Proc. NatL Acad. Sci. USAVol. 78, No. 3, pp. 1527-1531, March 1981Biochemistry

Transformation of mouse fibroblasts to methotrexate resistance bya recombinant plasmid expressing a prokaryotic dihydrofolatereductase

(eukaryotic expression vector/dominant marker/gene transfer)

K. O'HARE, C. BENOIST, AND R. BREATHNACH*Laboratoire de Ge'netique Moleculaire des Eucarvotes du Centre National de la Recherche Scientifique, Unit6 184 de Biologie Mol6culaire et de GCnie G6n6tiquede l'Institut National de la Sante et de la Recherche MWdicale, Institut de Chimie Biologique, Facult6 de MWdecine, Strasbourg 67085 C6dex-France

Communicated by A. Frey-Wyssling, December 1, 1980

ABSTRACT A recombinant plasmid has been constructed forthe expression of inserted DNA sequences coding for polypeptidechains using the simian virus 40 early promoter and splicing andpolyadenylylation signals from the rabbit f-globin gene. The cod-ing regions for two prokaryotic methotrexate-resistant dihydro-folate reductases were introduced into the expression vector.When mouse fibroblasts were exposed to these recombinant plas-mids, it was possible to select methotrexate-resistant clones thathad integrated the plasmids and produced a chimeric RNA codingfor the prokaryotic enzyme.

The development of systems both in vivo and in vitro in whichgenes are faithfully expressed is one of the major aims of currentresearch in eukaryotic molecular biology. While methods havebeen described for the transfer of DNA into cells in culture (1,2), the proportion of cells that take up the foreign DNA is sosmall that a selection procedure is required in order to obtainsufficient material to analyze expression of the foreign DNAsequences. Much of the pioneer work in this direction has usedthe thymidine kinase (TK) gene from herpes simplex virus asa selective marker, but this could only be used with recipientTK- cell lines (3-5). A dominant selective marker that couldbe used with any type of cell would represent a considerableadvance because most of the cell lines into which it would beinteresting to transfer genes (e.g., steroid hormone-responsivechicken genes into a hormone-sensitive cell line) are not avail-able as TK- derivatives.

By using total chromosomal DNA from hamster cell lines thatproduce a methotrexate (Mtx)-resistant dihydrofolate reductase(H2folate reductase) it has been possible to select for Mtx re-sistance as a dominant marker in DNA transfer experiments(6, 7). A cloned marker that could be propagated in largeamounts in Escherichia coli would be convenient, but becausethe cellular H2folate reductase gene is at least 40 kilobases (kb)long (8) it is inappropriate for this purpose. An alternative wouldbe to construct a vector in which a cDNA for a Mtx-resistantenzyme would be expressed in cells in culture, much as hasbeen done for f-globin cDNAs (9, 10), but as yet only the cDNAfor a Mtx-sensitive enzyme has been cloned (11). It should benoted that an increased copy number of genes for Mtx-sensitiveenzymes also leads to Mtx resistance (12), so that elevatedexpression of the Mtx-sensitive cDNA in a suitable vector couldbe sufficient for use as a dominant marker (see Discussion).An alternative source of DNA sequences coding for Mtx-re-

sistant H2folate reductases is available, namely prokaryotic Rfactors (13), so we decided to construct recombinants in which

the prokaryotic enzymes might be expressed after introductioninto cells in culture. We report herein the construction of aeukaryotic expression vector designed to express inserted DNAfragments and its use for production of the prokaryotic H2folatereductases in mouse fibroblasts, leading to their transformationto Mtx resistance.

MATERIALS AND METHODSPlasmids pFE364 and pFE373 (14) were gifts from L. Elwell.Plasmid Z- pCRI/Rchr ,/-G-I (15) was a gift from C. Weissmanand his colleagues. RNA-directed DNA polymerase (reversetranscriptase) from avian myeloblastosis virus was a gift fromJ. Beard. Sources of other enzymes were as described (16).Simian virus 40 (SV40) DNA was prepared from CV-1 monkeycells, propagated and infected as described (17). Chicken ovi-duct RNA was a gift from M. LeMeur.

