Gene,46 (1986)277-286

Elsevier

217

GEN 01762

Cosmid vectors for high efficiency DNA-mediated transformation and gene amplification in mam- malian cells: studies with the human growth hormone gene

(Human recombin~t DNA library; neo and DHFR selection; gene expression)

K.H. Chooaeb*, G. Filby”, S. GrecoC, Y.-F. Lauaqd and Y.W. Kana**

U Howard Hughes Medical institute and ~iv~~on of Genetics and ~oiecular HematoIo~ of the repayment of ~ed~~in~: d repayment of Physiolo~, ~nivers~y of ~all~o~ia, San Francisco, CA 94143 (U.S.A.) Tel* (41~)4~b-~841: ’ Birth Defects Research Institute, and c Department of Clinical Biochemistry, Royal Children’s Hospital, Melbourne (Australia 3052) Tel. (03)345-5522

(Received February 14th, 1986) (Revision received June 30th, 1986) (Accepted July 17th, 1986)

SUMMARY

We have constructed two new recombinant cosmid vectors that can be used for direct expression and amplification of genomic DNA in mammalian cells. The vectors allow cloning of DNA fragments up to 40 kb in size. Each carries two dominant selectable markers: the bacterial neo gene and the mouse DHFR gene. In the first vector, pCVOO1, the neo and DHFR genes are regulated by the SV40 early promoter, and in the second, pAVCV007, by the avian sarcoma virus LTR promoter. The neo gene served as a dominant marker for the selection of transformants in all mammalian cell types, and we demonstrate here that the LTR promoter significantly improved the efficiency of DNA-mediated transformation of a human cell line. We isolated the human growth hormone genes from genomic libraries prepared in these cosmid vectors and used these recombinant cosmids for direct transfections of cultured cells. Selection of transformants in increasing concen~ations of methotrexat~ led to the out~o~h of resistant cell populations carrying opine copies of the DHFR marker. A 40-IOOO-fold coamphfication of the hGH genes was observed in the different transfected cell lines, along with a corresponding increase in transcription and translation activity of the hGH gene. Gene amplification could be achieved in both DHFR deficient or normal cell lines. Higb level expression of a cloned gene mediated by gene amplification should facilitate characterization of DNA sequences, as well as isolation of specific gene products for biochemic~, functions, and ph~acolo~c~ studies.

* Present address: Birth Defects Research Institute, Royal Chil- dren’s Hospital, Flemington Road, Parkville (Australia 3052) Tel. (03)345-5522.

** To whom correspondence and reprint requests should be addressed.

Dulbecco’s modified Eagle medium; FCS, fetal calf serum; hCS, human chorionic somatomammotropin; hGH, human growth hormone; IU, international unit(s); HSV, herpes simplex virus; kb, 1000 bp; LTK-, mouse LM cetls deficient in thymidine kinase; LTR, long terminal repeats; MTX, methotrexate; neo, aminoglycoside phosphotransferase II gene; nt, nucleotide(s); PolIk, Klenow (large) fragment of E. co/i DNA polymerase I; R,

Abbreviations: Ap, ampicillin; bp, base pair(s); CHO, Chinese resistance; RIA, radioimmunoassay; SV40, simian virus 40; Tk, hamster ovary; DHFR, dihydrofolate reductase; DME, thymidine kinase.

0378-I 119/86/%03.50 0 1986 Else&x Science Publishers B.V. (Biomedical Division)

278

INTRODUCTION

DNA-mediated gene transfer into cultured mam- malian cells is an important tool for studying the function and expression of eukaryotic genes and their flanking sequences (Szybalska and Szybalski, 1962; Graham and Van der Eb, 1973; Hamer and Leder, 1979; Gorman et al., 1982; Robins et al., 1982; Duesberg, 1983). Although a number of recombinant vectors have been constructed to serve as vehicles for such experiments (Subramani et al., 1981; Southern and Berg, 1982; Murray et al., 1983; Gorman et al., 1983), most cannot be used effectively as vectors for genomic library construction. Vectors allowing genomic library preparation as well as direct DNA-mediated gene transfer for expression studies would facilitate the functional analysis of cloned genes.

Recently, several cosmids were developed that can serve as genomic cloning and expression vectors (Grosveld et al., 1981; 1982; Ish-Horowitz and Burke, 198 1; Lau and Kan, 1983). They contain mammalian selectable markers such as SV2-gpt, SV2-DHFR, SV2-neo, and HSV-TK genes (Gros- veld et al., 1982; Lau and Kan, 1983) which permit direct transfection of cultured mammalian cells with isolated DNA sequences from bacterial hosts. Since the SV2 series of genes can be used in dominant selection systems, a wide range of cells may serve as hosts for these selectable markers. Because cosmids can accommodate inserts of up to 45 kb, their use increases the likelihood of isolating intact large genes, gene families, or DNA sequences flanking genes for subsequent expression studies.

