Vectors for the direct selection of cDNA clones corresponding to mammalian cell mRNA of low abundance

Gene, 35 (1985) 45-54 Elsevier

45

GENE 1264

Vectors for the direct selection of cDNA clones correspomlmg to mammalian cell mRNA of low abundance (R~ornb~~t

DNA library; bacte~op~~e

fl ; single-stranded DNA; human flbroblasts)

Jacek Kowalski, James H. Smith, Nancy Ng and David T. Denhardt * Cancer Research Laboratory, Universityof Western Ontario, London, Ontario (Canada N6A 5B7) (519)679-2361 (Received December 7th, 1984) (Accepted January Znd, 1985)

SUMMARY

We have constructed two cDNA cloning vectors which (1) carry the intergenic region of phage fl and (2) permit efficient cloning (by the Okayama-Berg procedure) of full-length copies of mammalian mRNA in either orientation. Infection of cells harboring these vectors with fl phage results in the encapsidation of single-stranded (ss) plasmid DNA carrying either the sense or the anti-sense sequence of the cDNA inserts. The complementary nature of the cDNA inserts in two such cDNA libraries facilitates preparative hybridization procedures. These vectors have general app~cab~ty to any eukaryotic system where changes in the abundance of mRNA transcripts are to be measured and the co~es~nd~g cDNA clones isolated.

INTRODUCTION

One of the useful aspects of cDNA cloning technology is that it allows us to focus attention on mRNA species of low abundance. About 30% of the mRNA in a cell consists of sequences represented only 15 times per cell, each constitu~g < 0.01% of the mRNA (Williams, 1981). It is this class of transcripts that is the most interesting from the point of *To whom all correspondence

should be addressed.

Abbreviations: AMV, avian myeloblastosis virus; bp, base pair; Cot, product of DNA concentration in mol of nucleotide per litre and time in s; dNT.Ps, deoxyribonucleoside triphosphates; kb, kilobases or kilobase pairs; L, Luria; nt, nucleotides; pfu, plaque-fo~~g unit; RF, replicative form; Ret, product of RNA con~ntration in mol of nucleotide per litre and time in s; SDS, sodium d~e~Isu~ate; ss, single-stranded; u, units. 0378-l 119/85/$03.30 0 1985 Eisevier Science Publishers

view of cell regulation. Messenger RNA species responsible for the rapid adaptation of a cell to changing conditions may be short-lived and therefore rare. Several laboratories have analyzed mRNA sequences on the basis of their increased presence in one particular type of cell, or in cells undergoing a particular physiological or developmental process. These include SV40-transformed cells (Williams et al., 1977), growing cells (Williams and Penman, 1975), and difTerentiating cells (Blumberg and Lodish, 1980; Timberlake, 1980). Differential screening of cDNA library colonies with 32P-labelled cDNA (Crampton et al., 1980) provides information about clones corresponding to mRNA species representing > 0.1% of the total mRNA (Williams, 198 1; Scott et al., 1983); however, various enrichment procedures before or after generation of a cDNA

46

library have allowed the isolation of sequences comprising O.Ol-0.1% (Scott et al., 1983) and 0.001% (Hedrick et al., 1984) of mammalian mRNA. To extend the limits of sensitivity of these techniques, we have constructed cDNA vectors with novel properties. They allow the cloning of cDNA so that either the sense or the anti-sense strand of the cDNA insert in the plasmid is packaged into phage particles. This provides the large amount of ss DNA required to drive the hyb~d~ation reaction necessary to identify specific clones. F~e~ore, the presence of cDNA sequences in ss DNA makes possible its straightforward use both for hybridization and for transformation.

