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Modification of the bacteriophage vector M13mp2: introduction of new restriction sites for cloning
Gene, 15 (1981) 167-176
167
Elsevier/North-Holland Biomedical Press
Modification o f the bacteriophage vector M13mp2: introduction of new restriction sites for cloning (Synthetic adaptors; cloning vehicle; single-stranded DNA; ~ite-specific mutagenesis)
Rodney Rothstein and Ray Wu * Department o f Microbiology, CMDNJ - New Jersey Medical School, Newark, NJ 07103, and * Section on Biochemistry, Cell and Molecular Biology, Cornell University, Ithaca, N Y 14853 (U.S.A.}
(Received October 27th, 1980) (Revision received May 22nd, 1981) (Accepted June 5th, 1981)
SUMMARY
The construction of two new derivatives of the bacteriophage cloning vector M13rap2 is described. One derivative, mWJ22, contains a new HindIII site while the other, mWJ43, contains a new BamHI site. These new sites were both introduced at the EcoRI site at amino acid five of the 145 amino acid-long fragment ofEscherlchla coli /3-galactosidase within the phage. The new restriction sites do not disrupt the blue color detection system of Ml3mp2; therefore insertion of cloned fragments results in colorless plaques on indicator plates for the new derivatives.
INTRODUCTION
The fdamentous single-stranded DNA phages of E. coli offer numerous advantages as cloning vehicles. Both large amounts of replicative form I DNA and single-stranded DNA can be isolated. Only one strand of the cloned DNA is packaged depending on the orientation of insertion into the phage vector. This results in the purification of that strand. The high virus titer permits the isolation of large amounts of any cloned DNA fragment. Finally the complete nuAbbreviations: bp, base paks; DTT, dithiothzeitol; IPTG, isopropyl-~-D-thiogalactopyranoside; PEG, polyethylene glycol; RF, repficative form ofMl3 DNA; X-gal, $-bromo4-chloro2-indolyl-~-D-galactoside.
cleotide sequence of the MI3 genome is known (Van Wezenbeek et al., 1980). Puri~ed single-stranded DNA has many current applications. It is useful for both purification and analysis of RNA transcripts since there is no complementary DNA strand competing with the DNA: RNA hybridization. In addition, a purified singlestranded template greatly facilitates the dideoxynu. cleotide sequencing method of Sanger et al. (1977). Several single-stranded DNA phage have been modified as cloning vehicles (Gronenborn and Messing, 1978; Barnes, 1979; Boeke et al., 1979; Hermann et al., 1980; Hines and Ray, 1980). The RF of one, Ml3mp2, hasa 789 bp insertion ofE. coli DNA that codes for the first 145 amino acids of the ~f,alactosidase gene (Gronenborn and Messing,
0378-1119/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
t68
1978). When Ml3mp2 infects an E. coli strain that contains an appropriate partial deletion of/~galactosidase, the phage-encoded peptide fra~gment can complement the host fragment (a-compler~ntation), resviting in blue plaques on indicator medium. The vector has an EcoRl site at the nucleotides corres p o n ~ g to the fifth amino acid of the ~gahctosi~se ~. When a foreign EcoRI fragmem is cloned into the RF, the resultant phage makes a colorless plaque after transfection. Short oligonucleotide primet~ for the DNA sequence analysis of cloned fragments have also been made for this phage (Heidecker et al., 1980; Anderson et al., 1980; Wu et ~., 1380). These features of Ml3mp2 make it an excellent systeF for cloning. In this report we will present some modifications that we have engineered into MI _~m?2 to make it a more versatile cloning vector. First, we eliminated the natural BaraHl site of M13m?2 by cl~emical mutagenesis since this s~te is in an essential gene and cannot be used for cloning. Next ~e introduced either a HindIll or a EamHl site at the EcoRl site using synthetic oligonucleotide adaptors. The resultant two new vectors, mWJ22 and n~WJ43, are both colorless when a ltindlll or a BamHl fragment is cloned into them, re:pec~ively.
MA]~ERIALS AND METHODS
(a) Purification of DNA fragments from agarose DNA fragments were purified from agarose gels by two methods. The first is essentially the method of Chen and Thomas 0 9 8 0 ) as described in Yang et al. (1979) in which the gel slice is dissolved in 4.5 M 5.0 M sodium perchlorate. The second method of DNA purification of agarose gel fragments was by electroelution (McDonell et al., 1977).
(b) Preparation of self-complementary oligonucleotides for ligation 200 pmol of the oligonucleotide sequences H O GGAATTCC-OH (Collaborative Research), H O ACAAGCTTGT-OH and HO-CCGGATCCGG-OH (gifts of Saran Narang) were each mixed ,vith 300
pmol of [~-aep]ATP (Amersham, diluted to 100 Ci/ mmol) and 0.8 uaits polynucleotide kinase (Biogenics or Boehringer-Mannheim) in 55 mM Tris-HCI, pH 8.0; 10 mM MgCl2.10 mM DTT. The unreacted triphosphate was removed by G-50 Sephadex (Pharmacia) column chromatography with sterile water as the ehant. The fractions containing the phosphorylated oligonucleotide were pooled and dried in an Evapo-Mix, resuspended at a fmal concentration of 10 pmol//d in TE (10 mM Tds- HCI, pH 7.4, 1 mM EDTA) and stored by freezing at -20°(2.
(c) Joining DNA fragments with T4 DNA iigase Blunt-end ligation of self-complementary oligomers (Rothstein et al., 1979) was catalyzed by T4 DNA ligase (New England BioLabs, MA). Phosphorylated oligomers were mixed at 1-10 #M in 5 ~1 of 50 mM Tris- HCI, pH 7.4, 10 mM MgCI2, 10 mM DTT and 66 /~M ATP. The mixture was heated to 65°C and slow-cooled to 4°C. Between 0.5 and I unit of T4 DNA ligase was added and the mixture was incubated for 24 h at 4°C. The reaction was monitored by electrophoresis on a 15% polyacrylamide gel. The ligation of cohesive ends was carried out essentially the same way, but at a lower concentration of DNA and DNA ligase. For example, linear DNA molecules recovered from a gel were ligated at 10 nM concentrations to promote intramolecular cyclization. For a 10 ~1 reaction 0.05-0.2 units of ligase were used and the mixture was incubated for 2 to 6 h at 14°C.
(d) In vitro mutagenesis with sodium bisuifite The bisulfite mutagenesis reactions were carried out in very small volumes following the procedure outlined by Shortle and Nathans (1978). BamHldigested Ml3mp2 DNA at 2.4 pg in 4 0 / d of TE was reacted with 120 gl of 4 M sodium bisulfite in a 6 mm X 50 mm siliconized glass test tube. Mineral oil was layered on top. The various dialyses required were carried out in that tube. A small piece of dialysis tubing was fitted over the top of the tube with a 3 mm length of tight-fitting tygon tubing acting as a rubber band. The test tube was inverted and centrifuged for a minute to invert the reaction mix and oil layers. This inverted tube was moved through the vat-
169 ious dialysis solutions required for the procedure. Care was taken when the 37°C dialysis was done since the coefficient of expansion for the tygon tubing is different from that for glass. The temperature of the test tube was slowly raised to 37°C.
(e) Transfection of MI 3mp2 and its derivatives The E. coli strains 71-18 (Gronenbom and Messing, 1978) and LE392 (Leder et al., 1976) were made competent using the RbC1 regime (Bolivar and Backman, 1979) and were stored at -70°C (Morrison, 1979). Although 71-18 contains the proper genoWpe for directly detecting plaque color with M13mp2 and its derivatives, we rarely obtained transformation frequencies greater than l0 s transformants//ag of transforming DNA/109 E. coil cells. To increase transformation frequencies, we often used LE392 which gave frequencies as high ~ 5 × l 0 6 transformants//zg/lO 9 cells. Since LE392 transfected cells are F-, phage are only produced within the transformed cells - neighboring cells are not reinfected. In order to view plaques, we also added 0.1 ml/plate of a saturated culture of 71-18. This procedure does not allow the immediate determination by color indication of whether a fragment is cloned, since LE392 is itself/acZ* wild type. Plaques can, however, either be picked or pooled and replated on the appropriate strain and indicator medium to permit color development. Plating of phage with color indication is achieved by mixing 0.1 ml of the appropriate phage dilution in 10 mM Tris (pH 7.4), 10mM MgCI2, 0.01% gelatin with 0.1 ml of a saturated culture of 71-18. After 15 rain at 37°C, 50/al of a 2% solution of X-fal dissolved in N,N-dimethyl formamide and 10/A of a 100 mM solution of IPTG were added. Next 3 ml of L-B soft afar (45°C) were added and the cells and phage were plated on M9 minimal plates supplemented with 5 /ag/ml thiamine. Plaque color develops after 9-12 h. The NIH guidelines require a transfer-deficient host for cloning with MI3 derivatives and certain eukaryotic DNAs.
