1976). The two-dimensional maps of the two oligonucleotides and the a s s ~ e n t of various spot~ are shown in Fig. 2, which verify the sequences of the two synthetic decsdeoxynucleotides.

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Fill 2. Two-dimudoml map o f a ~ i a l snake venom phosphodiesterase digest of (A) the ~ueleoti~ d(~~,A-T-C~~) and (n) the deeanueleotide d(A-C-A-A-G~-T-T-G-T). D~mion i,e b on eeUulm seetate ~rip , t p H 8.5, dimension H. h o , oehromatol~phy on DP~~..llulose thin-layar plate run in Homomixture VI (Jay et aL, 1974). The • s values a d mobility shift values (S°me end S°°s) are U described by Tu et al. (1976).

8ynthais of duplex DNA linker molecules and their digestion by restriction enzymes ChemicaUy synthesized decadeoxynucleotides (400 pmoles) were phosphor:

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(Wu et at,,

pl o f l O O m M O°C and t h e n _ _Leotide was

86

formed by slowly cooling the sample to room temperature and then to 4°C, Baml linker when digested with BamI restriction endonuclease (Wilson a n d Young 1975) gave the expected trinucleotide 3=pC-C-G as the labeled product. The duplex HindIII linker molecule, when digested with AluI (Roberts et al., 1976 and Jay and Wu, 1976) gave the expected pentanucleotide 3=pA~-A-A-G as the labeled product. ~

Insertion of lee operator sequence into pMB9 plusmid DNA by the use of chemically synthesized linker molecules and blunt-end ligation As shown in Fig. 3, the synthetic duplex linker (B) (10 pmole) and the synthetic/ac operator (A) (0.6 pmole) were joined end-to-end (Sgammella et al., 1970) by incubation with 3 units of T4 DNA ligase in 50 #1 of a solution containing 20 mM Tris-HCl (pH 7.5), 10 mM dithiothreitol, 10 mM MgCI2 and 35 #M ATP at 20°C for 6 h to produce molecule (C). The solution was heated at 70°C for 5 min to inactivate the ligase and cooled slowly to room temperature. 2 vol. of ethanol were added, and after 12 h at -20°C the DNA was pelleted at 10 000 g for 1 h. The pellet was dissolved in 50 #1 of a solution containing 6.6 mM Tris--HCl (pH 7.5), 6.6 mM MgCI~ and I mM dithiothreitol. To this solution was added 1 #g of pMB9 DNA (Rodriguez et al., 1976) and 2 units of the restriction endonuclease (BamI or HindIII for the corresponding linker). The sample was incubated at 37°C for 12 h to produce molecule (D) and linear pMB9 DNA.~The samples were then heated at 70°C for 5 min, cooled slowly to room temperature, and the DNA was precipitated with 2 vol. of ethanol as before. The DNA pellet was dissolved in 50 #1 of a solution containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCI2, 10 mM dithiothreitol and 35 #M ATP. Three units of DNA ligase were added and the samples were incubated at 12.5°C for 24 h to produce the hybrid/a¢-pMB9 DNA. After heating at 70°C for 5 rain, and slow cooling to room temperature, the hybrid /ac-pMB9 DNA was directly used for the transformation step. Transformation and selection of la¢-pMB9 hybrid plasmids The hybrid/ac-pMB9 DNA was used to transform competent E. coil HB129 cells (Rodriguez et al., 1976). The procedure for transformation was exactly as that described in an earlier paper by Marians et al. (1976). The cloneswere screened for/~-galact0sidase e s c ~ synthesis ( M ~ e t al,, 1976) by using the reaction with ONPG or X-gal (lVliller, 1972). The DNA from the clones showing positive color reactions due t o over-production of O-galactosidase was isolated and tested for/ae repressor binding by the millipore filter assay (Riggs et al., 1970). RESULTS AND DISCUSSION

Chemically s y n t h e s i ~ oligonucleotideswith defined sequences have a great potential for the study of ~ o u s problems in m o l e ~ biology-. In this communication we.describe ameth~:, that exploits, the usefulness-of synthetic

