Purification and cloning of a mouse ribosomal gene fragment in coliphage lambda

Gene, 2 (1977) 173--191 173 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands

PURIFICATION AND CLONING OF A MOUSE RIBOSOMAL GENE FRAGMENT IN COLIPHAGE LAMBDA (RPC-5 chromatography, ~gtWES cloning system, restriction endonucleases; R-loop mapping; electron microscopy; EcoRI; BamI; SalI; SstI; HindIII) D.C. TIEMEIER, S.M. TILGHMAN and P. LEDER Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014 (U.S.A.") (Received April 5th, 1977) (Accepted June 1st, 1977)

SUMMARY

We have formed and characterized a recombinant between the EK2 vector

~gtWE8.~C and a portion of the mouse ribosomal genes. A 6.6 kb endoR.Eco RI fragment was purified from total mouse DNA using RPC-5 ion exchange chromatography and then cloned and detected twice among 183 hybrid phage screened. In situ hybridization of restriction fragments of the hybrid phage DNA revealed that the inserted fragment contained both 18S and 28S RNA sequences. Electron microscopic analysis further suggested that most, if not all, of the 28S RNA sequence was present in the insert. The orientation of the 28S sequences in the hybrid phage was such that the "sense" of the inserted fragment should be under the control of the leftward promoter of ~,. INTRODUCTION

The ability to select an interesting DNA fragment from a large and complex array is one of the most impressive strengths of recombinant DNA technology. Detailed molecular studies are simplified by considering a small genetic sequence rather than the entire mammalian genome. Our own experience indicates that the ability to clone a specific fragment depends largely upon the efficiency of the vector system and the power of the screening technique. Furthermore, sufficient amounts of a partially purified DNA fragment must be prepared to satisfy the demands of the vector system and the practical limits of screening. Here we have assessed these factors using an EK2 vector system. Abbreviations: DTT, dithiothreitol; PEG, polyethylene glycol.

174 The EK2 vector, ~gtWES.~C (Enquist et al., 1976), was used to clone an endoR.EcoRI fragment containing a segment of the mouse ribosomal genes. The parental phage, ~,gt-~,C, was earlier used by Davis and his associates (Thomas et al., 1 9 7 4 ; Kr-mer et al., 1976) to clone ribosomal sequences from yeast and McClements and Skalka (1977) have recently used the EK2 derivative to clone a chicken ribosomal gene segment Because of the complexity of the mouse genome, we have used the EK2 system in conjunction with an RPC-5 column chromatography procedure which permits the large-scale purification of DNA fragments derived from total mouse DNA (Hardies and Wells, 1976; Landy et al., 1976). Screening fewer than 200 recombinants yielded two ribosomal hybrids.

MATERIAII~ AND METHODS Preparation of phage vector DNA The construction of ~,gt Warn403 Eam1100 8amlOO.kC has been described (Enquist et al., 1976). Lysogens of the phage in E. coil C600 supE (LE 325) were prepared and induced essentially as described by Schrenk and Weisberg (1975). An overnight culture of the lysogen was diluted 1 : 1 0 0 in 3001 LB (1% tryptone, 0.5% yeast extract, 0.5% NaCI) and grown at 32"C to a cell density of 2 • 10 s celis/ml. The culture was shifted to 42"C and incubated with vigorous aeration for 15 rain. The culture was shifted to 370C and grown for an additional 2.5 h. Chloroform was added to 0.1% and the culture incubated for an additional 10 rain to complete lysis. (Although Warn403 and Earn1100 are efficiently suppressed in this strain, the phage mutation in the lysis gene Sam100 is poorly suppressed. The resulting delay in lysis promotes yields of approx. 10 z° plaques/ml.) NaCI was added to 0.5 M and MgSO4 to 10 mM and cell debris was removed by centrifugation. PEG was added to the supernatant to a concentration of 10% and the suspension incubated overnight at 4°C. The precipitate (223 g) was collected by centrifugation and resuspended in a total volume of 1.5 1 20 mM Tris--HCl (pH 7.5), 10 mM MgSO4. The suspension was extracted with an equal volume of chloroform to remove the PEG and CsCI was added to a density of 1.2 g/cm 3. As needed, phage were concentrated on a cushion of CsCI and then banded to equilibrium as previously described (Enquist et al., 1976). [The presence of the mutations Wam403, Eam1100, SamlO0, ninS, clts857 was confirmed for the phage stock as described in Enquist et al. (1976)]. Phage DNA was isolated as described in Enquist et al. (1976). Approx. 100 mg of phage DNA were obtained from the 300 1 lysate. Isolation of mouse genomic DNA High molecular weight DNA (> 30 - 106 daltons) was prepared from frozen MOPC 41 myeloma tumor using the method of Blin and Stafford (1976). EndoR.EcoRl restriction EndoR.EcoRI was purified from Kcoli BRY 13.3 (1.100.5) kindly provided