MATERIALS AND METHODS

(a) Cells, DNA-damaging treatments and preparation of ~i~A)+~A All poly(A)+ mRNA used to make the cDNA libraries was prepared from the WI-38 line of human diploid flbroblasts (Hayflick, 1965). Conditions of cell culture and treatment with DNA-damaging agents will be described elsewhere (J.K. and D.T.D., in preparation). Cells treated with various doses of each agent at various times before harvest were pooled. Total cytoplasmic RNA was prepared essentially as described by Goldenberg and Raskas (1979), and stored as a precipitate in 70% ethanol at -20°C. Poly(A)+ RNA was selected on columns of oligo(dT)-cellulose (Aviv and Leder, 1972) and was stored as a precipitate in 70 % ethanol at -20 ’ C. For preparation of the experimental (X) library, equal amounts of poly(A) + RNA from ultraviolet, N-methyl-N’-nitro-N-nitrosoguanidine, and bleomycintreated cells were combined and annealed to vectorprimer. For preparation of the normal (N) library, poly(A)+ RNA was prepared from WI-38 cells grown under culture conditions identical to that for the treated cells. (b) Piasmids, vector-primers, and linkers The plasmids used as vector-primers to construct the two cDNA libraries were derived from pBR322SV40 (map units 0.71-0.86) (Okayama and Berg,

1982). This plasmid was cleaved at the unique EcoRI site and ligated together with the puritied 1248-bp EcoRI fragment containing the fl intergenic region from pD4 (Dotto et al., 1981). After transformation of Escherichiu colz’RRl, the resultant recombinants were screened for the orientation of the fl fragment in the plasmid by restriction enzyme analysis. A BumHI site was inserted at the position of the 7% 1111 site by blunt-end ligation of a BumHI IO-bp linker to the product of T&l111 digestion treated with large fragment of DNA polymerase I. M~tiple insertions of linker were selected against by extensive BumHI digestion and membrane dialysis of the vector prior to re-ligation at low DNA concentration and transformation into E. coli RRl. The resultant plasmid was designated pSS24 (see Fig. 1). The plasmid pSS25 was derived from pSS24 by removing most of the SV40-derived sequences. This was achieved by cutting pSS24 with HindIII, blunting the ends with T4 DNA polymerase, and treating with bacterial alkaline phosphatase. This DNA was then ligated with a phospho~lated IO-bp K@zI linker, cleaved with KpnI, m-ligated at low DNA concentration, and transformed into E. co& RRl. Transformants were screened for plasmid DNA of the appropriate size. The preparation of vector-primers and linkers was modelled on Okayama and Berg (1982). Linker DNA fragments were prepared from two different plasmids, depending on the orientation in which the cDNA was being cloned. When pSS25 was used as the vector-primer, the linker was derived from the 180-bp fragment of pSS24 or pSS25 between the KpnI site and the BumHI site (see Fig. 1). When pSS24 was used as the vector-primer, the linker was derived from pBR322SV40 (map units 0.19-0.32) as previously described (Okayama and Berg, 1982). The plasmids pMN and pMX were produced by circularizing vector and linker without the introduction of a cDNA insert; these plasmids provide minimal-size markers for the libraries. Vector DNA was cleaved with KpnI and tailed with 15-30 dC residues in a terminal transferase reaction as described below. It was then cut with the appropriate restriction enzyme to remove one dC homopolymer tail (see Fig. 2) and annealed and ligated witb linker under the conditions described above. This was used directly to transform R4 (see section d below).