(f) Screening colorless plaques for inserts Plaques can be screened for inserts by hybridizing a labeled probe to a nitrocellulose filter, either from a master plate of plaques or from random plaques
(100-2 O00/plate) by the procedures described for ~,. plaque screening (Benton and Davis, 1977). Mini-screening of RFI DNA from a plaque was accomplished by either of two procedures. We have adapted these procedures, originally described fo~ plasmid nfini-screens, for phage RFI isolation. This was accomplished either by growing 10 ml of the E. co~ strain 71-18 to an A6oo of 1 and infecting with 101° phage or by putting a single plaque (as a plug using a sterile pasteur pipette) into 10 ml of L-B medium with 0.1 ml of a saturated culture of 71-18 cells. In the first case the cells were infected for 45 rain after which chloramphenicol at 30 ~g/ml was added. 45 rain later the cells were harvested. In the second case the cells were harvested at a density (Aeoo) of 0.5 to 1 (4 h) and treated as in the plasmid screening procedure previously described (Bolivar and Backman, 1979). We found that by doing two ethanol precipitations, the DNA was suitable for restriction-enzyme digestions. The final pellet was resuspended in 150 pl of TE and 10-15 /~1 was used for each digest. The other DNA mini-screen was that described by Davis et al. (1980).
(g) Large-scale preparation of RFi Any convenient method for preparing a cleared lysate of a plasmid preparation can be adapted to preparing RFI from M13 (Rothstein, 1979). To maximize the RFI yield we grew E. coli strain 71-1~g at 37°C to an A600 of about 1. The cells were infected with 2-3 X 109 pha~/ml of culture. After 1 h, ch~oramphenicol was ad'.ied to 150 ~/ml. The cells were harvested 45 min later and RFI was isolated from the infected cells (yield approx. 1 rag/liter).
(It) End-labeling of small inserts and DNA sequence analysis DNA molecules with 5' protruding ends can be end-labeled by reverse transcriptase and the addition of one or more radioactively labeled tripho',phate (Bah1 et al., 1977). Both the chemical method (Maxam and Gilbert, 1977) and the dideoxynue1-~otide method (Sanger et al., i977) were employed for DNA sequence determination. For chemical sequencing, the fragments were first labeled at their 3' ends and single end-labeled fragments were isolated following gel electrophoresis (Rothstein, 1979).
~70 Single-st.randed phage DNA to be used as template for the chain torminator method was isolated by the method of Schreier and Cortese (1979), or by our plasmid prefLx is pWJ, therefore our MI3 clones The primer for sequencing was either the synthetic 19-met (Wu et al., 1980) or that described by Heidecker et al. (1980). We have used either the method of Sanger et al. (1977), or substituted reverse tr~scfiptase for polymerase ! (Smith, 1980). (i) Nomenclature
We have chosen to use a lower case m as the prefix for M 13 clones. This is followed by the same upper ca~ letters that we have registered with the Hasraid Reference Center, Stanford, CA. For example, our plasmid prefix is pWJ, therefore our M 13 clones are prefixed mWJ.
RESULTS (a) In vitro chemical mutagenesis of the BamHlsite of M i 3mp2
To construct a BamHl vector, we first needed to mutate the natural BamHl site of M 13rap2 since this site lies within an essential gene of M 13 (Van Wezenbeeket al., 1980). Since sodium bisulfite mutagenesis is thought to be restricted to deoxycytosine residues on single-stranded DNA (Shortle and Nathans, 1978), we used this method to mutate the natural deoxycytosine residues on the exposed cohesive ends of the BamHl recognition sequence with bisulfite. This procedure converts deoxycytosine to deoxyuracil. This conversion can take place at either of the two cytosine residues or at both of them in the BamHl cohesive ends. After chemical mutagenesis, the linear molecules were re-itgated with T4 DNA ligase. Along with the ligation of the unaltered cohesive ends, guanosineuracil bases are expected to form. a stable base pair and give: G-A-T-U C-T -A-G The G - U mismatch can be tolerated in this position since the. stack/ng interaction amongst the bases
is favorable after mutation. The iigated cohesive ends can result in the four configurations shown in Table I. The first example, A, shows the religation of unreacted cohesive ends. The configurations in B and C are expected when one or the other cohesive end is converted by bisulfite and subsequently ligates with the unreacted end. Finally, D shows the configuration after the ligation of two reacted ends. The ligated molecules were next redigested with BamHl and transfected into E. coll. This digestion lineartzes the type A molecules (Table I), while the B, C and D configurations are not digested due to the altered sequence. Since uncut DNA circles transfect with a greater efficiency than linear molecules, molecules containing altered BamHl sites were enriched after transfection into competent E. coil cells. Based on these assumptions and the change in transfection frequency before and after BamHl digestion, we calculate that 3% of the molecules were altered (i.e., resistant to BamHl digestion). The plaques obtained after BamHl digestion and transfection were pooled (40 individual plaques). RFI DNA was purified after infection with the phage pool. The pooling was undertaken because the initial plaques with the presumptive BamHl mufations were each formed from a heteroduplex molecule (G. U base pair). After heteroduplex segregation each plaque has a mixed population of phage both with and without a mutated BamHi site. After extensive digestion of the pooled RFI molecules with BamHl, a large fraction of the molecules (approx. 25%) remained resistant to BamHl digestion. Circular DNA was isolated after agarose gel electrophoresis from this fraction and was transfected into E. coli cells. Several plaques were purified, and the RFI DNA from
TABLE 1 Nucleotides 2220-2225 a (BamHl site) after ligation of bisulfite-teacted molecules (A) (B)
(C) (D)
...GGATCC... CCTAGG ...GGATUC... CCTAGG ...GGATCC... CUTAGG ...GGATUC... CUTAGG
umeacted top strand reacted bottom strand reacted botlt strands reacted
a Nucleotidenumber frora Van Wezenbeeket al. (1980).
171
F.;g. 1. DNA sequence of the mutated natmal BamHl site of M13. Dideoxynucleotide sequencing of the mutated BamHl site at position 2220 was w.imed by isolating a 133 bplong Haelll-Hpalll fragment (position 2247 to 2380). [A] The natural BamHl sequence -GGATCC- is shown for mWJll. [B] The mutated sequence -GAATCC- is found in mWJ22. In addition, nucleotide 2229, G, has also been muta-~ed to A in mWJ22. Note that mWJ43 contains this same mutated sequence (mutated bases ate underlined). each isolate was resistant to BamHl digestion. We determined the alteration caused by the bisulrite mutagenesis by sequencing the DNA surrounding the BamHl site. A 133.nucleotide primer (Haelll2247 to Hpall-2380) was isolated and used to sequence both the parental BamHl site and the mutated site. As can be seen in Fig. 1, a G -* A change at nucleotide 2224 has resulted in the BamHl resistant mutation. It is worth noting that an additional G-* A change has occurred at position 2229. Although this region is in the double-stranded DNA and is supposed to be protected from bisulfite mutagenesis, it is possible that the mutagenesis may have occurred during a transient single-stranded phase resulting from the natural "breathing" of the ends of linear DNA molecules. (b) Introduction o f a new Hindm restriction site into M l 3 m p 2 *
M13mp2 contains an E. coli DNA fragment which encodes for the first 145 amino acids of//-galactosi* J. Messing and B. Gronenborn (personal communication) have also constructed a Hindlll phagewhich they have cal!ed M13mpS. This phage however has multiple EcoRI sites in addition to a unique Hindlll site.