Procedures for construction of recombinant DNA plasmids,transfection of bacteria, and preparation and sequence deter-mination of plasmid DNA were as described (16, 18). MouseLMTK- cells were grown in monolayer culture and transformedwith plasmid DNA by using the calcium phosphate coprecipi-tation technique essentially as described (19). Selection withmedium containing Mtx (Sigma) at 0.4 jLM was begun 24 hr aftertransfection, and clones were subsequently grown in Mtx-con-taining medium.The analysis of DNA of mouse cell clones by blotting was as

described (20). Cytoplasmic RNA was isolated from cells by lysisin Nonidet P40, pelleting of the nuclei, and phenol/chloro-form extraction of the supernatant (21). Electrophoresis of RNAon formaldehyde/agarose gels, transfer to diazobenzyloxymeth-yl-paper (DBM-paper), and hybridization to probes was ac-cording to published procedures (22, 23).

Procedures for DNA labeling by nick-translation and 5'-endlabeling with [y-32P]ATP and phage T4 polynucleotide kinasewere as in refs. 16 and 20. For determining the sequence ofRNA from the clone HG33, a primer (see text) was preparedas described (16), using a "thin gel" (24). It was then hybridizedto cytoplasmic poly(A)-RNA (50 ,ug) from clone HG33 and ex-tended with reverse transcriptase exactly as detailed in ref. 21.The sequence of the resulting cDNA was determined by theMaxam and Gilbert procedure (25), using a "thin" 8% acrylam-ide/7 M urea gel (24). For determination of the location ofthe 5' end of clone HG33 RNA, the coding strand of a HindIII/

Abbreviations: kb, kilobase(s); bp, base pair(s); TK, thymidine kinase;H2folate reductase, dihydrofolate reductase; Mtx, methotrexate; m.u.,map unit; SV40, simian virus 40; DBM-paper, diazobenzyloxymethyl-paper.* To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

1527

1528 Biochemistry: O'Hare et al.

Hpa II fragment of SV40 [0.725-0.648 map unit (m.u.)] end-labeled at its HindIII end was isolated on a strand-separationgel (25). This probe was hybridized to cytoplasmic RNA fromclone HG33 or from a rat cell line transformed by wild-typeSV40, and hybrids were treated with S1 nuclease exactly asdescribed in ref. 21. S1 nuclease-resistant hybrids were ana-lyzed on an 8% acrylamide/7 M urea thin gel.

Biohazards associated with these experiments were exam-ined by the French National Control Committee.

RESULTSConstruction of Expression Vector. The construction of the

expression vector pKCR is outlined in Fig. 1. Its main featuresare: (i) The SV40 early gene promoter region and nucleotidescoding for the 5'-untranslated region of the SV40 early mRNAs(Hpa II/HindIII fragment, 0.725-0.648 m.u.). There are noinitiation codons downstream from the SV40 early cap sites inthis region. (ii) A unique BamHI site into which sequences canbe inserted for expression under the control of the SV40 earlypromoter. (iii) Donor and acceptor splice sites from the rabbitP-globin gene. Extrapolation from experiments with anSV40-mouse f-globin recombinant (26) suggests that thesesites should be functional. (iv) A polyadenylylation site from therabbit A3-globin gene. (v) A polyadenylylation site from the SV40early region, lying downstream from the globin polyadenylyla-tion site (Hpa Ib/BamHI fragment, 0.169-0.144 m. u.). (vi) Anorigin of replication and ampicillin resistance gene frompBR322 (EcoRI/BamilI fragment).The construction of pKCR is rather convoluted because we

hoped initially to be able to use pBR2 as an expression vector.It contains the SV40 early promoter and polyadenylylation sig-