47

(c) Preparation of cDNA libraries

Restriction enzymes were from either Bethesda Research Laboratories, Boehringer-Mannheim, or New England Biolabs. Terminal transferase was from either New England Nuclear or BoehringerMannheim. E. coli DNA ligase and RNase H were obtained from P.L. Biochemicals, and DNA polymerase I was from Boehringer-Mannheim. AMV reverse transcriptase was purchased from Life Sciences Inc. Poly(A) + RNA (16 pg in 16 ~1 H,O) was treated with 1.5 ~1 100 mM CH,HgOH for 10 min at room temperature, followed by 1.5 ~1 of 1 M dithiothreitol for 5 min at 0’ C. Vector-primer (4 pmol) and H,O were added to bring the volume to 60 ~1. To this was added 60 ~1 of a solution of 100 mM Tris * HCl (pH 8.3 at 42”C), 20 mM MgCl,, 4 mM each of dATP, dGTP, dCTP, dTTP, 60mM KC1 and 1 &i/p1 [ 3H]dTTP. AMV reverse transcriptase was added (120 units) and the reaction was incubated 15 min at 42’ C. Aliquots were removed for determination of acid-insoluble radioactivity before addition of enzyme and every 5 min thereafter. The reaction was terminated by the addition of EDTA to 25 mM and SDS to 0.1% . It was extracted once with water-saturated phenol-chloroform (1: l), several times with butanol to reduce the volume to about 75 ~1, and three times with ether. It was then dialyzed overnight at 4’ C against 10 mM Tris * HCl (pH 7.9) and 1 mM EDTA on a Millipore Type VM filter. This step reduced the high level of nucleoside triphosphates to a level suitable for the subsequent homopolymer tailing. Agarose gel analysis of the product of a similar reverse transcription with unlabeled nucleotides indicated that essentially all of the 3H-labeled vector-primer DNA had been annealed to the excess RNA and extended in the reaction. The DNA was then mixed with an equal volume of a solution of 240 mM K * cacodylate, 60 mM Tris. HCl (pH 6.9) 125 PM dCTP, and 4 mM CoCl, to give 25-50 pmol/ml of 3’ ends. Poly(A) (1 pg) was added, and the sample was heated at 65°C for 3 min and at 37°C for 5 min. Terminal transferase was added to 1250 units/ml, and incubation was carried out at 37°C for a predetermined length of time until about 15-30 nt had been added per 3’ end. The reaction was terminated, the DNA was extracted as described above for reverse tran-

scription, and it was precipitated overnight at -70 ’ C by the addition of NH, * acetate to 2 M and 2.5 vol. ethanol. The DNA was centrifuged at 15 000 x g for 10 min, washed once with 70% ethanol, and dissolved in 75 ~1 of the appropriate restriction buffer. It was then digested with Hind111 or BumHI (30-40 units/pm01 of vector-primer) for 60 min at 37 ’ C. Cleavage was monitored by agarose gel electrophoresis after digestion of the product with PstI, (to detach the heterogeneous cDNA : mRNA portion from the vector; see Fig. 2). DNA was extracted and precipitated as described above, redissolved and quantified via the radioactivity. Recovery of vector-primer through all the above steps was 35-50x * The vector-primer mRNA : cDNA with dC residues at one end and a 4-base 5’ overhang at the other end was annealed with linker as described by Okayama and Berg (1982). RNA was replaced with DNA in an RNase H-driven nick-translation reaction. The sample was adjusted to 50 PM dNTPs, 0.15 mM NAD, 6 pg/ml E. coli DNA ligase, 5 u/ml RNase H, and 75 u/ml DNA polymerase I. Incubation was at 15°C for 1 h, at room temperature for 1 h, and at 65°C for 15 min (Gubler and Hoffman, 1983). The mixture was then used directly for transformation. (d) Transformation

of E. coli and library formation

Both libraries were generated in the R4 derivative of RR1 by the procedure of Hanahan (1983). R4 was constructed from the strain RR1 (Bolivar et al., 1977) by mating with FPL (Bukhari and Metlay, 1973) and selection for male F’pro + Zuc+ transconjugants on minimal glucose plates containing streptomycin and leucine (our strain of RR1 required both proline and leucine for growth on minimal medium). Several independent annealings and transformations were performed to produce each cDNA library. The ratio of recombinant DNA to cells was varied to obtain the optimal number of transformants. Generally, a 50 ~1 annealing reaction was employed, resulting in 500 ~1 after ligation and replacement of RNA. A 40-~1 aliquot of this was added to each of eight standard transformations. Each of these contained 2 ml of cells in transformation buffer derived from 25 ml of culture at 4 x lo7 cells/ml (Ham&an, 1983). This was spread onto two dry 150 mm L-agar plates