We have developed a new cosmid vector contain- ing both the SV2-neo and SV2-DHFR genes that permits dominant selection with the neo-resistant gene as well as amplification of transfected sequences with the DHFR gene and methotrexate selection. Additionally, in another vector we have modified the cosmid so that both selectable markers are driven by the avian sarcoma virus LTR to increase the trans- formation efficiency in mammalian cells. Such clon- ing and amplification should enhance gene expres- sion and make available large quantities of the gene products. We constructed two human genomic libraries using the new cosmids and, to demonstrate their utility, we have isolated recombinant cosmids containing the hGH gene. The selectable markers in

these vectors have enabled us to transfect, amplify, and express the hGH gene at high levels in trans- formed cell lines.

MATERIALS AND METHODS

(a) Vectors and bacterial strains

The construction of the cosmid vector pCV108 has been reported previously (Lau and Kan, 1983). Plasmids pSVZDHFR and Escherichiu coli ED8767 were obtained from P. Berg and R.A. Flavell, respec- tively. Plasmid pAV3, in which the BstEII site at the 3’ end of the LTR promoter is converted to a BumHI site (Luciw et al., 1983; Hudziak et al., 1982), was obtained from P.A. Luciw.

(b) Construction of cosmid vectors

Cosmid pCVOO1 was constructed by inserting the modular mouse SV2-DHFR gene from pSV2- DHFR (Subramani et al., 1981) into the cosmid pCV108 (Lau and Kan, 1983) which contains the SV2-neo gene and its structure is shown in Fig. 1. The procedure for constructing the cosmid pAVCV007 is outlined in Fig. 2. In this vector, as in pCVOO1, the LTR-neo and LTR-DHFR genes were both oriented in the same direction of transcription.

(c) Preparation of cosmid libraries and screening of

colonies

The cosmid library was prepared according to methods previously described (Lau and Kan, 1983). High-M, human leukocyte DNA was prepared from a normal male individual according to standard procedures (Maniatis et al., 1982). The DNA was partially digested with Mb01 and fragments of 35-45 kb obtained by fractionation on a 10-30x sucrose density gradient. The DNA preparation was checked on gel immediately prior to use to ensure intactness of the DNA molecules. The DNA was cloned into the BumHI site of pCVOO1 and pAVCV007 cosmid vectors and packaged into bac- teriophage (Lau and Kan, 1983). Libraries con- structed with pCVOO1 and pAVCV007 (designated CVOO 1K and AVCV007K, respectively) were

279

screened for recombinant clones at high density on Millipore nitrocellulose paper according to the pro- cedure of Hanahan and Meselson (1980). Human growth hormone gene probe (DeNoto et al., 1981) (a gift from F.M. deNoto) was labeled to high specific activity by nick translation (Rigby et al., 1977).

(d) Cell culture, transformation, and methotrexate

selection

Mouse LTK (from J.M. Bishop) and human Heia cells (from R.A. Farrell) were cultured in DME medium supplemented with penicillin-strepto- mycin, and 10% FCS. CHO-Kl/dhfr- cells (from L. Chasin) were grown in Hams F-12 medium supplemented with 10% FCS, 1.50 pg glutamine/ml, and 21 pg praline/ml.

DNA was transfected into cultured mammalian cells using a modified calcium phosphate coprecipi- tation procedure (Lau and Kan, 1983). The transfor- mation efficiency varied between one colony per 103-lo4 cells. Surviving transformant cells were

pooled and subjected to stepwise selection (two-fold increases) in MTX. The starting MTX concentra- tions for cells transfected with pCVOO1 and pAVCV007 clones were 0.05 and 0.3 PM, respec- tively. MTX selection oftransfected CHO-Kl/dhfr - cells was performed in DME medium supplemented with 10% FCS and 21 pg/ml proline. Cells were maintained at each MTX level for approx. 2 weeks, with the medium changed twice weekly, and were subcultured once at a given drug concentration prior to proceeding to the next level.

(e) Analysis of gene amplification and gene expres-

sion

Gene amplification was assayed by standard nitrocellulose filter hybridization (Southern, 1975). Gene expression at the transcriptional level was determined by transferring total RNA onto nitro- cellulose filters, followed by hybridization of [ 32P]DNA (Dobner et al., 1981; Thomas, 1980). hGH polypeptides were assayed by standard RIA on medium maintained in a subconfluent culture for 5 days. The assay was performed according to stan- dards set by the WHO First International Reference Preparation for hGH (66/127), and has a sensitivity of 2 IUjl (or 1 ng/ml).