175

by Dr. Julian Davies, essentially as described by Modrich and Zabel (1976) through hydroxylapatite chromatography. This enzyme preparation was treated with the sulfhydryl zeagentp-chloromercuribenzoate (Sigma) which does not affect endoR.EcoRI activity but does inactivate an exonuclease-like activity which otherwise lowers the capacity of restricted fragments to be sealed by T4 ligase. The concentration of ~-mercaptoethanol in the enzyme preparation was reduced by dialysis against 25 vol. 10 mM Tris--Cl, (pH 7.5), 0.1 M NaCI. 1.0 mM EDTA, 20% glycerol with one buffer change. The dialyzed endoR. EcoRI was incubated with 1.0 mM p-chloromercuribenzoate for 30 rain at 37°C. DTT was added to a concentration of 10 mM to stop the reaction and the enzyme preparation was dialyzed for 24 h against 25 vol. 30 mM Tris-HCI, (pH 8.0), 0.3 M NaCI, 0.3 mM EDTA and 50% glycerol. Starting with 250 g cells this procedure yielded sufficient endoR.EcoRI to digest 3--6 g DNA under standard conditions (Enquist et al., 1976).

RPC-5 chromatography of DNA RPC-5 was prepared as described by Pearson et al. (1971). Plaskon beads used for the preparation of RPC-5 were obtained from Allied Chemicai Co., but are no longer manufactured by them. Stocks of these beads are generally available from research groups involved in the isolation of tRNAs. Forty A260 units of endoR.EcoRI-treated MOPC 41 DNA in 10 ml of 1.25 M sodium acetate (NaOAc), 50 mM Tris--HCl (pH 7.5), 1 mM EDTA were applied to a 30 ml RPC-5 column which had been equilibrated at 200 psi in the same buffer. The DNA was eluted in 5 ml fractions with a 500 ml gradient of 1.45 M to 1.65 M NaOAc. Aliquots of 1.5 ml were dialyzed extensively against H20, lyophflized, dissolved in 25/~1 H20, and heated at 100°C for 10 rain. To each fraction was added 25 ;d of 2 M NaCI, 20 mM Tris--HCI (pH 7.5), 2 mM EDTA and 1400 cpm of 12sI rRNA, specific activity 5 • 10 v cpm/~g. The hybridization reactions were incubated at 68°C for 36 h, followed by RNAase A treatment as previously described (Melli et at., 1971). The left and right arms of )~gtWES.7~C DNA were separated from the ?,C fragment by chromatography of 10 A26o units of endoR.EcoRI-treated DNA on a 15 ml RPC 5 column using a 1.48--1.54 M NaOAc gradient in 50 mM Tris--HCl (pH 7.5) and I mM EDTA. Portions of individual 2-ml fractions were precipitated in 2 volumes of ethanol and analyzed for the absence of the kC fragment on 1% agarose slab gels. In situ hybridization of restriction fragments o f DNA Restriction fragments of DNA were subjected to electrophoresis in 1% agarose gels as described previously (Helling et al., 1974). After visualization of the DNA bands by ethidium bromide staining, the DNA was eluted from the agarose gel onto a Millipore filter using the blotting technique of Southern (1975). The filters were baked at 80°C for 16 h and incubated for 20 h at

176 68°C in the presence of 12sI RNA, 2--10 • 10 s cpm/ml in 6 × SSC (1× SSC is 0.15 M NaCI; 0.015 M Na citrate), 0.1% SDS. After the completion of the hybridization reaction, the filters were washed for several hours in 2 X SSC at room temperature.