48

cont~~g 100 rug/ml c~b~ni~~. A total of 50 to 75 plates were used to generate each library. We purposely grew the transformants as independent colonies on plates rather than in mass liquid culture in order to reduce enrichment by faster growing transformants. Culture conditions here and in subsequent manipulations of the libraries were chosen to minimize the number of divisions that the cells underwent, so as to reduce alterations in the relative abundance of difIerent cDNA clones. After the tr~sfo~~t colonies reached a size of about 1 mm they were counted and scraped from the plates into L broth containing 100 @g/ml carbenicillin and 20% glycerol and stored at -70” C. Since the number of colonies per plate varied among transformations, the transformations were pooled and adjusted to a concentration of lo3 colonies/ml to preserve the relative abundance of mRNA species. (e) Preparation of phage and plasmid DNA from the libraries For the preparation of phage DNA the glycerol stock of the library was inoculated at 10 - 3 dilution into L-broth at 37°C containing 100 ,&nl carbenicillin When the culture reached 5 x 10’ cells/ml (about 10 divisions) it was infected with the IRl mutant of fl phage (Dotto et al., 1981) at lo-20 pfu/cell and grown for 4 h. The supernatant was prepared and the phage DNA purified as described by Zinder and Boeke (1982). We routinely obtained 2gg ss DNA per ml of culture. Phage [ 32P]DNA was prepared by growing cells and phage in medium containing dephosphorylated Casamino acids and Tryptone (Difco) in the presence of [ 32P]orthophosphate at 50 pCi/ml. Plasmid DNA was prepared by standard procedures from cells grown to a density of 5 x lO’/ml. (f) Hybridization and Sl assay Single-stranded DNA was prepared from either the pXcDNA library or the pNcDNA library. Phage DNA was similarly prepared from R4 infected with fl. The DNA was then adjusted to the following conditions for hybridization. All samples contained 0.3 M NaCI, 0.03 M Na, *citrate, pH 7, and 2 mg/ml unlabelled sonically sheared ss pNcDNA + fl DNA. The [32P]DNA was either fl

5 x 105 cpm/pg) or ss pXcDNA + fl DNA (10 pg/ml, 2 x lo5 cpm/pg). Ohgomers dT,,_,, and dC,2_,8 were included at 600 pg/ml and 80 pg/ml, respectively. This is a 20-fold excess over the amount of dA and dG present in the hybridization in the pNcDNA, based on the assumption that dA tracts average 150 nucleotides and dG tracts 20 nucleotides in length. Samples were sealed in 50-~1capillary pipettes, heated at 100°C for 3 min, and incubated at 65 “C for 32 h to a C,t of 700. After hybridization, two equal portions (10% each) were removed; S 1 nuclease was added to one in an amount previously determined to just produce a limit digest in 30 min at 37°C. Both aliquots were incubated and the acid-insoluble radioactivity was determined. The remainder of each hybridization was then digested with Sl nuclease, extracted once with phenol-chloroform and precipitated together with carrier tRNA by 2 ~01s. of ethanol. Acidsoluble Sl digestion products were removed by two precipitations with NH, * acetate and ethanol (Okayama and Berg, 1982). The Sl-resistant DNA was then electrophoresed in a 5% polyacrylamide gel and detected by autoradiography.

DNA

(10pug/d,

RESULTS

(a) Const~ctiw of vectors and libraries The Okayama-Berg vector pBR322-SV40 (0.71-0.86) was modified (Fig. 1). The alterations were designed so as to make it possible to clone cDNA in either orientation and to allow the production of ss DNA in vivo by use of a helper phage. Note the symmetric distribution of sites: a single site resulting in 3’ overhangs (QnI) is bounded by two sites generating blunt ends (HpaI and PvaII), which in turn are bounded by two sites generating 5’ overhangs (Hind111 and BarnHI). Just as the Okayama and Berg (1982) procedure utilized the sites K&rI, liipa1 and Hind111 to clone cDNA in one orientation, the @zI, PvuII and BarnHI sites can be used analogously to clone cDNA in the opposite orientation (see below). The second alteration consisted of insertion of an EcoRI fragment carrying the functional origin of fl phage replication into the EcoRI site of the plasmid.