EcoR I HMndM

(fHpqp Tetj ,

Mp.Ue indII

EcoRI HindM EcoRT BamHImiI 1 EcoR!I

2poi! BamHI3Hi~ndlfl pSV4 X f(psDRin

2pol I3 Ligation with

lHpall Barn linker2poi1 4BamHIM3Hind 1BamHi

2 poll

pal TaqI 1 TaqI. BamHl

pa7tEari.i poll

EcoRI BamMI

% S

Hpal a

BamH paI b Hpala

BamHI EcoRI!9111 Pvull3i l b

BamHl+HpaIBamHI. Pvull

Rabbit globin ATGEcoRI BamMI

_T AA H~folatereductase EcoRI

Pst IpG/pMCG fragment pKCR

FIG. 1. Outline of construction of the expression vector pKCR andplasmids pHG and pMCG (see text). Amp and Tet indicate ampicillinand tetracycline genes, and pol I indicates repair of restriction enzyme5' protruding ends by E. coli DNA polymerase I. The numbers 1, 2, etc.indicate the order in which the operations involved in each step of theconstruction were performed. The map of SV40 shows only thatHindIIIsite used to generate the Hpa II/HindEI fragment (0.725-0.648 m.u.)inserted into pBR322 to generate pSV4. The SV40 sequences used inthe construction of the recombinant plasmids are shown as filled-inblocks, while rabbit 3-globin exons are shown as open blocks. Gener-ation of pHG and pMCG from pKCR was by insertion of H2folate re-

ductase fragments (Hae Ill/Alu I of pFE364 and Hinfl/Alu I ofpFE373, respectively, shown underlined in Fig. 2 a and b). The H2folatereductase fragment is shown as a hatched block, with initiation andtermination codons indicated.

0 lbp

Sau3AI Sau96O mHoe Sau3AI Hinf II I I

Alul

I ~~~~~~~~~~Ia) pFE364 G TAA

M E R S S N E V S N P V A G N F5 '. .. C1EACATA~jUCAACTAGGTTUCACAGAAGGTTCGGjUAAGGAACGAGTAGCAT6GAAGTCAGTAATCCAGTCGCTGGCAACTTT...

towdi Sou3AI Seu96l HinflHinfI Sou3AI HinfI Alul

ATG TAAb) pFE373

B6 K S S G E A N A P V A G Q F5' .. TlTbCTTGTGTCGCCATAATAGATTACAA GArTCGACATGGGTAAAAGTAGCGCGAAGCCAACGCTCCCGTTGCAGGGCAGTTT...

HInf I Hinfl

FIG. 2. Restriction enzyme maps of the regions of pFE364 (a) andpFE373 (b) containing the sequences coding for their H2folate reduc-tases. Above each map is given the strategy used for sequence deter-mination: Arrows extend from the sites used for end-labeling and in-dicate the extent of the sequence read. Below each map is given partof the sequence obtained, and its translation into an amino acid se-quence (the one-letter code is used). The positions of the protein ini-tiation codon as deduced from the sequence are marked on the map.The positions of the termination codons as deduced from the size of theprotein are also shown. the Haefll/Alu I fragment of pFE364 and theHinfl/Alu I fragment of pFE373 used for inserting into pKCR to gen-erate pHG and pMCG, respectively, are indicated by lines under themaps. A scale in base pairs is provided.

nals, as well as the splice sites corresponding to the t splice ofSV40 (Taq I/BamHI fragment 0.566-0.144 m.u.). Integrationof various fragments into the BamHI site of pBR2 did not leadto production of detectable levels of the corresponding RNAswhen the recombinant plasmids were introduced into mousefibroblasts. Because the same fragments, when introduced intopKCR, did produce high levels of RNA, we speculate that thefailure of pBR2 may be because the t splice is not occurring.

