CA1218949A - Use of eucaryotic promoter sequences in the production of proteinaceous materials - Google Patents

Abstract

THE USE OF EUCARYOTIC PROMOTER SEQUENCES
IN THE PRODUCTION OF PROTEINACEOUS MATERIALS
ABSTRACT OF THE DISCLOSURE

This invention concerns the use of inducible promoter sequences in the production of elevated amounts of pro-teinaceous materials by transformed eucaryotic cells.
Eucaryotic cells which have been cotransformed with DNA
which includes a gene coding for a desired proteinaceous material such as insulin, human or bovine growth hormone, or an antibody and an inducible promoter sequence and with DNA coding for a selectable phenotype will, in the pres-ence of an inducing agent for the promoter, produce ele-vated quantities of the desired protein. This approach can be used in combination with gene amplification tech-niques to produce particularly high amounts of desired proteins.

This invention also provides methods for the therapeutic treatment of genetically defective eucaryotic cells and of persons suffering from genetically-based diseases or ill-nesses such as sickle cell anemia.

Description

~L29L~ 9 THE USE OF EUCARYOTIC PROMOTER SEQUENCES
IN THE PRODUCTION OF PROTEINACEOUS MATERIALS

Field of the Invention This invention concerns the introduction of DNA which includes a gene or genes coding for, regulating or otherwise in~luencing the production of proteinaceous materials into eucaryotic cells, that is, cells of organisms classified under the Superkingdom Eucaryotes incIuding organisms of the Plant and Animal Kingdoms.
lS It also concerns the expression o genes which have ~een introduced into eucaryotic cells. Such genetic intervention is commonly referred to as.genetic engineer-ing and in certain aspects involves the use of recom-binant D~A technology.
The invention disclosed is to be distinguished from the introduction of DNA into organisms of the Superkingdom Procaryotes including particularly bacteria. This distinction is based in part upon the basic differences between eucaryot~ nd procaryotic cells, the former being characterized by true nuclei formed by nuclear envelopes and by meiosis and the latter being charac-terized by the absence of well-defined nuclei and the absence of meiosis. Moreover, at the genetic level many genes in eucaryotes are split by non-coding sequences which are not continuously colinear, whereas in procaryotes, the genes are continuously colinear.

B ckground of the_Invention Although advances in the understanding of procaryotic organisms, particularly bacteria, have for the most part proceeded independently of advances in the under-standir~ of eucaryotic organisms, it may be helpful to an appreciation o the present invention to set forth certain developmen~s involving procaryotes.

1~ In 1944, Avery reported transformation of a procaryotic cell using DNA-mediated transfer of a cellular gene.
Avery, O~., et al., J~ Exp. Med. 79:137-158 (1944).
Thereafter, reports of procaryotic transformation appear~d in the litera~ure. In 1975, Cohen and others repo~cea results involving first transformation, then cotransformation of the procaryote Escherichia coli.
Kretschmer, P.J., et al., J. Bacteriology 124: 25-231 (197S). In these experiments the authors disclosed cotransformation of procaryotic cells using plasmid DNA, that is, extrachromosomal DNA which occurs natur-ally in many strains of En~erobacteriacae. They found that pa~ticular cells in a CaC12-treated bacterial population are preferentially competent for transforma-tion~ However, the frequency of transformation and the stability of the transformants was low, possibly because the plasmid is not incorporated into the chromosomal DNA. As a result, cotransformants lost acquired traits after several generations. In addition, these experi-ments required addition of a gene promoter to the trans-forming DNA in order to obtain expression.

Meanwhile, experiments with eucaryotic cells proceededsubstantially independently. In 1962, Szybalska, E.H.
and Szybalski, W. PNAS 48:2026 (1962) reported trans-3t4~
formation of mammalian cells, but with such low transfor-mation frequency that is was impossible to dist;inguish transformants from cells which had undergone spontaneous reversion. As with procaryotic cells, further reports s of eucaryotic transformation appeared in the literature, but were oftentimes not reproducible by others. In addition, low frequencies of transormation, lack of understanding of the molecular basis for gene expression and lack of molecular hybridization probes contributed to the lack of progress in this area. As a result, studies on the transformation of eucaryotic cells were essentially restric~ed to viral genes. Graham, F.L., et al_, Cold Spring Harbor Symp~ Quant. Biol. 39:637-650 (1975) and McCutchen, J~H. and Pagano, J.S., Journal National Cancer Institute, 41:351-357 (1968).

~ore recently, eucaryotic cells, specifically mammalian cells, were ~ransformed with foreign DNA coding for a selectable phenotype~ Wigler, M., et al., Cell 11:223-232 (1977)~ The present invention has resulted from extending this work~ It has been discovered inter alia that eucaryotic cells can be cotransformed to yield transfor-mants having foreign DNA integrated into the chromosomal DNA of the eucaryotic cell nucleus~ Moreover, it has unexpectedly been discovered that such foreign DNA can be expressed by the cotransformants to generate func-tional proteins. In addition, by contrast with procary-otic transformants, the foreign DNA is stably expressed through hundreds of generations, a result that may be attributable to integration of the foreign DNA into the chromosomal DNA.

This invention provides major advantages over bacterial systems for the commercial preparation of proteinaceous ~2~89~9 materials, particularly proteins of eucaryotic origin such as interferon protein, antibodies, insulin, and the like. Such advantages include the ability to use unaltered genes coding for p~ecursors. Ater cellular !3ynthesis, the precursor can be further processed or converted within the eucaryotic cell to produce the desi~ed molecule of biological slgnificance. This phenomenon is well known for insulin which is initially produced in the eucaryotic cell as preproinsulin, then converted within the cell to active insulin. Since procaryotic cells lack the requisite cellular machinery for converting preproinsulin to insulin, the insertion into a procaryotic cell of the eucaryotic gene associa-ted with insulin will result in production of prepro-~5 insulin, not insulin. In the c-ase of insulin, a rela-tively small and well characterized protein, this di~ficulty can be overcome by chemical synthesis of the appro~riate gene~. However, such an approach is inherently Iimited by the~ level of understanding of the ~o amino acid sequence of the desired protein~ Thus, for interferon protein, clotting factors, antibodies and uncharacterized enzymes, for which the exact amino aoid sequence is not yet known, a procaryotic system may not prove satisfactory. By contrast, a eucaryotic system 7~ is not associated with such disadvantages since the eucaryotic cell possesses the necessary processing machinery. It is thus one important object of the invention to provide a process for producing desired proteinaceous materials such as interferon protein, insulin, antibodies and the like which does not require a detailed molecular understanding of amino acid sequence.

In addition to the problem of precursors having additional amino acids which must be removed to produce active ~2~L8~4~
protein, important biological materials may be modified by chemical additions after synthesis and cleavage.
For example, human-produced interferon is a glycoprotein containing both sugar molecules and protein. If produced in a bacterial cell, the interferon lacks the sugar molecules which are added when interferon i5 produced in a human cell. Finally, pro~eins produced within bacteria may be contaminated by endotoxins which can cause inflammation if the protein is administered to a mammal without significant purification. By contrast~
interferon produced in a eucaryotic cell would be free o endotoxins~ It is therefore another important object of this invention to provide a process for producing compounds which include both non-proteinaceous and L5 proteinaceous moieties such as ~lycoproteins which cannot be produced in bacterial cells.

~2~ 9 SI~MMARY OF THE INVENTION

A oreign DNA I to which an inducible promoter DNA III
sequence has been linked can be introduced into a suitable eucaryotic cell by cotransforming the cell with a DNA molecule which includes this combination of foreign DNA I and DNA III and with unlinked DNA II
coding for a selec~able phenotype not otherwise expressed by the`eucaryotic cell. The cotransformation is carried out under suitable conditions permitting survival or identi~ication of eucaryotic cells which have acquired .. the selectable phenotype.

Proteins and protein-containing products may be produce*
by cotransforming eucaryotic cells as described herein and maintaining the cotransformed cells. under suitable conditions including the presence o~ an agent.capable o~ inducing the promoter DNA IrI sequence so that DNA I
is repeatedly transcribed.to form mRNAs and the mRNAs so ~ormed are transIated to form protein or protein-20 . containing products.

Cotransformation with a DNA. molecule whi.ch i.ncludesforeign DNA I and a promoter DNA III may be combined with gene amplification using as DNA II an amplifiable gène for a dominant selectable phenotype not expressed by the eucaryotic cell. The cotransformation is carried out under suitable conditions including the presence of an antagonist permitting survival or identification of eucaryotic cells which have acquired the dominant selectabl~ phenotype and the presence of an inducing agent for promoter DNA III.

Finally, methods for the therapeutic treatment of gene-tically defective eucaryotic cells and for the treatment -7~

of patients suffering from genetically-based illnesses or diseases are provided.

~2~L894~
BRIEF DESCRIPTION OF THE DRAWINGS
-FIG. 1 is a schematic flow diagram illustrating the co-transformation process.

FIG. 2 is a schematic flow diagram illustrating a process for recovering foreign DNA I from cotransformed cultured cells using double selection techniques.

FIG. 3. Recominant hGH clones. ~ ~OA is a Charon 4A
clone with a 14kb Eco RI fragment. Exons are shown by solid bars. A ~ore detailed map of the sequenced 2.6 kb Eco ~ragment, "wtGH", shows the five exons (hatched bars) interrupted by introns A-D.

~5 FIG. 4. Recombinant plasmid pGHtk contains an hGH-tk fusion gene. Plasmid pGHtk contains an 0.5 kb Eco RI/Bam Hl fragment of hGH (I ) (from the 5' end of the 2.6 kb Eco RI fragment) inse~ted at the BgL II site of ptk, replacing the tk promoter. Tk information includes the entire coding sequence of the tk gene ) as well as 1.7 kb of 3' 1anking DNA sequences ~ æ~). The initiator AUG is located 50 nucleotides 3' to the Bgl II site. The Bam HI site of the hGH
fragment including the putative promoter is 3 nucleo-tides beyond the transcription initiation site of hGH,De Noto, F.M., et al., Nucl. Acids Res. 9:3719-3730 (1981~.

~ 9~9 Detailed Descri~tlon of the Inventlon Prior to setting forth the invention, it may be hel~
tQ a~ understanding thereof to set forth definltions of certain terms to be used hereinafte~.

Transformation means th~ process for changin~ the genotype~ of a recipient celt mediated by the introduction of purifiea DNA~ ~ransformation i5 typicaLly det~cted ~y al stabl~ an~ h.exitable change in the phenotype of the rec~pie~ cell that results from an aLteratiorL.
either the biochemical or morph~logical propertLes o th~ recipien~ cell..

2~ Cotransforma~ion means the. process for carrying out _ transormations o a recipient cell wi~h more than one different gene. Cotransformation includes ~oth simultaneous and se~uential changes ln the genotype o a ~ecipient cell mediated ~y the introduction of DNA corresponding to either unlinked Gr linked genes.

Proteinaceous material means any hiopolymer formed from amino acidsO

-lo- ~2~ 9 y~ me~n~ the genetlc consti.tution of an organism as di.stinguished rom lts physical appearance..

~ Ye~ means the observable prope~ies of an organism as produced by th.e genot~pe in conjunction.
with the environment.

SeIecta~le phe otype~ is a phenotype which confers upon an organism the ability to exist under conditions which kill of~ all organisms not possessïng the phenotype. ~xamples include drug resistance or ~he a~iLlty to synthesi2e some molecul~ necessary to cell metabolism in a given growth medium. As used herein, selectahle ph.enatypes.also include identifiable L5 phena~ypes~ su~h a~ the production of materials which p~s~ ~rum or are secrete.d by the cell and can ~e de~ected a~ new, phen~type~ei~hex by functional, immunologic or biochemicaL assays.

20 ~9~ e~ means the pro~ei~aceous part of the glycoprotei~ interferon, tha~ is, the p~rtion remaining after remaval of th.e sugar portion. It includes the protei~ portion of interferon derived from human leukocyte, fibroblast or lymphoblastoid cells.
Chromosomal DNA means the DNA normally associa_2d with histone i~ the form of chromosomes residing in the nucleus of a eucaryotic cell.

Transcription means the iormation of a RNA chain Ln accordance with the genetic information contained in th e DNA .

~ tneans the process whereby the genetic 35 information in an mRNA molecule directs the order of specifïc am~no acids during protein synthesis.

L8~9 rn accordance with the present ~ention, foreign DN~ I
can be inserted into ar~ eucaryoti;c cell by cotransformin~
th:~ c:ell. Wit~:L DNA r and w, tI~ unli:nked ~ore- gn DN~ I:l:
which incIudes: a gene codi n~ for a se:Le~table phenotype no~ expressed by the cell unless ac~u:ired ~y trans-formatio~ The co~rans:E~ormation is carr~ed 011t in a su~table~ yrowt~ medium atld 1~ th~ presenc~ oi~ selective-condi~ on~ s.uch that t~ onLy c~Lls whic~ survive :1~. or are ot~e~ise altered are those ~hich have required th~ selectable phenatype~ 5ee Eig.. 1..

~Ithougk the ex~eriment~ discussed hereina~e~ conce . cuLture~ eucaryotic ce11s, o mammalian orig~n such as L5 ~uma~ blood cells., mous~ ibro~1ast ceL1s, chLnese hamst~r o~ary ceIlsi an~ mouse teratocarcinoma ce~ls, Lt'i5: ctea~ ~hatthe ~rocess: descri~ed i5: ~enerall~
-appL~ca~L~ to a1I eucar~tic cell~ inc1ud1ng, ~or exampla~, cells ~o~ bird~ suck a~ c~ickens,. ce11s --2~ ~rom yeast and ~ungi, a~ ce11s. rom ~Iants includin~
grai~s and flowers~. ~hereore-~ it.is to b~ ~nderstood:
tha~ the.i~e~t~on ~ncomp~sses alI eucaryot~c cells even thougk the in~ention may uLtimately be most useuL in co.transformin~ mammalian cells~
~5 ~he present invention is especially use~ul in co~nection with the insertion into eucaryo ~ic cells of foreIgn DNA
which includes genes wh.ich code for proteinaceous materials not associated with selectable phenotypes.
Since such pxoteinaceous materials are characterized by the ~act that ~hey are not associated wlth a selectal:lle phenotype, cells which contain DNA coding the~efore can~ot be identified exceptby destruction o~ the txansformed cell and examînation of its contents.

--L~--4~

Exa~nples- oi~- prote~l~ace~us materials, the ~enes ~or w~ch may ~.~ lnserted into and expressed ~y eucal:yotic cel Ts u~ln~ ~h~ co~rans~ormatio~r 2roce~. .incl ude-~te~eron pr~teiIr,. ~nsu~i~, gro~ ElQrmones, clotti:n~
~ac~ars ,. vi~ral a~ gens,. enzymes ana antibodies _ ough~ some cases tkLe r~ and DNA II may not need ts:
be~ E~ur~ied to: o~n i~tegr~ti o~ ar~d: expression, it is af~ten~es E?ref~ 1e that the; ~N~ h~ ~ur~f~Led:
r~ use i~r cotrans:i~o::ming ceIls ~;uc}~ pur1:f~ica.ti;or limi;ts tke poss,ibilit:~ o~ spurlol1~ re;ult~; due to the E~re5e~ce oi~ co~am~t~ and; ~Ilcrease~ ~e proE~ lTt t~rat c~tr~ncQrmed ceLIs caT~ be içien~ie~C a~d~ sta~ly- :
cuL~re~ Iscr,. ~l~thoug}:L not~ essentiaL,. it i5 somet:~mes dQ iirabl~ that D~ r an~/or DN~ Ir }~av~ beeTr ob.ta~ned~
by ~:estr~cto~ e~3nuc~ease c~ea~ge~ Q~ Ch:l:OmQSOma~ dc:lnOr .
DN~,~ such; a~r ~~ ~E?~e~ restr~c~cln~ endonucleas~-dea~g~ of~ eucæ~ c chromosomal l:)N~, AaditiorsalI~
Lt i5 ~re~ abl~ that DNP~. r and~ D~ Ir ~ treated wi c~lcilmt phos}?hat~ ~rior t~ use ir~ cQtrans~orming euc~y~te: czlI~. ~h~ procedure? ~or so trea~ng~ D~
wi~l caI;cium ph~sphat~ is s~t fort~ r~ fully hereina~ter.
~inaLl~, it is ~refera~l~ thatth¢ forelg~ DNA I be pr_sent durin~ catxansormatio~ in a~ amo~ _ relativ~ t~ DNA II
cc~ding- for a sel ectable E?henotype which constitutes an.
exces~-of the former, suc~ as an amount Ln tle range ~rom about 1:1 to a~out ~00,000:1..

I~ a~ pre~erred e~odiment o:E the ~Yention, the :Eoreign D~ r and/or the foreign DNA I~ are attached to bacterial pLasmid or phage-DNA prior to use i~ cotransforming eucar~otic cel:ls. In a particularly prom~sing emhodiment, oreig~ DNA I and/or DNA II are attached to phage DN~ and the~ encap~idated in phage particles prior to cotransfo~ma~
tion.
.

--~3-- .

ALt~Qugh any DNA. Il codl~q f.or a selectahle phenotypQ
wo.~ ~ use~ul in the^ cotran5form~tion process oi~
esent ~er~tio~,. ~e exE?er~nentaL detai ls~ set ~ort}i: particularly conc~ L ~ u~ af. a gene ~or t~dine kiIIase oh~a:~ned ~rom ELQLpes~ sLmplex virus ;~ Cli~ a ge~e ~ adenLne l?ho~phori~osyL
s~erase~ 3:n a~tio~, a DNA. Ir ~ ch iIIcludes a~ gen~ codislg~ fio~ ~ seLecta;~le ~?heno.type~ assccia~ed LO~ w,i~r- d~ug resista:~ce,. e~_g~, a mllta~t ~yd~:ofoLate reduc:tase gene whic~r renaers. ceIIs~ resis.tarLt to met~trexate greatly exten~. ~ a~?~?licabili~y o:E ~e pwcess:

L~ ccar~nce: ~rl~ a; Preferre~ embodiment,~ ~se co-~si~o~:matio~: i~voLves DN~ :CwhicE~ hy~icaII
chemically urll~ke~ D~IA Ilr a~& the DNP~ r i5 stab.L~
: ~rated: irL~ t}~ c~ro~som~L DNl~ e r~uc:leu~
Q i~e~ cQ~sor~ed~ eucz~ot:~c cell_ za C~ran~orm~L~ioD: i~ ccorda~Lc~ ~ith thi~ i~ventio~
mæ~ rried out iSL any suita~le m~iu~ limited.
or~ly i~ that cotransorme: ce~Is ~ capabI~ o~ survi;vaL
and/or identiication on the medium. Merely b~ way o '~Ç exampLe~ a s~ abLe mediu~ r mou~ f~roblast ceLI~
which ~ave ac~ulred th~ th~midine. kinase ge~e.is EAT
described more ~uLly hereinater.. Also, the cotrans-~ormatio~ is carried ou~ in the presence of selective conditions ~hlch permi t survival andJor ldentiication o those cells which ha~e acquired the selectahle phenotype~ Such conditions may include the presence o~ nutrients, drug or other chemîcal antagonists,.
temperature and th~ like .. ..~

~.~3l8~114~

Eucaryotlc cells cotrans~o~med in ac ordanc~ with this ~n~re~tio~ co~ oreigr~. DNP.. I codin~ ~or desired mæt~i:a-L~ ~:ich can. 1~ reco~er~d ~ro~ the- cells~ usiny 5: tec~ques ~elL know~ ir~ the a:~t~ Ad~tiona:Lly, celI5 can hç~ p~tted~ t~ transcri~L~ DNA I t~ ~
mR~ wh~c}~ ur~ ï~ transIa~e~l ~o. urm l?roteirl or oth:er desl~e~ mater~ which ma~ b~ recovere~ aga~rr }cno~ techniques_ ~nally, the. cells car~
L~ be gro~ ~ culture, har~ested ar~ rQteirI. or other desired~ materi L recovered t3~Ye~rom.
i ~thoug~ t}~e! aesi~ed~ Pr~tei~ce~ materIals; identiied~
herei~ ave are natl;~ maLteria:ls,~ t:he ~rbces~ can: b~
qual.Ly u5eul ~ thë~ E?roduction of ~yntElet~c ~io~oLymers;
cs~i~r ~yn~e~c genes a~e a~ cted_ ~hus ,. th~
i:lsta~ ren tO~S: prcn~i~e~ æ prGces~ ~Qr ~?rod~g~ n~eL
prc~tei~I2s ~sst yet ~: ~xi~terI 1~0~aXlYr ~:t ~?ro~s~e&-~ l~roce5s. ~o~ Esmduc~g~ }2r~t~in5 W~Itch~r altt~oug}~ e~ ~ .
e~t:l;~ existr d~ sa L~ such~ ~t~ c~ es Ol:in: suc~ impure. ~or~r that ~h~ isolati~on and/or Lder~ ca'ci:o~ cannot otherwIse; he~ e~ect:ed~ }?inally, l:he in~ention ~ro~i~es ~k pæocess ~Qr producing. pa~:tially prote-~naceaus products such as t}~e glyco~?r~teins and other 2~ products~ the synthesis o which is~ geneti~cally directed-AnQther aspect o the in~ntio~ Lnvolves processes for Lnserting muLtlple copiec of genes lntD eucaryotic c~lLs in order to increase the am~unt o~ gen~ product formed ~ithin th~ ceIl. On~ prac~s for insexting a multipLicity o~ foreign DNA ~ mole~ules into a eucaryotic celI compri~e~ c~trans~orming ~ cell with multiple DNA I molecules and wi~h multiple.,::unlinked foreign DNA II molecules corresponding ~o m~ltiple copies o an ampliiab1e gene ~or a dominant selectable ~ ' ~

--~5--4~
~?henotyp~ not ot~.erwise expre~sed by the cell . Thi 5 cotransorma~ior~ process LS- carrie~ out in. a su~ta~Le med~u~ and~ presence o~ a~ age~t pe~t~ing 5~ su~v~ val and/~r identi.flcatlo~r o~ ceLIs whicll ac:~uir~
th ~ dom¢nant ~eLectab le ~h en~ typ~ ~ rei~erab ~y ,. ~s.
is~ da~e. ~ e~erLce:. Q:~ suc~essivel~ hïgEler COnCesrt~ atIOn5 oi~ 5UC~ arr agent sc t31at only- thos~
c~lls ac:q~ th~ ~i:ighest nur~er o~ a~lifiabl~
l o d~m~na~t:: genes ~D~. III 5111~1V~ and~c~r ~rQ identified_ se cel~; t~e~ also contairc multil?l~ coE?ies o:E DNA r ~l?E?rC~ac~ ~s ~?articu~ y appropr~te for th~
i~sert:~o~ ~i~;tiE?Ie~ co}~Les o~. amplifia}:~Ie genes w~r;c~
c~ner ~g~ re~tanc~ upc~. ~ cell:r e g r t~e muta~Lt X.5 a~yd~:QfoIa~t~ r~tuctase~ gen~ w~ ender~ c.ells ~:e~i~tarLtt~ m~thotrexate_.