49

phagc 11

intwawsic

Fig. 1. cDNA cloning vector. The prototype cloning vector, pSS24, consists ofportions of pBR322(open segmentand AmpR gene), SV40 and fl phage DNA. See MATERIALS AND METHODS, section b, for details.

Plasmids that carry the ColEl origin of replication, as well as this segment of fl DNA, are capable of two modes of replication (Dotto et al., 1981). Normally, replication originates from the ColEl origin and gives rise to double-stranded progeny DNA that is maintained within the cell at a copy number dictated by the plasmid replication genes. However, when the cell is infected with fl phage, phage gene products recognize the fl origin of replication and initiate a single-stranded mode of DNA synthesis. This gives rise to large numbers of progeny plasmid ‘plus’ strands that are packaged into ‘virus’ particles. These can be visualized on a gel, since the plasmid DNA is generally smaller than the 6.4-kb fl viral DNA that is also produced and packaged in similar numbers. Since fl is a filamentous phage, singlestranded circles of any size can be encapsidated. We tested our constructs for this property and found that plasmids carrying the EcoRI fragment in one orientation gave rise to more ss DNA than those with the opposite orientation (not shown). The plasmid producing more ss DNA was designated pSS24. The strategy for construction of the two cDNA libraries is diagrammed in Fig. 2. For the pXcDNA library [ poly(A) + mRNA from DNA-damaged WI-38 cells] the strategy was identical to that described by Okayama and Berg (1982) in that it utilized the same restriction cleavage sites of the

vector (pSS24) and the same SV40 fragment as a linker. To implement the strategy for the construction of the pNcDNA library it was necessary to modify the vector further. Numerous attempts to generate transformants in this orientation in pSS24 resulted in very low efficiencies of transformation. We reasoned that any sequences present in the pNcDNA construct but absent in the Okayama-Berg construct could be responsible for poor maintenance of this library in E. coli. Consequently we constructed a plasmid, pSS25, in which we deleted from pSS24 the SV40 region from KpnI to HindIII, but retained the KpnI site (see Fig. 1). We also used as a linker the pBR322 fragment originally present in the cloning vector. This last modification is particularly important to avoid interference with the modulation of activity of the neighbouring ColEl origin of DNA replication. The efficiency of the cloning is summarized in Table I. The efficiency relative to RNA is about 35-60x of that reported by Okayama and Berg (1982) for this procedure. The efficiency of transformation by the recombinant DNA is only 40-400fold lower than that of supercoiled form I plasmid DNA. The linker dependence is high, indicating an efficient and specific circularization of the vector by the linker. Our pNcDNA and pXcDNA libraries consist of 4.4 x lo5 and 2.5 x lo5 clones, respectively. It has been calculated (Williams, 1981) that a cDNA library of 1.7 x lo5 clones has a 99% chance of including mRNAs represented in only 15 copies per cell. We believe that our cDNA libraries are reasonably complete. (b) Characterization

of the libraries

Plasmid and phage DNA produced from the libraries was characterized by electrophoresis on agarose gels. Plasmid DNA was prepared from the two libraries and from the two marker plasmids (which represent the equivalent of vectors without inserts (see MATERIALS AND METHODS, section b) and cleaved with CM to prepare linear DNA that is amenable to size analysis. The electrophoretic analysis of the CM-cleaved DNA is presented in Fig. 3a. Both libraries show clear evidence of cDNA inserts in the cloning vector. Estimation of the maximum size of inserts is complicated by the small proportion of the largest clones and by the fact that these