RESULTS AND DISCUSSION

(a) Construction of cosmid

genomic libraries

Construction of genomic

vectors and human

recombinant DNA libraries with cosmid vectors permits the isolation of DNA fragments up to 45 kb in size, and increases the likelihood of isolating intact genes or gene clusters and their flanking sequences for analysis. The inclusion of a mammalian selectable gene in the cosmid prior to cloning facilitates direct transfer of cloned DNA sequences into cultured mammalian cells for functional assays. The two new cosmids described herein further extend the application of these versatile vectors for gene isolation and expres- sion analysis, and increase the chances of obtaining adequate expression through gene amplification.

The structures of the two cosmid vectors con- structed in this study are shown in Figs. 1 and 2. The

EcoRl BamHl Clal

Ava I

Fig. 1. Structure of cosmid vector pCVOO1. Cosmid pCV108 was

modified by inserting the modular mouse SVZ-DHFR gene that

was excised by double digestion of pSV2-DHFR with

BamHI + PvuII and isolated by gel elution. The BamHI site was

converted into a blunt end by tilling in with PolIk. This fragment

was ligated into the Sal1 site of pCV108, which had been tilled

in to give blunt ends. In the resultant cosmid vector pCVOO1, the

SV2-neo and SV2-DHFR genes were both oriented in the same

direction of transcription. Ori, origin of replication; ApR, ampi-

cillin-resistance gene; COS, cohesive end site of bacteriophage

1. The open box represents the two selectable marker genes. The

thick lines represent the position of the indicated sequences.

280

1. Digest with Sal 1 2. Partial digest with Hind 3. Isolate large frafyt( - 4. Pill-in with PO1 5. Ligate with LTR promoter

111 7.2kb)

1. 2. 3. 4.

BamHl

Digest with Hind 111 6 Pvu 11 Isolate large fragment( - 4.6kb) Fill-in with Pal I k

Li$ate with LXX promoter

1. Digest with Baa Al 6 Pvu 11 2. Isolate DHFR fragmcnt(-2.2kb) 3. Fill-in with PO1 T k

1. Digest with Sal 1 2. Fill-in with Pol I k

Fig. 2. Construction of cosmid vector pAVCV007. The LTR promoter segment was prepared from plasmid pAV3 (Luciw et al., 1983) by &II and BarnHI double digestion. The 594bp fragment containing the promoter was eluted from a gel and GIled-in with Pollk. This LTR fragment replaced the SV40 promoter in the setectable gene markers in pCVIO8 and pSV2-DHFR. The resulting LTR-DHFR gene was isolated and inserted into the modified pCVlO8 (pAVCVneo) to produce the new cosmid vector pAVCV007. Both LTR-neo and LTR-DHFR were oriented in the same direction of transcription. as indicated by the arrows. Restriction enzyme sequences preserved at the site uf insertion are designated by asterisks.

281

vectors are approx. 10 kb in size, which allows for cloning DNA of up to 40 kb. Each vector carries two markers - the bacterial neo gene and the mouse DHFR gene. In pCVOO1, both the neo and DHFR genes are regulated by separate SV40 early pro- moters, whereas in pAVCV007 these promoters are replaced by separate avian sarcoma virus LTR pro- moters. These two vectors, when used in combi- nation, allow dominant selection with the neo-

resistance gene to introduce exogenous DNA into the host cells, and amplification of transfected DNA by the DHFR gene after MTX selection. The expres- sion of the amplified gene is thus greatly enhanced.

Human genomic libraries were prepared from each of the two vectors. The CVOOlK and AVCV007K libraries contained 1.4 and 2.4 x lo6 independent recombinant colonies, representing the human genome more than 10 and 20 times, respec- tively. Restriction endonuclease analysis of ran- domly picked colonies indicated that they contained inserts of 35-40 kb. Isolation and restriction enzyme analysis of specific clones such as the human cc-globin cluster revealed no rearrangement of the DNA inserts (results not shown).

(b) Isolation of hGH gene

A total of lo6 bacterial colonies from the AVCV007K library were screened with radiolabeled hGH gene probe. Of the 10 positive clones found, restriction analysis of 7 revealed 6 different over- lapping patterns (not shown). Two of these clones (designated hGH-a and hGH-b) were used in expression and amplification studies.