87

oligonucleotides to create cohesive ends at the termini of any duplex DNA molecule so that the latter can be inserted into cloning vehicles. We have synthesized two decadeoxynucleotide fragments, d(C-C-G-G-A-T-C-C-G-G) and d(A-C-A-A-G-C-T-T-G-T). Each synthetic flragment is self-complementary and can form a duplex molecule which contains the recognition sequence of the restriction endonuclease BamI (Wilson and Young, 1975) and HindHI (Old et al., 1975), respectively, The synthetic fragments can serve as linker molecules* for the insertion of any duplex DNA molecules into cloning vehicles. The principle of this general method is illustrated in Fig. 3. Its feasibility has been tested 5 ' pA-A-T-T-G-T-G-A-C,-C-C-G-A-T-A-A-C-A-A-T-T 3t

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Fig. 3. A general scheme for introducing cohesive ends at the termini of any duplex DNA molecule for molecular cloning. Short dotted lines represent the recognition site of a restriction end©,nuclease; long double tines represent the 21 nucleotide-long duplex of/ac operator DNA. *These synthetic oligonucleotides are also useful model substrates for studying the mechanism of cleavage of DNA by a number of restriction endonucleases. For example, the synthetic decanucleotide d(C-C-G-G-A-T-C-C-G-G) in the duplex form also contains the recognition sites for HpalI (Garf'm and Goodman, 1974) and MboI (Roberts, R., personal commun.) restriction enzymes. Similarly, the oligonucleotide d(A-C-A-A-G-C-T-T-G-T) contains the recognition sequence for A|uI (Roberts et al., 1976; Jay and Wu, 1976). Moreover, the sequence d(C~-G,G-A-T-C-C-G-~) as a duplex can be polymerized by T4 ligase and all the ~ents, direst onwards will contain restriction endonuclease recognition sites for HaeIII (Middlctonet al,, X972) and B ~ (Roberts, R., personal commun.) endonucleases. A study onthe cleavage Of a synthctic octanucleotide, d(T-G-A-A-T-T-C-A), by E¢oRI endonuclease has been reported by G r i n et al. (197§).

88

" " ~" t by i• n t r ~ c i n" g a synthetic lac operator duple.x, into pMB9 plasmid. ~ the step, ~ . ~ = i ~ molecule (B)is enzymatically l i ~ t e d to ~ t h ends o f the /uc ope~tot DNA duplex (A) to produce molecule (C), ~ latter is cleavedby the proper restriction enzyme to produce molecule (D)which contains the desired cohesive ends at both ends. The DNA module (D) is .thenhybridized to ~ B 9 DNA which ~ been made line~ ~ the same restriction enzyme and thus contains t h e stone cohesive e n ~ . The hydrogen~bonded molecules are enzymatically li~ted to form hybrid ~c, pMB9iDNA~ The]atter m ~ to transform E. eoli cells ~ tetracycline resistant. T h e resulting colonies am screened for the over-production of ~-galactosidase, which indicates the insertion of the luc operator DNA. The cells which contain the hybrid kzc.pMB9 DNA are purified by streaking on agar plates which contain tetracycline and X-gal (Miller, 1972; Marians et al., 1976). The hybrid Zac-pMB9 DNA was isolated, after amplification by the addition of chloroamphenicol to the bacterial culture, labeled by nick translation (Maniatis et aL, 1975), and studied for bzc repressor binding properties. Typical binding curves are shown in Fig. 4. The inhibitory effect of IPTG shows that the binding is specific, and thus confirms that a ~c operator has been inserted into the plasmid. The hybrid plasmid DNA was further characterized by digestion with the appropriate restriction endonuclease (Baml or Hindlll corresponding to the linker used) and then labeled at the 3'~nds by repair synthesis in the presence of [~-32P] dNTP (Wu et al., 1976). It gave two fragments on polyacrylamide gel electrophoresis as shown in Fig. 5. The large fragment corresponds to the linear pMB9 DNA. The small fragment corresponds to the kzc operator since it binds specifically to the/ac repressor.