dase. The fusion of the E. coli fragment into Haelll (position 5867) of M13 results in three additional amino acids at the end of the ~-galactosidase fragment before a termination signal is reached (Messing, 1979; R. Rothstein, unpublished observations). This phage can complement the appropriate E. coli host deletion of ~-galactosidase giving blue plaques on indicator medium. The phage has an EcoRl site corresponding to the DNA encoding for amino acid 5 of the /~-galactosidase fragment. Our plan was to utilize available adaptors to introduce a small number of nucleotides containing a Hindlll site at this EcoRl site. hi addition we wanted to preserve the blue color complementation of the phage. By combining an octamer EcoRl adaptor with a decamer Hindlll adaptor we could, by the scheme shown in Fig. 2, produce the desired result. By inserting a multiple series of Hindlll adaptors into the EcoRl site we first disrupted the phage//-galactosidase gene by creating a frame-shift within the protein. We next removed the extra Hindlll adaptors resulting in a net addition of 18 nucleotides to the gene. This restores the reading frame and the addition of six amino acids still permits the wcomplementation of the E. coli host deletion. Blue plaques are again produced. We mixed the phosphorylated EcoRl octamer pGGAATTCC-OH with the phosphorylated Hindlll decamer pACAAGCTTGT-OH in an effective rado of I : 10. The products of the ligat!~.n were analyzed by first digesting two small aliqvo~s with EcoRl and Hindlll. After Hindlll digestion fr:~grnentcmainly 10 and 20 bp long were produced. 12re EcoRl digestion left most of the multimers intact. This meant that more HindllI adaptors were figated than EcoRI adaptors. The multiple Hindlll adaptors between EcoRI cohesive ends produced after EcoRI digestion were ligated into RF of M 13rap2. Approx. 30 pmol of EcoRl-digested adaptor mixture was added to 0.1 pmol EcoRI-digested Ml3mp2 vector. After ligation, the mixture was transfected onto E. coli strain 71-18. Approx. 3% of the plaques after transfection were colorless on plates containing the inducer IPTG and the color indicator X-gal. This indicated that an insertion had occurred. Several of these colorless plaques were chosen for further analysis. RFI DNA was isolated from four phages (mWJ1mWJ4). DNA from each phage was digested with H/ndlll. Each sample generated a linear band with the
172 EeoBI
Hi__~In
octaser (S)
~.,G~TT~
decaser (H)
pACkeT
+
~ liEation
creates many multimers H
R
H
H
H
with excess HindIII sites
R
_
EcoRI digestion
H
R
R
H
H
H
R
R m
M13ap2
EeoRl
"I
transfection ' '
~
colorless plague wi~h a 38-bp insertion
~
HindIiI digestion; religation
H
trarL~fection "
blue plaque with ~" a 18-bp insertion
Fig. 2. A scheme to inUoduce a HindIll site (H) into phage Ml3mp2 at the EcoRl site (R). The double-sUandedDNA or c,~Jgonucleofide molecules shown here are drawn with gaps to emphasize the sotuce and location of individualadaptors in the iigated products (the gaps represent the points where ligafionoccurred).
mobility of the parental Ml3mp2. This confirmed that Hindlll-containing fragments had been cloned into the parental phage. We next purified the single DNA band of each phage from the agarose gel by dissolving the small gel slice in 5 M sodium perchlorate (Yang et al., 1979). The DNA was religated and once
again tmnsfected into cells o f E. coli 71-18. All the plaques produced were blue on indicator medium. This result suggested that we had successfuny excised the extra HindHI adaptor sequences with the net result of adding 18 n~cleotides to the vector (Fig. 2); One blue plaque from each of the four Hindlll reli'
173 • ration mixtures was purified and RFI DNA was pre, pared. Each phage maintained both the EcoRl and Hindlll digestion pattern of its parent. A more detailed analysis of the configuration of the inserted DNA fragments was carried out for mWJ3, mWJ4, and mWJ22. Replicative form DNA isolated from mWJ3, mWJ4, and mWJ22 phage was digested with EcoRl and the exposed cohesive ends of each EcoRI frag. merit were labeled w i t h radioactive [a-32p]dATP (Bald et al., 1977). The resultant products were electrophoresed on a 15% polyacrylamide gel with a 5% spacer gel. The gel pattern in Fig. 3 shows that for mWJ3 (lane a) a 38 bp-long EcoRI-Hindlll mixture cloned into Ml3mp2 caused the disruption of a-complementation and thus the colorless phenotype. The mWJ22 phage that was formed after HindlIl digestion and religation had only one 18 bp-long adaptor added (lane d). The analysis of the phage mWJ4 is included since this phage contains three inserts of the 18 bp-long EcoRI-Hindlll-EcoRI adaptor sequence as shown by the very dark lower band (lane b). Therefore, the RF-DNA of this phage is a ready source of this 18 bp-long sequence. The utility of this adaptor will be discussed later. The nucleotide sequence of the inserted 18 bplong adaptor in mWJ22 was confirmed by DNA sequencing (Narang et al., 1980). Thus, in mWJ22 there are two EcoRl sites and a Hindlll site within a 24 bp-long region. The resulting six amino acids insertion at amino acid five of/~-galactosidase does not disrupt a-complementation: TCC ACA AGC TTG TGG AAT DNA sequence Se r - T h r - S e r - L e u - T r p - A s h - amino acid sequence Therefore, mWJ22 which is capable of accepting HindlIl fragments, will form a colorless plaque indicaring those phage with inserts. (e) Introduction of a new BamHl restriction site into Ml3mp2
A similar s~ries of experiments was undertaken with a BamHI decamer adaptor. The sequence p-CCGGATCCGG-OH was substituted for the H/ndIH decamer sequence. Again we joined the BamHI decamer with the EcoRl octamer by blunt-end ligati~. By procedures identical to those described for the
• ii ¸
Fig. 3. Sizing of fragments inserted into M13mp2. The derivatives mWJ3, mWJ4, mWJ22, mWJ39,and mWJ43 were each digested with EcoRI and 3' end-labeled with reverse transcriptase and [a-32P]dATP. In each case, approx. 10 000
cpm weze loaded onto a 15% polyaewlamide gel. Exposure was for2 h. (a) mWJ3 (b) mWJ4 (c) mWJ39 (d) mWJ22 (e) mWJ43. The positions on the autoradiogzam where 18- and
38-bp fragments ~ a t e axe shown. Note that the intervening EcoRI-Hindlll fragment in mWJ4 is approx. 34 bp long rather than a size expected in the series 28, 38, 58 etc. This smaller fragment probably arose during the cloning of this phage.
Hindlll phage, several colorless plaques were purified (mWJ39 in Fig. 3). Replicative form DNA from each phage was isolated and digested with BamHl. Tl~s generated a linear molecule that was purified, religated and transfected into E. coli 71-18. Again, blue plaques were formed indicating that the additional six amino acids resulting from the 18-bp insertion did not disrupt the a-complementa~ion (mWJ43 in Fig. 3,lane e):
TCC CCG GAT CCG GGG AAT DNAsequence - S e r - P ~ o - A s p - P r 0-431 y - A s n - amino acid sequence
t74 remit w ~ confn~med by DNA sequencing. The phage mWJ43 can be used to clone DNA fragments can3'ing BamHl cohesive ends. Since several other enzymes cleave DNA to give the same 5' cohesive ends ( G - A - T - C ) , DNA fragments digested with Mbol, Sau3A, Bg/ll, Bc/l and Xholl can also be cloned.