.. . C~trans~ned; eucary~tic celIs wh~ ave~ ac~r~
m~t~?le! ca~lefi o~ D~. I~ ~La~ t}I~; }:-e~ used. tQ p:~:oduceA
2~ L~creased amourlts oi~ t}~ gen~ product ~or ~ic~ Dl~ r ccsdes~ th~ sam~ manner as descri;}:~ed he~:einabove_ ALterna~veLy,. muLti~?le copiei~ o ~oreigr~ genei~ car~ be generated: i~ and ilL~imately expressed by eucaryoti.c cells; b~ t:ra~5 ~,.,m~ t~Le eucaryotic cells, WLth~ D~
mQLe~l e5, each a whic:h has been. formed by ~inking `
a foreig~ D~. T to a foreig~ DN~ r~ which corresponds t~ an~ ampLi~iab1~ gene for a dominant se1ectab1e phenotyp~ ~o~ normaLly expressed by the eucaryotic cell.
3Q T~e Iinkag~ ~etween DNA r and DNA I~ is preferably in the form o~ a chemica1 bond, paxticularly a ~ond formed ac a; resu~t o~ enzymatic trea1:me~t ~1t~. ~ ligase.
Trar~sformatiorL with such hy~:Lrid DN~ molecules so formed is- then carried out in a suitab1e growth medium and in the presence of successîvely elevated concentrations, ~ 9 e.g., amounts ranging from 1:1 to lQ,000:1 on a molari~y ~asis,o~ an agent. wh~ch permits survival and/or identi-ficatio~ o those eucaryotic cells which have acquired S a su~iciently hlg~ num~er of copies o~ the amplifiable ge~e~ Using this approach, eucaryotic cells which have acquired multipl~ copies o~ the ~mpLiXiable gene for a dominan~ s~lectable phenotype not otherwise e~pressed by the cell survive and~or are ldentifiable in the L0 presence o elevated concentratlons of a~ agent comple-mentary to the ampliiab1~ gene which would otherwise result in death or inability to ide~tlfy the celLs:.

ALthou~h ~rario~ ampli~iahle genes ~or dominant selectable lS phenotype~ are us-e~ul in the practices a~ this invention, genes. associated~with drug r~sistance, e.g., the gene far d~hydroolate reductase which renders cells resistant to me~thotrexate, are ~ arLy suitable.

20 By usin~ either o~ th~ tWQ approaches just described, muLtiple coE?ies o~ }?roteinaceous or other desired lecuies ca~ be produced wi~hin eucaryo~ic cells. Thus, for exam~le, muLtiple molecules of lnterferon protein, insulin, growth hormone, clotting factor, viral antigen or antiboay or of interferon per se can be produced by eucaryotic cells, p~i ~ arly mammalian cells, which have been transformed using hybrid DNA or cotransformed.
using purified DNA which has been treated wlth calcium phosphate in the manner described hereinafter. Thus, ~his invention pro~ides a process for producing highly desired, rare and costly proteinaceous and other biological materials in concentrations not ohtainble using conventional techniques.

8~
Still another aspect of the present invention involves the preparation of materials normally produced within eucaryotic cells in minute amounts such as glycoproteins including interferon, which are in part protein but additionally include other chemical species such as suyars, ribonucleic acids, histones and the llke. Although the method or methods by which cells synthesize compli-cated cellular materials such as the glycoproteins are poorly understood, one may by using the process of the presert invention synthesize such materials in commerically useful quantities. Specifically, after inserting a gene or genes for the protein portion of a cellular material such as a glycoprotein, which includes a non-protein portion, into a eucaryotic cell of the type wn~ch normally produces such material, the cell will nok only produce the corresponding proteinaceous material but will utilize already existing cellular mechanisms to process the proteinaceous materials, if and to the extent necessary, and will also add the appropriate non-proteinaceous material to form the complete, biologi-cally active material. Thus, for example, the complete biologically active glycoprotein interferon may be prepared by first synthesizing interferon protein in the manner described and additionally permitting the cell to produce the non-proteinaceous or sugar portion of interferon and to synthesize or assemble true inter-feron therefrom. The interferon so prepared may then be recovered using conventional techniques.

In accordance with the present invention and as described more fully hereinafter, eucaryotic cells have been stably transformed with precisely defined procaryotic and eucaryotic genes for which no selective criteria exist.

The acldition of a purified viral thymidine kinase (t~) gene to mouse cells lacking this enzyme results in the appearance of stable transforma~ts whlch can be selected by ~leir a~ility to grow in HAT medium. Since these biochemical transformants represent a subpopulation of competent cells which are li~ely to integrate other unli~ked genes at fre~uencies higher than the general population, co~ransformation experiments were perormed with the viral tk gene and bac~eriophage ~X174, plasmid pBR 322 or cloned chromosomal human or rabb~ globin gene sequences. T~ transformants ~ere cloned and analy2ed for cotransfer of addi~ional DNA sequences by blot hybridizatIo~. In this manner, mouse cell lines were iden~i~ied which contain multiple copies of ~X, pBR 322, or human and rabbit 3-globin sequences. From one to more than 50 cotransformed se~uences are integrated into high molecular weight DN~ isolated from independent clones. Analysis of subclones demons~rates that the cotransformed DNA is stable through many generations -in culture. This cotransformation syst~m allows the introductlon and stable integration of virtually any defined gene into cultured eucaryotic cells. Ligation to either vira} vectors or selecta~le biochemical mar~ers is not required.

Cotransformation with dominant-acting markers will in principle permit the introduction of virtuaLly anv cloned genetic element into ~ild-type cultured eucaryotic cells. To this end, a dom~nant acting, methotrexate resistant, dihydrofolate reducatse gene from CHO A29 cells was transferred to wild-type cult~red mouse cells.
By demonstrating the presence of CXO DH~R sequences in transformants, definltlve evidence for gene transfer was provided. Exposure of these cells to elevated f ~ --- 19 - ~ %~49 levels of meth.otrexate resul~s in enhanced resistance to this drug, accompanied by amplification of the newly transferred gene. Th.e mutant DHF~ gene, therefore, has been used as a eucaryo~ic vector, by ligating CHO A29 cell DNA to pBR 322 sequences prior to ~ransformation. ~mpli~i-cation of the DHFR sequences results in amplification of the p~R 322 sequences. The use of this gene as a dominant-acting vector in eucaryotic cells will expand the repe.toire o~ potentially transformable cells, no longer restricting these sort of studies to available mutants.

Using the techniques descrlbed, the cloned chromosomal rabbit ~-globin gene has been introduced into mouse fibroblas~s by DNA-mediated gene transfer. The cotrans-formed mouse fibroblast conta~~ning thi.s gene provides a uni~ue opportunity to study the expression and subsequent processing of these sequences in a hetero-20 logous host. Solution hybridization experlments inconcert with RNA blotting techniques indicate that in at least one transformed cell line rabbit globin se~uences are expressed in the cytoplasm as a polyadenylated 9S
species. These 9S sequences result from perfect splicinq 25 and removal of the two intervening sequences. Th.ese results therefor suggest that nonerythroid cells from heterologous species contain the enzymes necessary to correctly process the intervening sequences of a rabbit gene whose expression is usually restricted to erythroid cells. Surprisingly, however, 45 nucleotides present at the 5' terminus o mature rabbit mRNA are absent from the globin mRNA sequence detected in th.e cytoplasm of the transformants examine. These studies indicate the potential value of cotransformation sys~ems in the an.alysis of eucaryotic gene expression. The introduction of wild - 20 ~ 4~

type genes along wi~h native and in vitro constructed mutant genes into cultured cells provides an assay for the functional significance of sequence organization.

Recombinant DNA technology has facilitated the isolation of several higher eucaryotic genes for which hybridization probes are avallable. Genes expressed at exceedingly low levels, with mRNA transcripts present at from one to 20 copies per cell, such as those genes coding for essential meta~olic functions, cannot be simply isolated by conventinal techniques involving construction of cDNA clones and the ultimate screening of recombinant libraries. An alternative approach for the isolation of such rarely expressed genes h~s there~ore been developed utili2ing transormation in concert with a procedure known as plasmid rescue. This schema which is currently underway in the la~oratory is outlined below. The apxt gene of the chic~en is not cleaved by the enzyme, Hin III
or Xba, and transformation of aprt mouse cells with cellular DNA digested with these en2ymes results in the generation of aprt clonies which express the chicken aprt genes. Ligation of Hin III-cleaved chicken DNA with Hin III-cleaved plasmid pBR 322 results in the formation o~ hybrid DNA molecules in which the aprt gene is now adjacent to plasmid sequences. Transformation of aprt cells is now performed with thls DNA. Transformants should contain the aprt gene covalentlv linked to pBR 322 with this entire complex in~egrated into hign molecular weight DNA in the mouse cell. This initial cellular transformation serves to remove the chicken aprt gene from the vast majority of other chic~ sequences. This transformed cell DNA is now treated with an enzyme, Xba I, which does not cleave either pBR 322 or the aprt gene.
The resultant fragments are then circularized with ligase.

-21~ 9~

One such fragment should contain the aprt gene covalently linked to pBR 322 sequences coding for an origin of replication and the ampicillin resistance marker. Trans-fc.rmation of a bacterium such as E. _oli with these circular markers selects for plasmid sequences from eucaryotic DNA which are now linked to chicken aprt sequences. This double selection technique should permit the isolation of genes expressed at low levels in eucaryo-tic cells for which hybridization probes are not readily obtained.
It has become increasingly clear that the introduction of genes coding for highly differentiated functions inl.o the mouse fibroblast which normally does not express these ~unctions results in low level transcription which is either quantitatively or qualitatively inappro~
priate. Examination of the role of specific sequences in controlling gene expression therefore requires intro-duction of genes into more appropriate cellular environ-ments. To this end, one may introduce the human globin genes into cultured murine erythroleukemia cells. This line is particularly amenable, since globin genes are inducible in the presence of DMSO and in the fully induced state represent over 20~ of the total mRNA. In preliminary studies, a number of tk derivatives of MEL
cells were created which facilitated the performance of cotransformation studies with cloned human globin genes.
It has been observed that the tk Friend cell is extremely refractory to transformation with a cloned viral tk gene and can only be transformed at levels from 10 5 to 10 6, the frequency observed in mouse L cells. Nonetheless, this difficulty has been overcome and numerous tk transformants have been obtained which retain and express the donor viral gene. A series of cotransformation / ~ ---22~

experiments have been performed with wild type chromosomal clones containing adult human globin genes and 5-10 kb of 5' and 3' flanking information. A series of mouse cotransformants have been identlfied which now contain 1-5 copies of the human ~ globin gene. A series of experiments were carried out to determine whether this human gene is expressed after induction in these murine cells. Since these heterologous human globin genes can be expressed in this system, a series of exceedingly interesting experiments may be performed utilizing in vitro constructed mutants along with natural mutants of genes derived from thalassemic individuals to determine:
a) the role of flanking 5' and 3' sequences in the induction process; b) the significance of a linked gene arrangement observed in this locus in controlling gene expression; and c) the metabolic nature of the defect in the variety of ~ and ~ thalassemic states.

Thus, one may identify and separately recover a promoter DNA sequence for the globin gene which, in the presence of DMSO, mediates the increased synthesis of RNA coding for globin. A desired gene such as a gene coding for insulin, inter~eron protein or the like may be used in the cotransformation of a eucaryotic cell after being linked with the promoter sequence. Cotransformed cells will then produce in the presence of DMSO markedly elevated levels of the protein for which the gene codes.

FUL thermore, one may combine this approach with the gene amplification approach described hereinabove.
Thus, the promoter sequence, joined to a desired gene, e.g., for an antibody may be used in cotransformation with an amplifiable gene for drug resistance. The cotransformed cells may then be cultured both in the presence of successively elevated concentrations of the -23~

appropriate drug and in the presence of DMSO to produce high levels of protei~aceous material.

Another illustration of an inducible promoter sequence involves the human growth hormone gene. The human growth hormone gene in pituitary cells is tightly contr^~led by levels of thyroid hormone and corticosteroid. The addition of these hormones results in a dramatic inductive effect which at least in part is reflected by an enormous increase in the appearance of translatable mRN~. The molecular mechanisms responsible for hormonal induction, however, remain obscure. Tentative evidence suggests that induction results at leas~ in part from the inter-action of specific hormone receptor complexes with the chromosome which may result in transcriptional activat on.
lS Strong evidence in support of this attractive hypothesis, however, is lacking at present. Transformation may provide the means to discern whether specific sequences reside close to structural genes so as to render these genes responsive to hormone induction. An illustrative system studied in the laboratory involves induction of the human growth hormone gene. Rat pituitary cells, ~H-3, synthesize increasing amounts of rat growth hormone in response to the addition of corticosteroids or thyroid hormone. Therefore, one may introduce the cloned human growth hormone gene into these cells and place it under hormonal control in its new cellular environment. One may then begin to alter this gene, creating a series of deletions of DNA from the 5' and 3' termini of the transcript to discern whether hormonal induction is maintained in these deletion mutants. In this way, one may localize potential sequences which render a gene responsive to hormone action. These sequences may then be genetically transplanted to appropriate loci on ~ "
-24- ~ 9~9 other genes such as globin genes and in this way defini-tively prove the regulatory effect of such sequences on gene expression. Thus, this s~stem provides a promoter which is inducible in the presence of hormone and can be used in cotransformation, with or without ampli~ica-tion, in the manner described hereinabove.

Finally, cotransformation provides an approach to genetictherapy. Specifically, one may therapeutically treat and perhaps cure genetically defective eucaryotic cells in order to alleviate associated symptoms by cotrans-forming the defective cells with DNA which includes a gene for a selectable marker and with DNA which includes a genetically correct gene.

Thus, for example, one may treat human sickle cell anemis by removing the genetically-based defective cells from the patient's bone marrow~ The defective cells may then be cotransformed with a competent globin gene and with a gene conferring drug resistance being used as a marker. The cotransformation is carried out in the presence of the drug. The cotransformed cells may then be transplanted back into the patient, and the patient maintained on dosages of the drug such that only transformed cells survive. Since the cells will be identical in all respects except for the additional gene, the transplanted cells should not be rejected as is the case with transplants using foreign cells. This approach may lead to a cure for sickle cell anemia and for other genetically-based illnesses.
In order to assist in a better understanding of the present invention, the results of various experiments are now set forth.

EXPERI~133NTAL DETAILS
FIRST SERIES OF hXP~RIMENTS
_ The identiIication and ïso~a~ïorA o~ cells trans-for~ed wlth genes ~hich do not code or selectable markers is problematic since current transfo~,ation procedures are highly inefficient. Thus, experiments were undertaken to determine the feas~bility of cotrans-formin~ cells with two physically unlinked genes. In these experiments it was determined that cotransformed cells could be identified and i~olated wh~n one o the genes codes for a selectable mar~er. Viral thymidine kinase gene was used as a selectable marker to isolate mouse cell lines whlch contain the tk gene along with either bacteriphage ~X 1 74, plasmid p~R 322 or cloned rabbit 3-globin gene sequences stably integrated into cellular DNA. The results of these experiments are also set forth in Wigler, M., et al., Cell 16: 777-785 (1~79) and Wold, 3. e~ al., Proc. Nattl. Acad. Sci. 76:
5684-5688 (1979) are as follows:

E erimental Desi n xp The addition of the purified thymidine kinas~ (tk.l gene from herpes simplex virus to mutant mo~e cells lac~ing tk results in the appearance of stable trans~
formants express~ng the viral gene which can be seLec~ed by their ability to grow in HAT. Maltland, N. J. and McDougall J. X. Cell, 11: 233-241 (1977); ~igler, L~. et al., Cell 11: 223-232 (1977). To obtain cotransformants, cultures are exposed to the tX gene Ln the presence of an excess of a well-defined DNA sequence for which hybridiza-tion Drobes are available. Tk transformants are isolated and scored for the cotransfer of addltional DNA sequences by molecular hybridization.

-26~
~2~8~

Cotransformatïon of Mcuse Cells wlth ~174 DNA

~ X174 D~ was ini tially use.d in cotrarLsformation experi--ments with the t~ gene as the selec~able mar~er. ~X
replicative form DNA was cleaved with Pst 1, which recognizes a single site in the circlllar genome. Sanger, F. et al., Natnre 265: 687-695 (lq77~. 500 pg of the purified tk gene were mixed ~ith 1-10. ~g of Pst-cleaved ~X replicative form DNA. Thi:s DNA ~5 then added to mouse Ltk cells using the transformation conditions described under Me~hods and Materials hereinafter.
After 2 weeks in selective medium ~H~T), t~+ tra~sformants were observed at a frequenc~ of ona colony per 10 cells per 20 Pg of purified gene. Clones were pîcked and grown to mass culture.

It was then asked ~he~her tk transformants also contained ~X DNA sequences. ~igh molecular weig~t ~MA ~rom the tran5foxmants was cleaved wlth t~e restriction Pndo-nuclease Eco RI, which recognizes no sites in the ~X genome. The DNA was ~ractionated ~y agarose gel electrophores;s and transferred to nitrocellulose filters, and these ~ilters were the~ annealed ~ith nic~-translated P-~X DNA (blot hy~ izationl. Southern, E. M., J.
Mol. Biol. 98: 503-517 (1975); Botchan, M., et al., Cell 9: 269-287 (1976); Pellicer, A., et al. Cell 14: 133-141 (19781. These annealing experiments d~monstrate that six of the seven transforma~ts had acquired bacteriophage

3~ sequences. Since the ~X genome is not cut by the enzyme Eco RI, the number of bands observed re~lects the mlnimum number of eucaryotlc DNA fragments-containing informa-tion homologous to ~X. m e clones contain variable amounts of ~X sequencesO C}ones ~Xl and ~X2 reveal a single annealing fragment whîch is smaller than the ~X genome. In these clones, therefore, only a portion of the ~ransforming sequences persis~. There -27- :~2~

was al50 observed a t.k~ transformant (clone iX3~ with no detectable ~X sequences. Clones ~X4, 5, 6, and 7 reveal numerous high moleculax weigh.t bands which are too closely spaced to count, indicatïng th.at th.ese clones contain multïple ~X-specific ~ragments. These experiments demonstrate cotransformation of cultured mammalian cells with the viral tk gene and ~X DNA.

Selection i5 Necessar~_to identify ~X Trans~ormants It was next asked whether transformants with ~X DNAjwas restricted to the population of tk cells or whether a significant proportion of the origlnal culture now contained ~X sequences. Cultures were exposed to a mixture o~ the tk gene and ~X DNA in a molar ratio of 1:2000 or 1~20,000. Half of the cultures ~ere plated under selective conditions, while the oth.e.r half were plated in neutral media at low density to facilitate cloning. Both selected (tk ) and unselected (tk ) col-onies were picked, grown into mass culture and scored for the presence of ~X sequences. In this series o experi-ments, eight of the nine tk selected colonies contained phage information. As in the prevlous experiments, the clones contained varying amounts of ~X DNA. In contrast, none of fifteen clones pic~ed at random from neutral medium contained any ~X informatlon. Thus, the addition of a selectable marker facilitates the identification of those cells which contain ~X DNA.
~X Se uences are Inte rated into Cellular DNA

Cleavage of DNA from ~X transformants ~ith Eco RI
generates a series of fragments which contain ~X DNA
sequences. Th.ese ~ragments may reflect multiple inte-gration events. Alternati~ely, these fragments could -28~

result from ~andem arrays of complete ~r partial ~X
sequences which z,re not ln~egrated into cellular DNA.
To distinguish between ~hese possiDllities, transformed cell DNA was cut wïth BAMHI or Eco RI, nelther of which cleaves the ~X genome. If the ~X DNA sequences were not integrated, neither of these enzymes would cleave the ~X fragments. If the ~X D~A sequ~nces were not integrated, neïther of these en~mes would clea~e the ~X fragments. Identical patterns would be generated from undigested DNA and from DNA cleaved with either of these enzymes. If the sequences are integrated, then BAM ~I and Eco RI should recognize different sites in the ~la~ ng cellular 3NA and generate unique restriction pa~te~rns. DNA rom clones ~X4 and ~XS was cleaved with BAM III or Eco RI and analyzed by Southern hy~ridization~
In each inst~nce, the annealing pattern wlth Eco RI
fragments differed from that observed with the B~ HI
fra~ments. Furthermore, the profile obtained with undigested DNA reveals annealing only in very high molecular weight regions with ~o discrete fragments obser~ed. Similar observations ~ere made on clone ~Xl.
Thus, the most of the ~X sequences in these three clones are integrated into cellular DNA.

IntraceLlular Localization of the ~X Sequences , The location of ~X sequences in transformed cells was determined by subcellular fxactionation. Nuclear and cytoplasmic fractio~s was prepared, and the ~X DNA
sequence content of each was assayed by blot hybridization.
The data indicate that 95% of the ~X sequences are located in the nucleus. ~igh and low molecular weight nuclear DNA was prepared by Hirt fractionation. Hirt, B. J., Mol. Biol. 26: 365-369 (1967). Hybridization wi~h DNA
from these two ~ractions indicates that more than ~5% of the ~ information co-purifies with the hi~h molecular - 29~ L8~9 weight DNA raction . Th.e small amour~ of hyhridi zation ol:: served in the supernatant fraction re-Je~' s a profile identical to ~hat of the h~gh molec~lar welgh.t DNA, suggesting conta~nination of this ~ract:ion wi~h high m~le~nl ar weight DNA.

Extent o~ Se uence Re resentation of th.e ~X Genome The annealing profiles of DNA from transfo~ned clones digested w~ih enzymes that do not cleave the ~X
genome provide evidence that i~tegra~^on o~ ~X
sequences has occuxred and allo~ us to estimate th~ number of ~X sequences integrated. Annealing pr~iles of DN~ from transformed clones digested with }S enzymes which cleave within the ~X genome allow us to determlne what proportion of the genome is present a~d ~ow these sequences are arranged following integratio~. Cleavag~ of ~X with th~ e~zyme ~pa I
generates three ~ragments for each i~egratio~ event:
two "internal" fragments o:l~ 3. ~ and 1.3 kb which together comprise 90% of the ~X genome, and one "bridge"
~ra~ment of O.5 kb which spans the Pst I cleavage site. In the annealing profile observed when clone ~X4 is digested with Hpa I, ~wo intense bands are obser~ed at 30 7 and 1.3 ~b. A less intense series of bands of higher molecular weight is also obser~ed, som~
of which probably represent ~X sequences adjacent to cell-ulax DNA. These results indicate that at least 90% of the ~X genome is present in these cells. It is worth noting that th~ internal 1.3 kb Hpa I fragment is bounded by an Hpa I site only 30 bp from the Pst I cle vage site Comparison o the intensities of th~ ~nternal bands with known quantities of ~pa I-cleaved ~X DNA suggests that this clone contains approximately 100 copïes of the ~X
genome. The annealing pattern of clone 5 D~A cleaved with Hpa 1 ls more complex. If Lnternal fragments are pr~sent, ~hey are markedly reduced in inte~ity; lnstead, multiple bands of varying molecular weight are.obse~ed. The 0.5 kb Hpa I fragment which brïdges the Pst 1 cleavage s.ite is not ohs~rved for e~ther clone ~X4 or clone ~X5.

A similar analysis of clone ~X4 and ~X5 was performed with the enzyme Hpa II. This enzyme cleaves the ~X genome five times, thus senerating four "internal"
fragments of 1.7, 0.5/ 0.5 and 0.2 kb, and a 2.6 ~b "bridge" fragment which spans the Pst I cleavage site.
The annealing patterns for Hpa II-cle~ved DNA from ~X
clones 4 and 5 each show an intense 1.7 kb band, consistent with the retention of at least two internal Hpa II sites.
qhe a .5 kb internal fragme~tscan also be observed, but they are not sh~wn on this gel. Many additional fragments, mostly of high molecular weight, are also present in each clone. These presumably reflect the multiple inte-gration sites of ~X DNA in the cellular genome. The2.6 kb fragment bridging the Pst I cleavage site, however, is absent from clone ~X4. Reduced amounts of annealing fragments which co-migrate with the 2.6 kb Hpa II bridge fragment are obser~ed in clone ~X5.
Similar observations were made in experiments with the enzyme Hae III. The annealing pattern of Hae III-digested DNA from these clones was determined. In accord with previous data, the 0.87 kb Hae III bridge fragment spanning the Pst site is absent or present in reduced amount in transformed cell DNA. Thus, in general, I'internal'' fragments of ~ are found in th.ese transfor-mants, while "bridge" fragments which span the Pst I
cleavage site are re.duced or absent.