Construction o f recombinant phage. In accordance with the NIH Guidelines, the following experiments were performed under 1)3 physical containment conditions. ~gt Wam403 Eam1100 8amlO0.~C is a certified EK2 vector with the condition that the left and right arms produced by endoR.£coRI digestion be first purified from ~C fragment before use. (i) Ligation. Mixtures of ?,gt WE8 arms and mouse endoR.EcoRI DNA fragments enriched for the 6.6 kb fragment containing 18S and 28S ribosomal DNA sequences were incubated in 0,015 ml at 45°C for 10 rain. This disrupted sequences annealed by the four base endoR.EcoRI cohesive end without affecting the interaction of ~ cohesive termini. The reactions were chilled on ice, adjusted to 0.10 M Tris--HCl (pH 7.8), 50 mM NaC1, 12 mM MgC12, 10 mM DTT, 0.10 mM ATP, 50 #g/ml bovine serum albumin, 0.10 mM EDTA and 2.5 U/ml T4 polynucleotide ligase (Miles) and incubated at 4°C for 24 h. A second addition of ATP to 0.10 mM and ligase to 1.0 U/ml was made and the reactions incubated for an additional 24 h at 4°C. Under these conditions, 50% of the ~gtWES arms were sealed into larger molecular weight forms. The concentration of DNA yielding optimal hybrid formation was 37.5 #g/ml ~gtW£8 arms plus 12.5 #g/ml of mouse endoR.£coRI digested DNA. (ii) Transfection. Hybrid DNA molecules constructed in vitro were used to transfect an K coil derivative (Wood, 1966) ED 8656, a gift of N. Murray. This strain, designated here as LE392, is hsd M+ hsd R'supE supF gal'. Cells competent for transfection were prepared essentially as described by Cameron et al. (1975). Aseptic technique and sterile solutions were used for all steps and except where noted all steps were performed at 4°C. An overnight culture of LE392 was diluted 1/100 in fresh LB + 50 #g/ml thymidine and grown to a density of 2. l 0 s cells/ml (Asg0 = 0.60) at 37°C with vigorous aeration. The cells were washed with an equal volume of 25 mM Tris--HCl, (pH 7.5), 10 mM NaCI. The cells were resuspended by gentle swirling in 1/2 vol. 25 mM Tris--HC1 (pH 7.5), 10 mM NaC1, 50 mM CaC12 and incubated on ice for 20 rain. The cells were collected by centrifugation for 10 rain at 2000 g and resuspended in 1/5 voL of the same CaCI2 solution. CaCl2-treated cells were used within 30 min. For transfection 0.4 ml competent cells were added to 0.2 ml 25 mM Tris--HCl (pH 7.5), 10 mM NaCI 50 mM CaC12 containing 2--5/~1 of an in vitro recombination reaction such that the final DNA concentration was 0.1--0.3 #g/ml. The suspensions were incubated on ice for 60 rain, shifted to 37°C for 60 sec and then plated with 1 drop of a fresh LE392 overnight culture. Plates were incubated overnight at 38.5°C. Under these conditions the trans-

177

fection efficiency of intact ~ D N A p1~lues/10 s D N A molecules).

is 5 • 10 s to 2 • 10 6 plaques/~g (2.5--10

Plaque hybridization Recombinants containing ribosomal gene sequences were identified using a plaque hybridization technique essentially as described by Kramer et al. (1976). Plaques obtained by transfection were amplified by transferring them with toothpicks to a fresh lawn of LE392 and incubating the plates for 8-15 h at 38.5°C. The plates were inverted over chloroform for 60 min to kill the bacterial cells and the plaques were transferred with Pasteur pipettes (as agar plugs) or with wooden applicators to microtiter dish wells containing 0.10-0.20 ml TMG (10 mM Tris--HC1 (pH 7.5), 10 mM MgSO4, 0.1% gelatin). These individual hybrid phage suspensions were spotted with capillaries onto millipore filters (HAWG 047000) impregnated with the indicator LE392. The use of capillaries permitted the rapid preparation of replica filters for each set of hybrids. The filters were incubated on LB plates for 9--15 h at 38.5°C and processed as described (Kramer et al., 1976) to yield arrays of denatured DNA representing the individual hybrid phage. The filters were hybridiT.ed at 70°C with [12sI] mouse reticulocyte polysomal RNA (10 cpm/pg, preheated for 10 rain at 70°C in 50 mM Tris--HCl pH 7.5) in a modified Denhardt solution of 0.09 M sodium citrate, 0.9 M NaCI, 0.5% SDS, 0..02% ficol, 0.02% polyvinyl pyrrolidone, 0.02% bovine albumin (Denhardt, 1966) to a Cr0t = 0.10. The filters were washed with 2 X SSC and positive clones were identified after autoradiography on Kodak X-ray film SB-5.