50

pSS24-

pSS25 - Kpnf - dT

KpnI - dT

Born

PvuIl

jf

I ompi

TTT

$TTT Pvu 11

HpoI Cut + Purity

J [YJlTfT

\ TTTB

Annrei Rovorre

X-u&NIL

potyA*mRNA Tronroriba

N - mRNA

c

t

I::;

“**“**‘*-

z’z.--7

;;;

‘SJ

dC Toil

Anneot Ligot*

RoploteRNA

%OMfFicellr

t cGGG

Linkrr Hind

m

Cut

t

Purify

t

dG Toil

t PItI

Barn HI

t

cut

t pBR322(SV40

l

l9ObP

Kpnf

t 0.19-0.32)

DSS24

Or pSS75

Fig. 2. Cloning strategy for generation of two cDNA libraries with complementary inserts. The Kpni-cleaved, oligo(dT)-tailed vectors are diagrammed at the top (not to scale). The plus strand of the fl intergenic region (fl ori) is indicated by a thick line. N-mRNA and X-mRNA denote ~oly(A)+mRNA preparations derived from normal (untreated) and experimental (treated) cultures of human cells, respectively. The cloning steps follow the procedure of Okayama and Berg (1982). Sense strand: dotted tine; anti-sense strand: dashed line.

“quick” plasmid preparations are contaminated by traces of E. coli DNA. However, since a restriction enzyme was used to linearize the DNA, cleavages within large inserts would lead to an underestimate of the maximum size of inserts even in a highly purified preparation. Lane 4 of Fig. 3a also reveals that a proportion of the clones in the library of normal cDNA consist of deletions.

Another estimation of the sizes of the inserts can be obtained from an analysis of the size of the ss plasmid DNA that is encapsidated upon infection with fl phage. DNA was purified from phage particles present in supernatants of cells infected with f 1, and analyzed on the agarose gel shown in Fig. 3b. Two bands are visible in each lane. The slower twang band is the ss circular DNA of fl(6.4 kb).

51 TABLE I cDNA cloning efficiencies Library

Treatment Vector Linker Polarity of insert Amount of poIy(A) + RNA Number of inde~ndent transfo~~ts Linker dependence” Cloning efficiencyb per pg poly(A) + RNA per pg vector Transformation et?iciencyC

N (normal)

X (experimental)

none pss25 180 bp (pSS25) sense

UV, MNNG, bleomycin pSS24 272 bp (SV40) anti-sense

7.5 Pg 4.4 x lo5 50x

7-I Pg 2.5 x 105 30x-75x

6 x 104 8 x lo5 0.3-1.6 x lo*

3.5 x 104 1.8 x lo5 3-l x 107

* Linker dependence was calculated by comparing transformants generated by DNA annealed in the presence or absence of the linker. b Cloning efficiency is expressed per pg of starting RNA or per pg of vector annealed to linker. a The range of efficiencies, calculated per pg of the supercoiled standard [pBR322/SV40 (0+71-0.86)] which was used to monitor each transformation.

al I

b) 2345

6

I

2

3

4

5

6

6.6. 4.7.

if:;: 22-

64 56 .39 ‘36

The faster migrating band is that of the plasmid present within the infected cell. There is no evidence of deletions in the pNcDNA library that was seen in Fig. 3a, lane 4, suggesting that those deletions that are present in pNcDNA extend into the fl origin fragment and render it inactive as an origin of ss DNA synthesis. Inserts in the experimental library extend up to at least 3 kb in size, a size range that includes some 90% of the cellular poly(A) + RNA (Bishop et al., 1974). Fig. 3b also illustrates the effect of growing the libraries to saturation. The DNA in lane 5 was obtained from cells infected with fl after tihd

(See

AND

Onij’