-31~ 9 Stability of the Transformed Genotype Previous observations on ~he transfer of selectable biochemical markers indicate that the transformed pheno-type remains stable for hundreds of generations if cells are maintained under selective pressure If maintalned in neutral medium, the transformed phenotype is lost at frequencies which range from 0.1 to as high as 30 per generation. Wigler, M~, et ~., Cell 11: 223-232 ~1977); Wigler, M. et ~., PNAS 75: 5684~5688 (1979).
The U52 of transformation to study the expression of foreign genes depends upon the stability of the transformed genotype. This is an important consideration with ~enes ~or which no selective criteria are available.
It was assumed that ~e presence of ~X DNA in transformants co~ers no selective advantage on the recipient cell.
Therefore, the stability of the ~X genotype was examined in the descendants of two clones after numexous generations in culture. Clone ~X4 and ~X5, both containing multiple-copies of ~X D~A, were subcloned and six independent subclones from each clone were pic~ed and grown into mass culture. DNA from each of these subclones from each original clone were picked and grown into mass culture. DMA from each of these subclones was then digested with either Eco RI or Hpa I, and the annealing profiles of ~X-containing fragmen~s were compared with those of the orlginal parental clone.
The annealing pattern observed for four of the six ~X4 subclones is virtually identical to that of the parent. In t~o subclones, an additional Eco RI fragment appeared which is of identical molecular weight in both.
This may have resulted from genotypic heterogeneity in the parental clone prior to subcloning. The patterns obtained for the subclones of ~,X5 are again virtually identical to the parental annealing profile. These data indica~e that ~X DNA is maintained within the ten subclones examined for numerous generations without sianificant loss or translocation or information.

Integration of pBR322 into ~ouse Cells The observations in cotransformation have been extended to the EK2-approved bacterial vector, plasmid pBR322.
pBR322 linearized with BAM HI was mixed with the purified viral t~ gene in a molar ratio of 1000:1. T~ trans-formants were selected and scored for the presence of pBR322 sequences. Cleavage of BAM HI linearized pBR322 DNA with Bgl I generates two internal fragments of 2.4 and 0.3 kb. The sequence content o~ the pBR322 transformants was determined by digestion of transformed cell DNA with Bgl I followed by annealing with 32P-labeled plasmid DNA. Four of five clones screened contained the 2.4 kb internal fragment. The 0.3 kb fragment would not be detected on ~hese gels.
From the intensity of the 2.4 kb band ïn comparison with controls, we conclude that multiple copies of this fragment are present in these transformants. Other bands are observed which presum~bly represent the segments of pBR322 attached to cellular DNA.

Transformation of Mouse Cells with the Rabbit 3-Globin Gene_ _ _ Transformation with purified eucaryotic genes may provide a means for studying the expression of cloned genes in a heterologous host. Cotransformation experiments were therefore performed with the rabbit~ major globin sene which was isolated from a cloned library of rabbit chromosomal DNA (Maniatis, T., et al., Cell 15: 687-701 (1978). One 3-globin clone designated R~G-l consists of a 13 kb rabbit DlaA fragment carried on the bacteriQ~hage cloning vectors Chazon 4a. Intact from thi~ clone (R~-l) wa~ mixed with the viral tk D~aA at a molar ratio of 100~1, and tk~ transformants were isolated and 5 examined for the presence of rabbit globin sequences~
Cleavage of R~G-l with the enzyaTe Kpn I generates a 4.7 kb fragment which contain~; the entire rabbit ~-globin gene. This fragment was purified by gel electrophore-~is and nicktranslated to generate a probe f or subse-lo quent anneal ing experiments. The ~globin genes ofmouse and rabbit are partially homologous, although we do not observe anneal ing of the rabbit ~globin probe with Kpn-cleaved mouse DNA under our experimenkal con-dition~. In contrast, cleavage of rabbit 1 iver DNA
15 with Rpn I generate~ the expected 4 . 7 kb globin band.
Cleavage of transformed cell DNA with the enzyme Rpn I
generates a 4.7 kb frag~aen~ con~aining globin-speci~ic information in six of the eight tk+ transformants exam-ined. In two of the clones, additional rabbit ylobin 20 bands are observed which probably resul;;* fro~ the loss of at least one of the gpn sites during transforma-tion~ The number of rabbit globin genes integrated in these transformants is variable. In comparison with controls, BOI112 clones contain a single co~y o the 25 gene, while others contain multiple copies of th;s heterologous gene. These result~ demonætrate that cloned eucaryotic genes can be introduced into cultured mammal i an cel 1 s by cotr an sf orm at i on .

30 Tran~f~ tion Competence is ~ot Stably Inh~ ed Our data suggest ~he existence of a subpopulation of 'cransformation-competent cells within the total cell population. If competence is a ~tably inherited trait, 35 then cells selected for transformation should be better recipient~ in ~ub~equent gene tran6fer experiment~ than ~2~89~

their parental celis. ~wo results indicate that as in procaryotes, competence is not sta~ly heritable. In the first series of experiments, a double mutant, Ltk aprt ( deficient in both tk and aprt), was transformed to either the tk~ aprt or the tk aprt phe~otype using cellular DNA as donor. Wigler, M. et al., Cell 14: 725-731 (1978); Wigler, M. et al., PNAS 76: 5684-5688 (1979). These clones were then transformed to the tk+ aprt+ phenotype. The frequency of the second transformation was not significantly higher than the ~irst. In another series of experiments, clones ~X4 and ~XS were used as recipients for the transfer of a mutan~ ~oLate reductase gene whIch renders recipient cells resistant ~o methotrexate (mtx). The cell line A29 Mtx I contains a mutation in the structural gene for dihydrofolate reductase, reducIng the affinity of this enzyme for methotrexate. Flintoff, W. F~ et al., Somatic Cell Genetic 2: 245-261 (lg76). Genomic DNA
from this line was used to transform clones ~X4 and ~XS
and Ltk cells. The frequency of transformation to mtx resistance for the ~X clones ~as identical to that observed with the parental Lt~ cells. It is therefore concluded that competence is not a stably heritable trait and may be a transient property of cells.

Discusslon In these studies, we have stably transformed mammalian cells with precisely defined procaryotic and eucaryotic genes for which no selective crlteria exist. Our chosen design derives from studies of transformation in bacteria which indicate that a small but selectable subpopulation of cells is competent in transformation. Thomas, R.
Biochim. Biophys. Acta 18: 467~481 (1955); Hotchkiss, R.

PNAS 40: 49-55 (195~); Tho~lasz, A. and Hotchkiss R.
PNAS 51: 480-487 (1464); Spizizen, J. et al~, Ann Rev. Microbiol. _: 371-400 (1966). If this is also true for animal cells, then biochemical transformants will represent a subpopulation of competent cells which are likely to integrate other unlinked genes at freque~cies higher than the general population. Thus, to identify trans~ormants containl~g genes which provide no selectable trai~, cultures were cotransformed with a physically unlinked gene which provided a selectable marker. This cotransformation system sh.ould allow the introduction and stable integration of virtually any deined gene into cultured cells. Ligation to either viral vectors or selectable biochemical markers is not required.

Cotransformation experiments were performed using the HSV tk gene as the selectable biochemical ~arker. The addition of this purified ~k gene to mouse cells lacking thymidine kinase results in the appearance of'~table transformants which can be selected by their ability to grow in HATo Tk transformants were cloned and analyzed by blot hybridization for cotransfer of additional DNA
sequences. In thls manner, we have constructed mouse cell lines which contain multiple copies of ~X, pBR322 and rabbit 3-globin gene sequences.

The suggestion that these observations could result from contaminating procaryotic cells in our cultures is highly improbable. At least one of the rabbit ~-globin mouse transformants expresses polyadenylated rabbit ~-globin RNA
sequences as a discrete ~S cytoplasmic species. The elaborate processing events required to generate 9S globin RNA correctly are unlikely to occur in procarvotes.

The ~X cotran~formants were ~tudie~ in greate~t detailO
The frequency o~ co~ransforma~ion is high: 14 of 16 tk~
tra~formants contain ~X ~equen oe ~ e ~X ~equen oe ~ are integrated into high molecular weight nuclear DNA. The 5 number of integration events varies from one to more than fifty in independent clones. ~he extent of the bacteriophage genome present within a given transformant i~ also variable; while some clones have lo~t up to half the genome, other clones contain over 90~ of the ~X
L0 sequen oe s. Analysis of ~ubclones demonstrates that the ~X
genotype i~ stable through many generations in culture.
Similar conclusions are emerging from the characteriza-tion of the pBR322 and globin gene cotransformants.

~5 Hybridization analysis of re~triction endonuclease-cleaved transformed cell DNA allows one to make 6ame prel~minary ~tate~nts on the nature o~ the integration intermediate.
Only two ~X clones have been examined in detail. In both clones; the donor DNA was Pst I-linearized ~X DNA. At-20 tempts were made to distinguish be ween the integration ofa linear or ~ircular intermediate. ~f either precise circularization or the formation of linear concatamers had occurred at the Pst ~ cleavage site, a~d if inteyration occurred at random point~ along thi~ DN~, one would expect 2s cleavage maps of transformed cell DNA to mirror the circu-lar ~X map. The bridge ~ragment, however~ i8 not ob~erved or is present in reduced amounts in digests of transformed cell DMA with three different restriction endonucleases.
The fragments observed are in accord with a model in which ~X DNA integrates as a linear molecule. Alterna-tively, it is possiU e that intramolecular recombination of ~X DNA occurs, resulting in circularization with dele-tions at the P~t termini. Lai, C. J. and Nathans, D. Cold Spring ~arbor Symp. Quant. BiolO 3g: 53-60 (lg74).

-37- ~
~2~

Random integration of this circular molecuie would generate a restriction map similar to th~t ob.served for clones ~X4 and ~5. Other more complex models of even~s occurring be ore, during or after integration can also be considered.
Although variable amounts of DNA may be delet~d from termini during transformation, most copies of integrated ~X sequences in clone ~X4 retain the ~pa I site, which is only 30 bp from the Pst I cleavage sLte. Whatever the mode of integrat-on, it appears that cells can be stably transformed with long stretches of donor DNA. Transformants have been observed containing continuous stretches of donor DNA 50 kb long.

There have been attempts to identify cells transfoxmed with ~X sequences in the absence of selective pressure.
Cultures were exposed to ~X and tk DNA and cells were cloned under nonselective conditions. ~X sequences were absent from all fifteen clones picked. In contrast, 14 of 16 clones selected for the tk+ phenotype con~ained ~X DNA. The simplest interpretation is that a subpopula-tion of cells within the culture is competent in the uptake and integration of DNA. In this subpopulation of cells, two physically unlinked genes can be introduced into the same cell with high frequency. At present one can only speculate on the biological basis of competence.
Competent cells may be genetic variants within the culture; however, these studies indicate that the competen~
phenotype is not stably inh.erited. If one can extrapolate from studies in procaryotes, th.e phenomenon of competence is lik.ely to ~e a complex and transient property reflecting the metabolic state of the cell.

Cotransformants contain at least one copy of the t~ gene and variable amounts of ~X DNA. Although transformation was performed with ~X and tk sequences at a molar ratio of {~ ~

1000:1, the sequence ratio o~served ïn the transformants never exceeded la o: 1 . There may be an upper .Limit to the number of ïntegraticn events that a cell can tolerate, beyond which lethal mutations occur. Alternatively, it i5 possible that the efficiency of transformation may depend upon ~he nature of the transforming fragment.
The t~ gene may therefore represent a more efficient transforming agent than phage DNA.
In other studies there has been demonstrated the co-transfer of plas~id pBR322 DNA into Lt~ aprt cells using aprt cellular DNA as donor and apxt as selectable marker. Furthermore, the use of domilldnt acting mutant lS genes which can confer drug resistance will extend the host range ~or cotransformation to virtually any cultured cell.

The stable transfer of ~X 3N~ sequences to mammalian cells serves as a model system for the introduction of defined genes for which no selectlve criteria exist.
The tk cotransformation system has been used to transform cells with the bacterial plasmid pBR322 and the cloned rabbit 3-globin gene. Experiments which indicate that several of the pBR transformants contain an uninterrupted se~uence which includes the re~licative origin and the gene coding for ampicillin resistance (~-lactamase), suggest that DNA from pBR transformants may transfer ampicillin resistance to E. coli. Although preliminary, these studies indicate the potential value of cotrans-formation in the analysis of eucaryotic gene expression.

SECOND SERI~:S OF E~PERIMEN~S

Cotransformed mouse fibroblasts containing the rabbi~
3-globin gene provide an opportunity to study the expression and subse~uent processing of these sequences in a heterologous host. In these experiments, we demonstrate the exp~ession of the transformed rabbit B-globin gene generating a discrete polyadenylated 9S
species of globin RNA. This RNA results from correct processing of both intervening sequences, but lacks approximately 48 nucleotides present at the 5' terminus of mature rabbit ~-globin mRNA

Transorma~ion of Mouse Cells with the Ra~bit 3-Globin Gene ._ _ We have performed cotransformation experiments with the chromosomal adult rabbit ~-qlobin gene, using the purified herpes virus t~ gene as a biochemical marker. The addition of the tk gene to mutant Ltk mouse fibroblasts results in the appearance of stable transformants that can be selected by their ability to g~ow in hypoxan~hine/
aminopterin/thymidine (HA~) medium. Cells were cotrans-formed with a ~-globin gene cl~ designated ~ Gl, which consists of a 15.5-kbp insert of rabbit DNA carried in the bacteriophage ~cloning vector Charon 4A. The purified tk gene was mixed with a 100-fold molar excess of intact recombinant DNA from clone ~ ~ his DNA was then exposed to mouse Ltk cells under transformation conditions described herein under Me~ods and Materials. After 2 weeks in selective medium, tk transformants were obser~ed at a frequency of one colony per lQ6 cells per 20 pg of tk gene. Clones were picked and grown into mass culture.

It was then asked if the tk+ transformants also contain rabbit 3-globin sequences~ High molecular weight 8~9 DNA from elght transforman~s was cle~ved with the restric-tlon endonuclease Knp I. The D~i~ ~as fractionated by agarose gel electrophoresis and transferred to nitocellulose filters, and these filters were then annealed wi~h nic~-translated globin [32p] DNA hlot hybridization. Southern, E. M., J. Mol. Biol. 98: 503-517 (1975). Cleavage of this recombinant phage with the en~yme ~pn I generates a 4O7-kpb fragment that contains the entire adult 3-globin gene, along with 1.4 kbp of Sl flankïng information and Z.O kbp of 3' flanking information. This ~ragment was pt~ified by gel electrophoresis and nick translated to generate a hybridizatior. probe. Blot ~ybridization experimsnts showed that ~he 4.7-k~p Kpn I fragment containing the globin gene was present in the DMA of six of the eight t~+ trans~ormants. In ~hree of the clones additional rabbit globin bands ~ere obser~ed, which probably resulted from the loss of at least one of the Kpn I sites during transformation. The number of rabbit globin genes integrated in these transformant~
was variable: some cLones containeda single copy of the gene, whereas others contained up to 20 copies of the heterologous gene. It should be noted that the ~-globin genes of m~use and rabbit are paxtially homologous.
However, we do not observe hybridization of the rabbit ~-globin probe to Kpn-cleaved mouse DNA, presumably because Kpn cleaveage of mouse DNA leaves the ~-gene cluster in exceedingly high molecular weight fragments not readilv detected in these experiments. These results demonstrate the introduction of the cloned chromosomal rabbit B-globin transfer.

Rabbit ~-Globin Sequences are Tran~cribed in Mouse Transformants The cotransformation system we have developed may provide a functional assay for cloned eucaryotic genes ~2~ 9 if these genes are expressed in ~he heterologous recipient cell. SLX transformed cell clones were therefore analyzed for the presence of rabhit ~-globin RNA sequences. In initial experLments, solution hybridization reactions were performed to determine the cellular concentration of rabbit glo~in transcript, in our transformants. A
radioactive cDNA copy of purified rabbit ~- and ~-globin mRNA was annealed with the vast excess of cellular RNA.
Because homology exists between the mouse and rabbit globln sequences, it was necessary to detenmine experimental conditions such that the rabbit globin cDNAs did not form stable hybrids with mouse globin mRNA ~ut did react completely with hom~logdus rabbit se~uences. At 75C in the presence of 0.4 M NaCl, over 80% hybridiza~ion was observed with the rabbit globin mRN~, whereas the heterologous reaction with purified mouse globin mRNA
did not exceed 10% hybridi2ation. The Rotl/2 of the homologous hybridization reactlon was 6 x 10-4, a ~alue consistent with a complexity of 1250 nucleotides con-tributed by the ~- plus 3-globin sequences in our cDNA
probe. Axel, R., e~ al., Cell 7: 247-254 (~976).

This rabbit globin cDNA was used as a probe in hybridization reactions, with total RNA isolated from six transfor~ed ~5 cell lines. Total RNA from transfor~ed clone 6 protected 44~ of the rabbit cDNA at completion, the value expected if only ~-gene transcripts were present. This reaction displayed pseudo-first-order kinetics with Rotl/2 of 2 x 10 . A second transformant reacted with an Rotl/2 of 8 x 103. No significant hybridization was observed at ROts ' 10 with total RNA preparations from the four additional transformants.

We have characterized the RN~ from clone 6 in greatest detail. RNA from this transformant was fractionated into nuclear and cytoplas~ic populations to determine f ~ `~

~21~39~3 the intracellular localization of the rabbit globin RNA. The cytoplas.mic RNA was ~urther fractionated by oligo (dT)-cellulose chromatography into poly (A)+
and poly (A) RNA. Poly (~)+ cytoplasmic RNA from clone 6 hybridizes with the rabbit cDNA with an Rotl/2 of 25. This value is 1/8~th of the Rotl/2 observed with total cellular RNA, consistent with the observation that poly (A)~ cytoplasmic RNA is 1-2~ of the total RNA in a mouse cell. ~ybridization is not detectable with either nuclear RNA or cytoplasmic poly (A) RNA at Rot values of 1 x 104 and 2 x 104, respectively.
The steady-state concentration of rabbit 3-globïn RNA
present in our transformant can be calculated from the Rotl/2 to be about ~ive copies per cell, with greater than 90~ localized in the cytoplasm.

Several independent experiments argue that the globin RNA
detected derives from transcriptïon of the rabbit DNA
20 sequences present in this transformant~ cDNA was prèpared from purified 9S mouse globin RNA. ~his cDNA does not hybridize with poly (A) ~NA from clone 6 at Rot values at which the reaction with rabbit globin cDNA _ complete (ii) Rabbit globin cDNA does not hybridize with total cellular ~NA obtained with t~ globin transformants at Rot vlaues exceeding 10 .
(iii) The hybridization observed doe.s not result from duplex formatlon with rabbit globi.n DNA ~ossibly contamin-ating the RNA preparations. Rabbit cDNA was annealed with 30 total cellular RNA from clone 6, the reaction product was treated with Sl nucelase, and the duplex was subjected to e~ulllbriu~ density centrifugation in cesium sulfate under conditions that separate DNA-RNA hybrids from duplex DNA The Sl-resistant cDNA banded at a densïty of 1.54 g/ml, as expected for DNA-RNA hybrid structure.s. These data, along with the observation that globin RNA is poly-r~

- 4 3--31.23l~ Lg ad~nylated, demonstrate that the hybridization ohserv~d with RNA preparations does not result from contaminating DNA sequences.

Characterization of Rabbit Globin Transcripts in Transformed Cells In rabbit erythroblast nuclei, ~he 3-globin gene sequences are detected as a 14S precursor RNA that reflects transcription o two in~ervening sequences that are subsequently removed from this molecule to generate a 9S
mess2..ger RNA. It was therefore of interest to determine whe ~er the globin transcrlpts detected exist at a discrete 1~ 9S species, which is likely to reflect appropriate splicing of the rabbit gene transcript by the mouse fibroblast. Cytoplasmic poly (A)-containing RNA from clone 6 was electrophoresed on a methyl-mercury/agarose gel, Bailey, J. & Davidson, N., Anal. Biochem. 70: 75-85 (1976), and transferred to diazotized cellulose paper.
Alwine, J. C. et al., Proc. Natl. Acad. Sci. USA 74:
5340-5454 ~1977). After transfer, the RNA on the ~ilters was hybridized ~ith~N~ from the plasmid p~,l, which contair~ rabbit ~~globin cDNA sequences. Maniatis, T., et al., Cell 8: 163-182 (1976). Using this 32P-labeled probe, a discrete 9S species of RNA was observed in the cytoplasm of the transformant, which o~sra ~ with rabbit globin mRNA isolated from rabbit erythroblasts.
Hybridization to 9S RNA species was not observed in parallel lanes containing either purifïed mouse 9S globin RNA or poly (A)-containing cytoplasmic RNA from a t~
transformant containing no rabbit globin genes.

In these experiments, it was not possible to detect the presence of a 14S precursor in nuclear RNA pop-ulations from the transformants. This is nct surprising, because the levels expected in nuclear RNA, '~ "

-44- ~2~

given tne observed cytoplasmic conc~ntration, are likely _o be be]ow the limits of detection of this techniques.
The S' and 3' ~oundaries of the rabhit globin sequences expressed ïn transformed fibroblasts along with ~e internal processing si~es can ~e defined more accurately by hybridizing this RNA with cloned DNAs, followed by Sl nuclease digestion and subsequent gel analysis of the ~NA products. Ber~, A. J. & Sharp, P. A.,Cell 12:
721-732 (1977). When ~-globin mRN~ fror rabbit ery-~roid cells was hy~ridized with cDNA clone p 3Gl under appropriate conditions, the entire 576-~ase pair insert of cDNA was protected from Sl nuclease attac~. When the cDNA clone was hybrldized with RNA from our transformant, surprisingly, a discrete D~A band was observed at 525 base pairs, but n~t at 576 base pairs. These results suggest that, in this transformant, rabbit globin RN~ molecules are present that have a deletion in a portion of the globin m~NA sequence at the 5' or 3' termini. To distinguish between these possibilities, DNA of the ~ clone, R ~Gl, containing the chromosomal rabbit ~ globin sequence hybridized with transformed fibroblast RNA. The hybrid formed was treated with Sl nuclease, and the protected DNA fragments were analyzed by alkaline agarose gel electrophoresis and identified by Southern blotting pro-~_~ures. Southern, E. ~., J. Mol. Biol. 98: 503-517 (1975). Because the rabbit 3-globin gene is interrupted by two intervening sequences, the hybridization of mature rabbit mRNA to ~ Gl DNA generates three DN~ fragments in this sort of analysis: a 146-base pair fragment spanning the 5' terminus to the junction of the small inter~ening sequence, a 222-base pair internal fragment bridging the small and large interveni~g se~uences, and a 221-~ase pair fragment spanning the 3' junction of the large intervening sequence to the 3' terminus of the mRNA molecule. When transformant RNA was analyzed in this fashion, a 222-base -~ 5~ 9~L9 pair rraglnen~ WlS observed as well as an aberrant fragment of 100 blse pairs ~u~ nc 1~6-base pair fragment.
~ybridization with a specific 5' probe showed that the internal 222 base pair fragment was present. The sum of the protected le~gths eaualed the length of the DNA
fragment protected by using the cDNA clone. Taken together, these results indicate that although the intervening sequences expressed in transformed mouse fibro~last are removed from the RNA transcripts precisely, the 5' termini of the cytoplasmic transcripts observed do not contain about 48+ 5 nucleotides present in mature 9S RNA of rabbit erythro~lasts.
DISCUSSION

In these studies, mouse cell lines have been constructed that contain the rabbit 3-globin gene. The ability of the mouse fibroblast recipient to transcribe and process this heterologous gene has then been analyzed. Solution hy-bridization experiments in concert with RNA blotting techniques indicate that, in at least one transformed cell line, ra~bit globin sequences are expressed in the cyto-plasm as a polyadenylylated 9S species. Correct processing of the rabbit ~-globin gene has also been observed in tk mouse cell transformants in which the globin and tk plasmids have been ligated prior to transformation. Mantei, N., et al., Nature (London) 281: 40-46 (1970). Similar results have been obtained by using a viral vector to introduce the rabbit globin gene into monkey cells. Hamer, D.H. & Leder, P., Nature ~London), 281: 35-39 (1979); Mulligan, R.C., et al., Nature (London) 277~ 108-114 (1979). Taken together, these results suggest that nonerythroid cells from hetero-logous species contain the enz-mes necessary to correctly process the intervening sequences of a rabbit gene whose expression usually is restricted to erythroid cells.

