Construction of an SV40-derived cloning vector

Gene, 11 (1980) 63-77 © Elsevier/North-HollandBiomedicalPress

63

C o n s t r u c t i o n o f an S V 4 0 - d e d v e d cloning v e c t o r (Recombinant DNA; restriction endonucleases; SV40 transcription)

Nicholas Muzyezka Department oflmmunology and MedicalMicrobiology, University of Florida, College of Medicine, Gainesville, FL 32601 [U.S.A.)

(Received January 10th, 1980) (Accepted Maxch3td, 1980)

SUMMARY A new reiterated variant of simian virus 40 (SV40; dl1142) has been constructed. It should be useful for the purpose of cloning foreign pieces of DNA in SV40 virions. Up to 80% of the SV40 genome has been made available for substitution with foreign DNA and the vector contains a number of unique (single-cut) restriction sites which will facilitate cloning. The 5' and 3' regions of both the SV40 early and late messenger RNAs are included in the vector. Prokaryotic DNA has been successfully cloned in the early region of the vector. The transcriptional properties of the recombinant have been studied, and it was found that both the vector and insert DNA are transcribed, mainly as non-adenylated RNA.

INTRODUCTION Recently a number of laboratories have investigated the possibility of using SV40 as a cloning vector. Such a vector would allow one to study the expression of eukaryotic genes in mammalian cells as part of an SV40 replicon. Two approaches have been tried. In the first approach a portion of the late region of

Abbreviations: bp, base pairs; form I DNA, covalently closed circular duplex DNA; form II DNA, open circular duplex DNA; MEM, minimal Eagle's medium, m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; p.Lu., plaqueforming units; SV40, simian virus 40.

SV40 is substituted with foreign DNA and the recombinant molecule is propagated using a helper virus with a temperature-sensitive mutation in the early gene (Goff and Berg, 1976; Mulligan et al., 1979; Hamer et al., 1977; Hamer and Leder, 1979). In the second approach, foreign DNA is attached to an SV40 fragment which contains the SV40 origin of replication and the recombinant DNA is propagated as a reiterated variant along with wild-type SV40 helper virus (Ganem et al., 1976; Nusbaum et al., 1976). In principle, the second approach should work with even a single SV40 origin of replication and therefore has the potential for a maximum replacement of the SV40 genome with foreign DNA. In this publication we describe an SV40 derived variant designed for this purpose.

64 MATERIALS AND METHODS

(a) Viruses and cells Wild type SV40 was the plaque-purified, smallplaque virus, strain 776 (Todaro and Takemoto, 1976), grown in the BSC-1 line of African green monkey kidney cells in MEM with 2% fetal calf serum, penicillin, and streptomycin. SV40 mutants ev 1114 and ev 1119 (Brockman and Nathans, 1974; Brockman et al., 1975) were grown in the same medium but in BSC-40 cells (Brockman and Nathans, 1974) at 40°C.

(b) Preparation of DNA restriction fragments Wild type SV40 DNA as well as the SV40 variants described in this paper were labeled with 32p as described by Danna and Nathans (1971)and isolated by the method of Hirt (1967).)~ DNA was isolated as described previously (Muzyczka, 1979). Restriction fragments were separated by ¢lectrophoresis in 1.4% agarose or 4% acrylamide as described by Danna et al. (1973). DNA was extracted from agarose gels by the method of Blin et al., (1975) as modified by Kelly (Wu et al., 1976). Fragments on acrylamide gels were recovered by electrophoresis into dialysis sacs.

(c) Enzymes All restriction enzymes used in this study were purchased from either New England Biolabs (Beverly, MA) or Bethesda Research Laboratories (Rockville, MD). Digestion conditions were as previously reported (Muzyczka, 1979). E. coli ligase was purified according to Modrich et al. (1973) from the l o p l l overproducing strain; the fraction VI enzyme was used. E. coli DNA polymerase I was obtained from Boehringer-Mannheim (Germany).

(d) Preparation of recombinant molecules The minicircles of 34% SV40 length were prepared by incubating 1.2 /ag of the e v l l l 9 fragment and 1/ag of the evl 114 fragments (see RESULTS) at 5°C for 48 h with E. coli DNA ligase. In addition to the two restriction fragments the ligation mixture contained in 0.1 ml: 30 mM Tris • HC1 (pH 8,0), 1 mM

EDTA, 10 mM (NH4)2SO4 (pH 7.0), 10 mM MgCI2, 0.3/~M NAD, 50 gg/ml bovine serum albumin and 20 to 40 units of E. coli DNA ligase, fraction VI. The extent of the reaction was assayed by electron microscopy. The ligation mixture was extracted with phenol, precipitated with ethanol and dialysed against 0.1 ×SSC (SSC=0.15 M NaCI, 0.015 M sodium citrate, pH 7.0). dll 142 ::?~was similarly prepared except that 19 #g of 32p-labeled ?~ HindlII-5 (1 × 103 cpm/#g) and 20 #g of 32P-labeled dll142 HindlII fragment (1.5 × 103 cpm/#g) were incubated with E. coli ligase in a volume of 0.5 ml for 16 h at 15°C. After incubation, the ligation mixture was extracted with phenol and precipitated with ethanol. It was then centrifuged to equilibrium in a cesium chloride-ethidium bromide gradient with a density of 1.56 g/ml. Ethidium bromide was removed by passage through a Dowex 50 column (Biorad). The RFI DNA isolated from the gradient was electrophoresed in a 1.4% agarose gel to separate the different recombinant molecules. (e) DNA transfections DNA transfections were according to the method of McCutchan and Pagano (1968). A subconfluent monolayer of BSC-1 cells in a 0.6 cm dish was washed once with 0.2 ml of PBS and then transfected with 0.025 ml of a mixture containing 1 #g SV40 DNA, 10 #g recombinant DNA, MEM, DEAE-dextran (250 #g/ml), and Tris.HC1 (pH 7.5, 0.05 M). After incubation at room temperature for 30 min, the DNA mixture was removed, and the cells were washed once with MEM supplemented with 2% fetal calf serum. Stocks were harvested after 10 to 14 days of incubation at 37°C in MEM supplemented with 2% fetal calf serum. Transfections of larger dishes of BSC-1 cells were scaled up proportionately. Plaque isolates were obtained and screened as described by Brockman and Nathans (1974). All experiments with cells carrying recombinant genomes were performed under conditions of P3 physical containment [(1975) Federal Register 41, No. 131,27902-27943 ].