Hybrid DNA preparation and analysis For rapid DNA analysis, the phage hybrid of interest was plaque-purified on LE392. A single isolated plaque was transferred to 2.0 ml LB in a 125 X 15 mm glass screw cap tube and one drop of a fresh overnight culture of LE392 was added. The suspension was incubated with shaking at 38--39°C to lysis (6--15 h). Two drops of chloroform were added and the lysate shaken for an additional 5 rain at 38°C. Debris was removed by centrifugation for 10 rain at 2000 g and the supernatant (1.5 ml) was removed to a second screw cap tube. Deoxyribonuclease I was added in 1.5 ml 20 mM Tris--HCl (pH 7.5), 20 mM MgSO4 to 10 ~g/ml and the suspensions were incubated at 20°C for 15 rain and then chilled on ice for an additional 30 rain. Sodium chloride was added to 0.5 M and PEG to 10% and the suspensions were held on ice for 60 min. The PEG-precipitated phage was collected by centrifugation for 10 min at 10 000 g and resuspended in 0.3 ml 20 mM Tris--HCl (pH 7.5), 10 mM MgSO4. Residual PEG was removed by extraction in 2.0 ml Eppendorf tubes with an equal volume of chloroform. Sodium chloride was added to 0.25 M and EDTA to 25 mM. Phage DNA was extracted first with an equal volume of phenol saturated with 20 mM Tris--HCl (pH 7.5), 0.25 M NaCI, 1.0 mM EDTA and then with an equal volume of chloroform. The

178

aqueous phase was transferred to a new Eppendorf tube and carrier E. coli tRNA was added to 50/~g!ml followed by three volumes of 95% ethanol. After 60 rain at - 20°C, phage DNA was collected by centrifugation for 5 rain in a Brinkmann 3200 microcentrifuge and resuspended in 50/~1 of sterile deionized water. This method yielded 0.1--2.0 t~g of DNA. Approx. 10% of hybrids failed to yield detectable DNA presumably due to premature or incomplete lysis in the initial growth step. However, as many as 25 hybrids have been easily screened within 2 days by this procedure.

Electron microscopy of DNA R-Loops were formed between hybrid phage DNA and 28S RNA as described by Westphal et al. (1976). Hybrid DNA at 20 ~g/ml and 28S RNA at 100/~g/ml were incubated in 70% formamide, 0.1 M tricine NaOH (pH 7.5), 10 mM EDTA, 0.5 M NaCI at 55°C for 16 h in sealed capillary tubes. The hybridization mixture was diluted 20-fold in the same buffer and spread onto a hypophase of 10% (v/v) formamide, 0.1 mM EDTA, 0.01 M Tris-HCI, pH 8.5. The DNA was picked up on parlodion-coated grids, stained with uranyl acetate and shadowed with platinum-palladium. The grids were viewed with a Philips 300 electron microscope, using grating replica for length calibration. RESULTS

Efficiency of the cloning system EK2 certification of ~gtWES.~C requires that the central endoR.EcoRI fragment of the phage, ~,C (encoding ~,-mediated recombination functions), be deleted biochemically prior to use of the vector. While a number of techniques serve this purpose, we have found RPC-5 chromatography (to be described below) most convenient. Agarose gel analysis of the two larger endoR.EcoRI fragments, the outer "arms", separated from the ).C fragment, is shown in Fig. 1, lane b. The very low frequency with which parental type recombinants ~ e found among a population of hybrid phage (< 1%) indicates that the arms are greater than 99% free of the ~,C fragment. Purification of the larger fragments reduces the transfection efficiency of the ligated cloning mixture about 10-fold (Table I): 1--2 • 103 pfu//~g unpurified arms. However, purification eliminates parental phage from the recombinant population. Since the outer endoR.EcoRI fragments of ~gtWES •XC provide insufficient DNA for phage packaging (Thomas et al., 1974), essentially all the resultant phage are true hybrids, containing fragments of foreign DNA. In practice, we obtain 3--6 • 103 hybrid phage per/Jg of foreign endoR.EcoRI fragment DNA.

Purification of genomic DNA: RPC-5 chromatography Given that mouse genomic DNA would be expected to generate approx.

179 a

b

Fig.1. Agarose gel eleetrophoresis of kgtW£$.kC endoR-EcoR[ fragments. An endoR. EeoRI digest of kgtWE8.~C DNA (lane a) and ~,gtWE8left and right fragments purified using RPC-5 chromatography (lane b) were eleetrophoresed on a 1% agarose slab gel. 10 ~ endoR.EcoRI fragments, some degree of fragment purification is obviously necessary in order to select a specific, rare fragment. Recently, Hardies and Wells (1976) and Landy et al. (1976) have shown that the qua.. ternary amine reverse phase medium, RPC-5, provides a high capacity, high resolution chromatographic system for the isolation of DNA restriction fragments. Because this system readily accommodates the mg amounts of DNA necessary for m a m m a l i a n studies, we tested its ability to resolve mouse ribosomal gene fragments in total genomic DNA as a measure of its usefulness with DNAs of this complexity. EndoR.EcoRI fragments o f mouse DNA were, therefore, adsorbed to an RPC-5 column and the DNA eluted in a broad profile with a 1.45 M--1.65 M