Localization of H2folate Reductase Genes. The plasmidspFE364 and pFE373 are based on pBR322 and contain insertsof 2700 and 2200 base pairs (bp), respectively (14), which codefor Mtx-resistant (greater than 5 mM) H2folate reductases ofprokaryotic origin (27). The enzyme coded for by pFE364 is atype II reductase and the sequence of its 78 amino acids hasbeen determined (28). The enzyme coded for by pFE373 is ofundetermined type. We converted the amino acid sequence ofthe type II enzyme coded for by pFE364 into a nucleotide se-quence and noted that the sequence could contain sites for therestriction enzymes Sau3AI and HinfI separated by 90 bp. Thesites for Sau3AI and HinfI were therefore mapped in the 2700-bp insert of pFE364. The map obtained did indeed show Hinfland Sau3AI sites corresponding to those predicted from theamino acid sequence of the pFE364 reductase (Fig. 2a). Wetherefore determined the sequence of this region and locatedthe nucleotide sequence coding for the first 60 amino acids ofthe protein (Fig. 2a), including the initiation codon. From thesize of the protein, the entire protein-coding sequence mustbe contained within the Hae III/Alu I fragment shown in Fig.2a. The reductase initiation codon is the first ATG triplet in thisfragment (Fig. 2a).The reductase coded for by pFE373 is about 80% homologous

in sequence to the pFE364 enzyme (14). Thus it seemed likelythat their genes would share some restriction sites. We there-fore mapped the restriction sites for Sau96I, Hinfl, and Sau3AIin the pFE373 insert. Comparison of the map obtained with thatof the pFE364 insert showed a region where the maps weresimilar (Fig. 2b). We determined the sequence of this regionand found a sequence of 90 bp that could code for part of a

Proc. Natl. Acad. Sci. USA 78 (1981)

Biochemistry:O'HareetaL~~~~~Proc.Nati. Acad. Sci. USA 78 (1981) 1529

protein very homologous to the pFE364 reductase (a small partof this sequence is shown in Fig. 2b), including a possible ini-tiation codon. If this is the initiation codon, then, judging fromthe size of the pFE373 reductase (about 80 amino acids), theentire protein coding sequences would be contained in theHinfI/Alu I fragment shown in Fig. 2b. The reductase initiationcodon would be the first ATG triplet in the fragment (Fig. 2b).We used the plasmids pKCR, pFE364, and pFE373 to con-

struct recombinant plasmids designed to express Mtx-resistantH~flate reductase genes. This was achieved by inserting the370-bp Hae III/Alu I fragment (underlined in Fig. 2a) ofpFE364 or the 270 bp HinfI/Alu I fragment (underlined in Fig.2b) of pFE373 into the unique BamHI site of pKCR to generatepHG and pMCG, respectively (Fig. 1). On introduction intoeukaryotic cells, these plasmids were expected to produce, un-der control of the SV40 early gene promoter, capped and poly-adenylylated transcripts that contained functional donor andacceptor splice sites from the rabbit /3-globin gene. These tran-scripts should be spliced by the cellular machinery [this maybe important, because Hamer and Leder (26) have shown thata splicing event can be important for the accumulation of cy-toplasmic RNA in infections of CV-1 cells with an SV40-mouse-,3-globin recombinant]. The spliced transcripts of pHG andpMCG should be exported to the cytoplasm, and being capped,should associate with ribosomes. According to the scanningmodel (29), the ribosome should scan down the messengersfrom the cap and initiate translation at the first AUG codonreached. For both pHG and pMCG, the first AUG codon of thecorresponding transcripts should be the initiation codon for theH~flate reductase whose entire coding sequence is carried bythe plasmid. Translation should then produce the appropriateenzyme. We tested these predictions by attempting to trans-form mouse fibroblasts to Mtx resistance by using pHG andpMCG.

FIG. 3. Analysis of DNA fromHGMtx-resistant mouse cell clones.DNA (20 y~g) from the clones was digested withEcoRl, electrophoresedon a 1.2% agarose gel, transferred to a nitrocellulose filter, and hy-bridizedto nick- translated plasmid pHG. Lanes C-E, DNA from clonesHG12, HG21, and HG33, respectively. Lane B, DNA from normalmouse fibroblasts. Lane A, EcoRI digest of pHG. The 1350-bp EcoRIfragment of pHG discussed in the text is indicated by an arrowhead.