(c) Transformation of cultured mammalian cells

The transformation efficiencies of the pCVO0 1 and pAVCV007 vectors were compared in LTK- , HeLa, and CHO-Kl/dhfr- cells. As shown in Table I, transformation frequencies ranged from 1 in lo4 cells to > 1 in lo3 cells. Similar transformation fre- quencies were observed for both vectors in mouse LTK - and hamster CHO-K l/dhfr - cells; however, in human HeLa cells the pAVCV007 vector con- sistently gave approximately tenfold more transfor- mants than pCVOO1. A similar increase in transfor- mation efficiency occurred in the HeLa cells trans- formed with DNA from the total genomic library

TABLE I

Efficiency of cell transformation with cosmid DNA”

DNA LTK- HeLa CHO-Kl/dhfr _

Cosmid vector

pcvoo1

pAVCV007

Cosmid library

CVOOlK

AVCVOO’TK

Cosmid clone

hGH-a or -b

4.1 1.3 NDb

4.1 12.0 ND

ND 1.3 1.0

ND 15.0 ND

3.6 16.2 1.2

a 3 x 10’ cells were trypsinized, pelleted, and resuspended in

6 ml of DNA-phosphate precipitate (25 pg DNA/ml without

carrier DNA) for 15 min. followed by the addition of 54 ml of

growth medium, then the cells were subcultured into three

150-cm* flasks and incubated overnight. The cells were then

treated with 25% glycerol in growth medium for 1 min, rinsed

twice with growth medium, and cultured for 48 h before selection

in 400 pg/ml of G418 (geneticin, GIBCO laboratories). Surviving

cell colonies were fixed, stained with Giemsa, and counted.

Results are presented as the number of G41gR colonies per

lo4 cells, A minimum of lo6 cells were plated out for each

selection.

AVC007K as compared to HeLa cells transformed with DNA from the CVOOlK library. DNA from the cosmid clone containing the hGH genes also trans- formed LTK - and HeLa cells at efficiencies similar to those obtained for the vector alone and total vectorgenomic libraries. Since the initial transfor- mant selection was carried out in G418, the higher transformation efficiency in HeLa probably reflects the strength of the LTR promoter in supporting the expression of the neo gene. This finding corroborates data of Gorman et al. (1983), who observed the same

phenomenon in a number of other cell lines. How- ever, unlike the previous data (Gorman et al., 1983) we have observed a higher transformation efficiency with the LTR promoter in HeLa cells than in mouse LTK- cells. Furthermore, following transfection of the hGH cosmid DNA in both LTK - and CHO-Kl/dhfr - cells, the transformants were imme- diately resistant to 0.3 PM MTX (a concentration that killed virtually all the control cells). Presumably, under the regulation of the LTR promoter the DHFR gene in pAVCV007 vector produced enough enzyme to overcome the high toxicity of the drug. It should therefore be possible to use the LTR-DHFR

282

modular gene as an alternate dominant marker for gene transfer experiments.

(d) Amplification of the hGH gene

The relationship between gene amplification and drug resistance in cultured cells has been well docu- mented in different systems (Schimke, 1982). Several groups have demonstrated that, when transfected in mammalian cells, cloned DHFR genes can also be amplified by MTX selection (Schimke et al., 1978; Ringold et al., 1981; Kaufman and Sharp, 1982a,b; Brown et al., 1982; Christman et al., 1982; Haynes and Weisman, 1983; McCormicket al., 1984; Wigler et al., 1980). Furthermore, DNA sequences linked to these cloned DHFR genes are coampli~ed in the same process (Lau et al., 1984).

To test the eficiency of the linked neo and DHFR markers in these cosmid vectors, we studied the expression and amplification of two cosmid clones containing the hGH gene: clones hGH-a and hGH-b. Restriction endonuclease analysis demon- strated that both carried the hGH-1 gene which is known to be responsible for hGH production (DeNoto et al., 1981; Martial et al., 1979; Phillips et al., 1981; Miller and Eberhardt, 1983). In addi- tion, the hGH-b clone contained the related hCS-5, located approx. 6 kb 3’ to the hGH-1 gene (Miller and Eberhardt, 1983). EcoRI digestion of the two cosmid clones and hyb~dization with hGH gene probe yielded a 2.6-kb fragment containing the hGH-1 gene (DeNoto et al., 1981) (Fig. 3A). Due to the high degree of nucleotide homology between hGH and hCS (Miller and Eberhardt, 1983), the hGH probe detected an additional 9.8-kb EcoRI fragment in clone hGH-b containing the hCS-5 gene (Fig. 3A).