180

TABLE I TRANSFECTION EFFICIENCY W~TH xgtWES- xC DNA These experiments were performed as described in Mate~lR and Methods. The values are expressed as plaque-forming units per mg of xgtWES arms DNA. DNA

Ligase Minus (pfu/~g)

(a) (b) (c) (d) (e)

xgtW£S-xC EndoR.EeoRI d i g e s t e d xgt WES .~C EndoR.EcoRI digested xgtWES.XC + mouse DNA xgtWE8 outer fragments xgtWES outer fragments

+ endoR-EcoRI digested mouse DNA

3 • 10s-1.5

Plus (pfu/~g) •

106

30--150

2-5 • 103

30--150 < 10

5--25 • 103 10-50

< 10

1--2 •

lOs

sodium acetate gradient. Fragments coding for 18S and 28S RNA, identified by hybridizing individual DNA fractions to ~2SI-rttNA, eluted as a sharp peak at the beginning of the DNA elution pattern and a more diffuse peak at the end of the column (Fig.2). The two peak fractions were analyzed by agarose gel electrophoresis (Fig.2, insert) and in both instances, a clearly d e f i n e d band was visible above the background DNA. When the DNA in each lane was transferred to Millipore filters using Southern's procedure (1975) and challenged with '2SI-rRNA, each visible band hybridized to the labeled probe. The sizes of the two ribosomal bands were 6.6 kb and 15--16 kb for the early and late peaks, respectively. From the distribution of total DNA, we can estimate that the extent of purification of the smaller fragment is approximately 35-fold. Assuming a reiteration frequency of 200 for the ribosomal cistron in the mouse, we would expect the endoR.EcoRI fragment to be present about once in every 100 molecules in the peak fraction. Cloning and the screening test

In order to assess the extent of purification of the ribosomal fragment and tile usefulness of RPC-5 chromatography, we formed hybrid phage by insertir~g mouse DNA fragments from the initial ribosomal peak (fraction No. 40, Fig. 2) into the isolated outer fragments of XgtWES'~C. 183 independently arising hybrids were transferred to microtiter wells and plated on bacteriasoaked filters in order to take advantage of the convenient in situ hybridization screening technique developed by Kramer et al. (1976). The filters, imprinted with DNA from the hybrid phage, were incubated with radioactive

181

;,.%

f~O0

~

a

b

c

d

e r~

1 200

30

I 40

50 FRACTION

| 60

~1 70

I 80

Fig.2. RPC-5 chromatography of endoR.£eoRI fragments of mouse genomic DNA and identification of fragments encoding ribosomal RNA. An endoR.£coRI digest of mouse genomic DNA was chromatographed on an RPC-5 column as described in Materials and Methods. e ~ $ , As6e; o m o , epm of I~SI-rRNAhybridized to individual fractions. Insert: agarme gel electrophoresis of an endoR.EeoRI digest of wild-type ~ ci857 DNA where migration is from top to bottom and the kb pair sizes of individual fragments are: 21.3, 7.36, 5.79, 5.40, 4.6g and 3.3 kb (lane a); ethidium bromide stain of DNA from fractions 40 and 59 (lanes b and d, respectively); autoradiogram of a Southern transfer of lanes b and d (lanes c and e, respectively), hybridized to lffiSI-rRNA. ribosomal RNA and, at a b o u t t h e incidence expected, two positive clones were identified (Fig. 3). Had the fragment of interest been rarer, several thousand hybrids could have been screened using this procedure w i t h o u t undue difficulty.

Characterization o f the ribosomal clone One advantage o f the ~gtWE8 system is t h a t we have been able t o develop techniques which permit us t o generate in one day sufficient DNA frora a single plaqu~seeded 2 ml lysate t o carry out several analyses including restriction-hybridization blots and electron microscopic mapping (Southern, 1975; Thomas et al., 1976; Westphal et al., 1976). This is obviously a strong advantage in screening a large group o f clones grown under physical containment conditions.

182

Fig.3. Identification o f r R N A clones by in situ hybridization. Individual x recombinant plaques were processed as described in Materials and Methods and grown on Millipore f'dters. The fdters were hybridized to 12SI-rRNA, washed, and autoradiographed as described.