(f) Electron microscopy Electron microscopy of DNA was done by the formamide method of Westmoreland et al. (1969) as described by Davis et al. (1971). Specimens were

65 stained with uranyl acetate and shadowed with platinum-palladium. Measurements of DNA were made from projections of electron micrographs using a graphics calculator (Numonics Corp.). Heteroduplexes were prepared by mixing two BarnHI digests in a total volume of 15 gl, namely, 15 ng o f d / l 1 4 2 monomer fragment and 7.5 ng of SV40 linear DNA in 0.1 M NaOH and 6 mM EDTA. After 10 rain at room temperature the heteroduplex mixture was adjusted to a total volume of 90/1l which contained (in addition to the reagents above) 10 mM Tris. HC1 (pH 8.6), 16 mM HC1 and 40% formamide (v/v). After the DNA was allowed to renature for 30 min at room temperature, 10/11 of cytochrome C (1 mg/ml) was added and the DNA was spread. All measurements of heteroduplex molecules were expressed as percent of full length parental strand in the same heteroduplex. Independent measurement of single-stranded DNA molecules on the same electron micrographs showed no significant difference in length between singlestranded and duplex DNA.

(g) Isolation of dl1142: :X virions by cesium chloride centrifugation dll142::~ virions were separated from SV40 virions as described by Scott et al., (1976)with modifications suggested by James M. Pipas (personal communication). 60 ml of cell lysate was frozen and thawed three times and chilled on ice. A 1 : 1 mixture of Triton X-100 (Sigma) and chloroform was added to a final concentration of 0.5% in Triton X-100 (v/v). The lysate was mixed vigorously and allowed to sit on ice for 5 min. The lysate was then extracted with an equal volume of ice-cold chloroform and 27 ml of the lysate was layered on a cesium chloride shelf gradient which contained 10 mM Tris pH 7.4, 10 mM MgSO4, 0.5 mM CaCI~ and cesium chloride at a density of 1.5 g/ml (5 ml) and 1.29 g/ml (5 ml). The gradient was centrifuged for 45 min in a Beckman SW 27 rotor at 4°C and 24000 rev./min. The virus band was removed and saturated cesium chloride (containing 10 mM Tris, pH 7.4; 10 mM MgSO4 and 0.5 mM CaC12) was added to a final density of 1.34 g/ml. The mixture was centrifuged to equilibrium (20 h; 38 000 rev./min, in a Beckman 50 Ti rotor at 4°C). The above step was repeated until the ratio of absorbance at 260 nm and 280 nm was consistent with a pure virus preparation. Generally two centri-

fugations were sufficient. In the case of dl1142::~, the recombinant virus will not form plaques or replicate by itself and so it was impossible to get an accurate estimate of the number of p.f.u, in the purified virions preparation. Under similar conditions wild-type virus preparations lose 90% of their infectivity. Comparison of the 260 absorbance of the purified dl 1142: :X preparation with comparably treated wild-type virus preparations leads us to estimate the infectivity of our dl1142: :X preparation at 6 × 10 a p.f.u./ml. When the virion preparation was plaque-assayed to estimate the level of wild-type contamination it contained 5 × 106 p.f.u./ml. By this reasoning our dl1142: :X preparation is 99% pure. (h) Isolation of SV40 RNA 10 cm dishes of confluent BSC-1 monkey cells were infected with SV40, d/l142::X plus SV40, or dl1142::X virion preparations (10 dishes each) at an m.o.i, of 1-2. The cells were incubated at 37°C in MEM containing 2% fetal calf serum. 24 h after infection the medium was removed, and the ceils were chilled on ice. The cells were then washed twice with ice-cold PBS and once with ice-cold isotonic buffer which contains 10 mM Tris pH 7.6, 100 mM NaC1, and 1.5 mM MgC12. The cells were lysed by addition of 0.5 ml/plate of isotonic buffer which contained 1% Nonidet P-40 (NP-40) and the lysates were scraped off the dish with a rubber policeman. The lysates were then extracted successively at room temperature with an equal volume of phenol (saturated with 0.01 M sodium acetate, pH5.1) and chloroform containing 1% isoamyl alcohol. The RNA was then precipitated by the addition of 1/10 vol. of 20% potassium acetate, pH 5.1 and 2.5 vol. of ethanol. The precipitate was dried under vacuum and dissolved in 20 mM Tris, pH 7.4, 10 mM NaC1, and 20 mM MgC12. RNase-free, pancreatic DNase (Worthington) was added to a final concentration of 20 #g/ml and the RNA preparation was incubated for 2 h at 37°C. Following the incubation the RNA was phenolextracted and ethanol-precipitated. (i) Isolation of polyadenylated RNA RNA was dissolved in 2 ml of binding buffer containing 10 mM Tris pH 7.5, 1.0 M NaCI, 1 mM EDTA, and 0.5% SDS. 10 000 cpm of 14C-labeled 35S poly-

66 adenylated polio RNA (the kind gift of Richard Rickles) was also added to the preparation. The RNA was passed through a column containing 0.3 g of oligo(dT) cellulose (Collaborative Research) which had been equilibrated with binding buffer. Typically 95% of the polyadenylated polio RNA was retained by the column. The column was then eluted with a buffer containing 10 mM Tris pH 7.4, 0.5% SDS and 1 mM EDTA. Fractions which contained 14C-labeled polio RNA were pooled, phenol-extracted, etherextracted and ethanol-precipitated. 100% of the polio RNA which bound to the column was eluted.