Two cell lines, CHO-Kl/dhfr - and LTK - were transfected with the hGH cosmid clones. Following initial selection in 400 pg/ml of G418, transformants were pooled and subjected to stepwise selection in medium containing MTX. Initial MTX concentra- tions of 0.05 or 0.1 PM produced no significant effect on the cells, although these drug concentrations killed a large portion of untransfected cells. Some cell death occurred at MTX concentrations above 0.3 FM, but dramatic cell killing was not observed until drug levels reached 2.5 PM. Culture media from resistant cell populations were assayed for hGH

TABLE II

Expression of hGH polypeptide” in transfected cells

MTX concentration

(PM)

Cell line

CHO-Kl/dh~-/hG~-a LTK - /hGH-b

0 >l 2-3 20 ND 32 40 750 45 80 1050 60

160 1350 NDb

a G418R and DHFR’ tr~sform~t cells were subjected to stepwise selection in MTX, starting at 0.05 and 0.3 FM for pCVOO1 and pAVCV007, respectively. Cells were maintained at least 2 weeks before each increase in MTX concentration. hGH polypeptide levels were determined by RIA according to World Health Organization standards. Rest&s are presented in ngiml of culture medium derived from an average of 5 x 10’ cells/ml. Untransfected CHO-Kl/dhfr-, LTK- and HeLa cells gave no detectable levels of hGH polypeptide. Sensitivity of the assay is 1 ng/mI. Average concentration of hGH in plasma is approx. 3 ng/ml (Slinksen and West, 1979). ’ ND. not determined.

polypeptide at different MTX concentrations using standard RIA (Table IQ. Prior to MTX selection, the transfected cells produced low or undetectable levels of hGH. At 80 PM MTX, the level of hGH polypeptide in the LTK- cells increased approx. 20-fold. A more dramatic effect was observed in the CHO-Kl cells. At 80 PM MTX the level of hGH increased more than lOOO-fold in these cells.

To monitor the amplification of hGH genes in the LTK ~ and CHO-Kl cells, genomic DNA was pre- pared from these cells and analyzed by DNA hybridization methods (Fig. 3A). Initially, the trans- formants incorporated about 20 copies of hGH gene per cell in LTK- and about one copy per cell in CHO-Kl cells, as determined by dot blot hybridiza- tion of serially diluted genomic DNA from the two cell lines using known amounts of cloned hGH DNA as standards (not shown). As the cells became resistant to 40-80pM MTX, the number of hGH genes increased dr~atic~iy, to about 400 copies (or 20-fold amplification) in LTK -, and > 1000 copies (or > lOOO-fold amplification) in CHO-Kl cells. No major DNA rearrangement occurred in the hGH gene since the restriction enzyme patterns of transfected DNA were essentially the same before

283

A

(I) hGH Probe (II) DHFR Probe

123456 8 9 10 123456 7 8 910

123

(I) hGH Probe (II) DHFR Probe

-23.6

-2.2 -1.9

-1.9

-23.6

12345 6 9 12345 6 7 8 9

-28 S-

-18S-

c

-28S-

-18S-

Fig. 3. Molecular analysis of the expression and amplification of hGH gene in transfected cell lines. Nitrocellulose filters were hybridized

with 3*P-labeled hGH probe (panels I) or DHFR probe (panels II). (Panels A) Lanes: 1, parental CHO-kl/dhfr- cells prior to

transfection; 2, CHO-Kl/dhfr- cells transfected with cosmid clone hGH-a before MTX selection; 3-5, after selection in 80 pM MTX

at 1: 25, 1: 5 and no dilutions of DNA, respectively; 6, hGH-a cosmid DNA showing the 2.6-kb hGH-1 gene band (larger bands are

due to incomplete digestion). The faint 4.4-kb band across all lanes is probably due to plasmid contamination. The inset shows a much

284

and after amplification. Analysis of these restriction

enzyme patterns by nitrocellulose filter hybridization

against [32P]DHFR probe showed the expected

amplification of the DHFR gene (Fig. 3A).

(e) Expression of the amplified hGH and DHFR

genes

Total RNA was isolated from cells grown at differ-

ent MTX concentrations for Northern hybridization

(Fig. 3B). Before MTX selection trace amounts of

hGH mRNA were detected in transfected LTK -

cells, but none was found in the CHO-Kl cells, even

after prolonged exposure of the X-ray film. The

nature of the small hGH mRNA seen in lane 1

(Fig. 3B-I) is not clear. This mRNA is presumably

not translationally active since it produced no

detectable hGH polypeptides (Table II). At

40-80 PM MTX, the concentration of hGH mRNA

increased dramatically, which corresponds with our

DNA analysis (Fig. 3A). Hybridization of the

DHFR [ 32P]DNA probe to Northern filters showed

that the level of DHFR mRNA was greatly

increased. The sizes of the mature hGH and DHFR

mRNA corresponded well with those previously

reported (Robins et al., 1982; Subramani et al.,

1981). The results in Fig. 3B further indicate that

DHFR gene transcription was significantly more

efficient than hGH gene transcription, especially in

the LTK - cells. Following a 20-fold DNA amplili-

cation, the hGH mRNA level increased approx.