Analysis of the specific insert confirmed the identity of the rDNA clones. DNA from one of the positive clones, A22A2,was digested with endoR.Eco RI and its electrophoretic mobility was compared to that of the mouse DNA sample from which it was cloned (Fig.4). The hybrid clone contains a single insert fragment of the same molecular weight (6.6 kb) as the fragment identified in mouse genomic DNA. The fragments were denatured in situ and transferred to Millipore filters by blotting for hybridization. Radioactive rRNA hybridized to the insert, but not to the separated arms or any of the fragments obtained from kgtWES.kC or clone A22A3, a hybrid prepared from the same DNA fraction but negative for the rDNA sequence by the initial plaque hybridization screening (data not shown). Finally, the hybrid was labeled with K coli DNA polymerase I (Maniatis et al., 1975) and used as a hybridization probe against the DNA fraction from which it was cloned (Fig.4). A fragment 6.6 kb in length was the only species to which this probe hybridized. Similarly labeled ~gtWES.~C and A22A3 failed to hybridize to this fragment (data not shown). The orientation of the mouse ribosomal fragment with respect to the phage genome was determined by identifying those endoR-SalI cleavage fragments of the recombinant which hybridize to purified 28S rRNA and to a mixture of 18S and 28S rRNA (Fig.5). kgtWE$.~C DNA contains two closely spaced endoR.SalI restriction sites corresponding to 67.3% and 68.3% on the wild type ~ map. The ribosomal hybrid contains an additional site 1.8 kb from the right end of the inserted fragment. Thus, endoR.SalI digestion of the hybrid generates three detectable fragments; 25 kb (from the left end of ~), 12.7 kb (from the right end of k), and 2.65 kb. The two closely _spaced endoR.SalI sites in the right fragment of k produce a small fragment

18~',

a

b

c

d

e

f

! if! { i ¸ i~ i 'i i~i~/iii ~

~i i~I/ i li

Fig.4. EndoR,E~oRI digestion of ribosomal DNA and the hybrid clone. Ribosomal DNA purified on RPC-5 columns (Fig. 2, fraction 40; lane b) and an endoR.EcoRI digest of hybrid clone A22A2 (lane d) were electrophoresed on a 1% agarose gel. The DNAs were transferred to Millipore filters and hybridized to "SI-rRNA using the Southern technique (1975) (lanes c and e). Hybrid DNA was labeled using E. eoli DNA polymerase I (Maniatis et al., 1975) and used to challenge DNA from lane b (lane f). Lane a contains an endoR,EdoRI digest of wild-type ~ as described in the legend to Fig.2.

not seen in this analysis. The 28S rRNA probe hybridizes only to the largest fragment. The 28S + 18S rRNA hybridizes to both the largest and smallest fragment indicating that the 18S sequence is located on the smallest visible 2.65 kb endoR.SalI fragment and is adjacent to the right arm of )~. A diagrammatic model of the hybridization is shown in Fig.5. The ribosomal fragment was further characterized by digestion with restriction endonucleases endoR.BamHI, endoR.SstI and endoR.HindIII (Fig. 6). Restriction sites corresponding to each of these enzymes can be oriented in the mouse ribosomal fragment by comparison to the cleavage pattern of the parent vector, kgtWE8.kC. EndoR.HindIII digestion generates four detectable fragments; 28.5 kb, 5.0 kb, 4.2 kb, and 3.75 kb in length, indicating the presence of a single endoR.HindIII site within the ribosomal insert. However, this site is so close to the right end of the inserted fragment as to be indistinguishable from the adjacent endoR.EcoRI site. (A G.3--0.4 kb endoR.HindIII fragment generated from the right end of k has run off the gel in this analysis.) Digestion with

184

a

b

,c

d

•

Fig.5. EndoR.Saff digestion of ~gtWES.xC DNA and rRNA hybrid DNA. DNA from ~,gtWES.~,C (lane b) and the rRNA hybrid clone (lane c) were cleaved with e n d o R ~ a | I (Hamer and Thomas. 1976) and electrophoresed on 1% agarosc gels. Lane a contains an endoR.E~oRI digest of wild-type ~ as described in the legend to Fig. 2. The hybrid clone DNA in lane c was transferred to Mil|ipore filters and hybridized against 12Sl 188 + 288 rRNA (lane d) or "sI 28S rRI~A (lane e). In the diagrammatic representation at the bottom of the page, the dark arrows indicate the sites of endoR-SaH cleavage in the hybrid DNA. The solid lines below represent endoR-SolI fragments which hybridized to the indicated RNA probe'~. Stippled boxes represent deleted portions of wild type X.

W~

S~

I

probes I'

'

I k----If,

. . . . . ." . . ". . . . . . .

I 18"1-28S

185

¸

¸¸¸¸~¸~ t ~

i,i,~i~i~iii,'i~!i,i,

i i~,!i~i~i~:I

~

I

....