(j) Transfer of DNA and RNA to f'dters DNA fragments which had been electrophoresed in agarose gels were denatured in situ and transferred to nitrocellulose filters as described by Southern (1975). Diazobenzyloxymethyl paper was made as described by Alwine et al. (1977). RNA was separated by electrophoresis in 1.4% agar0se gels (16.5 X 15 X 0.3 cm) essentially as described by Bailey and Davidson (1976). The electrophoresis buffer (pH 8.1) contained 50 mM boric acid, 5 mM Na2B4OT, 10 mM sodium sulfate, and 1 mM EDTA. The gel, in addition to agarose and electrophoresis buffer, contained 5 mM methylmercuric hydroxide (Alfa). RNA samples (10/ag/gel channel) were dissolved in electrophoresis buffer which contained 5 mM methylmercuric hydroxide, 5% glycerol and 0.1% bromophenol blue. When the electrophoresis was complete, the gel was soaked in 800 ml of 14 mM fl-mercaptoethanol (Eastman) and 1/.tg/ml ethidium bromide for 1 h. It was then soaked in two changes (250 ml each) of 200 rnM potassium phosphate (pH 6.5) for a total of 30min and four changes (200-400ml each) of 25 mM potassium phosphate (pH 6.5) for a total of 1 h. The transfer to diazobenzyloxymethyl paper was done in the same manner as DNA transfers except that 25 mM potassium phosphate (pH 6.5) was used for the elution. RNA was allowed to transfer for a total of 36 h.

probes were 0.5-1.5 X 108 cpm//ag. Nitrocellulose filters were hybridized according to Denhardt (1966). Hybridization mixtures contained in 10 mls: 3 × SSC (pH 7.0), 0.02% Ficoll, 0.02% polyvinylpyrolidone, 0.02% bovine serum albumin, 1 mg/ml denatured herring sperm DNA and 0.05 #g/ ml [32p]sv40 DNA or 0.075/~g/ml [32P]~k DNA. The hybridization was carried out at 67°C for 16 h. Following the hybridization, the t'dters were washed at 67°C t~or 1 h in hybridization buffer without [32p]. DNA, 1 h in 0.1 M potassium phosphate (pH 7.0), and 2 changes (1 h each) of 1 × SSC, 0.06% SDS (pH 7.0). The filters were then dried and placed in close contact with Dupont Cronex X-Ray film and a Dupont HiPlus intensifying screen at -70°C for a variable length of time (1-100 h). 0.05 ~g of digested DNA was loaded in each slot of the gel. The sensitivity of the method was sufficient to detect a 0.1% contaminant in our preparations of dl1142: :;~ DNA. Diazobenzyloxymethyl paper was hybridized essentially as described by Alwine et al. (1977). The paper strips containing transferred RNA were treated for 4 h at 37°C with a prehybridization solution containing in 20 ml: 5 X SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 1% glycine, 50% formamide, and 400/ag/ml herring sperm DNA. The herring sperm DNA had been boiled in 0.2 M NaOH for 20 min prior to use. The hybridization was carried out at 37°C for 16 h in a solution which contained all of the components of the prehybridization buffer except glycine and also contained 0.05 ~g/ml denatured [a2P]SV40 DNA or 0.1 ~tg/ml denatured [32p]k DNA. After hybridization the filter was washed at 68°C for 1 h in prehybridization buffer without glycine, 1 h in 0.1 M potassium phosphate (pH 6.5), and 2 h in 2 changes of 1 × SSC (pH 7.0), 0.6% SDS. After the filters were dried, the bands were discerned by exposure of X-ray film (see above).

RESULTS

(k) DNA hybridization

(a) Construction of the vector

a2P-labeled ?~ or SV40 probes were made by nick translation of the wild-type viral DNA as previously described (Muzyczka, 1979). Specific activities of the

In constructing a general purpose SV40 cloning vector, the three major concerns were: (1) to maximize the amount of space available for inserting

67

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Infect cells with minicircle and wt helper DNA~clone variants with tandemlyrepeated BamHI sites

BamHl

BamHl

Vector tetramer after additional cell mediated deletions

Hha , Hpa I 1 ~ ' ~ ~ I (/~lat e ~,~.