20-fold, whereas the mRNA level of DHFR in-

creased by as much as lOOO-fold. This large dis-

crepancy in the transcriptional activity of the two

genes may have occurred because the LTR promoter

used to regulate the DHFR gene is much stronger

than the natural hGH gene promoter in mouse cells.

hGH genes have previously been introduced into

cultured mammalian cells, either by calcium phos-

phate coprecipitation, or as infectious retroviral par-

ticles and bovine papilloma virus (Robins et al.,

1982; Doehmer et al., 1982; Pavlakis and Hamer,

1983; Miller et al., 1984; Karin et al., 1984). Once

the transfected genes were stably incorporated into

the host cells, their expression was properly regulated

by glucocorticoid hormones. hGH secretion to the

media by these cell transformants varied from 3 to

7.5 ng/ml/h. In this study, amplification of trans-

fected hGH genes into the host cells increased hGH

synthesis and secretion to about 11 ng/ml/h in

CHO-Kl cells without glucocorticoid induction.

This amplification process can therefore be used as

a direct approach for increasing the level of expres-

sion of cloned genes and their specific products.

The ability to obtain efficient expression should

facilitate the direct isolation of a functional gene by

transforming a cell population with total DNA pre-

pared from a cosmid library, as we have recently

described for the human thymidine kinase gene (Lau

and Kan, 1984). Although both the pAVCV007 and

pVCOO1 vectors can be used to transfect and amplify

a cloned gene, pAVCV007 has the advantage of

higher transformation efficiency in a wider range of

cell types, including primate cells. A further advan-

tage in transforming cells with cosmid rather than

genomic DNA is that the former can be readily

recovered by in vitro packaging reactions of the

cellular DNA. Amplification of cosmid DNA may

improve the efficiency of the recovery process by

providing more copies for such packaging reactions.

longer X-ray film exposure of lanes l-3 to reveal the faint 2.6-kb band in lane 2. Lanes: 7, parental LTK- cells prior to transfection

(this lane was not shown in part I; there was complete absence of hybridization of the L cell DNA to the hGH probe); 8, LTK- cells

transfected with cosmid clone hGH-b before MTX selection; 9, after selection in 40 pM MTX; 10, hGH-b cosmid DNA showing the

2.6-kb (hGH- 1 gene) and 9.8-kb (hCS-5 gene) bands, and a lower band on longer exposure (arrow). All samples were digested with EcoRI.

Except where stated in the dilutions, 5 pg of genomic DNA was used. (Panels B) Hybridization of total RNA. Lane 1, CHO-Kl/dhfr

cells transfected with cosmid clone hGH-a before MTX selection; lanes 2-5, after selection in 80 PM MTX at 1: 125, 1: 25, 1: 5 and

no dilutions of RNA respectively; lane 6, LTK - cells transfected with cosmid clone hGH-b before MTX selection; lane 9, after selection

in 40 PM MTX. Because the DHFR probe showed very intense hybridization to the LTK- cell RNA, the RNA samples from these

cells were also diluted 1: 25 (lane 7) and 1: 5 (lane 8). Except where stated in the dilutions, 10 pg of total RNA was applied onto each

track. The rRNA size markers (28s and 18s) were visualized by staining the filter with methylene blue (Maniatis et al., 1982) while

the 10s position was determined with reference to the size of mature a-globin mRNA. These sizes corresponded to RNA molecules

measuring approx. 4.5, 2.0, and 0.8 nt, respectively.

285

ACKNOWLEDGEMENTS

We thank D.M. Danks and R.G.H. Cotton for

their support in a substantial part of this work, and

P. Berg, R.A. Flavell, P.A. Luciw, and F.M. deNoto

for the vectors and clones. K.H.C. is a recipient of

a C.J. Martin Research Fellowship from the

National Health and Medical Research Council of

Australia. Y.W.K. is an Investigator and Y.-F. L. an

Associate Investigator of the Howard Hughes Medi-

cal Institute.

REFERENCES

Brown, P.C., Kaufman, R.J., Haber, D. and Schimke, R.T.:

Characteristics of dihydrofolate reductase gene amplification

in murine and Chinese hamster ovary cell lines. In R.T.

Schimke (Ed.), Gene Amplification. Cold Spring Harbor

Laboratory, Cold Spring Harbor, NY, 1982, pp. 9-13.

Christman, J.K., Gerber, M., Price, P.M., Flordellis, C.,

Edelman, J. and Acs, G.: Amplification of expression of

hepatitis B surface antigen in 3T3 cells cotransfected with a

dominant-acting gene and cloned viral DNA. Proc. Nat].