-46- ~2~8~

The level of exprassion of rabblt globin sequences in th2 S transformant is low: five sopies of globin RNA are present in the cytoplasm of each cell. The results indicate that the two interveninrg se~uences present in the original globin transcript are processed and removed at loci in-distinguishable from those observed in rabbit erythroid cells. Surprisingly, 45 nucleotides present at the 5' terminus of mature rabbit mRNA are absent from the 3-globin RNA sequence detectRd in the cytoplasm of the trans-forman~ examined. It is-~ossible that incorrect initiation of transcrip~ion occurs about the globin gene in this mouse cell line. Alternatively, the globin sequences detected may result from transcription of a long precursor that ul-timately must undergo 5 r processing to generate the mature ~5 species. rncorrect processing a~ the 5' terminus in the mouse ~ibroblast could be responsible ~or the results. At present, it is difficult to distinguish among these alterna-tives. Because the analysis is restricted to a single trans-formant, it is not known whether these observations are common to all transformants expressing the globin gene or reflect a rare, but in~eresting abberation. It shou}d be noted, however, that in similar experiments by Weissman and his colleagues, Mantei, N., et al., Nature (London) 281:
40-46 (1979), at least a portion of the rabbit globin RNA
molecules transcribed in transformed mouse fibroblasts re~
tain the correct 5 r terminus~
Several alternati~e explanations can be offered for the expression o~ globin sequences in transformed fibroblasts.
It i~ possible that constitutive synthesis of globin RNA
occurs in cultured fibro~lasts, ~umphries, S., et al., Cell 7: 267-277 (1976), at levels five to six orders of magni-tude below the level o~served in erythroblasts. The intro-duction of 20 additional globin DNA templates may simply increase this constitutive transcription to the levels ob-served in the transformant. Alternativaly, it is possible -4 7- ~ Z18~

that the homologous globin gene is repressed by factors S that are partially overcome by a gene dosage effect pro-vided by the introduction of 20 additional globin genes.
Finally, normal repression o~ ~he globin gene in a fibro-blast may depend upon the position o~ these se~uences in the chromosome. At leas~ some of the newly introduced genes are likely ~o reside at loci distant from the resident mouse globin genes. Some of these ectopic sites may support low level transcription. Present data do not permit one to distïnguish among these and other alternatives.

Although the number o~ rabbit globin genes within a given transformant remains Istable fo~ over a hundred generations o culture in hypoxanthine/aminopterin/thymidine, it has not been possible ~o prove that these sequences are covalently integrated into recipien~ cell DN~. In previous studies, however, it has ~een demonstrated that cotransformation of either ~X174 or plasmid pBR322 results in the stable in-tegration of these sequences into high molecular nuclear DNA. In the present study, the globin gene represents a small internal segment of the ~igh molecular weight con-catenated phage DNA used in the transformation. Analysis of integration sites covalently linked to donor DNA istherefore difficult. Preliminary studies using radioactive ~ sequences as a probe in DNA blotting experiments indicate that, in some cell lines, a contiguous stretch of recom-3~ binant phage DNA with a mïnimum length of 50 kbp has beenintroduced.

The presence of 9S glo~ïn RNA in the cytoplasm of trans-formants suggests that this RNA may be translated to give rabbit ~-globin polypeptide. Attempts to detect this pro-tein in cell lysates using a purified antï-rabbit ~-globin anti~ody have t~us fax been unsuccess ul. It is possible that the glo~ïn RNAs in t~e transformant are not translated or are translated with very low efficiency due to the ab-senoe of a functionnal ribo~anal bind:ing ~;ite. q~he cyto-pla~mic globin tran~cripts in ~che tr~nsformant lack about 48 nucleo~ide~ of untran~lated 5' ~equence, which include~
13 nucleotide~ wn to interact with the 40S ribo~omal

5 subunit in nuclease protection ~;~cuclie~. Efstratiadi~, A., et ~1., Cell ~ 571-585 (1977); I.egon, S,, J. Mol.
Biol. ~.: 37-53 (1976). Even if translation did occur with normal ef f iciency, it is pr obable that the pr o~ein would exi~t at level~; bel~w the limit~ of detectio31 of the 10 immunologic as~ay due to t~e lcw level of globîn RNA~ and the observation that the hal f 1 if e of ~ -globin in the absence of heme and 91 obin may be le~ than 30 min.
Mulligan~ R.C., et al., ~ature tLondon) 217: 108 114 (1979) .

These studies indicate the potential value o cotransfor-mation systems in ~he analysi~ of eucaryotic gene expre~-sion. The in~roduction of wild-type genes along with native and ~ vitro-constructed mutant genes into cultured 20 cells provides an assay for the functional signif icance of ~equence organization. It is obvious ~roan these studies that this analysis will be facilitated by the ability to extend the generality of cotransformation to recipient cell lines, such a~ murine erythroleukemia oell6, that 2s provide a more appropriate environment for the ~tudy of heterologou~ globin gene expression.

q~TRD SERIES ~F EXPERI MENT~

30 The cotransformation expperiments involving transformation of mouse cell~ with rabbit ,B-globin and with plasmid pBR322 and ~X-174 DNA were con~inued and extended with the follawing result~.

. ~

~8~9 ~X D~A wa~ used in cotransformatioIl e~periments with the tk gelle a~ the selectable marker. ~X repl icatiYe form DNA
was cleaved with Pst I, which r~cognizes a single site in the circular genome, Sanger, F. e~ alO, Nature ~:687-695 ~L2~L~9~3 (1977). Purified tk gene (500 pg) was mixed with l~lO ~g S of Pst-cleaved ~X replicative form DNA. This DNA was then added to mouse Ltk cells using the transformation condi-tions described herein and in Wigler, M., et al., Cell 16:777-785 (1979). After two weeks in selective medium (HAT), tk+ transformants were observed at a frequency of one colony per 106 cells per 20 pg of purified gene.
Clones were picked and grown into mass culture.

It was then asked whether tk+ transformants contained ~X DNA sequences. Hiqh molecular weight DNA from the transformants was clea~ed with the restric~ion e~do-nucLease Eco RI, which recognlzes no sites in the ~X
genome. The DNA was fractionated by agarose gel electro-phoresis and transferred to nitrocellulose filters, and these filters were then annealed with ~ick-translated 32 ~X DNA (blot hybrid;zation).

These annealing experiments indicated that 15 o 16 transformants acquïred ~acteriophage sequences. Since the ~X genome is not cut with the enzyme Eco RI, the number of bands obser~ed reflects the minimum num~er of eucaryotic DNA fra~ments containing information homologous to ~X.
The clones contain variable amounts of ~X sequences: 4 of the 15 positive clones reveal only a single annealing frag-ment while others reveal at least fifty ~X-specific fragments.
It should be noted that none of 15 clones picked at random from neutral medium, following exposure to tk and ~X DNA, contain ~X information. Transformation with ~X therefore is restricted to a subpopulation of tk transformants. The addition of a selecta~le mar~er therefore facilitates the identification of cotransformants.

Transforma~ion of Mouse Cells with the Ra~bit ~-Glo~in Gene Transformation with purified eucaryotic genes provides a means for studying the expression of cloned genes in a heterologous host. Cotransformation experiments wexe per-ormed with the rabbit B major globin gene which was iso-lated from a cloned library of rabbit chromosomal DNA.One ~-globin clone, designated R G-l consists of a 15 kb rabbit DNA fragment carried on the bacteriophage ~ cloning vector Charon 4A. Intact DNA from this clone (R~G-l) was mixed with ~he viral tk DNA at a molar ratio o 100:1, ~S and t~ transformants were isolated and examined for the prese~ce of rabbit globln sequences Cleavag~ o~ R~S-l with the enzyme Kpn I generates a 4.7 kb fragment whlch contains th~ entir~ ra~bit ~-~lo~in gene. This fragment was purified by gel electrophoresis and nick-translated to generate a probe for subsequent anneaLing experiments. The ~-globin genes o mouse an~ ra~i~ ar~ partially homologous, although we do not obse~ an~eali~g of the rab~it ~-globin probe with Kp~-cleaved mouse DNA, ~resumably because Kpn generates very large globin-specïfic fragments. In c~ntrast, cleavaqe of rabbit li~er DNA wïth ~pn I generates the expected 4.t kh globin band. Clea~age o~ transformed cell DNA with the enzyme Kpn I generates a 4.7 kb frasment containing ~lobin-specific information in six of the eight tk transformants examined. The num~er of rabbit globin genes present in these transformants is variable. In comparison with con-trols, some of the clones contain a single copy of the gene, while others may contain as many as 20 copies of this hetero-logous gene.

Rabbit ~-Globin Seque ces are Transcrib ~ ansform=
ants The cotransformation system developed provides a functional assay for cloned eucaryotic genes if these genes are expressed in the heterologous recipient cell. Six transformed cell -51- ~.2~9~

clones were analyzed for the presence of rabbit globin RNA sequences. In initial experiments, solution hybri.diza-tion reactions were performed to determine the cellular concentration of rabbit globin transcripts in transformants..

A radioac~ive cDNA copy o~ purified rabbit a and 3-globin mRNA.was annealed wi~h a vast exces~ of total cellular RNA
from trans~ormants under experimental conditions such that rabbit- globin cDNA does.not form a stable hybrid.with mouse.
se~uences.. Total RNA from transformed clone 6 pro~ects 44~
of the rabbit cDNA at completion, the value expectqd. if only ~ gene transcripts are presen~. This reaction displays ~
~seudo-~irst-~rder kinetics with an Rotl/~ of 2 X l~ ~ A
second transormant (clon~ 2) reacts with an ~otl/2 of 8 X.l~ . No signi~icant hybridization was observed with total RNA preparatio~s ~rom ~our other transformants.
Further analysis of clone. 6 demonstrates that virtually all a~ the rabbit ~-globin RNA detected in this transformant is;
polya~eny]:ate~ and exists at a steady state-concentration o about five copies per ceI1 with greater then 90% of the sequences locali2ed in.the cytopLasm.
Globin Se uences Exist as a Discrete 9S Species in Trans-q _ _ _ _ formed Cells __ _ In rabbit erythro~last nuclei, the ~-globin gene sequences are. detected as a 14S precursor RNA which reflects trans-cription of two intervening sequences which are subsequently spliced from this molecule to generate a 9S messenger RNA.
Our solution hybridization experiments only indicate that polyadenylated rabbit globin RNA sequences are present in the mouse transformant. It was therefore of interest to deter~ine whether the globin transcripts we detected exist as a.discrete 9S species, which is likely to reflect appropriate splicing of the rabbit gene transcript ~y the mouse fibroblast. Cytoplasmic poly A-containing RNA from clone 6 was denatured by treatment with 6~ urea at 70C, ~8~9 and electrophoresed on a 1~ acid-urea-agarose gel and transferred to diazotized cellulose paper. Following trans~er, the RNA f.ilters were hybridized with DNA from the.
plasmid r~ 3G_l containing rabbi~ ~-globin cDNA sequences.
Using this 3~-labeled probe, a discrete YS species of cyto-plasmic RNA is seen which co-migrates with rabbit globin m~N~ isolated from rabblt erythroblasts. Hybridization to 9S RNA species is not observed in parallel lanes containing either purified mouse 9S globin ~NA or polyadenylated cytoplasmic RNA from a t~l trans~ormant containing no rabbit globin genes.
~5 One is una~Le i~ these experiments to detect the presence o~ ~ 14S precursor i~ nuclear RNA populations ~rom the trans-formant... This is not surprising, since the levels expected in nuclear RNA, given the observed cytoplasmic concentration, ar~ likely to be beIow.the limits of detection of this tech~iqu~. Nevertheless., the results wlth cytoplasmic RNA
strongly suggest that the mouse fibroblast is capabLe of processing a transcript of the rabbit 3-globin gene to generate a 9S p~lyadenylated species which is indistinguish-able from the ~-globin mRNA in rabbit erythroblasts..

Rescue o~ pBR 322 DNA from Transformed M~) ~e~Cells Observations on cotransformation were extended to the EK-2 approved bacterial vector, plasmid pBR 322. Using the co-transformation scheme outlined herein, cell lines wereconstructedcontaining multiple copies of the pBR 322 genome.
Blot hybridization analyses indicate that -the pBR 322 se-quences integrate into cellular DNA without significant loss o plasmid DNA. pBR 3Z2 DNA linearized with either Hind III
or Bam HI, which destroys the tetracycline resistance gene, integrates into mouse DNA with retention of both the plasmid replication origin and the ampicillin resistance (3-lacta-mase) gene. It was therefore asked whether these plasmid se~uences could be rescued from the mouse genome by a second txansformation o~ bacterlal cells.

The experimental approach chosen ls outlined in Figure 20 Linearized pBR 322 DNA is introduced into mouse Ltk cells via cotransformation using the tk gene as a selectable marker. DNA i5 isolated from transformants and screened for the presence of pBR 322 sequences. Since the don~r plasmid is linearized, interrupting the tetracycline re-sistant gene, transformed cell DNA contains a linear stretc~
of plasmid DNA consisting of the replication origin and the ~-lactamase gene covaLently linked to mouse cellular DNA This DNA is~ cleaved with an enzyme such as- Xho I, which does not digest the plasmid genome~ The resulting ~ragments are circularized at low DNA concentrations in th~ presence of ligase. Circular molecules containins plasmid DNA are se}ected from the ~ast excess.o eucaryotic cir~Ies by transformation o~ col~ strain XI776.

This series of experiments ~as been carried out and a recombinant plasmid isolated from transformed mouse cell ~5 D~A which displays the following properties: 1) The rescued plasmid is ampicillin resistant, but tetracycline sensitive consistent with the fac~ _hat the donor pBR 322 was linearized by cleavage within the tetracycline re-sistance gene. 2) The rescued plasmid is ~.9 kb larger than pBR 322 and therefore contains additional DNA. 3) The rescued plasmid anneals to a single band in blot hybridiza-tions to Eco RI-cleaved mouse liver DNA, suggesting that the plasmid contains an insert of single copy mouse DNA.
~hese obser~ations demonstrate that bacterial plasmids stably integrated into the mouse genome via transformation, can be rescued from this unnatur~l environment, and retain their ability to function in ~acterial hosts.

-5'4- ~2~ 9 This result i~mediately suggests modified schemes utilizing S plasmid rescue to isolate virtually any cellular gene or which selective growth criteria are available. The aprt gene o~ the chicken is not cleaved by Hind III or Xho I and trans-formation o~ apr~ mouse cell_ with cellular DNA digested with these enzymes results in the generation o~ aprt colonies which express the ~k~en apr~ gene. Llgation of Hind III cleaved chicken DNA with Hind III cleaved pBR 3~2 resul~s in the formation of hybrid DNA molecules, in which the aprt gene is- now adjacent to plasmid sequences. Trans-formation of aprt cells ls now performed with this ~NA.
Transformants; should contain the aprt gene covalently linked to ~R 32Z, integrated int~ th~ mouse genome ~his trans-~ormed cell DNA is now treated with an enzyme whic~ does not cleave either pBR 322 or the aprt gene, and the resultant ~ragments ar~ circularized with ligase.. Transformation of E~ coli with thes~ circular molecules should select for .
plasmid.sequence~ rom eucaryotic DNA.and enormously enrich for chicke~ aprt sequenc~s. This. douhle selection technique pe~mits the isola~ion o genes expressed at low levels in eucaryotic c-ells, for which.hybridization probes are nct ~5: readily obtained.

DISCUSSION

The frequency with which DNA is stably introduced into com-petent cells is high. Furthe~more, the cotransformed se-quences appear to be inteqrated lnto high molecular weight nuclear DNA. The number of integration events varies from one to greater than f-ifty inindependent transformed clones.
At present, precise statements cannot be made concerning the nature of the integration intermediate. Although data with ~X are in accord with the model in which ~X DNA integrates as a linear molecule, it is possible that more complex intramolecular recombination events generating circular intermediates may have occurred prior to or during the in-tegration process. Whatever the mode of integration, it ~2~8~4~
appears that cells can be s~ably transformed with long stretches of donor DNA. It has been observed that trans-formants contain contiguous stretches of donor DNA 50 k~
long. Fur~hermore, the frequency o competent cells in culture is also high. At least one percent of the mouse Lt~ cell recipients can be ~ransformed to ~he tk pheno-type. Although the ~requency of transformation in natureis not known, this process could have profound physiologic and evolutionary consequences.

The in~roduc~ion of cloned eucaryotic- genes into animal cclls pr~vides an in vivo system to study the functional sig~ificance of variol~s features of DNA sequence organiza-tion. In these studies, stable mouse cell lines have been co~structed which contain up to 20 copies of the rabbit ~-globin gene. ~he ability of the mouse fibroblast re-cipient to transcribe and` process this heterologous geneh~s bee~ analyzed~ Solution hybridization experiments in concert with RNA blotting techniques indica~e that in at least one transformed cell line, rabbit globin sequences are expressed in the cytoplasm as a 95~species indistinguishable fro~ the mature messenger RNA o~ rabbit erythroblasts. These results suggest that the mouse fibroblast contains the en-zymes necessary to tr~ cribe and correctly process a rabbit gene whose expresseion is normally restricted to erythroid cells. Similar observations have been made by others using a viraL vector to introduce the rabbit globin gene into monkey cells.

These studies indicate the potential value of cotrans-formation systems in the analysis of eucaryotic gene ex-pressio~. The introductio~ of wild type genes along withnative and in _ constructed mutant genes into cultured cells provides an assay for the functional significance of sequence organization. It is obvious from these studies -56- ~218~

tha;. .h~s analysis will be facilitated by the ability to ext~nd the generality of cotransformation to recipient cell lines, such as murine erythroleukemia cells, which may pro-vide a more appropriate envlronment.for the study o heterologous glo~in gene expression.

FOURT~I SERI~S OF EXPERIMENTS

The ability to ~ransfer purified genes into cul~ured cells provides the uni~ue oppor~unity to s~udy the function and physical state of exogenous genes in the transfo~med host.
~he, deve~opme~t of a sys~em for DNA-mediated transfer o the ~SV thymidine ~lnase ~tk), gene to mutant mouse cell~, Wigler,. M , et al~, CeIl 11:223-232 (1977), has permitted ex~ension.o~ these studies to unique cellular genes. Wigler, M.,, et al., Cell 14:72s-73l ~1979) r It has been found that high.moIecular weight DNA ohtained ~rom tk tissues and cuLtured cells from a variety o~'eucar,yotic organisms~ can be used to transfer tk activity to mutant mouse cells de-~icient in this.e~zyme. The generality of the transformatio~
process has,been ~emonst~ated by the successful transfer o Z5. the cellular adenine phosphoribosyl transferase (aprt) gene and' the hypoxanthine phosphoribosyl transferase (hprt) gene. Wigler, ~ . et al., Proc. Nat. Acad. Sci. USA 76:
1373-1376 (1979); Willicke, K., et al., Molec. Gen. Genet.
I_ 179-185 (1979); Graf, L. Y., et al., Somatic Cell Genetics, in press (1979).

More recently, it has been demonstrated that cells trans-for~ed with genes coding for selectable biochemical markers also integrate other physically unllnked DNA fragments at high frequency. In this manner, the tk gene has been used as a markex to identify mammalian cells cotransformed with de-fined procaryotic and eucaryotic genes into cultured mammalian cells. Wigler, M., et al., Cell 16:777-785 (1979).

~2~ 9 Detection of gene tran~fer ha~ in the pa6~ rel~ed exten-sively on the use of appropriate mutant cell li~es. In some case~, cell~ resis~ant to mletabolic inhibitors contain dominant acting mutant gene~. Cotransfor~ation 5 with such dominant acting markers should in principle permit the introduction of virtually any cloned gene~ic element into wild type cultured cells. In this study, cells were transformed with the gene coding or a mutant dihydrofolate reductase (dhfr) gene which renders cells lo resistant to high concentrations of methotrexate ~mtx).
Flintoff, W. F., et al., Cell ~: 245-262 (1976).

Cultured mam~alian cells are exquisitely ~ensitive to the folate antagonist, methotrexate. Mtx resistant cell lines have been identified which fall into three catego-ries: 1) cells with decreased tran~por~ of this drugO
Fischer, G. A. Biochem. Pharmacol. 11: 1233-1237 (1962):
~irotnak, ~. M., et al., Cancer Res. ~: 75-80 (1968); 2) cells with structural mutations which lower the affinity of dhf r f or methotrexate. Fl intof f, W. F~, et al ~, Cell iL: 245-262 (1976); and 3) cells which produce inordinate-ly high levels of dhf r. Biedler, J. L., et al., C~ncer Res. ~L~ 153-161 ~197~); Chang, S. E., and Littlefield, J. W., Cell 1: 391-396 (1976). Where they have been 25 examined, cells producing high levels of dhfr have been found ~o contain elevated levels of the dhfr gene (gene amplification). Schimke, R. T., et al., Scienoe 202:
1051-1û55 (1978).