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"BamHI '~1~"Hindll early//~=Hinf j/

Hindlll/ ~ . _ _ . . / j Hinf dl 114 2 monomer structure ( 2 4 % of SV40 size) Fig. 1. Construction of d/1142, dl1142 was constructed from pieces of two evolutionary SV40 variants (ev1114 and e v l l l 9 ) which had been isolated and characterized by Brockman et al. (1975). A HpaII-BamHI fragment from ev1119, which contains a deletion in the early region of SV40 (0.24 to 0.54), and an HpaII-BamHI fragment from ev1114, which contains a deletion in the late region of SV40 (0.75 to 0.11 map units) were isolated by acrylamide gel electrophoresis. The two fragments were ligated in vitro to form a minicircle which was 34% of SV40 size and contained both the early and late deletions. The minicircle was propagated in monkey cells with wild-type SV40 helper DNA and the resulting virus stock was plated at low dilution. Plaques were screened for the presence o f variants with tandemly repeated BamHI sites and one of those which were found, d71142, was chosen for further study. During passage in monkey cells the 34% minicixele suffered additional deletions in both the early and late regions so that its size was reduced to 24% of SV40. In addition, the monomer minicitcle was reiterated to form two new variants 72% and 96% of SV40 size.

68 foreign pieces of DNA, (2) to facilitate cloning by incorporating a number of single-cut restrictionenzyme sites into the vector, and (3) to preserve as many of the SV40 transcriptional signals as possible. Fig. 1 illustrates the construction of the vector. Two evolutionary variants, evl 119 and evl 114 (originally isolated by Brockman and Nathans, 1974), were digested with the restriction enzymes B a m H l and HpalI. One of the HpalI.BamHI fragments from evl 119 contains a large deletion in the early region of SV40 (0.24 to 0.54 map units) while a HpalIBamHI fragment from e v l l l 4 contains a deletion in the late region (0.75 to 0.11 map units). These two fragments were joined together in vitro with E. coli DNA ligase to form a 34% minicircle con-

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taining no intact SV40 genes. A minicircle of this type should reiterate during propagation in mammalian cells (Fareed et al., 1974; Khoury et al., 1974) to yield a variant whose size is suitable for packaging in the SV40 capsid (70%-100% of full length size). The minicircle and wild-type DNA (as helper) were transfected into the BSC-1 line of monkey cells in the hope of obtaining a reiterated variant whose monomer length would be 34% of SV40. The stock obtained from the 'initial transfection was plaqued and the plaques were screened for the presence of variants with multiple BamHI sites by agarose gel electrophoresis. None of the plaques contained variants with a monomer length of 34% but a number of plaques contained variants whose monomer length was

.

ii¸ Fig. 2. a2P-labeled d/1142 DNA was digested with BamHI and the monomer fragment was isolated by electrophotesis on 1.4% agarose gels. The monomer fragment was then digested with the restriction enzymes indicated below and electrophoresed on 4% polyacrylamide gels. The following enzymes were used: HhaI, lane c; HaelII, lane d; Hinfl, lanes e and n; HinfI + HpaH, lane g;

BglI, lane k; HindIH, lane 1; HindII + HindIII, lane m;HpalI, lane o. a ~P-labeled wild-type SV40 DNA was similarly digested with HaelII, lane b; Hindll + HindlII, lanes a and i; Hinfl + BamHI, lane f; or HinfI + HpalI, lane h. Lane j contains undigested BamHI monomer fragment.

69 approx. 24% o f SV40 size. One o f these, dl1142, was chosen for further study. dl1142 is related to the in vitro constructed 34% minicircle in that it contains the same late deletion but has an additional 10% o f SV40 deleted from the early region. Two forms are found in our stocks (see Fig. 5B, lane 1), a trimer which is 72% o f SV40 size and a tetramer (96%). (b) Physical mapping o f dl1142

dl1142 was mapped b o t h b y restriction enzyme digestion and electron microscope heteroduplexing techniques. For the restriction enzyme analysis, the BamHI linear monomer fragment o f dl1142 was isolated b y agarose gel electrophoresis and digested with TABLE I Molecular lengths of restriction fragments Restriction enzyme a (BamHI+)

Fragment size (% SV40)

Sum of fragments (% SV40)

HindlII Hindll +Hindlll Hpal or Hindll Hhal HpaIl HinfI BglI HaelII HinfI+Hpall Hindlll +Hpall HpalI + BglI

12.5, 11.6 11.7, 10.1, 2.5 21.8, 2.5 18.5, 5.9 18.5, 6.0 12.6, 6.0, 5.8, (5.6) b 13.7, 10.5 7.7, 5.8, 5.3, 3.9 6.6, 5.9 c, 5.7, (5.6) b 12.3, 5.8 c 14.0, 6.0, 4.5

24.4 24.3 24.3 24.4 24.5 24.4 24.2 22.7 24.1 23.9 24.5

a 32p.labele d d11142 was digested with BamHl and the monomer linear fragment was isolated from 1.4% agarose gels. The monomer fragment was then digested with the indicated restriction enzymes and electrophoresed on 4% polyacrylamide gels. Fragment sizes were calculated by comparison with SV40 wild type fragments on the same gel obtained by HindlI + dill, HinfI or HaelII digestion. The size of marker fragments was obtained from the published sequence of SV40 DNA (Reddy et al., 1978 and Fiers et al., 1978). b This fragment which was also seen in wild-type digest patterns is most likely the result of incomplete digestion at theHinfl site at map position 0.199. c 2 mol of this fragment were seen per mol of fragment digested. d Three additional HaelII fragments would not have been retained on 4% aerylamide gels. The expected difference in size of the dl1142 monomer due to these fragments is 1.6%.

a variety o f restriction enzymes. Fig. 2 shows some o f the digest patterns obtained and Table I summarizes the sizes o f the different restriction fragments. The d/1142 monomer fragment (Fig. 2-j) is 24.3% o f SV40 full length size. The monomer has the following .unique restriction sites: BamHI, HpaII (Fig. 2-o), HhaI (Fig. 2-c), HindII (Fig. 2-m), HindIII (Fig. 2-1, m), HpaI (table I) and BglI (Fig. 2-k). The 5.9% small fragment obtained in BamHI, HpaII double digests o f d/1142 (Table I) is identical in mobility to the BamHI, HpaII fragment obtained from evl 114 for the construction o f the 34% minicircle. Apparently the additional sequences deleted during propagation o f the 34% minicircle occurred entirely within the fragment derived from evl119. This was confirmed b y electron microscope DNA heteroduplex measurements. Fig. 3 shows an example o f a heteroduplex obtained when wild-type SV40 linear molecules cut

Fig. 3. Electron microscope photographs of a heteroduplex formed between a wild-type SV40 linear molecule and a d11142 monomer linear, both of which had been digested with BamHI. The tracing of the photograph indicates the measurements made on 40 such molecules. See Table II.