D~LISSION
There are many advantages to using single-stranded DNA phage as cloning vehicles. The M13mp2 phage a~ :onstructed by Gronenbom and Messing (1978) offers an excellent system for detecting phage cont~g cloned EcoRl fragmer.rs due to the production of colorless plaques. The mr, lifications which we ir~troduced have extended its versatifity to allow cton~.g of H/ndIH fragments in mWJ22 and BamHl, Bgill, Bcil, Xholl, Mbol end Sau3A fragments in mWJ43. Fig. 4 shows the relevant restriction sites of these vectors. Recently, Messing et al. (1981) have used roWJ43 to add even more restriction enzyme sites for clo,ung. In our discussion, we will briefly describe several application.~ of this cloning system. MI 3 phage package only the plus strand of DNA in the mature virion. This allows for the purification of a single strand of a cloned fragment in MI3. DNA sequencing by the dideoxynucleotide chain-termination method of Sanger et al. (1977) is facilitated by a p~ified single-stranded template. Phage DNA can be parified by a simple and rapid procedure and several synthetic primers are available for this phage. Sequencing strategies using these primers have been described (Sanger et al., 1980; Wo et al., 1980;
Heidecker et al., 1980; Anderson et al., 1980; Messing et at., 1981). Purified single strands can be used to purify a complementary RNA molecule. Since more than 1013 phage particles are produced/ml of culture, over 40 mg of phage DNA can be isolated from a 1-fiter culture ~ and Holm, 1969). Thus large quantifies of purified single strands can be made for hybridization studies. Recently, in vitro genetic manipulation of DNA sequences has become a powerful tool for studying specific DNA fragments and their associated phenotypes. Among the techniques used are in vitro mutagenesis using sodium bisulfite (Shorfle and Nathans, 1978), which we used for the BamHl site mutagenesis. Bisulfite mutagenesis is thought to be specific for cytosine residues residing in single-stranded regions. A fragment cloned in MI3 can be mutagenized due to its single-stranded nature. Bisulfite specifically causes a C -* U conversion resulting in a G- C -* A- T transition. Specific sites can be selectively mutagenized by hybridizing adjacent "protecting" fragments, since double-stranded regions are mostly protected from the bisulfite reaction. The resultant mutated fragment can be rephcated in vitro using either reverse transcriptase or DNA polymerase Klenow fragment and the appropriate primer (i.e., the "protecting" fragment). Another in vitro manipulation of DNA involves a method for creating specific deletions in a singlestranded DNA (Humayun and Chambers, 1979). This technique can easily be applied to sequences cloned into MI 3. Creation of in vitro mutations may also be accomplished by the synthesis of small mutant oligonucleotide primers complementary to the cloned sequence.
r.g;olp
EcoR
Hindlll EcoRl~
OamHI roWJ22
mW143
BamHI
Fig. 4. The relevant zestxiction sitesof M13mp2,mWJ22and mWJ43. The thick segmentrepresents the region codingfor the 145 amino acid long N-terminal peptide of/~-galactosidase cloned in MI3. The BamHl site in both mWJ22and mWJ43 has been xemovedby nmtation fXsymbols).
175
By hybridizing a synthetic fragment to the template, a complementary minus strand can be synthesized. Upon transfection and subsequent segregation of the initial heteroduplex the mutant clone can be recovered. This method has proved successful with 0X174 (Hutchison et al., 1978; Razin et al., 1978) and with a yeast Wrosine tRNA gone cloned in mWJ43 (R. Rothstein, S.A: Narang and R. Wu, in preparation). Lastly, the presence of the adjacent restriction sites in the modified phages also provides a convenient means for converting restriction sites in DNA (Boeke et al., 1979). For example, mWJ22 can be used to convert a HindllI site at the terminus of a DNA fragment to an EcoRl site. First the Hindlll molecular end of a fragment is ligated to the HindllI site of mWJ22. After digestion with EcoRI restriction enzyme, nine nucleotides are transferred from roWJ22 to the end of the DNA fragment. This results in the conversion of the HindllI end of the fragment to an EcoRI end. Additionally, this strategy can be reversed by first digesting a phage such as mWJ4 (shown in Fig. 3) with EcoRl to release the 18 bplong adaptor. In the case of mWJ4 there will be three times as many adaptor molecules when compared to mWJ22. The isolated 18-mer can be ligated onto an EcoRl end of a molecule. Digestion with Hindlll will create a Hind.'ll cohesive end 9 bp from the original EcoRI site. In summary, purified single strands of a cloned fragment can facilitate many studies. The modifi. cations we have introduced into M13mp2 extend its versatility to include the cloning of HindllI fragments (mWJ22) and BamHl, Bglll, Bell, Xholl, Mbol and Sau3A fragments (mWJ43).
ACKNOWLEDGEMENTS
We would like to thank Barbara Hess and Cathy Betzel for excellent teclmical assistance in DNA sequence analysis. We also thank Dr. Riccardo Cortese for introducing us to the M13 cloning system. This work was supported by research grants GM24904 and GM27365 from the National Institute of Health, U.S. Public Health Service.
REFERENCE5 Anderson, S., Gait, M.J., Mayol, L. and Young, I.G.: A short primer for sequencing DNA cloned in the single-stranded phage vector M13mp2. Nucl. Acids Res. 8 (1980) 1 7311 743. Bahl, C.P., Wu, R., Stawinsky, J. and Narang, S.A.: Minimal length of the lactose operator sequence for the specific recognition by the lactose repressor. Proc. Natl. Acad. Sci. USA 74 (1977) 966-970. Barnes, W.M.: Construction of an M13 histidine transducing phage: A single-stranded cloning vehicle with an EcoRI site. Gone 5 (1979) 127-139. Benton, W.D. and Davis, R.W.: Screening ~,gt recombinant clones by hybridization to single plaques in situ. Science 196 (1977) 180-182. Boeke, J.D., Vovis, G.F. and Zinder, N.D.: Insertion mutant of bacteriophage fl sensitiv,: to EcoRI. Proc. Natl. Acad. Sci. USA 76 (1979) 2 699-2 702. Bolivar, F. and Backman, K.: Plasmids of Escherichia coli as cloning vectors, in Wu, R. (Ed.), Methods in Enzymology, Vol. 68. Academic Press, New York, 1979, pp. 245-267. Chen, C.W. and Thomas Jr., C.A.: Recovery of DNA segments from agarose gels. Anal. Biochem. 101 (1980) 339-341. Davis, R.W, Thomas, M., Cameron, J., St. John, T.P., Scherer, S. and Padgett, R.A.: Rapid DNA isolations for enzymatic and hybridization analysis, in Grossman, L. and Moldave, K. (Eds.), Methods in Enzymology, Vol. 65. Academic Press, New York, 1980, pp. 404-411. Gronenborn, B. and Messing, J.: Methylation of singlestranded DNA in vitro introduces new restriction endonuclease cleavage sites. Nature 272 (1978) 375-377. Heidecker, G., Messing, J. and Gronenborn, B.: A versatile primer for DNA sequencing in the Ml3mp2 cloning system. Gone 10 (1980) 69-73. Hermann, R,, Neugebauer, K., Pirkl, E., Zentgraf, H. and Sch~er, H.: Conversion of bacteriophage fd into an efficient single-stranded DNA vector system. Mol. Gen. Goner. 177 (1980) 231-242. Hines, J.C. and Ray, D.S.: Construction and characterization of new coliphage MI3 clo, ing vectors. Gone 11 (1980) 207-218. Humayun, M.Z. and Ch:tmbers,R.W.: Construction of a sitespecific delefion-~'rarleshift mutation in an essential gone of bacteriophage oX~ 74. Nature 278 (1979) 524-529. Hutchiso:.~ II1, C.A., l'hflHps, S., Edgell, M.H., Gillam, S., Jahnke, P. and Smith, M.: Mutagenesis at a specie position ~n a DNA sequence. J. Biol. Chem. 253 (1978) 6 551-6 56O. Laskey, R.A. and Mills, A.D.: Enhanced autoradiographic detection of 32p and 1251 using intensifying scr~ns and hype~sensitized film. FEBS Lett. 82 (1977) 314-316. Leder, P, Tiemeier, D. and Enquist, L.: EK2 derivatives of bacteriophage lambda useful in the cloning of DNA from highe~ organisms: The ?,gtlfE$ system. Science 196 (1976) 175-177.