30 ~n interesting methotrexate resistant variant cell line ~A293 ~as been identified that syntbesizes elevated levels of a mutant dihydrofolate reducta~e with reduced affinityh for methotrexate. Wigler, M., et al.~ Cell 1~:
777-785 (1979). Genomic DNA frcm this cell line has been 3s r used as donor in experiments to ~cransfer ~he mu'cant dhf r gene to mtx sen~itive cell~. ~xpo~sure of mtx resistan~
transfoxmed cells to increasing level~ of ~tx ~el-ects ~or cells which have amplified the trans-5 ferred gene. In this way, it is possible to trans-~o ~2~

~er and ampli~y virtual~y any genetic element in cultured mammalïan cells~

~rans~e~ o t~ Mu~ant Hamste~ Dïh drofolate Reduc~ase Gene ___ _ _ _ _ Y
to MQUS~ Celi~
LO
~lgk molecular weight ce~llular DNA was prepared ~rom wild-t~pe mtx sensitive CHO cells and ~rom A~9 cells, an-mtx resistant CH~ deriva~lve syn~hesïzing increased ~evels o~
æ muta~t dhr_ EIint~f~r w~ p et aI , CelI 2:245--262 (I9761_ ~h~ abiI~t~ o~ ~hes~ DNA pre~aratio~s to tr~nser either the dh~r gen~ or the t~ gen~ to tk mo~s~ L ce-lls~
.~ apr~ j ~a~ tes.te~ usins ~modificat~a~ o the caI~
~asphate^coprecipit~tTo~ metho~. WigLer, ~. r et ~ r Proc~..
Nat.~ ~ca~_ Sc~_ US~ ~6 L~ T3T6 ~9~9). DNA ~rom.both.
muta~t AZ9 ar1~.~ild-ty~e C~ ce~lls.~as;comp~tent .i~ trans-. .~e~L~ t~ tk ge~ t~ h~k ap~t celI~_ ~thctrexate re-sista~t coIo~ie~ wer~ o~serve~ onl~ ~Ilowïng t~eatme~t o~
ce~Is wit~ D~ ~ro~ A~9_ ~h~ data o~taine~ suggest that treatment of ~ethotrexa~e sensiti~e cells with A~9 DN~ ~e- -sul~ed L~ the-tra~s~er and expression of ~ mutant dhfr gene, thus renderin~ these cells Insensitive t~ eIe~ated leveIs o~
methotrexate~

In order to test this hypothe~is directly, molecular hybrid-ization studies were performed to demonstrate the presence o the hamster dhfr gene in DNA from presumed transformants.
A mouse dhf~ cDNA clone (pdfr-21), Chang, A.C.Y~, et al., Nature 275:61~-624 (19781, that shares homology with the structural gene sequences o the hamster dhfr gene was used to detect the presence of this gene in our transformants.
Restriction analysis of the dhfr gene from A29, from pre-sumed transformants, and from amplified mouse cells, was performed by blot hykridization. Southern, E . M., J . Mol.
Biol~ 98:503-517 (1975). DNA was cleaved with restriction endonuclease Hind III, electrophoresed in agarose gels, ~nd transferred to nitrocellulose filters. These fi:Lters were ~2~ ~ ~4~
the~ hybridized w~th high specific actlvity, 3~-labeledS nic~-translated ~dhfr - ~1 and dev~Loped by autoradlography.
rocedur~^ v~sualizes ~estriction fragmentc ~ genomic DNA_homologous ~Q t~e dhf~ probe~ Promu.nent ba~ds are obser~ed ~t 1~ ~b.~ 3:~ k~. an~ 3 kb ~or ~lous~ DNA and lT k~r r_g~k~, 3:~t kh an~ L.~ k~ ~o~ hamste~ D~1A_ ~he restrictio~
LO ~rQ~iles-~twee~ thes~ two species are sufficlently differ -ent to. permit orIe to. distin~ish ~h~ hamster gen~ in the.
presenc~ oi~ endogenous mous~ gen~.. Five L ce~lL trans-~orma~ts~ reslstant to methotrexat~ were therPore exam~ne~
. ~y }:llot hybri~Eizatio~_ Irr eac~ trans~ormed c~l~ lin~, one I5~ ohse~rved~ t~se expe~ted~ ~?ro~l l e o~ ~a~ds resultin~ ~rom cd:~s~a~e.. Qf th~ endogenous mouse dl~ gen~ an~ series; o ad~ia~al ~a~nds whose~ molecuIar weights ar~ identlc~L
~o thas~ o~se~red~ ul?or~ clea~ag& o hamster DN~ The~ :L7~g l~
~_n~ kh-and L..4- }~ ban~s o~se~e~: i~ hamster DNA~ are~ dias--Za ~o~t-ic i~ t~ E~si~nce-o th~ ~amste~ dh~r qen~ and a:re-E?r~sesL~ ~r~ aII ~s~orma~ts~

I~ ~sit:i.aL exl?erIments,. t~ 10t~Fest. concentra~on of metho--trexate.(.O~_L ~g ~ mL)~ was.~chos-en w~:ich would decreas~
~5 sur~v~L o~ Ltk aprt c.eLIs ta Iess than 10 ~.~ Pre~ious studies,. Flin~o~, W~ F., et al~ CeIr 2~.2.45-~62 (1976), suggested that ~he presence o~ a single..mutant dhr gene can render cells..resistant ta this concentration o methotrexate..
Comparison o~ the intenslty o the hamster dhfr gene frasments.
o trans~orme~ cell DNA with those of wlld-type hamster DNA
suggest that our ~ransformants contain one or at most.a ew methotrexate resis~ant hamster genes.. By cbntrast, donor A~9 cells, which have been shown to produce elevated levels o the. mutant dhfr, Flinto~ F.., et al., Cell 2:245-262 (1976),. appear to contain muLtiple copies of this gene.

Amplificatlon_of the Transferred dhfr Gene Initial transformants were selected for resistance to reIati~ely low levels of mtx (O.l ~g/ml). For e~ery clone , 12~89~
however, it was possible to select cells resistart to elevated levels of mtx by exposing mass cultures to successively increasin~ concentrations of this drug. In this manner, we isolated cultures resistant to up to 40 ug/ml of methotrexate starting from clones th~t were ini-tially resistant to 0.1 ~g/ml. We next asked i~ increased resistance to methotrexa~e in these transformants was associated with amplification of a dhfr gene and, if so, whether the endogenous mouse or the newly transferred ham-ster gene was amplified. DNA from four independent isolates and their resistant derivatives was examined by blot hy-bridization. In each instance-, enhanced resistance ta m~thotrexate was accompanied by an increase in the co~y number a~ the hamster gene. This is most readily seen by ~omparing the intensities of the 1.5 kb band. In no in-stance have we detected amplification of the endogenous mouse dhfr ~ene. Lastly, it is noted that not all lines selected~ at equiv~Tent methotrexate~concentrations appear ta have the same dhfr gene copy number.

The dhfr Gene as a ~eneralized Transformation Vector __ __ _ _ .
Selectable genes can be used as vectors for the introduction of other genetic elements into cultured cells. In previous studies, it has ~een demonstrated that cells transformed with the tk gene are likely to incorporate other unlinked genes.
Wigler, M., et al., Cell 16:777-785 (1979). The generality of this approach was tested for the selectable marker, the mutant dhfr gene. 20 ~g of total cellular DNA from A29 was mixed with l ~g of Hind III-linearized pBR 322 DNA. Re-cipient cells were exposed to this DNA mixture and, after two weeks, methotrexate resistant colonies were picked. Genomic DNA from transformants was isolated, cleaved with Hind III
and analyzed for the presence of pBR322 sequences. Two in-dependent isolates were examined in this way and in ~oth ~2~a8~9 cases multiple copies o~ psR322 se~uences were present in these methotrexate transformants.

An alternate approach to generalized transformation in-volves ligation of a nonselectable DNA sequence to a selectable gene. Since the muant dhfr gene is a dominant acting drug resistance factor, this gene is an ideal vector.
Furthermore, i~ should be possi~le to ampLify any genetic element ligated to this vector by selecting cells resistant to elevated levels of mtx. To explore thls possibility, re-strictio~ endonucleases ~hat do no~ destroy the dh~r gene of A29 were ide~ti~ied by transformation assay. One such restriction endonuclease, Sal I, does not destroy the trans-~ormation potential of A29 DNA~ SaI I-cleaved A2g DNA was there~ore Ligated to an equal mass o~ 5al I-linearized pBR32~. This ligation product was subsequently used in tra~sformation experiments Methotrexate resistant colonies were picked and grown into mass culture at0 1 ~g methotrexate/
ml Mass cultures were subsequently exposed to increasing concentrations of methotrexate.

~5 DNAs were obtained from mass cultures resistant to 0.1, ~, 10 and 40 ~g/mL methotrexate, and the copy number of pBR322 and dhfr sequences was determlned by blot hybridiza-tion. Six independent transformed lines were examined in this fashion. Five of these lines exhi~ited multiple bands homologous to pBR322 sequences. In four of these transforme~
clones, at least one of the pBR 322-speciflc bands increased in intensity upon amplification of dhfr. In SS-l, two pBR322-specific bands are observed in DNA from cells re-sistant to 0.1 ~g/ml methotrexate. These bands increase several-fold in i~tensity in cells resistant to 2 ~g/ml.
No further increase in intensity is observed, howe~er, in cells selected for resistance to 40 ~g/ml. In a second line, SS-6, all pBR 322 bands present at 0.1 ~g/ml continue to ~8~9 increase in intensity as cells are selected first at 2 ~g/
ml and then at 40 ~g/ml methotrexate. Curiously, new pBR322-speci~ic bands appear after selection at higher methotrexate concentrations. I.t was estimated that there i5 at least a fity-fold increase in copy number for pBR32Z sequences ln this cell line. In a ~hird cell line, HH-l, two pBR322-speci~ic bands increase in inte~sity upon ampli~ication, others remain constant or decrease in inten-sity. Thus, the pattern o~ ampl fication of pBR322 se-quences observed in these cells can be quite varied. Never-1~ theless, it appears that the mutant dhfr gene can be usedas vector for the introduction and ampli~ication o define~
DN~ sequences into cul.tured anïmal cells.

DISCUSSION

The.~otentiaL useulness.of DNA-mediated transformation in th~ study of eucaryotic gen~ expression depends to a large extent on i~s generality_ Cellular genes coding ~or select-able biochemical functions have previously been introducted intQ mutant cultured cells, Wigler, M , et aI., Cell 14:725-731 (lg79); Wigler, M., et al., Proc.. Nat.. Acad. Sci. U5A
~6:1373-1.376 (1979); W~llecke, K., et al., Molec. Gen. Genet.
170:179-185 (1979~; Graf, L. H~, et al., Somatic Cell Genetics, in press (1979). In the present study, a dominant acting, methotrexate resistant dhfr gene has been transferred to wild-type cultured cells. The use of this gene as a vector in cotransformation systems may now permit the introduction of virtually any genetic element into a host of new cellular environments.

In inltial experiments, DNA from A29 cells, a methotrexate resïstant CHO derivative synthesizing a mutant dhfr was added to cultures of mouse L cells, Methotrexate resistant colonies appeared at a frequency of one to ten colonies/
5 X 105 cells/20 ~g cellular DNA. No colonies were observed upon transformation with DNA obtained from wild type, methotrexate sensi~ive cells, althDugh. this DNA was a competent donor cf the thymidine ki.nase gene. Definitive evidence that we hav~ effected tran~fer of a mutant hamster dhfr gene was obtained by demonstrating the presence of the.hamster gene in mouse transformants.
The restriction maps o~ ~he mouse and hams~er dh.fr genes are signlficantly different and.permit one to distinguish these genes in blot hybridiza~ion experiments. In all transformants examined, one obser~es two sets of restrictian fragme~ts homalogous to a mouse dhfx cDNA
clone- a s~ries of bands characteristic.of the endogenous mause gene and a second series characteristic of the L5 dQnor hamster gene.

~h~ utiLi.ty o transformation of the ahfr locu~.is. a ~unction Q~ th~ relativ~ fre~uencies~both o~ transformatio~
and o spantaneaus. resistance- to mtx~ The demonstration that all mtx res~stant ~ cells plc~ed result~from trans-formation.rather than amplification o endogenous genes suggests that ampli~ication o dhfr is a rare event i~ this cel~ line. Attempts were made to transform other cell lines, including mouse teratoma and rat liver cells and, in these instances, hybridization studies reveal that the acquisition of mtx resistance results from amplification o~ endogenous dhfr genes. The use of a purified dhfr gene is likely to overcome these difficul.~ies by enormously increasing the frequency of transformation.
The ahfr copy num~er obser~ed in ini~ial transformants is low. This observation is consistent with previous studies ~.uggesting that a single mutant dh~r gene is capable of rendering cells mtx resistant under 35 selective criteria (0.1 ~g/ml mtx). Flintoff, W. F., et al., Cell 2: 245-262 (19763. Exposure of these initial mtx resistant transformants to stepwise increases in drug concentration results in the selection of cells with enhanced mtx resistance resulting from amplification of S thenewly transferred mutant hamster dhfr gene. In no transformants has amplification of the endogenous mouse gene been observed in response to selective pressure.
It is likely that a single mutant gene affords signifi-cantly greater resistance to a given concen~ration of mtx than a sin~le wild-type gene. I~ the frequency of the amplification i~ low, one is merely selecting resistance variants having the minimum number o ampLification events., It is also possible that newly transferred genes may be ampLiied more readily than endogenous genes.
LS
~he mutant dhr gene has been used as a dominant transfer vector t~ introduce nonselectable ge~etic elements into cuLtured cells. One experimental approach exploits the observation made previously, Wigler, M., et al~; Cell I6-7t7-785 ~1979), that competent cells integrate other physlcally unlinked genes at high frequency. CuItures exposed to pBR32~ DN~, along with the genomic DNA
containing the mutant & fr gene give rise to mtx resistant cell lines containing multiple copies of the bacterial plasmid.

An aLternative approach to genetic vectoring involves ligation of pBR322 sequences to the selectable dhfr gene prior to transformat~ns.This procedure also generates transformants containing multiple pBR322 sequences.
Amplification of dhfr gene$ results in amplification of of pBR322 sequences, but the patterns of amplification differ among cell lines. In one instance, all pBR322 sequences amplify with increasing mtx concentrations.
In other lines, only a subset of the sequences amplify.

~`~

-65- ~Z~8~

In yet other lines, sequences appear to have ~een lost or rearranged. In some lines, amplification proceeds with increasing m~x concentrations up to 40 ~g~ml, whereas in others, amplifica~ion ceases at 2 ~ CJ/ml . At present, the ampliication process ls not under~;tood nor ~as the ampli~ication unit ~een defined. Whatever the mechanisms responsible for these complex eve~ts, it i5 apparent that.
they can be ~xpolïted to control the dosage of virtually any gene introduced into cultured cells.

~0 ~%~ 49 FIFTE SERIES 0~ EXPERIMENTS

Mouse teratocarcinoma (TCC) stem cells provide a unique vector for the introduction o~ specific, predeter..nined, genetic changes. into mice.Mintz, ~. & Illmensee, K., Proc. Natl Acad. Sci. 72 3585-3589 (1975):; Mintz, B., Brookhaven Symp.. Biol. 29: 82-85 (1977). These cells los~ their neoplastic proper~ies and undergo normal differentiation when placed in the environment o~ the early embryo. There they can contribute to formation of alL soma~ic tissues in a mosaic animal comprising both donor- and hast-derlved cells, and also to the germ line, ~rom which the progeny ha~e genss o the tumor strain in alL their cells. Thus, during initial propagation of TCC stem cells in culture, clones with expe~imentally selected nuclear, Dewey, M_ J., et al., Prac..Natl. Acad_ Sci~r t4 : 5564-5568 (~977), and cytoplasmlc, Watanabe, ~-~, et al., Proc_ Natl~ Acad..
Sci~, 75 5II~-51I~ (1978), gene mutations have been . obtaine~ and the cells have proved.capable o pa~ticipat-in~. in embryogenesis.

The effective application of this sys~em in probing the control of gene expression durins differen~iation would be greatly enhanced if, as proposed, Mintz, B., Differentiation 13: 25-27 (1979), precisely defined genes, either in native or modified form, with known associated sequences, could be introduced into develop-mentally totipotent TCC cells prior to their develop-ment ln ivo. DNA-mediated gene transfer into cultured mouse cells has now been reported for a variety of viral and cellular genes coding for selectable bio-chemical functions. The purified viral thymidine kinase (tk; ATP: thymidine 5'-phosphotransferase, EC
2.7.1.21~ gene has provided a model system for gene -67- ~2~g~g :ransfer, Wigler, M. et al., Cell 11: 223-2~2 ~1977), and has been followed by the DNA-mediated ~ransfer of the cellular genes coding for thymidine kinase, Wigler, M~, e~ al., Cell 14: 725-~31 (1978), hypoxa~thine ~hosphoribosyltransferase, Willecke, K., e~ al., ~olec. Gen. Genet. 170: 179-185 ~1979); Graf, L. H., et al., Somat Cell Genet., in press (197'~), adenine phosphoribosyltransferase, Wigler, M., et al., Proc.
Natl. Acad~Sci. US~, 76: 1373-~376 (1979), and dihydrofolate reductase, Wigler, M., et al., Proc.
Natl. Acad. Sci, in press (1980); Lewis, W. ~., Pt al., Somat_ Cell. Genet., in press (1979). In this report is d monstrated the contransfer o th~ cloned Herpes ~ ~SV) thymldine kinase gen~ ~long with the h~man ~-~lobin gene into mutant (tk ) teratocarcinoma stem cells in cuLture. ~hese transformed cells, when tested by subcu~aneou~ inoculation into mice, retain their deveIopmentaI capacities i~ the tumors that are produced, and exhibit the ~iraI-specific tk enzymatic activity for numerous cell generations ln v _ Transfc ~ ~ a Cells.

The addition of plasmid DNA containing the ~SV thymidine kinase gene to cultures of attached mouse L tk cells yields L tk+ tran~formants in ~AT at a frequency of one colony per 100 pg of DNA per 5 X 105 cells. Under identical transformation procedures, tk teratocarcinoma cells showed a strikingly lower transformation efficiency.
Based on the average of three independent experiments, one surviving colony was obtained per 4 ~g of plasmid DNA per 5 X 10 cells, a value four to five orders of magnitude below that of the L tk cells. This ~elatively low efficiency was confirmed when the DNA was added to TCC tk cells in suspension. Addition of 10 ~g of Bam -68~ g Hl-restri.c+ed ptk~l 2NA to 7 X 106 cells resulted in only ~our transformants in HAT. With identical trans-formation condition~, ~ tk cells gave 3 X 10 tk colonies per ~07 cells per L.5 ~g of ptk~l DNA. While high concentrations of gene are thus required to effect transformation in this teratocarcinoma celL line, the availability o~ clon d DNA no~ethel.ess allows numerous tk transformants to be obtained.

Ex ression of HSV tk Activit in Transformed Teratocarcinoma.
P _ . ~. , , . Y , ~
Cells~ ~

~o ascertain whether the tk phenotypes of the TCC
clones were indeed att~i~utah~e to expression of the viral tk gene, seven.colonies were picked from independen~
culture dishes and grown into mass cultures for testing.
The activit~ of. fiv~ clones.were characterized by serological, and o two by biochemical, techniques. ..
Th~ ~er~es-type antigenic.identity of tk was verified zo by assayinq the; ability of HS~- tk-specific antibody to neu~r~lize enzymatic activlty. Over 90% lnhibition of tk.activity was in fac.t observed when immune serum was reacted with extracts of each of the five trans-ormed clones chosen (Table I). The low residual activity remaining after neu~ralization of transformed-cell extrac~s may represent mitochondrial tk activity, which by itself is unabl~ to aford survival in HAT. Cell extracts from the other two TCC tk clones ~hosen were testad for tk electrophoretic mobility because of the mar~ed difference be~ween th.e mouse and HS~ tk enzymes. While the TCC tk control, as expected, shows no major peak of actiYity, the transformants have ~he HSV tk ~haracteristic peak migrating ~ith an Rf of 0.45, as shswn for one o ~he clones.

f~, ``~
-69~ 3949 Table ~ . Spec1fic neutralization of Herpes thym~dine kinase in transformants Activity Wl~h Activity with Cell linepreimmune ser~m antiserum source ofUnits X 10 UnLts X 10 ~ ResiduaL
extractper ml per ml ac~ivity TCC wt* 2.8 3.0 107.0 TCC tk 0.05 0.06 100.0 LXB 2b* 3.4 0.06 2.0 TCC tk-l~ 2.1 0.1~ 8.0 TCC t~-3 5.5 0.43 8 0 TCC t3c-46 . l Q .. 15 ~. 5 TCC tk--53 ~ ~ O . 21 6 . O

30 ,000. X q supernatants ~f homogenates (S-30) from t~e indi-cated ceI~ lines were mixed ~ith preimmune serum or antiserum to ~l~ifiea HSV-1 tkr and tk activi~y was assayed as described in-~aterials an~ Method`. A~tivity is expressed as units per ml o the S 30 fraction.
*$C~ wt is a mouse teratocarcinoma feeder-independent cell line (6050P) with tk (wild-type) phenotype.
~TCC tk is a derivative of TCC wt that is resistant to BrdUrd and is tk-deficient.
~HB 2b is a mouse L tk cell line transformed to the tk phenotype with the He ~ thymidine kinase gene.
~TCC tk~ 3, -4, and -5 are HAT-resistant teratocarcinoma clones derived from TCC tk after transformation with the Her~s tnymidine kinase gene.

_70 ~2~94~
.
The Physlcal State of ~le tk Gene i~ Transformed Teratocarcinoma Cells .

The number o~ viral tk gene fragments and the location o~ these fragments in independent transformants were examined utilizing the blot hybridization t~chni~ue o~ South.ern, Southern, E. M., J. Mol. Biol., ~8: 503-517 (1975). The donor DNA was the recombinant plasmid, ptk-l, diges~e~ to completion wlth.Bam Hl. This plasmid contains a 3.4 kb ~ragment wi~h. the viral tk gene inserted at the single Bam Hl site wi~hin the tetracycline resistance gene of pBR32~.. Transformation with Bam-cleaved tk DNA results in integrati~n wi~h loss o:E the Bam si~es at t~ termini of the 3..4 kb fragment. Eigh ~olecular weight DNA rom transformants was cleaved with ~5 Bam Hl, ~ractionated by agarose gel el.ec.trophoresis, and transferred t~ nitrocell.ulos~ ~iLters.; the filters wer~ the~ anneaIed with nic~-translated 32P-tk DN~
In each ceLl clon~, a single a~nealing fragment was.
seen; there~ore, each clone c~ntain~ at leas~ one viral ~0 tk.gene. As expected,. each clone reveals a band of mol~
ecular weight greater than 3.4 kb. The molecular weights o~ the annealin~ fragments differ a.mong the transformed clones, a result suggesting that integration has occurred at dif~eren~ sites within the DNA of the respec~ive transformants.

Stabil~ of_the Transformed Phenotype in Culture To test the capacity of the TCC transformants to retain expression of the donor tk gene in culture in the absence of selective pressure, individual clones grown lnto mass culture in HAT selective medium were subcultured for various peri.ods in the ab.sence of the selective agent.
The fraction of cells that retained the t~ phenotype was determined ~y measuring cloning efficiencies in selective and nonselective media. Wide differences among clones became apparent (Table II). Some cell lines, -71~ 8~

Table II. In vitro stabilit~ of the transformed phenotype in teratocarcinoma cells.

~ _ _ _ _ Generations Relative cloning Rate of Ioss Clonal in ef~iciency in of ~k cell nonselectiveselective phenotype per line ~Peri- medium* mediumt generation~
ment TCC tk-l 1 28 0.45 2 ,150 0.50 ~O.OOL
TCC t~-~ 1 '28 a.~3 z ~5~ Q~o~ 0,~17 TC~ tk-3 i 28 O.47 ~ 150 0.~7 ~00 15TCC tk-4 1 2~ 0~26 ~ ~50 0 16 Q.003 TCC tk-5 I 28 O~L4 ~ 15~ 0.01 0.02~

*Clones were pic~ed and g~wn in ~T selective medium for 40 cell gener~tions. Cells were then arown in non~elective medium or 28 or 150 generations prior to ~etermining their cloning e~':ciencies under selectlve and nonselective condi-tions.
~5 tOne hundred cells were plated in triplicate into ~AT selec-tive and nonselective media. mhe relative cloning efficiency in selective medium is defîned as the ratio of the cloning efficiency under selecti~e conditions to the cloning efi-ciency under nonselective condi~ions (S0-70%).
$In these calculations it is assumed t~at for any given cell line the rate of loss of the tk phenotype is constant in each cell genexation. ~he rate of loss per generation may then be calculated from the formula FM (l-X)N M ~ FN, in which FM is the relative cloning efficiency ïn selectî~e medium after M
generations in non-selective medium; FN is similarly defined for N generations; and X is the rate of loss per c~l geN~tion.