A B CD

FIG. 4. Size analysis of RNA from clones HG12, HG21, and HG33.Cytoplasmic RNA (20 u.g) from the clones was electrophoresed on a1.5% agarose gel run in the presence of formaldehyde, transferred toDBM-paper, and hybridized with a nick-translated probe from a rabbit13-globin gene plasmid (the BamHI/Pvu II fragment used for integra-tion into pBR2; see Fig. 1). Lane A, lysozyme mRNA as size marker(580 nucleotides, see arrowhead). Lanes B-D, RNA from clones HG12,HG21, and HG33, respectively. The position of migration of other sizemarkers run on the gel (ovalbumin mRNA and 28S and 18S ribosomalRNAs) is not shown.

Transformation of Mouse Fibroblasts. Cultured mouse fi-broblasts were treated with pHG (linearized with Pst I) orpMCG, using the calcium phosphate coprecipitation technique.Selection of the treated cells in 0.4 AM Mtx led to the appear-ance of Mtx-resistant clones, clearly visible after 4 weeks. Nocolonies developed on the control plates. The efficiency oftransformation to Mtx resistance by pHG and pMCG was about1/10th (about 20 colonies per jig) relative to the efficiency weobtain for transformation to TK' by using a cloned herpes sim-plex virus TK gene (about 200 colonies per pig). Six independentpHG-induced clones were picked and taken for further analysis.The plating efficiency of these clones was not significantlychanged over Mtx concentrations ranging between 0.4 and 50A.M (higher concentrations were not tested).

Total DNA isolated from the six clones was digested withEcoRI, electrophoresed on an agarose gel, and transferred toa nitrocellulose filter. The filter was probed by using nick-trans-lated pHG. The DNA of all six clones contained sequences com-plementary to the probe. This is shown in Fig. 3 for three ofthe clones (lanes C-E). DNA from untreated cells showed nobands (lane B). The EcoRI patterns of the DNA from the clones(lanes C-E) and the DNA of the plasmid pHG (lane A) are con-sistent with random integration of the plasmid into the host-cellDNA. Each clone contains multiple copies of the pHG plasmid,a large proportion of which have an intact 1350-bp EcoRI frag-ment of pHG (arrowhead in Fig. 3, lane A) that contains all butthe 3' distal 270 bp of the region expected to be expressed inmRNA (see Fig. 1). Analysis of the DNA from three indepen-dent clones induced by pMCG led to similar conclusions (datanot shown).

Analysis of RNA of Transformed Clones. We found that thepHG sequences present in three Mtx-resistant clones used in

Biochemistry: O'Hare et al.

1530 Biochemistry: O'Hare et aL

the experiment shown in Fig. 3 (clones HG12, HG21, andHG33) are transcribed. Cytoplasmic RNA was isolated fromthese clones, electrophoresed on a denaturing agarose gel, andtransferred to DBM-paper. The blot was hybridized to a nick-translated BamHI/Pvu II fragment of the rabbit 3-globin gene(Fig. 1). A transcript of 650 ± 50 nucleotides was detected inRNA from all three clones (Fig. 4, lanes B-D) [the arrowheadbeside lane A indicates chicken lysozyme mRNA, 580 nucleo-tides long (30)]. Control RNA from mouse fibroblasts gave noband (not shown). The size of the transcripts corresponds to thatexpected if initiation of transcription uses the SV40 early gene

C Aa G T C C

rTCC

TCA

G A ........

__C

GC.