Fig. 3. Electrophoretic analysis of plasmid and ss DNA constituting the two libraries. (a) Agarose gel electropherogram of C&I-cleaved plasmid DNA. Lane 1: Hind111 digest of A DNA. Lane 2: pMX (pSS24 plus 272-bp linker circularized without a cDNA insert). Lane 3: pXcDNA. Lane 4: pNcDNA. Lane 5: pMN (pSS25 plus 180-bp linker circularized without a cDNA insert). Lane 6: HineD digest of phage 1. DNA. Fragment sizes are indicated on the left in kb. (b) Agarose gel electrophero~~ of DNA purified from supernatants of plasmid-bearing cells infected with fl phage. Lane 1: pD4 (as a size marker). Lane 2: pMX. Lane 3: pXcDNA. Lane 4: pNcDNA (second passage of library; see RESULTS section b. Lane 5: pNcDNA (first passage of library). Lane 6: pMN. Sizes are indicated to the right in kb.

sectiond). DNA obtained from cells infected with fl after growth to saturation (lane 4) shows a decreased amount of plasmid DNA relative to fl DNA. The strategy by which we constructed our two cDNA libraries (Fig. 2) predicts that the cDNA inserts will be complementary when one analyzes the ss DNA that results from fl infection. The following experiment illustrates this. S~~e-s~~d~ 32Plabelled DNA was prepared from the pXcDNA library infected with fl and from R4 infected with fl as a control. Unlabelled ss DNA was prepared similarly from pNcDNA. The unlabelled DNA (pNcDNA + fl) was used at a 200-fold excess to drive the hybridization of complementary sequences METHODS,

growth

MATERIALS

0.9.

52

TABLE II

Hybridization of single-stranded N and X cDNA libraries 3ZP-labelled phage or 32P-labelled phage plus ss plasmid DNA from the X library was hybridized with a 200-fold excess of unlabelled phage-plus-plasmid DNA from the N library (“driver”) to a Cct of 700 as described in MATERIALS AND METHODS, section f. Aliquots were digested with S 1 nuclease, and the percentage of resistant acid-precipitable radioactivity was determined. Sample 1 was not incubated under hyb~dization conditions. Sample 4 contained a 20-fold excess of dT,Z_,s and dC,,_,, over the expected mount of poly(dA) and poly(dG) in the pNcDNA ss driver DNA. Sample No.

Labelled DNA

Input

1 2 3 4

fl (100°C + OOC) fl fl + pXcDNA fl + pXcDNA

1.0 x 2.3 x 1.0 x 1.0 x

oligo( dC) + oligo(dT)

cpm

lo5 10’ lo* 10s

in the two DNAs (ss pNcDNA and ss pXcDNA). The extent of hybridization was estimated by measuring S 1 nuclease resistance and by visualizing the resistant DNA on a polyacrylamide gel. Table II shows the quantification of S 1 resistance. The background of Sl resistance in fl DNA (samples 1 and 2) is about 3.5%. Since this level of resistance is present in both hybridized and unhybridized samples, we assume that it is the result of a few set-complements sequences in fl DNA. Hyb~dization of pXcDNA under these conditions (sample 3) increases the level of Sl resistance by about 5 %. This increased S 1 resistance is not caused by the poly(dT) and poly(dC) tracts in pXcDNA that are complementary to poly(dA) and poly(dG) tracts, respectively, in pNcDNA (see Fig. 2), because inclusion of dT,,_,, and dC,,_,, at a 20-fold excess over that of poly(dA) and poly(dG) in the DNA driving the hybridization (i.e., at a 4000-fold excess over that of the same sequence in the labelled DNA) did not reduce the level of Sl resistance. These results indicate that most of the Sl resistance observed is due to complementarity between the cloned inserts. The remainder of samples 2 to 4 was digested with S 1, pnrilled, and analyzed on a 5 % polyacrylamide gel. The autoradiogram of this gel is shown in Fig. 4. Two broad regions of S l-resistant DNA are observed in the samples containing pXcDNA and are absent in the sample containing the fl DNA alone. These extend from about 500 to 200 bp and from about 150 to 100 bp. The maximum size of S l-resistant hybrids is about 500 bp. This reflects the fact that