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Fig. 5. An autoradiograph of gel electrophoresis of hybrid lac-pMB9 plasmid. (A) pMB9 plasmid (as control) digested by BamI restriction endonuclease. (B) Hybrid plasmid constructed withBamI linker and digested by BamI restriction endonuclease. (C) Hybrid plasmid constructed with HindHI linker and digested by HindIII restriction endonuclease. Experimental detaik are the same as described by Marians et aL, 1976. In the insertion of the 21 nucleotide-long duplex/ac operator into pMB9 plasmid, the length of the linker molecules was so designed that after linking, three extra nucleofides were introduced on either side of the lac operator. This leaves the reading frome for pMBg-directed protein synthesis unchanged. R~zet al, (1976) reported that HindIII and B a m I restriction endonuclease sites in pMB9 are located in the gene which confers tetracycline resistance. We found that the insertion of 27 nucleotides to produce the hybrid/ac-pMB9 DNA does not change the tetracycline resistance of cells harboring this hybrid plasmid. This observation can have two interpretations. First, the B a m I site and HindIH sites are not in the tetracycline resistance gene. Alternatively, insertion of 27 nucleotides does not alter the activity of the protein for which it codes. Chemically synthesized linker molecules are extremely useful tools in molecular cloning since the same oligonucleotide linker can serve to introduce any double-stranded DNA molecule into cloning vehicles at specific sites. The double-stranded DNA may be obtained by cleavage with any restriction enzyme or by other means. If the ends of the DNA are even, they can be joined directly to the linker molecule. If the ends are uneven, they can be digested with Aspeq~llus nuclease $1 (Ando et al., 1966; Ghangas and Wu, 1975) to produce even~nd DNA molecules. Another advantege of this method is the facility with which two different

90 Lacoper4tor $t G-A-T-C . . . . . .

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Fig~ 6. A general scheme for linking any DNA molecule with kzc operator and pMB9 plasmid to make a hybrid DNA(X)-~c-pMB9which can be easily screened for the overproduction of ~-galaetosidase activity O/larians et aL, 1976). DNA molecules can be cloned ~)gether. One DNA (e.g. hzc operator) is chosen to allow for an easy screening for cells harboring hybrid plasmids, as shown by the following example. The ~¢ operator sequence with the BomI cohesive ends (such as molecule D in Fig. 3) can be ligated in vitro to another DNA molecule (X) conta/ning similar cohesive ends (Fig. 6). After separation of the unreacted ~z¢ operator from the hybrid DNA (X)-kz¢ operator molecule (molecule E), the latter can then be joined to a cloning vehicle. The lactose operator sequence in the hybrid DNA (X).kzc-pMB9 should make the cells constitutive for ~.galactosidase (Marians et al., 1976) which are easy to screen. In this way, the presence of DNA (X) in the hybrid p l u m i d can be easily detected. The purification of hybrid DNA (X)-hzc-pMB9 can also be carried out by binding to ~¢ repressor on a mill/pore filter and eluting with IPTG ( R i ~ s et al., 1970; Marians et al., 1976). Once the hybrid DNA (X)-hzo-pMB9 is characterized, it can be produced in large amounts by the u s u ~ method for the amplification of pMB9. Thus, large quantities of DNA (X) can be obtained, after digestion with the same restriction enzyme used for its insertion, for DNA sequence analysis and other types of studies. In conclusion, the cloning procedure described in this paper has a general application for the cloning of a variety of DNA moleculeS, and will facilitate the solving of different problems in molecular biology. REFERENCES

Ando, T., A nuclease specific for heat~denatured DNA isolated from a product of Asper~llus oryzoe, Biochir~ Biophys. A ~ 114 (1966)158--168. Bah~ C.P,, Wu, R., Itakura, K., K a r a t , N. and Narang, 8.~_,Chemical and enzymatic synthesis of lactose operator of Escherich~ coli and its bindi~ to lactose repressor, Pro~ Natl. Acad. ScL USA. 73 (1976) 91-94.

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