TT

A

G

CTC ...A

GC

Rabbit_13-gloinlDN

EXON INTRON EXON

5 ..GGATCCTGAGAACTTCAGG TGAGTTTGGG.....TTTCCTACAS TCCTGGGCAACGTGCTG. ..3'3 ..CCTAGGACTCTTGAAGTCC CTCAACCC....AAGGATGTt ZGGCCCGTTGCACGAC ..5'

HG33mnRNA/5 '. . GGAUCCUGAGAACUUCAG UCCUGGGCAACGLJGCUGG . .3'

FIG. 5. Determination of the splice point in clone HG33 mRNA.(a) Cytoplasmic RNA from clone HG33 was hybridized to an EcoRI/Bgl fragment (Fig. 1) of the rabbit fglobin gene, 5'-end-labeled atits Bgl II terminus. This primer was then extended with reverse tran-scriptase, and the sequence of the resulting 5'-end-labeled cDNA wasdetermined by chemical degradation (25). Cleavages used are shownabove individual tracks of the sequencing ladder. P indicates the po-sition of migration of unextended primer. In some positions (arrow-heads) bands appear in every track. This is probably caused by se-quence-specific stops of the reverse transcriptase during extension,because these bands were also observed when uncleaved cDNAwas runon a similar gel. The position of the splice point is marked by an arrow.Nucleotides forming the BamHI site are bracketed. (b) Part of the se-quence of the cDNA deduced from a is converted into the sequence ofthe RNA template (lower line), which is then compared with that ofthe regions around the f3-globin splice points (15) (upper line).

A B C D E

FIG. 6. Determination of the 5' ends of RNA from clone HG33.RNA from clone HG33 or an SV40-transformed rat cell line was hy-bridized with a HindIII/Hpa II fragment of SV40 (0.725-0.648 m.u)5'-end-labeled at its HindIII terminus. Hybrids were treated with S1nuclease and displayed on a sequencing gel next to cleavage productsof the HindIII/Hpa II fragment (cleavage used was A + C). Lane A,cytoplasmic RNA from clone HG33; lane B, cytoplasmic RNA from anSV40-transformed cell line; lane C, A + C cleavage of the HindIII/Hpa II fragment; lane D, poly(A)+RNA from clone HG33; lane E,poly(A)-RNA from clone HG33. All samples are from the same gel buta longer exposure of lanes B and C is shown.

promoter, the ,3-globin intron transcript is removed, and poly-adenylylation occurs at the f3-globin polyadenylylation site [60nucleotides of SV40 sequence, 370 nucleotides from the HaeIII/Alu I fragment carrying the reductase coding sequence(Figs. 1 and 2a), and 240 nucleotides of 3-globin transcript]. Wedo not detect any read-through across the rabbit 13-globin poly-adenylylation site; such a read-through could lead to a transcriptof about 1150 nucleotides terminating at the downstream po-lyadenylylation site from the SV40 early gene region (Fig. 1).

To prove that the rabbit f3-globin intron transcript had beencorrectly excised from the cytoplasmic RNAs detected in theHG clones, cDNA was synthesized from the HG33 cytoplasmicRNA, using as primer the 80-bp Bgl II/EcoRI fragment of therabbit 83-globin gene (Fig. 1), 5'-end-labeled at the Bgl II site.Both sites map in the third exon of the globin gene, the EcoRIsite lying 78 bp from the intron-exon junction. Hybridizationof the primer to RNA from clone HG33 followed by extensionwith reverse transcriptase produced end-labeled cDNA, whosesequence was determined by the technique of Maxam and Gil-bert (25) (Fig. 5). The sequence obtained (Fig. 5a) is comple-mentary to that of the RNA template. We show in Fig. 5b thesequence of the RNA template itself, deduced from Fig. 5a.This sequence diverges from that of the chromosomal /3-globingene at the intron-exon junction but corresponds exactly to thatexpected if the f3-globin splice has occurred correctly (see Fig.5b).