70 TABLE II Heteroduplex measurements

BamHI linearized dl1142 monomers were heteroduplexed with wild-type SV40 BamHI-linearized molecules as described in MATERIALS AND METHODS. Measurements were made on 40 heteroduplexes and each distance, as specified in Fig. 3, was divided by the total length of SV40 DNA: i.e., %a = a/(a + b + c + d + e). The numbers given below are the mean of these measurements. Numbers in parentheses indicate one standard deviation.

%SV40 Map position

a

b

c

d

e

7.45 (0.54) 0.143-0.22

12.46 (1.21) 0.60-0.75

3.23 (0.31) 0.11-0.143

40.02 (1.75) 0.22-0.60

36.83 (1.56) 0.75-0.11

0.22 and 0.60 on the wild-type SV40 map. Fine structure restriction enzyme mapping of dl1142 revealed an additional small deletion of wildtype sequences. Fig. 2, lanes b and d show that a HaelII digest of dl1142 BamHI monomer fragment contained no fragments in common with a wild-type HaelII digest. If the region between the HindlII site of dl1142 (0.652) and the HpalI site (0.725) were intact the wild-type HaelII-g fragment should have been present. (See Fig. 4 for comparison of wildtype and dl1142 maps.) This suggested that an additional small deletion of wild-type sequences exists in dl1142. The small deletion was mapped by comparing

with BamHI were hybridized to dl1142 monomer BamHI linears (Table II). The short duplex end (segment c in Fig. 3) has the same length as that contained in e v l l l 4 (Brockman et al., 1975) between the BamHI site (0.143) and the beginning of the late deletion in ev1114 (0.11). The deletion loop immediately adjacent to the short duplex end (segment e) is the same size as the late deletion in evl 114 (Brockman et al., 1975) and maps at the same location on the wild-type genome (0.75 to 0.11). Therefore, the second deletion loop in Fig. 3 (d) and the length of the long duplex end (segment a) can be used to determine the position of the early deletion in dl1142. By this reasoning the early deletion maps between

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Fig. 4. Physical maps of the wild-type SV40 genome (left and d11142 (right). G, B, A and C indicate the wild-typeHindlI + l l I fragments from which d/1142 was derived. The numbers in the outer circle o f the d11142 map indicate wild-type DNA map positions. Map positions on d! are indicated by number 0 to 24.3%, and arrows on this c~cte indicate HaeIlI cuts. The 2% deletion in all1t42 has been mapped between the BglI and HpaII sites. Its exact position is not known.

71 Hinfl + BamHI and HinfI + HpalI double digests of wild-type SV40 DNA with similar digests of d/l142 (Fig. 2, lanes e, f, g, h). BamHI +HinfI digests of d11142 generate three bands (Fig. 2, lane e). The two smaller bands (6.0% and 5.8% of the SV40 genome) are derived from the early region of dl1142. (An additional band at 5.6% of SV40 is the result of incomplete digestion at the HinfI site at position 0.199). The large 12.6% fragment in Fig. 2, lane e contains what is left of the late region of SV40 in dl1142. When dl1142 is digested with HinfI + HpaII + BamHI (Fig. 2, lane g), the 12.6% fragment is cut into two fragments, 6% and 6.6% of SV40 size. The 6% fragment contains the 36% late deletion in dl1142. The 6.6% fragment represents the region between 0.725 and 0.642 and it is 2% smaller .than the corresponding 8.6% fragment obtained in HinfI, HpaII digests of wild-type SV40 DNA (Fig. 2, lane h). A similar 2% deletion was found in comparisons of HpaII, HindIII and HpaII, BglI double digests of dl1142 and wild-type SV40 DNA (data not shown). All the data discussed above are consistent with the physical map of dl1142 shown in Fig. 4. The position of the 2% deletion is not known exactly. We know only that it is somewhere between the HpaII and BglI sites.

were extracted from the agarose gel and transfected into monkey cells with wild-type SV40 DNA using the DEAE dextran technique (McCutchan and Pagano, 1968). The composition of each of the resulting stocks was analyzed by agarose gel electrophoresis and is illustrated in Fig. 5B. Most of the stocks conrain d11142 trimers and tetramers (72 and 96% of SV40 length, respectively). In addition stocks 10-14 also contain a 70% variant. Stock 14 which contained relatively little of the ~1142 variant was chosen for further study. Both of the variants in stock 14 were found to contain the ~ HindlII fragment as well as the dl1142 vector fragment. The larger of the two variants contained two copies per molecule of the dl1142 vector fragment (data not shown). Stock 14 was plaque-purified to eliminate the larger variant (Brockman and Nathans, 1974). In order to obtain a clean virus stock of the k-SV40 recombinant, the 70% dll142::kDNA species was then isolated from agarose gels and transfected into monkey cells along with wild-type SV40 DNA. Fig. 5C illustrates the composition of the stock (d/l142:A) obtained in this fashion and it was this stock which was used for the studies described below.