176 Marvin, D.A. and Holm, B.: Filamentous bacterial v ~ . Bac,etiol. Rev. 33 (1969) 172-209. Maxam, A.M. and Gilbet% W.: A new method for sequencing DNA. Proc. Natl. Acad. SCI. USA 74 (1977) 560-564. McDonelk M.W., Simon, M.N. and Studier, F.W.: Analysis of resiric~ton fragments of T7 DNA and determination of molec'~lar weights by electrophoresis in neutral and alkaline gels. | . MoL Biol. U 0 (1977) 119-146. Messing, J.: A multi-purpose cloning system based on the single-stranded DNA bactexiophage MI 3. Recombinant DNA Technical Bulletin, NIH Publication No. 79-99, 2, No. 2 (1979) 43-48. Messing, J., Crea, R. and Seeburg, P.H.: A system for shotgun DNA sequencing. Nud. Acids Res. 9 (1981) 309321. Morzison, D.A.: Transfmmatton and preservation of competent bacterial cells by freezing, in Wu, R. (Ed.), Methods in Enzymology, VoL 68. Academic Press, New York, 1979, pp. 326-331. Nazang, S.A., Brousseau, R., Hsiung, H.M., Sung, W., Scarpulla, .q., Ghangas, G., tau, L., Hess, B. and Wu, R.: Synthesis of the human iz~sulin gene, IV. New synthetic deoxyzibonucleotide adaptors and primer fog DNA cloning and sequence analyses. Nucl. Acids Res. Symp. Seties No. 7 (1980) 377-385. Razin, A., Hirose, T., Itak~a, J. and Riggs, A.D.: Efficient correction of a mutation by use of chemically synthesized DNA. l~oc. Natl. Acad. Sci. USA 75 (1978) 4 268-4 270. Rothstein, R.: Deletions of a tyzosine tRNA gene in $. cerevis~e. Cell 17 (I979) 185-190. Rothftein, R.J., Lau, L.F., Bahl, C.P., Narang, SA. and Wu, R.: Synthesis adaptors for clonin~ DNA, in Wu, R. (Ed.), Methods in Enzymology, VoL 68. Academic Press, New York, 1979, pp. 98-109. Sanger, F. and Coulson, A.R.: The use of thin ac~ylamide gels fog DNA sequencing. FEBS Lett. 87 (1978) 107-110.
Sanger, F., Coulson, A.IL, BatteD, B.G., Smith, A.J.H. and Roe, B.A.: Cloning in single,handed bacteriophage as an aid to rapid DNA sequencing. J. MoL BioL 143 (1980) 161-178. Sanget, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chain-terminating inMbitors, l~oc. Natl. Acad. Sci. USA 74 (1977) 5 463-5 467. Sclueier, P.H. and Cogtese, R.: A fast and simple method fo( sequencing DNA cloned in the single-stranded bacteriophage M13. J. MoL BioL 129 (1979) 169-172. Shortle, D. and Nathans. D.: Local mutagenesis: A method for generating viral mutants with base substitutions in preselected zegions of the viral genome, l~oc. Natl. Acad. Sci. USA 75 (1978) 2 170-2174. Smith, A.J.H.: DNA sequence analysis by primed synthesis, in Gzossman, L. and Moldave, K. (Eds.), Methods in Enzymology, VoL 65. Academic Press, New York, 1980, pp. 560-580. Van Wezenbeek, P.M.G.F., Hulsebos, TJ.M. and Schoenmakers, J.G.G.: Nucleotide sequence of f'damentous bacteriophage MI3 DNA genome: comparison with phage fd. Gene 11 (1980) 129-148. Wu, R., Lau, L., Hsiung, H., Sung, W., Bzousseau, R. and Natang, S.A.: New synthetic oligonucleotides for specific trimming and sequencing DNA, in Scott, W.A. and Wetnet, R. (Eds.), Mobilization and Reassembly of Genetic Information. Acadermc Press, New York, 1980, p. 419. Yang, R.C.A., Lis, J. anti Wu, R.: Elution of DNA from agarose gels after electtophozesis, in Wu, R. (Ed.), Methods in Enzymology, Vol. 68. Academic Press, New York, 1979, pp. 176-182. Communicated by H.O. Smith.
167
Elsevier/North-Holland Biomedical Press
Modification o f the bacteriophage vector M13mp2: introduction of new restriction sites for cloning (Synthetic adaptors; cloning vehicle; single-stranded DNA; ~ite-specific mutagenesis)
Rodney Rothstein and Ray Wu * Department o f Microbiology, CMDNJ - New Jersey Medical School, Newark, NJ 07103, and * Section on Biochemistry, Cell and Molecular Biology, Cornell University, Ithaca, N Y 14853 (U.S.A.}
(Received October 27th, 1980) (Revision received May 22nd, 1981) (Accepted June 5th, 1981)
SUMMARY
The construction of two new derivatives of the bacteriophage cloning vector M13rap2 is described. One derivative, mWJ22, contains a new HindIII site while the other, mWJ43, contains a new BamHI site. These new sites were both introduced at the EcoRI site at amino acid five of the 145 amino acid-long fragment ofEscherlchla coli /3-galactosidase within the phage. The new restriction sites do not disrupt the blue color detection system of Ml3mp2; therefore insertion of cloned fragments results in colorless plaques on indicator plates for the new derivatives.
INTRODUCTION
The fdamentous single-stranded DNA phages of E. coli offer numerous advantages as cloning vehicles. Both large amounts of replicative form I DNA and single-stranded DNA can be isolated. Only one strand of the cloned DNA is packaged depending on the orientation of insertion into the phage vector. This results in the purification of that strand. The high virus titer permits the isolation of large amounts of any cloned DNA fragment. Finally the complete nuAbbreviations: bp, base paks; DTT, dithiothzeitol; IPTG, isopropyl-~-D-thiogalactopyranoside; PEG, polyethylene glycol; RF, repficative form ofMl3 DNA; X-gal, $-bromo4-chloro2-indolyl-~-D-galactoside.
cleotide sequence of the MI3 genome is known (Van Wezenbeek et al., 1980). Puri~ed single-stranded DNA has many current applications. It is useful for both purification and analysis of RNA transcripts since there is no complementary DNA strand competing with the DNA: RNA hybridization. In addition, a purified singlestranded template greatly facilitates the dideoxynu. cleotide sequencing method of Sanger et al. (1977). Several single-stranded DNA phage have been modified as cloning vehicles (Gronenborn and Messing, 1978; Barnes, 1979; Boeke et al., 1979; Hermann et al., 1980; Hines and Ray, 1980). The RF of one, Ml3mp2, hasa 789 bp insertion ofE. coli DNA that codes for the first 145 amino acids of the ~f,alactosidase gene (Gronenborn and Messing,
0378-1119/81/0000-0000/$02.75 © 1981 Elsevier/North-Holland Biomedical Press
t68
1978). When Ml3mp2 infects an E. coli strain that contains an appropriate partial deletion of/~galactosidase, the phage-encoded peptide fra~gment can complement the host fragment (a-compler~ntation), resviting in blue plaques on indicator medium. The vector has an EcoRl site at the nucleotides corres p o n ~ g to the fifth amino acid of the ~gahctosi~se ~. When a foreign EcoRI fragmem is cloned into the RF, the resultant phage makes a colorless plaque after transfection. Short oligonucleotide primet~ for the DNA sequence analysis of cloned fragments have also been made for this phage (Heidecker et al., 1980; Anderson et al., 1980; Wu et ~., 1380). These features of Ml3mp2 make it an excellent systeF for cloning. In this report we will present some modifications that we have engineered into MI _~m?2 to make it a more versatile cloning vector. First, we eliminated the natural BaraHl site of M13m?2 by cl~emical mutagenesis since this s~te is in an essential gene and cannot be used for cloning. Next ~e introduced either a HindIll or a EamHl site at the EcoRl site using synthetic oligonucleotide adaptors. The resultant two new vectors, mWJ22 and n~WJ43, are both colorless when a ltindlll or a BamHl fragment is cloned into them, re:pec~ively.
MA]~ERIALS AND METHODS
(a) Purification of DNA fragments from agarose DNA fragments were purified from agarose gels by two methods. The first is essentially the method of Chen and Thomas 0 9 8 0 ) as described in Yang et al. (1979) in which the gel slice is dissolved in 4.5 M 5.0 M sodium perchlorate. The second method of DNA purification of agarose gel fragments was by electroelution (McDonell et al., 1977).