-72- ~2~49 such as TCC t~-l, were relatively stable and lost the tk phenotype at frequencles less than 0.1% per generation in nonselec~ive medium Other, less stable, lines (TCC
tk-2 ~nd TCC t~-5) lost tk+ e~pression at 2% per generation in the absence o~ selection.

Maintenance and Expression o~ the ~S~ t]c Gene in Vivo Durin~ Tis~ue Differentiatlon in Tumors The more critical question of retention of the foreign gene and of its expression during TCC cell differentiation in viv~ in t~e a~s nce of selection was examined i~ solid tumarC, ~lImors wer~ forme~ ~y inoculating syngeneic hosts ~usually two hosts per clone) su~cutaneous1y wlth 107 cells ~rom each o~ the same five transformed clones. DNA
from thes~ tumors was analyzed ~y blot hybridization.
Weutralization assays and electrophoretic mobility tests ~ o~ theftk enzyme were als~ carrIed ou~ to identify e~-pression o~ the viral gene~ In additlon, samples o~ the same tumors were fi~ed and ~xamined histologically for ~ evidence of differentiation.
The restriction fragment profiles of the viral tk gene demonstrated that the gene was retained in all nine tumors analyzed. When each tumor (grown without HAT selection) was compared with its cell line of origin (cultured under HAT selective pressure)l t~e num~er and location o the annealing fra~ments ïn seven of the tumors was identical to that of the corresponding cell line, Thus, the introduced tk gene was, in most cases, maintained for many cell generations spannïng at least three weeks-in ~ivo without sianificant loss or translocation. In two instances, however, a gene rearrangement had occurred, resulting from the loss of the original tk containing fragment and the appearance of a new fragment of different molecular weight, It is of interest that --73~
8~

these two tumors were prod~ced from the two TCC clones that lost th.e ~k+ phenotype ln vitro at hïghest fre quencles (Ta~le II).

The results of neutralization tests with HSV-tk-specific-antiserum demonstrated that at least three of the nine tumors ( including one from the TCC tk--~ clone) had viral type tk actlvity. (Th.e presence o~ hsst cells la in th~ tumors pro~ably con~ribut~d substantial amounts of .non-neutralized mouse tk in the remaining cases.) Anather sample o~ the tumor deri~ed ~rom the TCC tk-~line was aLso analyzed eLectroph.oretically for HSV
tk acti~ity; a predominant: peak migrating with an R~
15 0~ 0 .45 r characteristic of the viral e~zyme, was obser~red ..

HIst~logical speciments ~rom each o~ the tumors were ~r~pare~ an~ examined. In- additia~ ta the TCC stem cells~, 20 tumors contained an array o dif~erentiat~d tissues similar to those in tumors from the untransformed TCC w~
and 'rcc tk ceLL lines o origin. Include~ were muscle, neural formations, adipose tissue, some bone, squamous keratini~ing epithelium, and other epithelia, ducts, and tubules.

Cotransformation of Terat~carcinoma Cells with the _uman ~-Globin ~

Biochemical transformants of mouse L may constltute a competent subpopula~ion in which an unselectable gene can be introduced, along with an unlinked selectable gene, at freq~encies high.er th.an In the general popu-lation, Wîgler, M., et al., Cell 16: 777-785 (197~).
Cotransformation experiments h~v~ therefore been carried out in which the Her~es viral tk gene was used as a ~L2~L894~

selectable mar3~er to introduce the human 3-globin gene into tk TCC cells. A cloned ~ind III restriction _ endonuclease frasment of human chromosomal DNA containing the ~-globin gene (plasmid ph~-8) was cleaved with the enzym~ Hlnd III and mixed wl~h Hïnd III-linearized ptk-l.
Ater TCC tk cells were exposed ~o these genes, they were grown ~or ~wo weeks in HAT selection medium and tk transormants were cloned and analyzed by blot 1 hybridization for presence of human ~-globin ~equences.
~ 4.3 kb Bg~ II restrlction ~ragment containing the intact human ~-globi~ gene is entirely contained within the donor -E plasmid. ~igh molecular weight DNA from the trans~ormants was therefore cleaved wlth the ~ I~ enzyme and analyzed in blot hybridization using t~e P-labeled 4_3.kb ~ II fragment as an annealing probe~

In bw~o~ th~ ten TCC trans~ormants examlned, human.
~-gLobln sequences were detected. One of the transformants contains one to three copies of the 4.3 kb B~ II fragment;
i~ this.celL line., thereore, the globin gene is evider.tly intact. The other TCC isolate containing the human ~-~lobin gene displays an aberrant high moleculax weight annealing fragment, a result suggesting that clea~age and integration have occurred within ~he ~ II fragment.
These data demonstrate that those TCC cells that are competent fo~ uptake an~ e~pression of the t~ gene a~so integrate anotherunlinked and unselectable gene at high requency.

DISCaSSION

The experimental introduction of foreign DNA into early mammalian embryos, and its persistence and augmentation during development, were first reported some six years -75~

ago, ~aenisch., R. & Mintz, B., Proc. Natl. Acad. Sci.
71: 1~50-1254 (1974) . Purifïed (nonrecoml:).inant~ SV 4~
viral DN~ was microinjected into mouse }:lastocysts; they 5 gave. rise to healthy adults whose tissue DNA contained SV 40 gene sequences. Newer technologies such as described herein shollld allow a wide range o speclfic genes to he. incorporated into the genome of the emhryo for in vivo analyses o~ control of gene expression during differentia-10 tic~n. With the ad~rent of recombinant DNA, quanti ties o:~particular genes in natlve or specifi~ally modified form can ~e obtairs~d~ In the bio1Ogical spheré, the malignant ~tem cells o mouse teratocarcinomas have contri}:uted a novel ave.nue RfE intervention.. These cells can ~e.
15 grown in culture, selected for specific mu~ations, and microinjec.ted into blastocysts, where they lose their neopLas.tic properties and particïE~ate in development, Dewey, ~_, ~. et aL~, Proc. Natl.. Acad:, Sci. USA, 74-:
5564-5568 (1977~; Watanabe, ~., et al., Proc. Natl.
Acad. Sci.. , 75: 5113-511~ ~1978) . T~e cultured TCC celIs hav~ the~eore been viewed as vehicles for transmitting predetermined genetic changes to mice, Mintz, B., Brook-haven Symp., Bio.., 29:. 82-85, (1977); Mintz, ~., Differentiation 13: 25-27 (1979). Such changes obviously 2S miqht include genes acquired by uptake of DNA.

DN~-mediate~ gene transfer into cells of fibroblas~: lines has been accompLished in cult-~re, Wigler, M., et al., Cell 11: 223-232 (1977); Wigler, M., et al., Cell 14:
725-731 (1978~; Willecke, K~, et al., Molec. Gen. Genet.
1 : 179--185(1~ 79), Graf, L. H., et al., Somat. Cell Genet., in press (1979); Wigler, M., et al., Proc. Natl.
Acad. Sci. USA, 76: 1373-1376 (1979); Wigler, M., et al.
Proc . Natl. Acad. Sci., in press (1980); Lewis, W. H.
et al ., Somat. Cell Genet., in press (.lq79), and furnished the basis for similar attempts here with tera-~76-tocarcinoma lines. The TCC~cell route for gene transfer into embryos, as compar~d wi.th embryo injection of DNA, of~ers the advantage th.at transforman~s, i.e., cell clones in which the speclfic gene h.as ~een retained, can be identiied and Lsolated by selectio~ or screening.
In the case o unselec~ble genes, cotransfer with a selectable one has been found to occur ~ith relatively high frequency, Wigler~ M.,.et al., CelL 16: 777-785 L0 (l979), In the present study, tk teratocarcinoma cells have been treated with th~ clo~ed thymidine kinase gen~ of Herpes simplex and a number of HAT-resis~ant tk~ clones have bee~ obtained with a frequency of about one transformant per ~g o DNA.. The reason for the markedly lower fre~uency o~ ~CC transformants than of L-celL trans~ormants, Wigler,.
M..,. et al., Cell 14: 725-731 (1978-), is:obscllre since the basis or transformation competence in eucaryotic cells-remains unkncwn. The donor origi~ of the tX phenotypein the TCC transformants W~5. demonstrated by the HSV-~ype-electrophoretic mobiLity o~ their ~k enzyme, and also by neutralization of the tk activity ~y specific antiserum raised against HSV~ (Table I)~ ~urthermo~e, blot hybridization tests indicated that at least one intact copy of the viral tk gene was present and integrated into other DNA in the transformed cells. ~hese data support the conclusion that the ~k activity in the transformed clones is indeed attributable to presence and expression of the viral gene , .
A requirement for experiments involving the introduction of genes is that they remain stable ln vivo, even in the absence of sele~tive pressure, durin~ many cell generations.
Stability of the tk transformed phenotype was in fact not only in culture (Table II), but also in tumors arising ~2~9~

aftex subcutaneDus inoculation of ~.e stem cells into mlce. Th.ese tumors exhibited va~ious types of tissue di~erentiation, sLml.Lar to the.rang~ observed in the untransformed parent TCC line. ~ybridization experiments comparing each tumor with its transformed cell line of origin indicated that the donor t~ gen~ was maintained withou~.signiflcant loss or xearrangement in seven of nln~ tumors examïned.

Man~ genes o~ interect in a developmental context are not seLectable~ An.example is. the glo~in gene. Ac.in related exper,iments with ~-cells, Wi~ler, ~.., et.aL., CelL 16:
777-785 (,1979), a fr~gment o~ human genomlc DNA containing 1S an in~act B-globi~ gene,was adminLstered to TCC tk cells along wi~h the unlinked HS~ tk gene. Th.is proved to be a~ ef~ective method to ~btain TCC t~ clones, in which, ~ro~ h~ridization evidence, ~he human R-globi~ gene was present.
20.
The experiments described herein therefore ~strate that cultured TCC stem cells~ can accep~ e~ogenous genes and that such genes can.be stably retained as well as expressed duri~ _ vivo differentiation in tumors.
On ~his basis, experiments with a euploid TCC cell ~ine can proceed, for the purpose of creating in vivo mar~ers. appropriate for analyses of gene regulation during embryogenesis.

SIXTH SERIES OF EXPERIMENTS

The following experiments are being published in an article coauthcred by Richard Axel, Diane M. Robins, Inbok S Paek and Peter H. Seeburg entitled "The Regulated ExpressLon of Human Growth Hormone Genes in Mouse Cells".

Transcriptional activa~ion of many genes is likely to involve the interactio~ of regulatory molecules with specific DNA sequences. Steroid hormones regulate the expression of restricted sets of gene products in a tissue-specific manner, Palmiter, R.D., Cell 4:189-197 (197S); Ivarie, R.D. and O'Farrell, P~H., Cell 13:41-55 (19783_ Significant evidence has accumulated to suggest that steroid horm~nes may induce gene expression by first associating with a protein receptor to generate an active complex which then interacts directly with accessible and highly specific sites on the chromosome, Yamamoto, K.R.
and Alberts, B.M~, Annual Re~. Biochem. 45:721-746 (1976)_ Thus, cells with mutant glucocorticoid receptors, defective either in hormon~ binding or chromosome association, are no longer regulated by glucocorticoid, Yamamoto, K.R., et al.~, PNAS 71:3901-3905 ~1974).

A simple model of hormone action compatible with available data assumes that the interaction of steroid-receptor complex with appropriate DNA sequences enhances transcrip-tion. There is no direct evidence in support of this model. This problem has therefore been dissected into experimentally accessible questions: Are there specific s~quences about inducible genes which render them sensi-tive to hormonal induction? Are there specific sequences about inducible genes that show high affinity for hormone-receptor complex? And finally, how does the interaction of receptor with DNA lead to transcription activation?

79~ 89~

The introduction of hormonally responsive genes into cells provides an experimental system to determine whether in-ducibility is a property inherent in discrete nucleotide sequences. Thus, rat ~-2~ globulln genes, Kurtz, D.T., i~ature 291:629-631 11981), as well as mouse mammary tumor virus (MMTV) genes, Hynes, N.E., et al., PNAS 78:2038-2042 (1981~; Buetti, Eo and Diggelmann, H., Cell 23:335-345 (1981~, remain responsive to glucocorticoids following - transfer into heterologous recipients. Further, the fusion of the promoter element of MMTV to the structural gene encoding dihydrofolate reductase renders this gene inducible with glucocorticoids, Lee, F., et al., Nature 294:228-23~ (1981).

In this study, the expression of human growth hormone (hGH) genes introduced into the chromosome of murine fibro-blasts has been examined In vertebrates, growth hormone synthesis is restricted to the pituitary gland. rn cul-tures of pituitary-cells, either glucocorticoid or thyroid hormone generates a 3-fold increase in the level of GH
mRNA, when added together, a 10-fold induction is observed, Martial, J.A., et al., PNAS 74:1816-1820 ~1977); Tushinski, R.J., et alO, PNAS 74:2357-2361 (1977). Cotransformation has been used to introduce from 1 to 20 copies of the human growth hormone (hGH) gene into thymidine kinase deficient (t~ ) murine fibroblast cells which express functional glucocorticoid receptors, Lippman, M.E. and Thompson, E.B., J. Biol Chem. 249:2483-2488 (1974). The administration of hormones to these cotransformed cells results in a 2.5 to 5-fold induction of hGH mRNA and a similar induction in secreted growth hormone protein in over half of these cell lines. The DNA sequences respon-sive to induction reside within 500 nucleotides of DNA
flanking the putative site of transcription initiation.
Fusion of this segment of DNA to the tk structural gene -80~ 8~

renders the ~k gene responsive to hormone action.

RESULTS

lrhe Regulated Expression of ~uman Growth Hormone Genes~ in Mouse Cells The human growth hormone gene is a member of a small multi-gene family composed of at least five genes, each sharing significant sequence homology and each likely to have arisen by divergence from a common ancestor, Niall, H~D., et aL., PNAS _ :866-870 (1971~, DeNoto, F.M..~ et al., Nucl. Acids Res. 9:3719-3730 (1981)~ Two of these genes have. diverged. significantly to generate two distinct lS ~ormones, chorionic somatomammotropin and prolactin.
Several dif~erent recombinant phage containing hGH
sequences have been isolated from a library of human DNA.
canstructed in. the Charon.phage ~4A, Maniatis, T., et al., CelI lS:68~-701 (I9~8). Two different phages, designated ~20A and ~2C, contain the entire growth hormone gene within a 2.6 kb Eco. RI fragment (Fi~. 3)O The complete nucleo-tide sequence of this Eco RI fragment derived from ~20A
indicates that this fragment contalns the entire hGH gene along with 500 5' and 525 3' flanking nucleotides. The restriction ~ap of the 2.6 kb Eco RI fragment of ~2C is identical to that of A20A. Partial se~uence analysis oE
~2C from the 5' Eco RI site to the Bam HI site adjacent to the irst exon (Fig. 1) is identical to that of ~20A.
Both ~20A and ~2C, which presumzbly encode the major form of growth hormone, were utilized in these studies.

Mouse Ltk cells express functional glucocorticoid receptor, Lippman, M.E. and Thompson, E.B., J. Biol. Chem 249:2483-2488 (1974). A viral t~ gene has been utilized as a selectable marker to introduce the DNA from recombinant phages ~20A

9~
and ~2C into this cell line by DNA-mediated gene transfer, Graham, F.L. and van der Eb, A.J., Virology 52:456-467 (1973); Wigler, M., et al., Cell 16:777-785 ~1979).
Cotransformants were identiied by blo~ hybridization, Southern, E.M., J. Mol. Biol. 98:503-517 (1975) and tested for capacity to regulate the expression of exoyenous human growth hormone sequences. DNA was isolated from nine transformnats obtained following cotransfer to tk and hGH
DNA, restricted with the enzyme for Eco RI, and analyzed by blot hybridization utilizing highly radioactive hGH
probes. Three of the six transformants exposed to ~2C DNA
integrate at least one intact Eco RI fragment containing the hGH gene Only one of three transformants exposed'to ~2QA.~NA contains an intact gene. The hGH copy number in the~e lines is variable. Som~ lines contain a single integrated fragment, others contain 10-20 copies of hGH
DNA.

These cotransformants were then tested for their ability to regulate the expression of growth hormone mRNA. Poly A ~ RNA was isolated from cells grown in the presence or absence of 10 6 M dexamethasone for 48-72 hours. Northern blot analysis of these RNA preparations reveals that two cell lines synthesize an 825 bp mRNA homologous to the hGH RNA; a 3~4 fold increase in hGH mRNA is observed upon the addition of glucocorticoid. These two indicible cell lines derive from cotransformations with each of the two different clones. Anothex cell line synthesizes an 825 bp species and a 3 kb species constitutively; no induction with dexamethasone is observed. The origin of the larger transcript is unknown and may reflect either aberrant initiation, termination or splicing.

Growth hormone mRNA is not detectable in a fourth line.
Sufficient homology exists between the mouse and human growth hormone sequences to detect the expression of the - -82- ~2~89~

murine gene in ~hese cell lines, albeit at reduced sensi-tivity ~see below). Tk transformants which do not con~
tain intact hGH sequences do not synthesize detectable GH RNA either before or after induction. Thus, the fibroblast does not synthesize apprec:iable levels of endogenous mouse GH mRNA.

A more quantitative determination of hG~I mRNA levels was obtained by titrating hG~ sequences in RNA populations by dot blotting. A standard curve was initially generated with increasing quantities of huma~ pituitary ~NA. If radioactive dots are coutned, the pituitary standard remains linear from at least 1 to S00 pg of mRNA. This assay proved highly reproducible and allows the detection of as; little as 1 pg of specified hGH mRNA in 25 ~ g of totaL cellula~ RNA in a few hours of autoradiographic exposure. The results of this analysis confirm the less quantita~ive characterizations obtained by North~rn blotting-. Two clones appropriately regulate hGH RNA in res~onse to glucocorticoid and express about 125 and 25 copies of mRNA, respectively, per cell in the fully induced state (see Table III).

Localization of Regulatory Elements The previous experiments were performed with A clones which contain the hGH gene along with several kilobases of S' and 3' flanking DNA. It was desired to determine the minimal nucleotide sequence necessary to render a gene responsive to glucocorticoid action. In initial experi-ments, the 2.6 kb Eco RI fragment was subcloned into the plasmid, pBR325, and introduced into Ltk cells. Eleven tk transformants were obtained. Analysis of the DNA from these cell lines by blot hybridization revealed that nine of eleven tk~ colonies have integrated from 1 to 20 copies -83- ~Z~8~4~

of the intact hGH Eco RI fragment. In these gels, the endogenous murine hGH gene can be seen as a somewhat fainter high molecular weight Eco RI fragmen~ present in both control Ltk and transformed cell DNA.

Total RNA was prepared from these transformants and screened for the presence of hGH RNA by dot blotting. Seven of nineicotransformants express hGH RNA~ Poly A RNA was then prepared from five clones and examined by Northern blot analysis. These lines synthesize an 825 bp hGH mRNA, comigrating with the human pituitary transcript, which is inducible by the addition of glucocorticoid. In several of these lines, the inducible hGH mRNA appears as a dou-blet, while the origin of the larger transcript is unclearr a similar doublet in rat GH mRNA in GH3 cells is due to diferential polyadenylation. The levels of hGH mRNA, as well as the extent oE induction, differ among the different cell lines_ Some lines show a 3-fold induction, other lines show intermediate Ievels of induction. A rough correlation exists between the- amount of mR~A produced and the number of hGH copies integrated into transformed ceIl DNA. Thus, the line which synthesizes perhaps the least amount of mRNA has probably integrated only one or a few genes. Once again, control transformants lacking an hG~
fail to express detectable human or mouse GH mRNA. The observation that these cotransformants express elevated levels of GH mRNA upon exposure to glucocorticoid, even if only a single intact hGH gene is integrated into the chromosome, strongly suggests that the information required for induction resides within the 2.6 kb Eco RI
fragment. Therefore, hormonal control is not likely to result solely from the integration of hG~ DNA into pre-existing hormonally responsive sites in the recipient cell.

,,r~ '' ` ~
-84~ 8~9 Hormonal induction seems to be a property of the nucleo-tide sequence about responsive genes. Therefore, one would not expect induction of other cotransformed genes not normally regulated by glucocorticoid. A control was performed in which RNAs previously examined for hGH
sequences were subjected to Northern blot analysis and exposed to highly radioactive- ~k probe to test for the induction of tk gene expression in these cell lines.. As noted earlier, these cotransformants were inducible for th~ expression of GH mR~. In contract, these cells express distinct 1.3 and 0.9 kb tk mRNA transcripts (see below) whose level remains constant before and a~ter induction_ Glucocorticoid inducibility therefore is not a property of all cotransformed elements, but is: specific for the hGH gene~

rnduction of Growth Hormone Protein . _ It was. then asked whether the growth hormone mRNA sequences present within our transformants direct the synthesis of a secreted growth hormone polypeptide. In this manner, one could determine whether the levels of induction of mRNA
are reflected by a proportionate induction in the level o~
secreted protein.. Control cells and transformants con-taining hGH genes were grown either in the presence orabsence of hormone for five days without media change.
The media was then tested for hGH in radioimmune assays utilizing antibody-coated filter disks. As shown in Table III, cell lines which demonstrate significant levels of hGH mRNA also secrete significant amounts of hG~ into the tissue culture medium. The maximum secretion of hGH
is observed with cell line 9, which also exhibits a high level of mRNA in the series. This cell line synthesizes about 15 ~ g per liter in the absence of hormone and 60~ g of hGH per liter in the fully induced state. Thus, induc-8~4~
tion in the levels of hGH mRNA results in a concomitant induction in the levels of secreted proteinO

Regulation of an hGH-tk Fusion Gene It was asked whether the hGH sequence element responsi~Te to hormonal induction can impart hormone sensitivity when fused to other structural genes. Expression of the 2.6 kb Eco RI fragment is hormonally regulated in mouse cells.
In contrast, cotransformed tk gene expression is not regu-lated by glucocorticoids. A fusion gene consisting of the 5' flanking sequences of hG~ and the structural sequences encoding tk was therefore constructed to discern whether the tk gene in this configuratio~ is responsive to gluco-corticoid induction~ The 5' flanking region of hGH ~Aextending from the ~co RI site to the Bam HI site, 3 nucleo-tldes into the 5' untranslated region, was ligated to a fragment of tk DNA beginning in the 5' untranslated region o~ the tk message 60 nucleotides upstream from the trans-lation start site (Fig. 4). This tk fragment contains theentire structural gene but lacks all essential promoter elements, McKnight, S.L., et al., Cell 25:385-398 ~1981).
Thus, a fusion gene was introduced into Ltk cells and poly A was then isolated from resulting tk transform-ants. The patterns of the tk RNAs on Northern blotanalysis are complex.