I

,11-

ab

cd

ef

gh

Fig,6. Restriction endonuclease of ~gtWE$.~.C DNA and rRNA hybrid DNA. DNA from ~gtWE8.~C (lanes c, e, g) and the hybrid clone (lanes d, f, h) were cleaved with endoR. HindIII (Murray and Murray, 1975) (lanes c and d), endoR./~mHI (lanes e and f) or endoR.Sstl (Tiemeier et al., 1976) (lanes g and h). The arrows indicate the presence of fragments in lanes f and h not clearly visible in the photograph. DNA markers were provided by an endoR.EcoRI cleavage of wild type ~ (lane a, as described in the legend to Fig. 2) and an endoR.HaeIII digest of SV40 DNA (lane b). The 3 fragments visible in lane b correspond to fragments of 1.5, 0.9 and 0.6 kb length.

endoR.BamHI yields five fragments; 18 kb, 11.1 kb (from the right end of X), 6.4 kb, 5.4 kb (from the left end of k), and 1.3 kb in length, indicating the presence of two endoR.BamHI sites within the insert, 1.3 kb apart. Digestion with endoR.SstI generates three fragments; 21.3 kb (from the left end of k), 19 kb (from the right end of ),), and 1.4 kb indicating the presence of two endoR.SstI sites 1.4 kb apart. One of the sites is so close to the left end of the insert as to be indistinguishable from the endoR.EcoRI site by this analysis. The location of these sites within the ribosomal fragment is shown diagrammatically in Fig. 7. The hybridization of 12sI 28S and 18S + 28S r R N A to individual endoR.Sall fragments of the hybrid clone (Fig.5) placed the 28S r R N A

186 0

A.

,

;O0%X w-

.

,

_

clls

I ~.

0 5sl I

L3

~,Baml

2.6

4.8

Vl

6.6 ~.b

'~'f~ll

Sst I 1.4

Hind III

_ dh

~

A

,es_ A

~

~L

Fig.7. Diagrammetric representation of ~he orientation of the ribosomal DNA in phage and the position of restriction endonuclesse cleavage sites. (A) Representation of xgtWES and the position of the inserted ribosomal DNA fragment. Stippled boxes represent deleted portions of wild type X. (B) A restriction map of the inserted fragment, where the num]~ers above the arrows indicate the distance in kb from the left hand end of the insert. In the lower figure, the approximate positions o f 28S and 18S rRNA coding sequences are shown, along with the 5'--3' orientation. PLOL and PitOR represent the approximate positions and orientations o f the X leftward and rightward promoters, respectively.

coding sequences to the left of the 18S rRNA sequences. Previous data of Southern (1975) suggested that most, if not all, the 28S rRNA sequences were contained within the smaller fragment generated by endoR.EcoRI cleavage of mouse genomic DNA. In order to confirm the orientation of the insert and to estimate the length of the sequences encoding 28S rRNA, Rloops were formed by incubating hybrid DNA in the presence of 28S rRNA (Thomas et at., 1976). These R-loops were readily visualized and Fig. 8 illustrates a molecule typical of the over thirty analyzed containing a single R-loop consisting of single-stranded and double-stranded branches. This configuration indicates the presence of 28S rRNA displacing one DNA strand and hybridizing to the other. The position of the 28S rRNA R-loop at 53% k length places it proximal to the left arm of the hybrid DNA, and confirms the orientation determined with endoR.SalI. The average length of the R-loop was 9.5% k length, or 3.95 kb. Thus it is possible that the entire 28S rRNA sequence is contained within the inserted fragment. In support of this, we observed that the large 21.3 kb fragment generated by endo R-Sstl cleavage of the hybrid DNA which would include the entire left arm of kgtWES and a small portion of the inserted DNA failed to hybridize to ~2sI 28S rRNA. However, because the endoR-SstI site mapped so close to the endoR.EcoRI site separating the left arm from the insert, it is also possible that there were not enough sequences of 28S DNA in this fragment to form a stable hybrid.