Proc. Natl. Acad. Sci. USA 78 (1981)

Proc. Natl. Acad. Sci. USA 78 (1981) 1531

We established that the 5' ends of the RNA present in cloneHG33 are produced by the initiation of transcription at the SV40early promoter by an SI nuclease mapping experiment. Theprobe used was the HindIII/Hpa II fragment of SV40, 5'-end-labeled at the HindIII site (0.648 m.u.), which lies 55-60 nu-cleotides downstream from the major cap sites for SV40 earlymRNAs (31). As can be seen in Fig. 6, identical patterns wereobtained upon S 1 nuclease digestion of hybrids formed betweenthis probe and HG33 RNA (lane A) or RNA from a SV40-trans-formed rat cell line (lane B).A sample of RNA from clone HG33 was separated into

poly(A)-RNA and RNA not containing poly(A) by oligo(dT) cel-lulose chromatography and tested as above. The results (Fig.6, lanes D and E, respectively) show that more than 90% of thetranscript is polyadenylylated.

DISCUSSIONWe have constructed recombinant plasmids based on a eukary-otic expression vector (pKCR, Fig. 1) that transform mouse fi-broblasts to Mtx resistance. These plasmids consist of the SV40early gene promoter and cap sites, sequences coding for Mtx-resistant H2folate reductases of prokaryotic origin, with donorand acceptor splice sites and a polyadenylylation site from therabbit f-globin gene. That the plasmids pHG and pMCG trans-form the cells harboring them to Mtx resistance is strong evi-dence that the transcripts are being translated correctly. Wehave analyzed DNA and-RNA from isolated Mtx-resistant clonesobtained by using one of these plasmids (pHG, Fig. 1). Theclones contain copies of the plasmid DNA, probably integratedinto the host chromosome, and transcripts complementary tothe globin sequences of pHG. These transcripts initiate undercontrol of the SV40 early gene-promoter and have been splicedproperly at the tglobin gene splice junctions. Judging fromtheir size, they contain a complete transcript of the sequencescoding for the prokaryotic H2folate reductase, and they ter-minate at or close to the B-globin polyadenylylation site. Thetranscripts are mostly polyadenylylated.The success of the expression vector pKCR in expressing a

gene of prokaryotic origin suggests that it could be used to ob-tain production of any protein for which a suitable coding se-quence of DNA was available. In fact, we have recently suc-ceeded in transforming mouse fibroblasts to Mtx resistance byusing a Mtx-sensitive mouse H2folate reductase expressed inpKCR. However, when -ovalbumin coding sequences are in-serted in pKCR, and the recombinant is introduced into mousefibroblasts, the level of transcription of the ovalbumin se-quences is very low. This could be because the ovalbumin tran-script is very unstable in this heterologous system; such insta-bility may place a limitation on the usefulness of expressionvectors such as pKCR.Our aim in constructing.plasmids capable of transforming

cells to Mtx resistance lay in the fact that they should act. asdominant markers for the transfer of cloned genes into any cellby cotransformation. A variety of dominant markers shouldprove most useful in gene transfer experiments; besides themarker described here, Mercola et al. (32) have been able touse the herpes simplex virus TK gene as a dominant marker,and Mulligan and Berg (33) have shown that the E. coli xan-thine-guanine phosphoribosyltransferase gene when cloned inSV40-derived vectors may be used as a dominant marker. Theinitial studies we report in this paper have all used TK- mousefibroblasts, because these cells may be transformed with a highfrequency relative to many other cell types. The general use-fulness of pHG as a source of a dominant selective marker forcell transformation will become established only when we have

used it successfully to transform a range of cells to Mtxresistance.

We thank Drs. M. LeMeur, L. Elwell, and C. Weissmann. and hiscolleagues for generous gifts of material. We are grateful to Prof. P.Chambon for support, encouragement, and useful discussions. Thetechnical assistance of B. Boulay, K. Dott, M. C. Gesnel, D. Schmitt,and L. Thia-Toong is gratefully acknowledged. This work was supportedby grants from the Institut National de la Sante et de la RechercheMedicale (Action Thematique Programmee 72.79.104 and CL 801033),the Centre National de la Recherche Scientifique (Action ThematiqueProgrammee "Biologie Moleculaire du Gene" N0 006520/50), from laFondation pour la Recherche M6dicale Frangaise and la Fondation Si-mone et Cino del Duca. K.O'H. is a European Molecular BiologyOrganization Long Term Fellow.

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