_ +

%Sl resistance

3.2 3.6 8.7 8.4

the pNcDNA driving the reaction had been sonicated. We conclude that the two libraries have antiwells

2527

245 164

100

Fig. 4. Polyacrylamide gel electrophoresis of S I nuclease-resistant hybrids. The products of Sl digestion of samples 2 to 4, Table II, were purified by electrophoresis in a 5% polyacrylamide gel and autoradiographed. Each lane contains labelled S l-resistant DNA remaining from equal amounts ofinitial DNA. The positions of unlabelled duplex markers (&&II digest of M13mp8 RF DNA), together with their sizes in bp, are indicated to the left. Lane A: sample 2. Lane B: sample 3. Lane C: sample 4.

53

parallel and complementary inserts, as predicted by the cloning strategy (Fig. 2). (c) Applications The av~ab~ty of cDNA cloned in the plasmids we have described has general app~cabi~ty. It will greatly simplify and enhance the sensitivity of the selection of clones of mRNA specifc for a type or a physiological state of mammalian cells. Such procedures are dependent upon preparative nucleic acid hybridization to very high values of R& (Williams and Penman, 1975; Wiiams et al., 1977; Timberlake, 1980; Scott et al., 1983; Hedrick et al., 1984). This allows the isolation of a cDNA fraction from which the majority of sequences common to both control and expe~ent~ cells have been removed, thereby enriching for those sequences present specifically in the cells under study. Further enrichment is then achieved by annealing the cDNA to mRNA from the cells under investigation and selecting the cDNA that forms hybrids. This approach has one serious drawback: both elements in the hybridization (cDNA and mRNA) are not amplified. As a result, the sensitivity of the overall procedure is low, because the low abundance class of transcripts is lost. The vectors we describe here avoid these shortcomings. Both elements in the hybridization are amplifiable. The normal cell mRNA can be replaced by ss pNcDNA, which is amplifiable and easily prepared in large amounts. The experimental cell cDNA is replaced by ss pXcDNA. After each hybridization to pNcDNA the nonhybridizing pXcDNA can be rendered duplex and used to transform E. coti to generate a sublibrary of cDNA sequences enriched for sequences present specifically in treated cells (unpub~sh~ results). One of the disadvantages of using this vector with cloned anti-sense cDNA sequences instead of cDNA itself is that it cannot readily be used to probe colonies or phage plaques, since it contains vector sequences. However, since the selected sequences are present in a vector with transforming ability, screening of colonies or plaques should be unnecessary. If it is necessary to use pXcDNA as a probe, it is possible to label it to very high specific activity (IO’ cpm/pg) with [ Y-~~P]ATP and pol~ucl~tide kinase after controlled micrococcal nuclease diges-

tion (Chikaraishi et al., 1983). Such a single-stranded probe would be very efficient in positively identi&ing clones that do change in abundance. Large amounts of phage are easily prepared from single clones in our library. After labeling with 32P they could be used to screen pairs of RNA dot-blots or Northern transfers of normal and treated cell mRNA to ascertain whether a selected clone changes in abundance and, if so, by how much. These vectors offer many advantages over the present means of selecting cDNA clones corresponding to induced transcripts in mammalian cells. We are presently using them to detect and select low-abundance mRNA sequences present specifically in cells treated with DNA-damaging agents and in cells treated with tumour promoters.

ACKNOWLEDGEMENTS

We thank Martha Hohnan for skillfully maintaining cultures of WI-38 cells, performing the UV and drug treatments, and preparing poly(A) + mRNA. We thank Craig Parfett for help in cons~ct~g pSS24, Hiroto Okayama, Ken Horiuchi and George Chaconas for gifts of bacteria, plasmids and phage, and Dale Marsh and Linda Bonis for typing, graphics and photography. J.H.S. was supported by a Terry Fox Training Grant Award from the NCI. This work was supported by the Medical Research Council of Canada and the National Cancer Institute of Canada. We also thank NEN (Canada) and the Nelson Arthur Hyland Foundation for support.

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