(b) Preparation of self-complementary oligonucleotides for ligation 200 pmol of the oligonucleotide sequences H O GGAATTCC-OH (Collaborative Research), H O ACAAGCTTGT-OH and HO-CCGGATCCGG-OH (gifts of Saran Narang) were each mixed ,vith 300
pmol of [~-aep]ATP (Amersham, diluted to 100 Ci/ mmol) and 0.8 uaits polynucleotide kinase (Biogenics or Boehringer-Mannheim) in 55 mM Tris-HCI, pH 8.0; 10 mM MgCl2.10 mM DTT. The unreacted triphosphate was removed by G-50 Sephadex (Pharmacia) column chromatography with sterile water as the ehant. The fractions containing the phosphorylated oligonucleotide were pooled and dried in an Evapo-Mix, resuspended at a fmal concentration of 10 pmol//d in TE (10 mM Tds- HCI, pH 7.4, 1 mM EDTA) and stored by freezing at -20°(2.
(c) Joining DNA fragments with T4 DNA iigase Blunt-end ligation of self-complementary oligomers (Rothstein et al., 1979) was catalyzed by T4 DNA ligase (New England BioLabs, MA). Phosphorylated oligomers were mixed at 1-10 #M in 5 ~1 of 50 mM Tris- HCI, pH 7.4, 10 mM MgCI2, 10 mM DTT and 66 /~M ATP. The mixture was heated to 65°C and slow-cooled to 4°C. Between 0.5 and I unit of T4 DNA ligase was added and the mixture was incubated for 24 h at 4°C. The reaction was monitored by electrophoresis on a 15% polyacrylamide gel. The ligation of cohesive ends was carried out essentially the same way, but at a lower concentration of DNA and DNA ligase. For example, linear DNA molecules recovered from a gel were ligated at 10 nM concentrations to promote intramolecular cyclization. For a 10 ~1 reaction 0.05-0.2 units of ligase were used and the mixture was incubated for 2 to 6 h at 14°C.
(d) In vitro mutagenesis with sodium bisuifite The bisulfite mutagenesis reactions were carried out in very small volumes following the procedure outlined by Shortle and Nathans (1978). BamHldigested Ml3mp2 DNA at 2.4 pg in 4 0 / d of TE was reacted with 120 gl of 4 M sodium bisulfite in a 6 mm X 50 mm siliconized glass test tube. Mineral oil was layered on top. The various dialyses required were carried out in that tube. A small piece of dialysis tubing was fitted over the top of the tube with a 3 mm length of tight-fitting tygon tubing acting as a rubber band. The test tube was inverted and centrifuged for a minute to invert the reaction mix and oil layers. This inverted tube was moved through the vat-
169 ious dialysis solutions required for the procedure. Care was taken when the 37°C dialysis was done since the coefficient of expansion for the tygon tubing is different from that for glass. The temperature of the test tube was slowly raised to 37°C.
(e) Transfection of MI 3mp2 and its derivatives The E. coli strains 71-18 (Gronenbom and Messing, 1978) and LE392 (Leder et al., 1976) were made competent using the RbC1 regime (Bolivar and Backman, 1979) and were stored at -70°C (Morrison, 1979). Although 71-18 contains the proper genoWpe for directly detecting plaque color with M13mp2 and its derivatives, we rarely obtained transformation frequencies greater than l0 s transformants//ag of transforming DNA/109 E. coil cells. To increase transformation frequencies, we often used LE392 which gave frequencies as high ~ 5 × l 0 6 transformants//zg/lO 9 cells. Since LE392 transfected cells are F-, phage are only produced within the transformed cells - neighboring cells are not reinfected. In order to view plaques, we also added 0.1 ml/plate of a saturated culture of 71-18. This procedure does not allow the immediate determination by color indication of whether a fragment is cloned, since LE392 is itself/acZ* wild type. Plaques can, however, either be picked or pooled and replated on the appropriate strain and indicator medium to permit color development. Plating of phage with color indication is achieved by mixing 0.1 ml of the appropriate phage dilution in 10 mM Tris (pH 7.4), 10mM MgCI2, 0.01% gelatin with 0.1 ml of a saturated culture of 71-18. After 15 rain at 37°C, 50/al of a 2% solution of X-fal dissolved in N,N-dimethyl formamide and 10/A of a 100 mM solution of IPTG were added. Next 3 ml of L-B soft afar (45°C) were added and the cells and phage were plated on M9 minimal plates supplemented with 5 /ag/ml thiamine. Plaque color develops after 9-12 h. The NIH guidelines require a transfer-deficient host for cloning with MI3 derivatives and certain eukaryotic DNAs.
(f) Screening colorless plaques for inserts Plaques can be screened for inserts by hybridizing a labeled probe to a nitrocellulose filter, either from a master plate of plaques or from random plaques
(100-2 O00/plate) by the procedures described for ~,. plaque screening (Benton and Davis, 1977). Mini-screening of RFI DNA from a plaque was accomplished by either of two procedures. We have adapted these procedures, originally described fo~ plasmid nfini-screens, for phage RFI isolation. This was accomplished either by growing 10 ml of the E. co~ strain 71-18 to an A6oo of 1 and infecting with 101° phage or by putting a single plaque (as a plug using a sterile pasteur pipette) into 10 ml of L-B medium with 0.1 ml of a saturated culture of 71-18 cells. In the first case the cells were infected for 45 rain after which chloramphenicol at 30 ~g/ml was added. 45 rain later the cells were harvested. In the second case the cells were harvested at a density (Aeoo) of 0.5 to 1 (4 h) and treated as in the plasmid screening procedure previously described (Bolivar and Backman, 1979). We found that by doing two ethanol precipitations, the DNA was suitable for restriction-enzyme digestions. The final pellet was resuspended in 150 pl of TE and 10-15 /~1 was used for each digest. The other DNA mini-screen was that described by Davis et al. (1980).
(g) Large-scale preparation of RFi Any convenient method for preparing a cleared lysate of a plasmid preparation can be adapted to preparing RFI from M13 (Rothstein, 1979). To maximize the RFI yield we grew E. coli strain 71-1~g at 37°C to an A600 of about 1. The cells were infected with 2-3 X 109 pha~/ml of culture. After 1 h, ch~oramphenicol was ad'.ied to 150 ~/ml. The cells were harvested 45 min later and RFI was isolated from the infected cells (yield approx. 1 rag/liter).
(It) End-labeling of small inserts and DNA sequence analysis DNA molecules with 5' protruding ends can be end-labeled by reverse transcriptase and the addition of one or more radioactively labeled tripho',phate (Bah1 et al., 1977). Both the chemical method (Maxam and Gilbert, 1977) and the dideoxynue1-~otide method (Sanger et al., i977) were employed for DNA sequence determination. For chemical sequencing, the fragments were first labeled at their 3' ends and single end-labeled fragments were isolated following gel electrophoresis (Rothstein, 1979).
~70 Single-st.randed phage DNA to be used as template for the chain torminator method was isolated by the method of Schreier and Cortese (1979), or by our plasmid prefLx is pWJ, therefore our MI3 clones The primer for sequencing was either the synthetic 19-met (Wu et al., 1980) or that described by Heidecker et al. (1980). We have used either the method of Sanger et al. (1977), or substituted reverse tr~scfiptase for polymerase ! (Smith, 1980). (i) Nomenclature
We have chosen to use a lower case m as the prefix for M 13 clones. This is followed by the same upper ca~ letters that we have registered with the Hasraid Reference Center, Stanford, CA. For example, our plasmid prefix is pWJ, therefore our M 13 clones are prefixed mWJ.