(c) Use of dl1142 as a cloning vehicle

FormI dll142::~ DNA (Fig. 5C) was isolated from 1.4% agarose gels and digested with HindlII, HindlI or EcoRI. The digests were then electrophoresed in duplicate on 1.4% agarose gels and transferred in situ to nitrocellulose fdters according to the method of Southern (1975). To determine which restriction fragments contained ~ or SV40 sequences, the Fdter was cut in half and hybridized to either [a2p] ~ or [a2p] SV40 probe. Following the hybridization, the two halves of the filter were juxtaposed in their original orientation as shown in Fig. 6. As expected (Fig. 7), HindlII cuts the recombinant molecule into two fragments (Fig. 6, lanes b and e). One of these contains exclusively ~ sequences and co-migrates with authentic ~ HindlII-5 fragment. The other fragment which contains only SV40 sequences is the d11142 monomer vector fragment. A lower intensity band which is present in both the SV40 and X-probed channels is the partially digested linear 70% variant. Similarly, EcoRI should cut the recombinant molecule only once (Fig. 7). Fig. 6 shows only one

To test the feasibility of using dl1142 as a cloning vector we chose a piece of ~ bacteriophage DNA, the HindlII-5 fragment, whose size (2400 bp) was just large enough to allow encapsidation of the recombinant molecule in the SV40 capsid, dl1142 was digested to completion with HindlII and ligated in vitro to the k-HindlII.5 fragment. The products of the ligation were centrifuged to equilibrium in a cesium chioride-ethidium bromide gradient to separate covalently closed circles of all sizes (form I) from nicked circular and linear molecules (form II). Both the form I and form II species were then electrophoresed on a 1.4% agarose gel. Fig. 5A shows the results. Up to 25 different form I DNA bands were observed. Many of these bands were presumably the result of molecules with different numbers of superhelical turns. The band labeled number 1 in Fig. 5A and its immediate neighbors, for example, are all circular forms of the vector monomer fragment. The bands indicated in Fig. 5A (tracks 1-20)

(d) Physical characterization of the dl1142: :k variant

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....... dCn421x

d11142/k

Fig. 5. Steps in the cloning of the dl1142:: h hybrid molecule. 32P_labele d h Hin dlII-5 fragment and a 2 P-labeled dl1142 monomer HindlII fragment were isolated from 1.4% agarose gels and joined to each other in vitro with E. coli DNA ligase as described in METHODS. The ligation products were centrifuged to equilibrium in a cesium chloride-ethidium bromide gradient to separate covalently closed circular molecules (form I) from nicked circular (form II) molecules. When the form I and form II isolates were eleetrophoresed on 1.4% agarose gels the pattern seen in Fig. 5A was observed, Each form I band indicated in A above was then separately isolated. A 0.6 em dish of subconfluent BSC-1 monkey cells was infected with 10 ng of each form I band (1-20) and 1 ng of wild type SV40 DNA to make a virion stock of the DNA in each band. The composition of each stock was determined by isolating 32P-labeled DNA from a 0.6 cm dish of monkey cells infected with each stock and electrophoresing the DNA on a 1.4% agarose gel. When this was done, the pattern shown in Fig. 5-B was observed. (The position of the wild-type and variant bands is indicated only for the form II region of the gel.) The 70% SV40 variant seen in stocks 10-14 proved to contain both h and SV40 sequences. Two Jag of the 70% variant and 0.2 ug of wild type SV40 DNA were infected into subconfluent 10 cm dishes of BSC-1 monkey cells'to produce a virus stock whose composition is shown in Fig. 5C. The stock shown in 5C was used for the characterization of the rill 142:: h hybrid molecule. (The DNA shown in B and C was obtained from an ethanol precipitate of a Hirt (1967) supernatant which had been treated with pancreatic RNase.)

band in b o t h E c o R I channels which migrates as the 70% dl1142: :~ linear.

sequences is seen o n l y w h e n ~ probe is used. The remaining

fragments

contain

both

SV40

and

k

Finally, in order to d e t e r m i n e the orientation o f

sequences and are consistent w i t h the orientation

the ~ segment in the vector, a partial HindlI digest

s h o w n in Fig. 7. The restriction digests shown in Fig. 6 (as well as additional digests w h i c h we have n o t

was done and these lanes o f the gel (Fig. 6, lanes g and h) were deliberately o v e r e x p o s e d to visualize the partial digest products. As e x p e c t e d (Fig. 7), the one

shown) indicate that there was n o obvious deletion or rearrangement o f either the S V 4 0 or the ;k

HindlI fragment (12% o f SV40) which contains o n l y

sequences in the cloned D N A .

73

X % SV40

475--199----134~ 90--

A

SV40

B

C

D

E

F

%SV40

X

SV

G

H

%SV40

!! ii~Ii ~i/iilii !!++~ii~~i

--

~ii

i~i ~! ~i ~ i~~ ii ~

i

W

U

46 41---

IIIIS --34

,4

Z _._20

8.6

Fig. 6.0.5 #g of dll142::h hybrid DNA was digested with HindIII (lanes B and E), EcoRl (lanes C and D), or HindlI (lanes G and H). Each digest was split into two parts and run in duplicate on a 1.4% agarose gel as shown along with an SV40 HindIlI digest (lane F) and a h HindlII digest (lane A). When the electrophoresis was completed, the DNA fragments were denatured in situ and then transferred to a nitrocellulose filter according to the method of Southern (1975). The left half of the filter was hybridized to 32p-labeled SV40 DNA; the right half was hybridized to 32P-labeled h DNA. Following this the two sides were juxtaposed in their original orientation and the bands were visualized by exposure of X-ray film. Symbol in lanes G and H indicates form II DNA.