Acad. Sci. USA 79 (1982) 1815-1819.

DeNoto, F.M., Moore, D.D. and Goodman, H.M.: Human

growth hormone DNA sequence and mRNA structure: pos-

sible alternative splicing. Nucl. Acids Res. 9 (1981)

3719-3730.

Dobner, P.R., Kawasaki, E.S., Yu, L.Y. and Bancroft, F.C.:

Thyroid or glucocorticoid hormone induces pre-growth-

hormone mRNA and its probable nuclear precursor in rat

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

2230-2234.

Doehmer, J., Barinaga, M., Vale, W., Rosenfeld, M.G., Verma,

I.M. and Evans, R.M.: Introduction of rat growth hormone

gene into mouse fibroblasts via a retroviral DNA vector:

expression and regulation. Proc. Natl. Acad. Sci. USA 79

(1982) 2268-2272.

Duesberg, P.H.: Retroviral transforming genes in normal cells?

Nature 304 (1983) 219-226.

Emerman, M. and Temin, H.M.: Genes with promoters in retro-

virus vectors can be independently suppressed by an epi-

genetic mechanism. Cell 39 (1984) 449-467.

Gorman, C.M., Merlino, G.T., Willingham, M.C., Pastan, I. and

Howard, B.H.: The Rous sarcoma virus long terminal repeat

is a strong promoter when introduced into a variety of

eukaryotic cells by DNA-mediated transfection. Proc. Natl.

Acad. Sci. USA 79 (1982) 6777-6781.

Gorman, C., Padmanabhan, R. and Howard, B.H.: High effi-

ciency DNA-mediated transformation of primate cells.

Science 221 (1983) 551-553.

Graham, F.L. and Van der Eb, A.J.: A new technique for the

assay of infectivity of human adenovirus 5 DNA. Virology 52

(1973) 456-467.

Grosveld, F.G., Dahl, H.H., De Boer, E. and Flavell, R.A.:

Isolation of B-globin related genes from a human cosmid

library. Gene 13 (1981) 227-237.

Grosveld, F.G., Lund,T., Murray, E.J., Mellor, A.K., Dahl, H.H.

and Flavell, R.A.: The construction of cosmid libraries which

can be used to transform eukaryotic cells. Nucl. Acids Res.

10 (1982) 6715-6732.

Hamer, D. and Leder, P.: Expression of the chromosomal mouse

B”“j-globin gene cloned in SV40. Nature 281 (1979) 35-40.

Hanahan, D. and Meselson, M.: Plasmid screening at high

colony density. Gene 10 (1980) 63-67.

Haynes, J. and Weissman, C.: Constitutive, long-term production

of human interferons by hamster cells containing multiple

copies of a cloned interferon gene. Nucl. Acids Res. 11 (1983)

687-706.

Hudziak,R.M., Laski,F.A., RajBhandary, U.L., Sharp, P.A. and

Capecchi, M.R.: Establishment of mammalian cell lines

containing multiple nonsense mutations and functional

suppressor tRNA genes. Cell 31 (1982) 137-146.

Ish-Horowitz, D. and Burke, J.F.: Rapid and efficient cosmid

cloning. Nucl. Acids Res. 9 (1981) 2989-2998.

Karin, M., Eberhardt, N.L., Mellon, S.H., Malich, N., Richards,

R.I., Slater, E.P., Barta, A., Martial, J.A., Baxter, J.D. and

Cathala, G.: Expression and hormonal regulation of the rat

growth hormone gene in transfected mouse L cells. DNA 3

(1984) 147-155.

Kaufman, R.J. and Sharp, P.A.: Amplification and expression of

sequences cotransfected with a modular dihydrofolate

reductase complementary DNA gene. J. Mol. Biol. 159

(1982a) 601-621.

Kaufman, R.J. and Sharp, P.A.: Construction of a modular

dihydrofolate reductase cDNA gene: analysis of signals

utilized for efficient expression. Mol. Cell. Biol. 2 (1982b)

1304-1319.

Lau, Y.-F. and Kan, Y.W.: Versatile cosmid vectors for the

isolation, expression, and rescue of gene sequences: studies

with the human a-globin gene cluster. Proc. Natl. Acad. Sci.

USA 80 (1983) 5225-5229.

Lau, Y.-F. and Kan, Y.W.: Direct isolation of the functional

human thymidine kinase gene with a cosmid shuttle vector.

Proc. Nat]. Acad. Sci. USA 81 (1984) 414-418.

Lau, Y.-F., Lin, C.C. and Kan, Y.W.: Amplitic;ition and expres-

sion of human a-globin genes in Chinese hamster ovary cells.