Qo

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188

DISCUSSION

The cloning procedure. Cloning a non-reiterated endoR-EcoRI fragment from mammalian DNA requires that the cloning efficiency of the vector system, the purity of the fragment and the logistics of the screening technique permit the selection of one out of approx. 106 fragments. While more powerful screening techniques may soon be available (Davis et aL, 1977), the method employed in this study (Kramer et al., 1976) permits the analysis of as many as 5000 hybrid phage at one time. Therefore, if a unique DNA fragment can be purified 1000-fold (from 1/10 ~ to 1/103) in sufficient yield to generate several thousand hybrid clones and if it does not affect phage growth (Sternberg et al., 1977), it can be obtained as a cloned fragment. Given the cloning efficiency of the kgtWE8 system, one ~g of a 1000-fold purified fraction of mammalian DNA would, therefore, yield sufficient hybrids to identify the desired clone. The study described above demonstrates that a 200-fold reiterated sequence is readily cloned. We have already indicated that the screening technology may easily be applied to 10 times the number of hybrid phage analyzed here. It has been our recent experience that repeated chromatography of specific fragments of DNA on RPC-5 using increasingly shallow salt gradients yields fractions which are 1000-fold enriched, bringing them into the range of purity necessary for cloning. While the NIH Guidelines obviously affect the versatility of recombinant technology, it appears that many unique mammalian gene sequences are within reach of the ~gtWES.~C as well as the ~gtWES.~B EK2 vector systems (Tiemeier et al., 1976). The mouse ribosomal fragment. The combination of restriction endonucleaqe analysis and the Southern hybridization procedure (Southern, 1975) demonstrated that both 18S and 28S rRNA sequences are contained within the 6.6 kb insert of the ~gtWES.rDNA hybrid. That most, if not all, the 28S coding sequences are contained within the insert was suggested by electron microscopic R-loop mapping, where an estimated size of 3.95 kb was observed for the 28,°, loop. The organization of rDNA in Xenopus laevis has been extensively studied and has been shown to consist of repeating units, each of which contains a region encoding the transcribed 40S rENA precursor and a heterogeneous non-transcribed spacer region (Morrow et al., 1974; Wellauer and Dawid, 1974). The precursor transcript contains both 18S and 28S rRNA sequences plus ENA on either side of the 18S sequence which is lost during processing (Wellauer et al., 1974). EndoR.EcoEI cleaves Xenopus rDNA once in the 18S and once in the 28S rRNA sequences, yielding a unique fragment which contains 18S and 28S RNA sequences separated by a portion of the transcribed spacer and a set of larger fragments containing 18S and 28S RNA sequences plus the heterogeneous non-transcribed spacer region and a portion of transcribed spacer (Wellauer et al., 1976). Southern (1975) previously showed that endoR .EcoRI cleavage of mouse genomic DNA gave 2 fragments

189

which hybridized to labeled rRNA. The large fragment was heterogeneous in size, and is equivalent to the 15--16 kb fragments which we observed on RPC-5 chromatography (Fig.2). By analogy to Xenopus, the large size and heterogeneity of this fragment suggests that it contains the non-transcribed spacer region. Thus the smaller fragment observed by Southern (1975) which we have cloned should contain the transcribed spacer surrounded by 18S and 28S rRNA sequences.

Electron microscopic R-loop mapping and restriction analysis suggested that the orientation of the inserted fragment was such that the 28S sequence is adjacent to the left endoR.EcoRI fragment of k and that the 18S sequence is adjacent to the right ), fragment. If the mouse 18S sequences are on the 5' terminus of the 45S ribosomal precursor as in the Xenopus precursor (Reeder and Brown, 1970; Reeder et al., 1976; Dawid and Wellauer, 1976), the 3'--5' sense strand in the hybrid is in alignment with the early gene transcript of )~ which is under control of the left hand promoter-operator of (PLOL). A diagrammatic model of the fragment in this orientation is shown in Fig. 7. A hybrid clone constructed by McClements and Skalka (1977) co,taining the ribosomal gene fragment from chicken appears to have the same orientation. Both clones should prove useful in generating in vitro transcripts for the study of rRNA processing. A potential vector for endoR.SalI fragments. The kgtWES.mouse rDNA hybrid is not only a mode~ recombinant for studying gene organization and expression but may also be useful as a vector. If the internal fragment were inserted in reverse orientation, endoR.Sall.generated DNA fragments up to 14 kb in length could be inserted between the ribosomal endoR.SalI site and the endoR.SalI site of ~, (see Fig. 7). Incorporation of an insert fragment would be required for phage viability and formation of parental type recombinants could be prevented simply by initial digestion of the vector with endoR.SsH (Tiemeier et al., 1976) to destroy the small parental endoR.SalI fragment. ACKNOWLEDGEMENT

We are indebted to Dr. Lynn Enquist for his contributions to the development of the kgtWES cloning system and for his advice and suggestions during these studies. We are very grateful to Dr. Jacob V. Maizel Jr. and Mrs. Marjorie Sullivan for the invaluable advice and assistance they provided during the electron microscopic studies. We would like to thank Dr. Fred Polsky for his critical gift of the high molecular weight mouse DNA used in these studies. We would also like to thank Dr. Dolph Hatfield for kindly providing RPC-5 and his valuable advice in its use. We are, once again, most grateful to Ms. Catherine Kunkle for expert assistance in the preparation of this manuscript.

190 REFERENCES

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by A. Skalka.