RESULTS (a) In vitro chemical mutagenesis of the BamHlsite of M i 3mp2
To construct a BamHl vector, we first needed to mutate the natural BamHl site of M 13rap2 since this site lies within an essential gene of M 13 (Van Wezenbeeket al., 1980). Since sodium bisulfite mutagenesis is thought to be restricted to deoxycytosine residues on single-stranded DNA (Shortle and Nathans, 1978), we used this method to mutate the natural deoxycytosine residues on the exposed cohesive ends of the BamHl recognition sequence with bisulfite. This procedure converts deoxycytosine to deoxyuracil. This conversion can take place at either of the two cytosine residues or at both of them in the BamHl cohesive ends. After chemical mutagenesis, the linear molecules were re-itgated with T4 DNA ligase. Along with the ligation of the unaltered cohesive ends, guanosineuracil bases are expected to form. a stable base pair and give: G-A-T-U C-T -A-G The G - U mismatch can be tolerated in this position since the. stack/ng interaction amongst the bases
is favorable after mutation. The iigated cohesive ends can result in the four configurations shown in Table I. The first example, A, shows the religation of unreacted cohesive ends. The configurations in B and C are expected when one or the other cohesive end is converted by bisulfite and subsequently ligates with the unreacted end. Finally, D shows the configuration after the ligation of two reacted ends. The ligated molecules were next redigested with BamHl and transfected into E. coll. This digestion lineartzes the type A molecules (Table I), while the B, C and D configurations are not digested due to the altered sequence. Since uncut DNA circles transfect with a greater efficiency than linear molecules, molecules containing altered BamHl sites were enriched after transfection into competent E. coil cells. Based on these assumptions and the change in transfection frequency before and after BamHl digestion, we calculate that 3% of the molecules were altered (i.e., resistant to BamHl digestion). The plaques obtained after BamHl digestion and transfection were pooled (40 individual plaques). RFI DNA was purified after infection with the phage pool. The pooling was undertaken because the initial plaques with the presumptive BamHl mufations were each formed from a heteroduplex molecule (G. U base pair). After heteroduplex segregation each plaque has a mixed population of phage both with and without a mutated BamHi site. After extensive digestion of the pooled RFI molecules with BamHl, a large fraction of the molecules (approx. 25%) remained resistant to BamHl digestion. Circular DNA was isolated after agarose gel electrophoresis from this fraction and was transfected into E. coli cells. Several plaques were purified, and the RFI DNA from
TABLE 1 Nucleotides 2220-2225 a (BamHl site) after ligation of bisulfite-teacted molecules (A) (B)
(C) (D)
...GGATCC... CCTAGG ...GGATUC... CCTAGG ...GGATCC... CUTAGG ...GGATUC... CUTAGG
umeacted top strand reacted bottom strand reacted botlt strands reacted
a Nucleotidenumber frora Van Wezenbeeket al. (1980).
171
F.;g. 1. DNA sequence of the mutated natmal BamHl site of M13. Dideoxynucleotide sequencing of the mutated BamHl site at position 2220 was w.imed by isolating a 133 bplong Haelll-Hpalll fragment (position 2247 to 2380). [A] The natural BamHl sequence -GGATCC- is shown for mWJll. [B] The mutated sequence -GAATCC- is found in mWJ22. In addition, nucleotide 2229, G, has also been muta-~ed to A in mWJ22. Note that mWJ43 contains this same mutated sequence (mutated bases ate underlined). each isolate was resistant to BamHl digestion. We determined the alteration caused by the bisulrite mutagenesis by sequencing the DNA surrounding the BamHl site. A 133.nucleotide primer (Haelll2247 to Hpall-2380) was isolated and used to sequence both the parental BamHl site and the mutated site. As can be seen in Fig. 1, a G -* A change at nucleotide 2224 has resulted in the BamHl resistant mutation. It is worth noting that an additional G-* A change has occurred at position 2229. Although this region is in the double-stranded DNA and is supposed to be protected from bisulfite mutagenesis, it is possible that the mutagenesis may have occurred during a transient single-stranded phase resulting from the natural "breathing" of the ends of linear DNA molecules. (b) Introduction o f a new Hindm restriction site into M l 3 m p 2 *
M13mp2 contains an E. coli DNA fragment which encodes for the first 145 amino acids of//-galactosi* J. Messing and B. Gronenborn (personal communication) have also constructed a Hindlll phagewhich they have cal!ed M13mpS. This phage however has multiple EcoRI sites in addition to a unique Hindlll site.
dase. The fusion of the E. coli fragment into Haelll (position 5867) of M13 results in three additional amino acids at the end of the ~-galactosidase fragment before a termination signal is reached (Messing, 1979; R. Rothstein, unpublished observations). This phage can complement the appropriate E. coli host deletion of ~-galactosidase giving blue plaques on indicator medium. The phage has an EcoRl site corresponding to the DNA encoding for amino acid 5 of the /~-galactosidase fragment. Our plan was to utilize available adaptors to introduce a small number of nucleotides containing a Hindlll site at this EcoRl site. hi addition we wanted to preserve the blue color complementation of the phage. By combining an octamer EcoRl adaptor with a decamer Hindlll adaptor we could, by the scheme shown in Fig. 2, produce the desired result. By inserting a multiple series of Hindlll adaptors into the EcoRl site we first disrupted the phage//-galactosidase gene by creating a frame-shift within the protein. We next removed the extra Hindlll adaptors resulting in a net addition of 18 nucleotides to the gene. This restores the reading frame and the addition of six amino acids still permits the wcomplementation of the E. coli host deletion. Blue plaques are again produced. We mixed the phosphorylated EcoRl octamer pGGAATTCC-OH with the phosphorylated Hindlll decamer pACAAGCTTGT-OH in an effective rado of I : 10. The products of the ligat!~.n were analyzed by first digesting two small aliqvo~s with EcoRl and Hindlll. After Hindlll digestion fr:~grnentcmainly 10 and 20 bp long were produced. 12re EcoRl digestion left most of the multimers intact. This meant that more HindllI adaptors were figated than EcoRI adaptors. The multiple Hindlll adaptors between EcoRI cohesive ends produced after EcoRI digestion were ligated into RF of M 13rap2. Approx. 30 pmol of EcoRl-digested adaptor mixture was added to 0.1 pmol EcoRI-digested Ml3mp2 vector. After ligation, the mixture was transfected onto E. coli strain 71-18. Approx. 3% of the plaques after transfection were colorless on plates containing the inducer IPTG and the color indicator X-gal. This indicated that an insertion had occurred. Several of these colorless plaques were chosen for further analysis. RFI DNA was isolated from four phages (mWJ1mWJ4). DNA from each phage was digested with H/ndlll. Each sample generated a linear band with the
172 EeoBI
Hi__~In
octaser (S)
~.,G~TT~
decaser (H)
pACkeT
+
~ liEation
creates many multimers H
R
H
H
H
with excess HindIII sites
R
_
EcoRI digestion
H
R
R
H
H
H
R
R m
M13ap2
EeoRl
"I
transfection ' '
~
colorless plague wi~h a 38-bp insertion
~
HindIiI digestion; religation
H
trarL~fection "
blue plaque with ~" a 18-bp insertion
Fig. 2. A scheme to inUoduce a HindIll site (H) into phage Ml3mp2 at the EcoRl site (R). The double-sUandedDNA or c,~Jgonucleofide molecules shown here are drawn with gaps to emphasize the sotuce and location of individualadaptors in the iigated products (the gaps represent the points where ligafionoccurred).