Mol. Cell. Biol. 4 (1984) 1469-1475.

Luciw, P.A., Bishop, J.M., Varmus, H.E. and Capecchi, M.R.:

Location and function of retroviral and SV40 sequences that

enhance biochemical transformation after microinjection of

DNA. Cell 33 (1983) 705-716.

Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning.

A Laboratory Manual. Cold Spring Harbor Laboratory, Cold

Spring Harbor, NY, 1982.

Martial, J.A., Hallewell, R.A., Baxter, J.D. and Goodman, H.M.:

Human growth hormone: complementary DNA cloning and

expression in bacteria. Science 205 (1979) 602-607.

McCormick, F., Trahey, M., Innis, M., Dieckmann, B. and

Ringold, G.: Inducible expression of amplified human /?inter-

feron genes in CHO cells. Cell 4 (1984) 166-172.

Miller, A.D., Ong, E.S., Rosenfeld, M.G., Verma, I.M. and

286

Evans, R.M.: Infectious and selectable retrovirus containing

an inducible rat growth hormone minigene. Science 225

(1984) 993-998.

Miller, W.L. and Eberhardt, N.L.: Structure and evolution ofthe

growth hormone gene family. Endocr. Rev. 4 (1983) 97-130.

Murray, M.J., Kaufman, R.J., Latt, S.A. and Weinberg, R.A.:

Construction and use of a dominant, selectable, marker: a

Harvey sarcoma virus-dihydrofolate reductase chimera. Mol.

Cell. Biol. 3 (1983) 32-43.

Pavlakis, G.N. and Hamer D.H.: Regulation of a metallo-

thionein-growth hormone hybrid gene in bovine papilloma

virus. Proc. Natl. Acad. Sci. USA 80 (1983) 397-401.

Phillips III, J.A., Hjelle, B.L., Seeburg, P.H. and Zachmann, M.:

Molecular basis for familial isolated growth hormone defl-

ciency. Proc. Natl. Acad. Sci. USA 78 (1981) 6372-6375.

Rigby, P.W.J., Dieckmann, M., Rhoades, C. and Berg, P.:

Labeling deoxyribonucleic acid to high specific activity in

vitro by nick translationwith DNA polymerase I. J. Mol. Biol.

113 (1977) 237-251.

Ringold, G., Dieckmann, B. and Lee, F.: Co-expression and

amplification of dihydrofolate reductase cDNA and the

Escherichia coli XGPRT gene in Chinese hamster ovary cells.

J. Mol. Appl. Genet. 1 (1981) 165-175.

Robins, D.M., Paek, I., Seeburg, P.H. and Axel, R.: Regulated

expression of human growth hormone genes in mouse cells.

Cell 29 (1982) 623-63 1.

Schimke, R.T. (Ed.): Gene Amplification. Cold Spring Harbor

Laboratory, Cold Spring Harbor, NY, 1982.

Schimke, R.T., Kaufman, R.J., Alt, F.W. and Kellems, R.F.:

Gene amplification and drug resistance in cultured murine

cells. Science 202 (1978) 1051-1055.

Siinksen, P.H. and West, T.E.T.: Growth hormone. In Gray,

C.H. and James, V.H.T. (Eds.), Hormones in Blood, Vol. 1.

Academic Press, London, 1979, pp. 225-254.

Southern, E.M.: Detection of specific sequences among DNA

fragments separated by gel electrophoresis. J. Mol. Biol. 98

(1975) 503-517.

Southern, P.J. and Berg, P.: Transformation of mammalian ceils

to antibiotic resistance with a bacterial gene under control of

the SV40 early region promoter. J. Mol. Appl. Genet. 1(1982)

327-341.

Subramani, S., Mulligan, R. and Berg, P.: Expression of the

mouse dihydrofolate reductase complementary deoxy-

ribonucleic acid in simian virus 40 vectors. Mol. Cell. Biol. 1 (1981) 854-864.

Szybalska, E.H. and Szybalski, W.: Genetics of human cell lines,

IV. DNA-mediated heritable transformation of a biochemical

trait. Proc. Natl. Acad. Sci. USA 48 (1962) 2026-2034.

Thomas, P.S.: Hybridization ofdenatured RNA and small DNA

fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci.

USA 77 (1980) 5201-5205.

Wigler, M., Perucho, M., Kurtz, D., Dana, S., Pellicer, A., Axel,

R. and Silverstein, S.: Transformation of mammalian cells

with an amplifiable dominant-acting gene. Proc. Natl. Acad.

Sci. USA 77 (1980) 3567-3570.

Communicated by J.L. Slightom.