(e) Transcription o f the d l l 1 4 2 : : X recombinant in m o n k e y cells d11142::X virions were isolated from the SV40 wild type helper virus b y equilibrium cesium chloride Eco RI (.543) nd l[

Hind

III (.522) ~ I ~ " Hpa It, HhaI

/?Hind ~ ~

1~5531

I[, HpoI (.565)

I'" HindIll 1.5681 " B@mHI HindII, HpaI

Fig. 7. Physical map of dl1142::h hybrid DNA. Numbers in parentheses indicate h bacteriophage map positions as reported in Robinson and Landy (1977). Dotted arrow indicates direction of SV40 early transcription. Thin line: h fragment; heavy line: d11142 DNA.

centrifugation as described in Methods. Monkey cells were infected with d l l 1 4 2 : : X virions, wild type SV40, or a mixture o f the two viruses. Total cellular RNA was extracted from the infected cells and electrophoresed on 1.4% agarose gels which contained 5 mM methylmercuric hydroxide in order to denature the RNA. The gels were transferred in situ to diazob e n z y l o x y m e t h y l paper according to the method o f Alwine et al., (1977) and probed for the presence o f or SV40 RNA with either [32p]~ or [32P]SV40 DNA. Very little SV40 and no X RNA was detected in RNA preparations from cells infected with wild-type SV40 or the mixture o f dl1142::;~ and SV40 virions (data not shown). However, when d l 1 1 4 2 : : X virions alone were used for infection, a significant amount of b o t h X and SV40 transcription was seen. Fig. 8 shows that total RNA preparations contain a number o f SV40 RNA species (lane D) and at least one h-containing RNA (lane A) which can be distinguished b y this technique. (Small amounts o f additional X and SV40 bands which are not apparent in Fig. 8 were

74

I

x A

B

sv C

D

-35S

fractionation on the oligo(dT)-cellulose column. 95% o f the polio RNA was retained by our column and all of the RNA which was retained could then be eluted. When this poly(A)-enriched fraction was probed for the presence of SV40 sequences only one SV40 band could be discerned (Fig. 8, lane C). Its size is approx. 19S and it can also be seen in the total RNA preparation as a relatively minor band. No poly(A)-containing RNA was seen which also contained ~ sequences (Fig. 8, lane B). The amount of poly(A)-enriched RNA loaded in Fig. 8 is equivalent to three times the amount of total RNA which was loaded in the adjacent channels.

DISCUSSION

--I 8 S We have constructed a reiterated variant of SV40

(dl1142) which should be useful as a cloning vector.

--4S Fig. 8. Transcription of dl1142::h in BSC-I monkey cells ("Northern" blot), d11142::h virions were separated from wild-type SV40 helper virus by centrifugation to equilibrium in cesium chloride as described in Methods. Ten 10-cm dishes of confluent BSC-1 monkey cells were infected with the purified hybrid virions and total cellular RNA was isolated 24 h after infection. 10 #g of this RNA was electrophoresed in duplicate on a 1.4% agarose gel which was 5 mM in methylmercuric hydroxide (tracks A and D). The remainder of the RNA was mixed with 14C-labeled 35S polio RNA and passed through an oligo(dT)-cellulose column to isolate polyadenylated RNA species. The polyadenylated RNA was electrophoresed on the same gel in duplicate (tracks B and C). After the electrophoresis was completed, the RNAs were transferred in situ from gels to diazobenzyloxymethyl paper according to the method of Alwine et al. (1977). The left half of the blot was hybridized to 32p-labeled ~ DNA; the right side to 32P-labeled SV40 DNA. Following hybridization, the two sides of the gel were juxtaposed in their original orientation and the bands were visualized by exposure of X-ray film. also seen; see DISCUSSION.) To determine whether any of this RNA was polyadenylated, total cellular RNA from dl1142::X infected cells was fractionated on an oligo(dT)-cellulose column. 14C-labeled polyadenylated polio RNA (35C) was added to the dl1142::~ RNA in order to follow the efficiency of

Recombinants can be propagated in monkey cells by attaching foreign DNA to a single copy of the monomer fragment of the variant and using wild-type SV40 as a helper. To facilitate cloning, d/1142 contains a number of unique (single-cut) restriction sites (HpalI, HhaI, HindlI, HpaI and BamHI). The vector monomer fragment is 24% of SV40 size and additional material can be deleted without affecting its usefulness as a cloning vehicle. Hamer et al. (1977) have shown, for example, that all o f the SV40 late region between the HpalI and BamHI sites (see Fig. 4) can be deleted and substituted with foreign DNA. If this were done with the rill 142 monomer, then in principle up to 80% of the SV40 genome (4200 bp) is available for substitution. In principle, the small HpalI, HindlII fragment of the dl1142 monomer (6%) should be sufficient for cloning purposes. We were unable, however, to propagate this fragment or the larger wild-type Hin4~ + D fragment as a reiterated variant (N. Muzyczka, unpublished results). D. Ganem (personal communication) had a similar experience with the Hin-C + D fragment. One possible explanation is a presence of an additional cis-acting SV40 sequence in what remains o f the SV40 early region in dl1142. Such a site may be involved, for example, in the packaging of SV40 DNA into capsids. A similar suggestion was made by Gutai and Nathans (1978). It is interesting that although there was no apparent difficulty in replicating and reiterating the 24% dll 142