Transformants containing a wild-type tk gene frequently express two tk mRNA transcripts, 1.3 and 0.9 kb. The 1.3 kb RNA is always expressed in transformants containing an intact tk gene and encodes the wild-type enzyme. The 0.9 kb transcript initiates internal to the tk gene, consists solely of tk structurai gene sequences, and encodes a truncated protein of diminished activlty. RNA from three tk transformants containing the hGH-tk fusion gene :~2~9gL9 reveals two species of tk RNA. If the hGH promoter diLected initiation appro~riately, one. expects a fusion transcript 1.25 kb in length consisting o three 5' nucleotides derived ~rom the hGX gene and the rer.ainder encoded by the viral tk gene. The size of the larger ~NA
i~ these cells is consistent with a fusion transcript..
The 0.9 kb RNA presumably represents the aberrant tk transcript. In two of three transformants containing the fusion.gene, one observes that the larger transcript is inducible upon glucocorticoid administration. The 0.9 kb transcript generated from an internal tk promoter provides a fo~tuitous internal control. Induction of RNP levels i5 obse.rved only for ~he ~usion transcript; the 0.~ kb re-mains unresponsive to glucocorticoid administration. It is apparent f~om these studies that sequences responsive to induction reside within the 500 nucleotides flanking.
the 5' terminus of the h~ gene_ Eusion of this element to other structural genes now renders: these sequences hormonally responsive.
DISCUSSION

The int~oduction of recombinant clones. containing the human growth hormone gene into mouse fibroblasts results in the regulated expression of hGH mRNA in over half of the cotransformants. These results, together with similar studies on MMTV, Hynes, N.E., et al., PNAS 78:2038-2042 (1981); Buetti, E. and Diggelmann, H., Cell 23:335-345 ~1981); Lee, F., et al., Nature 294:228-232 (1981); Huang, A.L., et al., Cell 27:245-255 (1981); Ucker, D.S., et al., Cell 27:257-266 (1981) and the ~-2~ globulin gene, Kurtz, D.T., Nature 291:629-631 (1981), indicate that sequence elements reside within these cloned genes which are sufficient to render them hormonally unresponsive. One such element is contained within the 5' flanking DNA of -87- ~2~

the hGH gene and can be fused to other structural genes which then become hormonally responsive.

These results suggest that the induction observed is based upon transcriptional activation rather than RNA stabiliza-tion. First, the hGH-tk fusion presurnably generates an inducible mRNA with very few 5' hGH nucleotides; the re-mainder of the RNA encodes tk enzyme. In control cells, wild-type- tk mRNA is not inducible by hormone. It is con-sidered highly unlikely that the short segment of hGH can confer stability on the remaining length of mRNA indepen~
dent o~ sequence or source. Second, hGH transformants syn-thesizing matuE~ hGH mRNA constitutively have been identi-fied. I~ induction results solely from stabilization, one would expect all transformants which synthesize hOEI RNA to ex~res~ enhanced levels in the presence of hormone. These resuLts do not exclud~ mRNA stability as a contributing factor in the inductive ~rocess, nor do they argue that transcriptio~ level control is the sole determinant of induction. The- data do indicate that an element present in 500 bp o~ 5' flanking DNA is sufficient to render a gene responsive to hormone and that this element most likely operates to enhance transcription. This is in accord with more direct tran~cription rates for glucocorticoid induc-ible genes, Ringold, G.M., et al., PNAS ~ 2879-2883 ~1977)-Evidence is accumulating for several genes that regulatory elements controlling the rate of transcription reside in 5' DNA quite close to the structural gene. Thus, MMTV, Lee, F., et al., Nature 294:228-232 (1981); ~-2~ globulin;
hGH; tk, McKnight, S.L., et al., Cell 25:385-398 (1981);
mouse globin, Chao, M., et al., manuscript in preparation;
metallothionein, Brinster, R.L., et al., Cell 27.223-231 (1981); and Drosophila heat shock, Corces, V., et al., PNAS

~ 9 78:7038-7042 (1981) genes remain responsive to widely differing inducing agents with lkb or less of 5' flanking DNA. It is impossible that such elements exert their effects locally and do not "transduce" information over S long distances. The hGH-tk fusion gene generates two mR~As; a 1.25-I.3 kb RNA presumabl~ initiated into GH
sequences and second 0.9 kb species initiating in the tk structural gene about 300 nucleotides downstream. However, the 5' terminus of this mRNA has not been precisely de-fined. Qnly the longer RNA is hormonally inducible,suggesting that the hG~ regulatory element acts locally to activate transcription and has little or no effect upon the frequency df close, but downstream initiations. This a~guments must be tempered by the fact that cell lines expressing the fusion gene have ,.Legrated multiple copies o~ gene. It is t~erefore possible that the smaller tran-script derives solely from genes which remain unresponsive to hormone action_ One striking observation is that newly-introduced hG~ genes ex~ress significant levels of mRNA and protein while the endogenous murine GH in fibroblasts remains inactive either in the absence or presence of glucocorticoid. Since GH
synthesis is restricted to the pituitary and is never expressed physiologically by fibroblasts, one is obliged to consider why newly-introduced GH genes function in the recipient cell. It should be noted that although the control fibroblasts do not synthesize significant levels of endogenous GH mRNA, it is not known whether the murine gene is active in transformants expressing exogenous GH genes.
However, in an analogous system, hormonal induction of exogenous rat ~-2~ globulin genes in a mouse fibroblast is not associated with activation of the endogenous mouse genes, Kurtz, D.T., Nature 291:629-631 (1981).

r~ ~ ~

lZ~
A pattern is emerging from gene transfer experiments which suggests that the mere introduction of exogenous genes into cells is sufficient to assure their expression. Thus, several genes, includiny globin, Mantei, N., et al., Nature 5 281:40-44 (1979); Chao, M., et al., PNAS 78:2038-2042 (1981) when introduced into cells, synthesize significant quantities of RNA while their endogenous counterpart genes remain transcriptionally silent. Further, if appropriate control signals exist in the recipient cell, expression of the newly-introduced genes may be properly regulated.
Exogenous hGH genes in the murine fibroblast containing receptors express inducible levels of mRNA. Finally, expression of endogenous G~ sequences in non-pituitary tissue is virtually never observed.
Studies of gene transfer together with studies of gene expression during normal development therefore define at least three states~ o~ genetic activity: "off", "on" and "regulated". The mere presence of a glucocorticoid re-ceptor complex is inadequa~e to activate genes in the "off"
state a~ is apparent for the endogenous GH gene in non-; pituitary cells. Maintenance of this state perhaps- re-flect~ the chromosomal location of the endogenous gene or alternatively may resu-lt from prior developmental events about this gene which are self-perpetuating through cell division. Whatever mechanism is responsible for mainte-nance of the "off" state, it appears to be "cis" acting; a single exogenous gene introduced into a cell in which the endogenous gene is "off" can function in a regulated manner. Transformed genes may there ore escape the developmental history of the cell and immediately conform to the "on" state, a state accessible to appropriate regulators. In this state, genes may be regulated if appropriate controlling elements exist within the cell.
The regulated state may therefore involve "trans" acting r~ -~8~4~

--so--~actors uch as steroid hormone-receptor complexes.

Table III. Quantitation of hG~ mRNA and Pro~ein in P~louse Cells .

Cellcpm/l~g .nRNA copies/b Fold Secreted ~ ior AG~-D --39z 35 l.. Z 1.0 (below c~ntrol ) ~473 4~ 3 . 8 AG~--E --91, .8 ~_9 1. 8 (belo~
co~trol ) t-26T 2~ 4~ g ~5 AGE--~ -2gL 2~ 4 ~a 5 . O
t-14 0 1 ~;2~ 1.9 . O

AGE--~ --30 ~- L. ~ N,D
ZQ~

w.tG~ 58 5 2.. 5 N.D~
~14 6. 13 'j WtG~l-4 --1147 10~ 1 7 19 . O
~2054 183 6~.0 wtGEL-9 -487 43 3 . 0 18 . 4 ~14S5 129 66 . 0 wtG~ 71 6 2.6 N.D.
+185 17 ~2 EGEND TO TABL:E III

aDete~mined from dot blots containing 3-5 concentrations of p~ly A~ RNA o~ each s ampl e .

bTh~e a~e Z X 105 mR~A molecules. per- L cell, assuming O.S
pg ~clly A+ RN~cell zmd an aver~g~ mR~A size of 1 kb. From s.ta~dard cu~vc. de~ved by do.~ blo~ing human pituitary R~A
i~L whic~ a~proximatE~ly 20% o~ t:he mRNA is hGH, there are 450 ~0 cpm~lûO rIq oi~ pituita~:y RNP., ~ .25 cpm/pg h~ mRNA.. All cpm' 5 have beer~ nc)rmalized to the same sltanda~d cur~..

CE~rom r.adioi~nun~ assay, in whic~s the negative control is Z.~- ng/ml..
~ç
N_~_ ~ Mc~ Oe!cermined_ .

_93~ 2~ g9L9 MAT RIl~,LS AND MET~IODS
CelL C s ~tk apr~,. a deriva~ive o~ Itk clone D, Kit, ~.. e~ al., Esp CelL Res 31:29l 312 (19~3), was ma:Lntained in 1~ Dulbecco's modiied Eagle~s medium (DME) con~alnin~
10% cal~ serum (F~ow ~a~oratories, Rock~ille, Maryland) and 50 ~g/ml o~ diaminopurine (DAP). Prior to trans-~ormation, cells were washed and grown or three gene~a-tions in th~ absenca o~ DAP. A Chinese hamster cell lln~
contai~ing a~ aLtexed dihydroolat~ reductase (renderin~
Lt re~lsta~t to m~th~xtrexat~) ~2g MtxgIIr,.F~into~
~_ ~ , ~t al~, Somatic C~lL Genetics ~:245-261 (19t6), was propa~ated i~ DME ~upplemented wit~ 3x non-essential amina.acidsr ~a% cal~ serum.and ~ ~g/ml amethopterin.. For ~Q th~ am~Liication exper ~entsr t~OE medium was additionally suppleme~ted wit~ Z0 ~q~mL o m~thotrexa~e~

Mur~n~ Ltk aprt celLs are adenine phosphoribosyltrans-erase-negati~e derivatives o L~k clorle D cells. CeLls 25 were maintained in growt~ me~ium and prepared f or trans -format~ A as described, Wigler, M., et al., PN~S 75:1373-1376 (197g).

~Ep~ (human), ~eLa (humarl), CXO (Chinese hamster ovary), and Lt~ cells were grown in growth medium. L~2b, a deri~ative o Ltk. transformed with hexpes simples virus tk DNA, was maintained in growth medium cc~ntaini~g hypoxanthine at 15 ~g/ml, aminopterln at 0.2 ~g/ml, and thymidine at 5 . O
~g/ml (XAT), Wigler, M., et al., Cell 1:223-232 (1977). All 35 culture dishes were Nunclon (Vang~ard International, Neptune, N. J. ) plastic.

The feeder-independent mouse tera~ocarcinoma cell cuLture line 6050P, Watanabe, T., et al., PNAS 75:5113-5117 (19781, ~ 94--~2~ 9~
obta~ned i~rom a tumor o~ ~he OTT 6050 transplant line, 5 was used as the wild-~ype, or tk~, parent and is here designated q~CC wt Thi.~ ~ 1~ o th~ X/O sex chromo--som~ type~ and has a modal num~er o:E 39 cl~romosomes with ohc.racte~ istics described ~ Watanabe, T., et al., (19~8) .
Th~ c~.lls were growrr ~ Dulbecco r S modified ~aqL~1 s 10 medium wlth: 10% ~etaL cal:~ se~.. Ate:~ 3 h:~ o exposur~
to 3 ~lg/ml o th~ mutagen N -rnethyl-N'--nitro N-nitrosoguarlL--din~, ~h~ cells wer~ a:llowed ~o recover for two days a~d wer~ then transferred to medium with 80 ~/mL o~ 3rdUrd.
A s~r:Les: oi~ reslstant clones wer~ isolated; one supplied 15 th~ clanaL ILne~ (TC:C ck ) used In the presenl: transi~orma--tio~ experiment~ This llne ~ad ~ reversio~ ~requenc~ t~

w~ ype~ ~ less then ~o 8~ ~h~ c~LL5 were mai~tai~e~ ~

me!diu~r with:. 3~ tlsjml o Brdl:lr~ d, p~:ior to tran.s~ormation, .

we~e washe~ an~ g~aw~ ~o th~e~ ge~erations in ~he. absence Zû c~ he dru~_ ~rr~sors~ orr ei~ic~ ~z wa.~ compared w~th:~
th~t o ~ tk-de~icient I~e, Ritr Ç~, et aL,,. Exp_ Cell.

R;es~. 31~:2,97--3I2 (1563:~ o mouse ~ cells (1; tk ) ~

~ - ~' DNA

~ h: molecular weight DN~ was obtained from cultured cells ~C~O, L~2b, and ~eLa) or ~rom ~roze~.rabbit livers as pre-viously descri~d. Wigler~ M , et al., Cell 14:725-731 (.19~ igh molecular weight salmcn sperm DNA was obtained rrom Warthington. Res~ric~io~ endonuclease clea~age (Bam I, ~lndIII, Kpn I, and Xba I) was performed in a buf~er contain-Lng 50 mM NaCl, 10 mM Tris oHCT ~ 5 mM MgCl~ ~ ~ mM mercapto-ethanol, and b~vine sexum al~umin at 100 ~g/ml (p~ 7.9).
3S ~he enzyme-to-DNA ratio was at leas~ two units/~g o D~A, and reaction mixtures were incubated at 37C for at least 2 hrs(one unit is the amount a~ enzyme that di~ests 1 ~g of DNA in 1 hr). To monitor the completeness of disestion, 1 ~1 of nic~-translated adenovlrus-2 [3 Pj~NA was incubated _95~ 49 wi~h 5 ul of reaction volume for at least 2 hr, cleavage ~roducts were separated by electrophores:is in L~ agarose g~lsr an~ digestio~ was m~nltored ~y exposin~ the dried.geL
t~l Cronex 2DC x-ray ~ilm..

Ihtact herpes sim~lex virus (RSV) DN~ wa.s isolated ~rom CV-l-Lnected ceIIs as ~re~iausly descri~ed. Pellicer, A , et aL_r Cell I4:133-L4~ (~978).. DN~ was digeste~ to com-le~io~ ~ith R~n r ~New EnsIand Biolabs) ln a buf~e~ con-taininq ~ m~ ~ris (pE 7 ~,.6m~ ~gCI~, 6 mM ~-mercap~o-ethanoL~ ~ m~ NaCI an~ 200 ~g/ml ~ovin~ serum a~bumin. ~he L5 restricte~ DN~ was ~actionate~ ~y electro~horesis th~ough Q_ ~ aqarose gels t~T ~ ~0 x ~.5 cm) ~or 24 hr a~ 70: V, an~
t~ 5.L ~ t~-contai~inq ~ragment was extracted rom the gel as describe~ by M2xam, ~_ M_ an~ ~ilbert, W_ PNA5 74:.560-~64 tlg~7) a~ W~ler, ~, ~ aL_, CeIL 14:t25-t3L ~1978) ~0 ~I74 ~. R~I D~ wa~ purchase fro~ Be~hesda ~search ~ab~ratories_ ~Iasml~ ~BR32Z D~A was grown in ~ coli ~
laL a~d puri~ie~ according to ~ method o~ Clewellr 3.`B., J.. Bac~erioL_.1~.66~-6~76 (19~72)~. ~h~ cloned ~abblt 3 ma.lo~
z~ globi~ gene L~ the ~ C~aron 4A deri~ative (R~G-L.) wa~ iden-tiied and isolated as previously described. Ma~iatis, T., et aL..,. Cell 15:687 70~(I9t8J.

In th~ amplifica~ion experiments,`the size o~ t~e high ~olecular weight DNA was determined ~y electrophoresis in O.3% agaros~ gels using ~erpes simplex vlrus DNA and its Xba r ~ragments as mar~ers. Only DNA whose a~erage size was larger tha~ 75 kb was found to possess transforming activity in th~ ampliication e~periments. In these experiments, plasmid DNAs were isolated from chloramp~enicol ampllfied cultures by isopycnic centrifugation in CsCl gradients con-taining 300 ~g/ml e~hidium bromide.

Transf ormation and Selection _ Th~ transformation protocol was as described in Graham, F
1~_ an~ van der Eh, A~ Vi~ ology, 52 456~45T (I9~) wlth the~ i~ol.lowing modiflcations_ One day prior to tra~sforma--tion~, c~lls wer~ seeded: at 0 T ~ 106 cells per dish_ Th~
o medium was change~ 4 h~ prior to t~:ansi~ormatior~. Sterile, et}Ia~sol-precipitated. higE~ molecular weight or restric~ion endonucleas~-cleave~ euca::yoti~ ~NA dissolved ~ L m~ Tris (pE T_3)/O_L m~q EDTA waC used to prepare DNA/CaC12~ which con~ains D~A at 40 ' ~Lg/ml an~ 250 m~ CaC12 (Malli~krod~) .
~wica-cor2c~n~rated E~epes-~uered; saline t2X E~:E35) was ~?r~--pare~,. it contains ,280 m~q NaCI, S~ . Hepes, an~ ~:.5 mD~
~u~ phosphat~r p~ a~juste~ to T.~0` + 0,05.. ûNA/CaC12, s~lutio~ was adde~L~ dropwise tcl a~ e~ual volume o~ steril~
~X ~S. ~ ~ I. steril~ pl~stic pi~tt~ w~th a cc~ o~ plug ~Q w~5. ~serte~t int~ tE~ tu15~ con:~aini~. 2X ~IBS, an~
:bleswe~ roduc d~ Eiy hIo~i~g~ w}~iTe~ th~ D~A was being adde~ 'rh~ caIcium phc)sphat~/DNA precipita~ was allowed~ to i~o~ witho.u~= agL~atiorr ~or30-4S ~ at: room temperature_ ~ precipitat~ w~ the~ mixed ~y qentl~ ~i~ettin~ with a 2S ~laqtic pi~ett~, an~ L ml a~ precipi~ate was added per plat~, directly to the 10 ml o~ g~owth medium that covered the re-cipien~ cells. A~ter 4-hr incuba~ion at 37~C, the medi~m was replaced.and the cells were allowed to incu~ate for an additionaL 20 hr. At that tLm~, selec~ive pressure was applied. For ~k+ selection, medium was changed to growth medium containing E~T. For aprt selection, cells were trypsinlzed and replated at lower density (about 0.5 X 106 cells per 10-cm dis~l Ln medlum containing 0 . 05 mM azaserine and 0~1 mM adenine. For ~oth tk+ and aprt~ selection, 3s selecti~e media were chan~ed t~e next day, 2 days a~er that, and su~sequently every 3 days for 2-3 weeks while trans~orm-ant clones arose. Colonies were pic~ed ~y using cloning cylinders and the remainder of the colonies were scored after formaldehyde ~ixation and staining with Giemsa. For characterization, clones w~re grown into mass culture under con~inued selec~i~e pressure. A reco~d was kep~ o the a~parent number o~ cell dou~lings for eac~ clone- isolated_ ~ethotrexate-resi~tan~ trans~ormants of ~tk aprt cells wer~ o~t ~ e~ ~oLlo~ing ~rans~ormation with 20 ~g ~ high 1~ molecular weight DNA fr~m A29 M~xR II celLs and selectio~
in DM~ containinq ~0% cal~ s2ru~ and 0 2 ~g/ml amethopterin_ Eror.tk~ s~le~tion, caLls were grow~ in aA~ medium; for re-sta~ce to methotrexat~, cells were selec~e~ in medium ~5 s~ppIemented ~tk ~ L ~s/m~L o~ methotre~ate. Col~ni.e~ were ~lo~e~ ~ro~ lndi~idual dishes t~. assl~re that each ~rans-c~rmant arose ~som an~ Lndependent event. l;igates between DN~ a~d lin~arized pB~32Z DNA were ~repared b~ incub~in~
æ ~-L ratio~tw/w) o~ 5aL r-cLea~ed DN~s with ~ ligas~
2~ etheisd~ Research ~abora~rIesl u~de~ ~he conditions ~e~
c~mmend:ea b~ the~ lier. ~ ca;Lc~ }?hosE~hat~ precipitate was ~repa~eL usi~g ~ ~g ligate. an~ ~8 ~g carrier/ml~ a~
adde~ to recipient cells (th~ amount o ligat~ was lLmited becaus~ o~ th~ o~serva~io~ that plasmid inhlbits trans~orma~
2S tio~l~ ~he DNA was aLlow~d to ~emain.i~ contac~ with the ceLls for 4-1~ hr and the medium was then aspirated and re-plac~d with ~resh DME~ Selective pressure was applied 24 hr ~ollowin~ expcsure to DNA, A~ter 2-3 wee~s, colonies were isolated using cloning cyli~ders~
In the mouse b~t~si=cma cell experiments, trans~ormatlon was performed as described previously except that the TCC
tk cells were seeded at.3 X 105 cells/plate one day prior to transformation. To each plate o~ attached cells was added a calcium phospha~e/DNA precipitate prepared with 4 ~g of the recombinant plasmid, Pt~ 1, digested with Bam ~1, in the presence of 20 ~ o~ high molecular weight DNA ob-tained from ~ tk aprt cells.

--98~
9~9 In addltion, some cells were treated in susper.s ion, WlllecXe, K e~ al. / Molec. Gen. Genet. 170:179_185 (L979) .
T X 106 ~reshly trypslniæed TCC t3~ cel~.s we::~ mixed with.
a calclum phospha~e/DN~ precipi~ate prepared wit~. ~0 of DNA ~rom the E~ cleave~ plasmid. ~ L and 150 ~lg o:~ ~ish molecuLar weiqht DN3~ from salmon sperm. Following LO celltriuqat~on, resuspension, and shak~nq, as described i$L Willec}c~, K e~ al~ ~19~9), the cells were aqain plated ir~ growt~s medium_ ~ter three days, the medium was re-pIaced ~t~ ~ medl~L and c~lor~ies oi~ transformants were isolated ater twc~ weeks~
15.
Cotrar~s~o:rmation experiments were pe~:orsned wit~ 4 ~ o ~am EL-digested ~tk~ DNA alonq with 4 ~q o~ E~in~ rII--c~Ieaved; pIasmid pEI~ contain ~ ~ the~ chromosomaL adul~
huma¢ ~-qlohL~ qen~, ~awn,. ~ M.,. et al., Cell 15^~5:~-~a LLT~ k txans~oxmants we~ selecte~ ~ g~owt~;
medi ~ containln~ O~ m~ h~poxanthine~0.4 ~ ~ nopteri~/
L5 ~ thymidin~ (~A~ Colonies were pic~ed with cIoninq cyLinders an~ were grown into mass cultures.
~, .
~5. ~ ~
Gerle Ltk aprt mouse cells were transformed with either 1 - 10 ~ o~ ~X174, 1 yg o pBR322 or ~ ~g o R8G 1 DMA in the presence o~ ~ ng of HSV-l t~ gene and 10-20 ~g of salmon sperm carrier DNA, as pre~iously described. Wigler, M. et al , PNAS 76:1373-1376 Cla79 ) . ~k transformants were selected in DME containin~ hyp~xanth1ne, aminopterin and thymidine (H~T) ~nd 10~ cal serum. Isolated colonies were 35 pic}~ed using cloning cylinders and grown into mass cultures.

Enzys~e s s~

Extracts were prepared by resuspending washed cell peLlets ~2~4~

(approximately 10~ cells) in 0.1 ml of O.0~ M potassium phosphate, pH T, con~aining 0.5% ~rito~ X-~OO. The super-nata~ (cytoplasm) obtained ater 25 mlN of ~00 X ~ cen~ri-fuga~on was used for th~ quantitatio~ o~ enzymatic activity an~ ~or electrophoresls. apr~ and protein we~e assayed a~
pre~iously aescribed.. C~asin, ~ A.., Cell 2:3t-41 (19~4).
LO Inclu.~ion oi~ 3 InM thymidine triphosphate, an inhibitor o ~
S' nucleotidase, ~urray, A~ W. and ~riedrichs, B., Biochem, J~ llL:83-89 (1969), i~ the ~eaction mixtur~ did not in-crease AMP rec~ery,. indicatin~ tha~ the nucleotidase was not i~ter~ering wi~h the measurement of aprt activi~y.. rSo-eLectric ~oc~si~q o aprt wa~ carrie~ out essentiaIly asd:escribed for hypaxan~h~n~ phosphoribosyLtrans:era5e, Chasi~rt.
L_ A. a~d~ Urlau}:,. G~ SomAt-celL Genet_ ~.453~467 (1976), wit~ ~
theS ~LIo~ln~ exce~tions. T~e p~Lyacrylamide gel cantained a~ AmphaIine. (LK~ mLxtur~ o~ 0.8~ pE 2.5-4, 0.8% pE 4-~, ~0 an~ 0~4~. ~ 5-7`~ For assay~n~ enzymatic act~ity, rz - EJ
a~e~ln~ t0_04 m~, 1 ~i/mmoL,. Ne~ England Nuclear ~ Ci =-3..~ X 101 ~ecquereLsl] was substituted ~or hypoxanthine.