mobility of the parental Ml3mp2. This confirmed that Hindlll-containing fragments had been cloned into the parental phage. We next purified the single DNA band of each phage from the agarose gel by dissolving the small gel slice in 5 M sodium perchlorate (Yang et al., 1979). The DNA was religated and once
again tmnsfected into cells o f E. coli 71-18. All the plaques produced were blue on indicator medium. This result suggested that we had successfuny excised the extra HindHI adaptor sequences with the net result of adding 18 n~cleotides to the vector (Fig. 2); One blue plaque from each of the four Hindlll reli'
173 • ration mixtures was purified and RFI DNA was pre, pared. Each phage maintained both the EcoRl and Hindlll digestion pattern of its parent. A more detailed analysis of the configuration of the inserted DNA fragments was carried out for mWJ3, mWJ4, and mWJ22. Replicative form DNA isolated from mWJ3, mWJ4, and mWJ22 phage was digested with EcoRl and the exposed cohesive ends of each EcoRI frag. merit were labeled w i t h radioactive [a-32p]dATP (Bald et al., 1977). The resultant products were electrophoresed on a 15% polyacrylamide gel with a 5% spacer gel. The gel pattern in Fig. 3 shows that for mWJ3 (lane a) a 38 bp-long EcoRI-Hindlll mixture cloned into Ml3mp2 caused the disruption of a-complementation and thus the colorless phenotype. The mWJ22 phage that was formed after HindlIl digestion and religation had only one 18 bp-long adaptor added (lane d). The analysis of the phage mWJ4 is included since this phage contains three inserts of the 18 bp-long EcoRI-Hindlll-EcoRI adaptor sequence as shown by the very dark lower band (lane b). Therefore, the RF-DNA of this phage is a ready source of this 18 bp-long sequence. The utility of this adaptor will be discussed later. The nucleotide sequence of the inserted 18 bplong adaptor in mWJ22 was confirmed by DNA sequencing (Narang et al., 1980). Thus, in mWJ22 there are two EcoRl sites and a Hindlll site within a 24 bp-long region. The resulting six amino acids insertion at amino acid five of/~-galactosidase does not disrupt a-complementation: TCC ACA AGC TTG TGG AAT DNA sequence Se r - T h r - S e r - L e u - T r p - A s h - amino acid sequence Therefore, mWJ22 which is capable of accepting HindlIl fragments, will form a colorless plaque indicaring those phage with inserts. (e) Introduction of a new BamHl restriction site into Ml3mp2
A similar s~ries of experiments was undertaken with a BamHI decamer adaptor. The sequence p-CCGGATCCGG-OH was substituted for the H/ndIH decamer sequence. Again we joined the BamHI decamer with the EcoRl octamer by blunt-end ligati~. By procedures identical to those described for the
• ii ¸
Fig. 3. Sizing of fragments inserted into M13mp2. The derivatives mWJ3, mWJ4, mWJ22, mWJ39,and mWJ43 were each digested with EcoRI and 3' end-labeled with reverse transcriptase and [a-32P]dATP. In each case, approx. 10 000
cpm weze loaded onto a 15% polyaewlamide gel. Exposure was for2 h. (a) mWJ3 (b) mWJ4 (c) mWJ39 (d) mWJ22 (e) mWJ43. The positions on the autoradiogzam where 18- and
38-bp fragments ~ a t e axe shown. Note that the intervening EcoRI-Hindlll fragment in mWJ4 is approx. 34 bp long rather than a size expected in the series 28, 38, 58 etc. This smaller fragment probably arose during the cloning of this phage.
Hindlll phage, several colorless plaques were purified (mWJ39 in Fig. 3). Replicative form DNA from each phage was isolated and digested with BamHl. Tl~s generated a linear molecule that was purified, religated and transfected into E. coli 71-18. Again, blue plaques were formed indicating that the additional six amino acids resulting from the 18-bp insertion did not disrupt the a-complementa~ion (mWJ43 in Fig. 3,lane e):
TCC CCG GAT CCG GGG AAT DNAsequence - S e r - P ~ o - A s p - P r 0-431 y - A s n - amino acid sequence
t74 remit w ~ confn~med by DNA sequencing. The phage mWJ43 can be used to clone DNA fragments can3'ing BamHl cohesive ends. Since several other enzymes cleave DNA to give the same 5' cohesive ends ( G - A - T - C ) , DNA fragments digested with Mbol, Sau3A, Bg/ll, Bc/l and Xholl can also be cloned.
D~LISSION
There are many advantages to using single-stranded DNA phage as cloning vehicles. The M13mp2 phage a~ :onstructed by Gronenbom and Messing (1978) offers an excellent system for detecting phage cont~g cloned EcoRl fragmer.rs due to the production of colorless plaques. The mr, lifications which we ir~troduced have extended its versatifity to allow cton~.g of H/ndIH fragments in mWJ22 and BamHl, Bgill, Bcil, Xholl, Mbol end Sau3A fragments in mWJ43. Fig. 4 shows the relevant restriction sites of these vectors. Recently, Messing et al. (1981) have used roWJ43 to add even more restriction enzyme sites for clo,ung. In our discussion, we will briefly describe several application.~ of this cloning system. MI 3 phage package only the plus strand of DNA in the mature virion. This allows for the purification of a single strand of a cloned fragment in MI3. DNA sequencing by the dideoxynucleotide chain-termination method of Sanger et al. (1977) is facilitated by a p~ified single-stranded template. Phage DNA can be parified by a simple and rapid procedure and several synthetic primers are available for this phage. Sequencing strategies using these primers have been described (Sanger et al., 1980; Wo et al., 1980;
Heidecker et al., 1980; Anderson et al., 1980; Messing et at., 1981). Purified single strands can be used to purify a complementary RNA molecule. Since more than 1013 phage particles are produced/ml of culture, over 40 mg of phage DNA can be isolated from a 1-fiter culture ~ and Holm, 1969). Thus large quantifies of purified single strands can be made for hybridization studies. Recently, in vitro genetic manipulation of DNA sequences has become a powerful tool for studying specific DNA fragments and their associated phenotypes. Among the techniques used are in vitro mutagenesis using sodium bisulfite (Shorfle and Nathans, 1978), which we used for the BamHl site mutagenesis. Bisulfite mutagenesis is thought to be specific for cytosine residues residing in single-stranded regions. A fragment cloned in MI3 can be mutagenized due to its single-stranded nature. Bisulfite specifically causes a C -* U conversion resulting in a G- C -* A- T transition. Specific sites can be selectively mutagenized by hybridizing adjacent "protecting" fragments, since double-stranded regions are mostly protected from the bisulfite reaction. The resultant mutated fragment can be rephcated in vitro using either reverse transcriptase or DNA polymerase Klenow fragment and the appropriate primer (i.e., the "protecting" fragment). Another in vitro manipulation of DNA involves a method for creating specific deletions in a singlestranded DNA (Humayun and Chambers, 1979). This technique can easily be applied to sequences cloned into MI 3. Creation of in vitro mutations may also be accomplished by the synthesis of small mutant oligonucleotide primers complementary to the cloned sequence.
r.g;olp
EcoR
Hindlll EcoRl~
OamHI roWJ22
mW143
BamHI
Fig. 4. The relevant zestxiction sitesof M13mp2,mWJ22and mWJ43. The thick segmentrepresents the region codingfor the 145 amino acid long N-terminal peptide of/~-galactosidase cloned in MI3. The BamHl site in both mWJ22and mWJ43 has been xemovedby nmtation fXsymbols).
175
By hybridizing a synthetic fragment to the template, a complementary minus strand can be synthesized. Upon transfection and subsequent segregation of the initial heteroduplex the mutant clone can be recovered. This method has proved successful with 0X174 (Hutchison et al., 1978; Razin et al., 1978) and with a yeast Wrosine tRNA gone cloned in mWJ43 (R. Rothstein, S.A: Narang and R. Wu, in preparation). Lastly, the presence of the adjacent restriction sites in the modified phages also provides a convenient means for converting restriction sites in DNA (Boeke et al., 1979). For example, mWJ22 can be used to convert a HindllI site at the terminus of a DNA fragment to an EcoRl site. First the Hindlll molecular end of a fragment is ligated to the HindllI site of mWJ22. After digestion with EcoRI restriction enzyme, nine nucleotides are transferred from roWJ22 to the end of the DNA fragment. This results in the conversion of the HindllI end of the fragment to an EcoRI end. Additionally, this strategy can be reversed by first digesting a phage such as mWJ4 (shown in Fig. 3) with EcoRl to release the 18 bplong adaptor. In the case of mWJ4 there will be three times as many adaptor molecules when compared to mWJ22. The isolated 18-mer can be ligated onto an EcoRl end of a molecule. Digestion with Hindlll will create a Hind.'ll cohesive end 9 bp from the original EcoRI site. In summary, purified single strands of a cloned fragment can facilitate many studies. The modifi. cations we have introduced into M13mp2 extend its versatility to include the cloning of HindllI fragments (mWJ22) and BamHl, Bglll, Bell, Xholl, Mbol and Sau3A fragments (mWJ43).
ACKNOWLEDGEMENTS
We would like to thank Barbara Hess and Cathy Betzel for excellent teclmical assistance in DNA sequence analysis. We also thank Dr. Riccardo Cortese for introducing us to the M13 cloning system. This work was supported by research grants GM24904 and GM27365 from the National Institute of Health, U.S. Public Health Service.
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