75 monomers (Fig. 5B), the original 34% monomer which we constructed did not grow well in monkey cells. We believe that this is probably due to the size difference between the two vectors. The 34% monomer could be reiterated during passage in monkey cells either to a 68% dimer or a 102% trimer. Both the dimer and the trimer are outside the range of DNA sizes which are efficiently packaged into SV40 virions. The recombinant dll142::k, which contained no SV40 genes and a single origin of replication, was propagated in monkey cells as a mixed stock with wild-type SV40 helper. Ganem et al. ( 1 9 7 6 ) h a d previously shown that a recombinant replicon which contained only the SV40 origin of replication could be propagated as a reiterated variant with multiple origins. We have shown here that a single origin of replication is sufficient for propagating recombinants. In addition we have shown (Fig. 5) that a clean recombinant virus stock can be obtained rather easily (and in the absence of selective pressure) by purifying the DNA products of the ligation mixture prior to infection of monkey cells. Since monkey cells are known for their ability to generate illegitimate recombinants in vivo (Brockman et al., 1975) we monitored the number of passages that could be tolerated before the stock developed additional variants. In our hands the stock developed a significant amount of a new variant after the third passage at an m.o.i, of 5 for the wild-type helper. The simplest way to maintain the purity of the stock was to regenerate it from a DNA infection in which the input ratio of recombinant to wild-type DNA was 10 : 1. dl1142 contains the 5' and 3' regions of both early and late SV40 messenger RNAs (Reddy et al., 1979; Thompson et al., 1979; Berk and Sharp, 1978; Ghosh et al., 1978). It does not, however, contain the splice junctions for either the early or late transcripts (Lai et al., 1978; Villareal et al., 1979; Reddy et al., 1979; Berk and Sharp, 1978; Khoury et al., 1979). The splice junctions have recently been implicated in the processing of nuclear transcripts into cytoplasmic messenger RNA (Laiet al., 1978; Khoury et al., 1979; Villareal et al., 1979). The k bacteriophage DNA cloned in dl1142 was inserted at the HindlII site at map position 0.649. In this position it is bracketed by the normal 5' and 3' ends of the SV40 early messenger RNAs which are located at 0.660 and 0.153, respec-

tively (Reddy et al., 1979). Because of the absence of the early SV40 intervening sequences we did not expect to find a cytoplasmic messenger RNA conraining k sequences. We were interested, however, in determining whether any transcription of k sequences occurred and whether such transcripts were polyadenylated at the same 3' position as normal SV40 early messenger RNA. Our results with wild-type and wild-type +dll 142: :k mixed infections were similar to those reported by Parker and Stark (1979). We saw no k RNA species in these infections, but we did see SV40 early message and a small amount of 16S late message (data not shown). When the dll142::k recombinant virions were isolated free of wild type, however, a significant amount of k transcription was seen. One major k band and three minor ~, bands (which are not seen in Fig. 8) were observed. Each k RNA species had an SV40 counterpart of the same mol. wt. when [32P]SV40 probe was used. This implies that both types of sequences, k and SV40, are contained in these RNA species. The smallest and most abundant k-containing RNA species (Fig. 8-A) was approximately the size of a complete transcript of dl1142: :k. TWO high mol. wt. RNA bands which contained only SV40 sequences were also seen. We have no simple explanation for their origin. The fact that we observed transcription of k sequences only in the case of cells which were infected with purified dl1142::k virions may be due to the fact that transcription of the SV40 early region is under negative control of SV40 T-antigen. It is known when a functional T-antigen is not made, early viral messenger RNA is overproduced (Reed et al., 1976; Khoury and May, 1977). Since no T-antigen is made in cells infected with dll142::k virions alone, there may be significantly more transcription of the k sequences which have been inserted in the SV40 early region than there is when wild type SV40 DNA is also present. An alternate explanation for the k transcription which we saw is that the m.o.i, was much greater in dll142::k infections than in dll142::k+SV40 infections. Since the dll142::k virion preparations could not be titered by plaque assay, we estimated the m.o.i, which we were using by comparison of the absorbance of the preparation with comparably treated wild-type preparations. It is possible, however, that the actual m.o.i, was much higher. Of all the RNA that we saw, only one relatively

76 minor RNA was clearly enriched after passage through an oligo(dT)-cellulose column. It contained only SV40 sequences and had the mobility of normal 19S early SV40 messenger RNA. We believe this messenger RNA is the result of a small amount (1%) of wild-type SV40 contamination in the d/1142::), virion preparation. We have not unequivocally ruled out the possibility that some of the transcripts, which contain h-coded RNA segments, are also polyadenylated. Additional experiments with d/l142::X and other types of recombinants could help identify the SV40 sequences necessary for polyadenylation in monkey cells. In sum, we have constructed an SV40-derived vector which should allow cloning pieces of DNA up to 4200 bp in length. This vector should be useful for cloning chromosomal genes and studying their expression. In addition, dl1142 may be a good prototype for conversion into other SV40-related vectors.

ACKNOWLEDGEMENTS I thank Daniel Nathans for his support and encouragement throughout this work; Keith Peden, Sandra Lazarowitz, James M. Pipas, George Michael, and Richard Rothenberg for technical assistance and helpful discussions; and Richard Rickles and James B. Flanegan for polio RNA and HeLa cell RNA. The author was supported by a training grant from the U.S. Public Health Service (T 32 CA 09 139-030031) during some of this work. The research was supported by grants from the U.S. National Cancer Institute (PO 1 CA 16519) and the American Cancer Society (VC-273).

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