AssaY o~ Th~ idlne. ~inase Ac~i~it~

For specific activity measurements, cells from monolayer cultures were scraped into phosphate buf~ered saline and washed~ ~he cell peIlet wa~ suspended in S volumes of ex-traction buf~e~ (0.0l M ~riso~Cl~ pH 7.5, 0.0l M KCl, lmM
30 MgC12, ~mM 2-me~captoethanol, and 50 ~M thymidine). The cell suspension was frozen and thawed three times and the KCl concentration was then adjusted to 0.15 M. ~fter s~nication, the cytoplasmic extract was obtained by centri-fugation at 30,000 X g for 30 min, and the supernatant was used for tk assays as described in ~igler, M. et al. Cell l6:777-785 (19191. Cytoplasmic extracts from tumors were obtained after disruption of the cells in a Potter-Elvejehm homogenizer. They were then treated as described above for cuLtured cells. One unit of thymidine kinase i5 defined as the amount of enzyme which converts one nanomole of thymi~

~2~39~9 dine into thy~dine monophospha~e per minute.

In enzyme neutrali2atio~r studies, anti-~SU--i t3c anti--s~tr or pre~un~ serum was mixed wit~ an equaL volume o-E
cytoplasm~c extra::t, and A~ arld magnesium were add~:~l to

6~T mM. Th~ e~zyme--antibady mix~ur~ was incubated or 30 min at room temperature, centri fused at ~, 000 X g for 10 m:Lrr, and ~h~ supernatant was assaye~ for t~ activity.

I~ an additionaL bioch~nical assay, 30,000 ~ 5 super-nata~ts oi~ homQgenatas i~rom c~ll cultures and from soli~
~-5 tumors w~re electro~horesed~ on 5% ~ lyac ylamide~ geLs which were ther~ cut irltQ L~ 6 mlrL slices a~d~ a~sayed i~or t~c activi~y as described~ Le~, L_ S_ arId~ Cheng, Y~ C_, J.
3iol. Che~, 251. 2.60û--~604 ~1976) _ 2~ ~ rsc1la~ion To~al R~A was isolated from loy~rithm~c--phase c~ltures o~
trar~s~orme~ r, cells }iy-successive ex~ractions with phenoL at pH 5~I, phenol~chloroform~isoamy~ alcohol (25.~:~, vol/vol), 25 and chloro~orm/iso~myl alcohoL (24:1, vol~vol).. Af~er Pthanol pre~ipit~tion, the RNA was digested with DNase, Maxwell, r. H., ~t al., Nucleic Acids Res. 4:241-24O (19t7) an~ precipitated with etha~ol.. ~uclear and cytoplasmic fractions were isolated as described in Wigler, M. et al;, 3Q PN~S 76:13~3-1376 (1979~ and RNAs were extracted ~s describ-ed above. Cytoplasmic polyad~nylylated RNA was isolated by oligo(dT)-cellulose chromatography. Axel, R. et al., Cell

7:247-254 (1976~.

cDNA S nthesis .

. Rab~it and mouse c~NAs ~ere prepared by using avian myelo-blastosis virus reverse transcriptase (R~A~dependent DNA
palymerase ) as described in Myers, J. C. and Spiegelman, S., PNAS 7S:5329--5333 (1978) .

8~9 Isolation of T:rans~ormed Cel.1 DNA
,, , ~

Cells wer~ har~7ested by scraping into P~S and c~ntriugin~
at 1000 X q ~or L0 mi~ he pellet was r.esuspe:rIded in 4û
vol ~ 10 mM TrIs--~Cl (ph 8 . 0 ), 150 mM Na~L, lO mM
EDTA], and~ SDS and pro~einas~ R were added to 0 . 2% an~ lOQ
. L0 ll~r/ml, respectively. The lysate was incubated at 3tC ~or ` S--10 h$: and then extracted se~uentially with buffer--saturated phenol arld C~C13 ~igh molecular weight ~r~A
isolat~d by mixing the a~ueous phase with 2 vol. of cold e~hanoL and i~ediately remo~ing the ~recipitate that L5 formed_ ~h~ DNA was washe~ with 70% ethanol~ and dissolved i~r L mM ~ris, Q.. L EDTA .

~ucLei an~ cyto~ asin froIIr cl.ones ~X4 an~L ~XS were. prepared as de~cri:l:ed by R~ngold:, G_ ~L.,. et al. CeLL lO:L9--26 (l~t7).
~Q ~r~e nucIear ~raction was ~u~ther fractionated into high and:
~a~ molecuI~ weig~t DN~ a~.d~scribe~ by ~irt, B~,. J. ~o~_ 3i~1. 26~365-369: ~1967~:.

DNA Filter ~ bridizatIons ~__ ,Y, ~
CelLular DN~ was digested wïth restrictlon endonucleases,.
electrophoresed on agarose sla~ gels, transfe~ d to nitro-cell~lose ~ilter sheets, and hy~ridized with 3 P-labeled DNA
probes as described ~y Wigler, M. e~ al., P~AS 76:1373-1376 (.1979).

D~A from transformed cells was digested with various re-striction endonucleases using the conditions speclied by the s~pplier (~ew England Biolabs or Bethesda Research Labora~ories). Digestions were performed at an enzyme to DNA ratio of 1. 5 U/~g for 2 hr at 37~C. Reactions were terminated by the addition o EDTA, and the product was electro~oresed on horizontal agarose slab gels in 36 mM

9~
Tris, 30 mM NaH~P04, L mM EDTA (pH 7.7). DNA fra~men-ts were trans~erred to nitrocellulose sheets, h~brldized and washe~ as previously describe~. Weinstock, R., et aL., ~NA5: 75:1299-L303 (1978) with two modiica~ions. Two ~it~ocellul~se ~ilterx wer~ used during ~rans~e~.
~e~reys, A. ~. an~ ~lavelL, R.. A~, Cell 12:109~-1108 ~ (1977). Th~ lower filter was discarded, and followinq hybrid~zation the filter was washed 4 times for ~0 min in 2 ~ SSC, 25 mM sodiu~ phosphate, 1~5 mM Na~P~O7, 0_05% SDS at 65C and the~ succes~ively Ln 1:1 and 1:5 dilution~ of this ~u~fer_ Je~freys~ A~ J. and FlavelL,.
~ ~r C~IL 12 :42~--439 (1977) I~ h~ am~liicatio~ ex~er1m2nts the prcbes wer~ either 3 ~ ~1c~ translate~ ~BR~2~ or pdh~r-2I, a cDNA c~py of mous~ dh~r mR~ Chan~r A~C~ t al~,. Nature ~5:6LT-~ 6Z4 ~1~7~)~

S~lu~ion ~vbridi~ations.

Z~-Labeled globL~ cDNAs (s~ecifi~ act~itles. o 2-9 X
108 cpm~us) were hy~ridized with excess RNA i~ 0.4 M
NaCl/25 mM 1,4~pipe~azinediethanesulfonic acid (Pipes), pH
6~5/5 mM EDT~ at 75C. Incubation times did not exceed 70 hr. Rots were calculated as moles o~ RNA nucleotides per liter times time in seconds. The fraction o cDNA rendered resistant to the sl~gle~strand nuclease Sl in hybridiza~io~
was determined as described. Axel, R. et al., Cell 7:247-254 (1976).

~NA Filter Hy~ridi~ation RNA was electrophoresed through 1% agarose slab geLs (17 X
20 X 0.4 cm) containing 5 mM methylmercury hydroxide as described by Bailey, J. and Davidson, N~, Anal. Biochem.

~2~

70:75-85 (1976). The concentration or RNA in e~ch slot was 0 . 5 ~g/~l..... E~lectrophoresis was at 110 V for 12 hr at room temperatu~:e .

RN~ was trans:~erred ~rom the gel ~o diazotized. c~llulose paper as descri~ed by Alw~ne, J.. C.. , et al., PNA5 74: 5350~
5354 (~979) by ~sing pE~ 4~0 citrate transfer }~u~fer. A:Eter transfer,. the RNA i~i.Lter was incubated or 1 h~ with trans-~er buf~er c~ntaining carrier RNA a~ 500 ~,q/mL. ~rhe RNA on the filters was hybridized with cloIIed: DNA probe at 5 0 ng/m~ led ~y 32E~nic3~ ~ransLati.o~:, We~nstoc~, R_, et al.. .
~5 ~NAS 75~ g9--I3Q3 (1978) to sE?eciic acti~ities o~ ~8 X.
L0~ cpm~ Reac~lio~ volume~; we~ Z5 pl/cm~ of~ ~iIter.
E~ybridi~atio~ was i~ 4~ standar~ saLine citrat~ (0.15 M
NaCI/0 ..GI5 ~ sod.ium citrate) /5~ orm~de at 57C :i~or er hybridizatior~r ~iLters wer~ soaked in twc~ changes o 2X ~a~dard saline citrate/25 mD~: sodium pho~pha~?~l. 5 m~
sodiu~ E~y~ophosphate/0.196 sodiu~r dodec:y~ sulf~te/5 m~ EDTA
at. 3~ac ~o~ 30 m~rs wlth shakin~ t~ remove formamide.
25 Successiv~ washes were at 68C with lX and .lX standard saline cltrate c~ntainin~ S mM EDTA and 0.1% sodium dodecvI
sulate for 30 min each.

Berk Sh~rE__nal~sis of Rab~ Globin RN~ in Transformed Mouse L Cells The hy~ridizations were carried.out in 80~ (~ol/vol) ormamide ~Eastman)/0.4 M Pipes, pH 6.5/0.1 mM EDTA/0.4 M
NaCl, Casey, J. and Davidson, N~, Nucleic Acid Res., 4:1539-1552 (1977~; Berk, A. J. and Sharp, P. A., Cell 1~:
721-732 (19771 for 18 ~r at 51C for the 1.8 kbp ~ha I frag-ment and 49aC for the Pst 1 fragment. The hybrids were treated with Sl nuclease and analyzed essentially by the procadure described by Ber~, A~ J. and Sharp, P. A. (1977).

Cell Culture; Hormonal Inductlon Murine Ltk cells were maintained in Dulbecoo's modified Eagle's medium (DME) supplemented with 10% calf serum (M.A. Bioproducts). Tk transformants were selcted and maintained in DME, 10% c~lf serum, 15 ~g/ml hypoxanthine, 1 ~g/ml aminopterin, 5 ~ g/ml thymidine (HAT), Wigler, M., et alO, Cell 11:223-232 (1977).

For hormonal induction, 10 6 M dexamethasone (Sigma) was added to sub-confluent cell cultures for 48 to 72 hours.
In initial experiments, 10 7 M thyroid hormone (Triiodo-L-thyronine, Sigma) was included: however, as it had no additional effect on the glucocorticoid induction of hGH
(even a~ter the remova~~ of T3 from serum) it was omitted from subsequent experiments. Calf serum was stripped of steroids by charcoal treatment, Kato, T. and Horton, R.J , Clin. Endocrinol. Metab. 28:1160-1170 (1968) for uninduced cell populations, there was no detectable difference in hGE mRNA content of cells grown in total or stripped serum (without additional dexamethasone).

Transformation of Ltk Cells With Growth Hormone Gene Ltk cells were transformed with 1 ng ptk and 1 ~g of either hgH ~ clone DNAs or hGH plasmid DNA per 10 cells, as described previously, Wigler, M., et al., Cell 16:777-785 (1979). In transformations with GH-tk fusion gene, 5-10 ng of the plasmid was used per 106 cells;
transformation efficiency of this construct is approxi-mately 10 fold lower than ptk.

Southern Blot Hybridization for hGH Experiment Cellular DNAs were digested with 2~ nuclease/~g for 2 hours. Samples were electrophoresed on 0.8% agarose gels (20 ~g DNA per slot) in 40 mM Tris, 4 mM NaAcetate, 1 mM
EDTA, pH 7.9. D~ fragments were transferred to nitro-cellulose, Southern, E.M., J. Mol. BiolO 98:503-513 (1973), and the filters were hybridized, washed and exposed to X-ray film as previously described, Weinstock, R., al., PNAS 75:1299-1303 (1978); Wigler, M., et al., Cell 16:777-785 (1979) DNA probe for the hGH gene was the 2.6 kb Eco RI fragment, nick-translated to a specific activity of 0O5-1.0 X 199 cpm~g with 32p deoxynucleotide triphosphates, ~einstock, R., et al., PNAS 75:1299-1303 (1978).

Northern Bl~t Hybridization for hOEI Experiment Total cellular RNA was prepared employing guanidine thio-cyanate and hot phenol extractions. Poly A selection was carried out as described by Aviv, H. and Leder, P., PNAS
69:1408-1412 (1972). Human pituitary RNA was prepared by the guanidine hydrochloride procedure, Chirgwin, J.M., et al., Biochem. 18:5294-5299 (1979). RNA was run on 0.8%
agaroseformaldehyde gels, Lehrach, H., et al., Biochem.
16:4743-4751 (1977) and blotted onto nitrocellulose and hybridi~ed under the conditions described by Goldberg, D.
PNAS 77:5794-5798 (1980).

Dot Blot Quantitation of hGH RNA

hGH mRNA in L cells was quantitated by dot blotting, Thomas, P., PNAS 77:5201-5205 ~1980) using a minifold filtration manifol~ (Schleicher and Schuell). Samples contained a total of 20 g RNA (specific RNA plus carrier ~2~ 8~

ribosomal RNA), in 2 X SSC in a total volume of 25~ L, and were loaded without vacuum. For optimal binding to nitrocellulose, the- filters were soaked at least 5 hours in 20 X SSC. After loading, vacuum was applied and .ndi-vidual wells were rinsed 3 X with 6 X SSC. Filters were then baked and hybridized exactly as for Northern blots.
Dots were visualized by exposure to X-ray ilm and sig-nals quantitated by punching out dots and liquid scintil-lation counting.
Although the instant disclosure sets forth all essential information in connection with the invention, the numer-ous publications cited herein may be of assistance ln understanding the background of the invention and the lS s~ate o~ the art~

Claims (46)

WHAT IS CLAIMED IS:

1. A process for inserting a foreign DNA I into a suit-able sucaryotic cell which comprises cotransforming said eucaryotic cell with a DNA molecule which includes said foreign DNA I and an inducible promotor DNA III therefor and with unlinked foreign DNA II which codes for a select-able phenotype not expressed by said eucaryotic cell, said cotransformation being carried out under suitable condi-tions permitting survival or identification of eucaryotic cells which have acquired said selectable phenotype, said foreign DNA I being incorporated into the chromosomal DNA
of said eucaryotic cell.

2. A process in accordance with claim 1, wherein said for-eign DNA I codes for proteinaceous material which is not associated with a selectable phenotype.

3. A process in accordance with claim 2, wherein said for-eign DNA I codes for the protein portion of interferon.

4. A process in accordance with claim 2, wherein said for-eign DNA I codes for insulin.

5. A process in accordance with claim 2, wherein said for-eign DNA I codes for human growth hormone.

6. A process in accordance with claim 2, wherein said for-eign DNA I codes for bovine growth hormone.

7. A process in accordance with claim 2, wherein said for-eign DNA I codes for a clotting factor.

8. A process in accordance with claim 2, wherein said for-eign DNA I codes for a viral antigen or an antibody.

9. A process in accordance with claim 2, wherein said for-eign DNA I codes for an enzyme.

10. A process in accordance with claim 1, wherein said for-eign DNA I is substantially purified.

11. A process in accordance with claim 1, wherein said for-eign DNA I or DNA II or both are attached to bacterial plasmid or phage DNA.

12. A process in accordance with claim 1, wherein said for-eign DNA I or DNA II or both are attached to phage DNA en-capsidated in a phage particle.

13. A process in accordance with claim 1, wherein said for-eign DNA I has been ligated to said inducible promoter DNA
III to form said DNA molecule.

14. A process in accordance with claim 1, wherein said DNA
molecule and said foreign DNA II are treated with calcium phosphate prior to use in cotransforming eucaryotic cells.

15. A process in accordance with claim 1, wherein said eucaryotic cell is a mammalian cell.

16. A process in accordance with claim 14, wherein said mammalian cell is an erythroblast.

17. A process in accordance with claim 14, wherein said mammalian cell is a fibroblast.

18. A process in accordance with claim 1, wherein said foreign DNA I is present in an amount relative to said foreign DNA II which codes for a selectable phenotype in the range from about 1:1 to about 100,000:1.

19. A process in accordance with claim 1, wherein said foreign DNA II which codes for a selectable phenotype is or includes the gene for thymidine kinase from herpes sim-plex virus.

20. A process in accordance with claim 1, wherein said foreign DNA II which codes for proteinaceous material which is associated with a selectable phenotype is or includes a gene for adenine phosphoribosyltransferase.

21. A process in accordance with claim 1, wherein said foreign DNA II which codes for, a selectable phenotype is or includes a gene associated with drug resistance.

22. A process in accordance with claim 21, wherein said gene associated with drug resistance is a gene coding for a mutant dihydrofolate reductase which renders cells resis-tant to methotrexate.

23. A process in accordance with claim 1, wherein said inducible promoter DNA III sequence is or includes a pro-moter sequence for the globin gene inducible in the pres-ence of dimethyl sulfoxide.

24. A process in accordance with claim 1, wherein said inducible promoter DNA III sequence is or includes a pro-moter sequence for human growth hormone gene inducible in the presence of thyroid hormone or corticosteroid.

25. A process for producing proteinaceous material which comprises cotransforming a suitable eucaryotic cell with a DNA molecule which includes foreign DNA I coding for said proteinaceous material and an inducible promoter DNA III
sequence using the process of claim 1, maintaining said cotransformed eucaryotic cell under suitable conditions including the presence of an agent capable of inducing said promoter DNA III so as to enable said foreign DNA I to be repeatedly transcribed to form complementary mRNAs and the complementary mRNAs so formed to be translated to produce said proteinaceous material, and recovering the protein-aceous material so produced.

26. A process in accordance with claim 25, wherein said proteinaceous material comprises the protein portion of interferon, insulin, human or bovine growth hormone, clot-ting factor, viral antigen, antibody or enzyme.

27. A process in accordance with claim 25, wherein said eucaryotic cell is a mammalian cell.

28. A process in accordance with claim 25, wherein said inducible promoter DNA III sequence is or includes a pro-moter sequence for the globin gene and said inducing agent is dimethyl sulfoxide.

29.. A process in accordance with claim 25, wherein said inducible promoter DNA III sequence is or includes a pro-moter sequence for human growth hormone gene and said in-ducing agent is thyroid hormone or corticosteroid.

30. A process for producing proteinaceous material which comprises cotransforming a suitable eucaryotic cell with a DNA molecule which includes a foreign DNA I coding for said proteinaceous material and an inducible promoter DNA III
using the process of claim 1, culturing or cloning said co-transformed eucaryotic cell under suitable conditions to produce a multiplicity of eucaryotic cells derived there-from, and recovering said proteinaceous material from the eucaryotic cells so produced.

31. A eucaryotic cell produced in accordance with claim 1.

32. A process for inserting a multiplicity of foreign DNA
I molecules corresponding to multiple copies of a gene cod-ing for a proteinaceous material into a suitable eucaryotic-cell with a multiplicity of DNA molecules, each of which includes said foreign DNA I and an inducible promoter DNA
III sequence and with a multiplicity of unlinked DNA II
molecules each of which is or includes a gene coding for a selectable phenotype not expressed by said eucaryotic cell, said cotransformation being carried out under suitable conditions permitting survival or identification of eucary-otic cells which have acquired said selectable phenotype and in the presence of an inducing agent for said promoter DNA III.

33, A process in accordance with claim 32, wherein said proteinaceous material comprises the protein portion of interferon, insulin, human or animal growth hormone, clot-ting factor, viral antigen, antibody or enzyme.

34 A process in accordance with claim 32, wherein said eucaryotic cell is a mammalian cell.

35. A process in accordance with claim' 32, wherein said foreign DNA I is present in an amount relative to said for-eign DNA II which codes for proteinaceous material associat-ed with a selectable phenotype in the range from about 1:1 to about 100,000:1.

36. A process in accordance with claim 32, wherein said foreign DNA II is or includes an amplifiable gene associ-ated with drug resistance.

37. A process in accordance with claim 32, wherein said inducible DNA III promoter is or includes a promoter se-quence for the globin gene and said inducing agent is di-methyl sulfoxide.

38. A process in accordance with claim 32, wherein said inducible DNA III promoter is or includes a promoter se-quence for human growth hormone and said inducing agent is thyroid hormone or corticosteroid.

39. A eucaryotic cell produced in accordance with the pro-cess of claim 32.

40. A method for producing proteinaceous material which comprises cotransforming a suitable eucaryotic cell with a multiplicity of foreign DNA I molecules coding for said proteinaceous material using the process of claim 32, main-taining said cotransformed eucaryotic cell under suitable conditions so as to enable said eucaryotic cell to produce said proteinaceous material, and recovering the protein-aceous material so produced.

41. A process for producing proteinaceous material which comprises cotransforming a suitable eucaryotic cell with a multiplicity of foreign DNA I molecules coding for said proteinaceous material using the process of claim 32, cul-turing or cloning said cotransformed eucaryotic cell in the presence of an antagonist permitting survival of only eucaryotic cells which have acquired said selectable pheno-type and in the presence of the inducing agent for said DNA
III promoter sequence in order to produce a multiplicity of eucaryotic cells, and recovering the proteinaceous material from the eucaryotic cells so produced.

42. A process for cotransforming a suitable eucaryotic cell with foreign DNA I to which an inducible promoter DNA III

has been joined and with DNA II, said DNA I and DNA II
being unlinked and said DNA II coding for a phenotype not expressed by said cell prior to cotransformation.

43. A process for producing a biological material, a portion of which is proteinaoeous, which comprises pro-ducing said proteinaceous portion within a eucaryotic cell in accordance with the processes of any of claim 25, 30 and 40, maintaining said eucaryotic cell under suit-able conditions to permit the eucaryotic cell to form, synthesize or assemble said biological material, and recovering the material so produced.

44. A process in accordance with claim 43, wherein said compound is interferon.

45 . A process for inserting foreign DNA I into a suitable eucaryotic cell which comprises cotransforming said eucaryotic cell with a DNA molecule which includes foreign DNA I and an inducible promoter DNA III and with unlinked DNA II coding for a phenotype not expressed by said eucaryotic cell, said cotransformation being carried out under suitable conditions permitting identification and recovery of eucaryotic cells which have acquired said selectable phenotype.

46. A process for producing a foreign proteinaceous material which comprises cotransforming a suitable eucaryotic cell using the process of claim 1, culturing or cloning said cotransformed eucaryotic cell under suitable -113a-conditions to yield a multiplicity of eucaryotic cells producing said foreign proteinaceous material and recovering said proteinaceous material from said eucaryotic cells.