Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~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
--5--
~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.
--8--
~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~.
1 0
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
.. ..~
--14--
~.~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,
-16-
~ 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.
-17-
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.
-18-
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.
- 2 5
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
-30-
L9
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
-32-
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
- --33--
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
-34-
~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.
-35-
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.
-36-
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
{~ ~
-38-
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.
--39--
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
-40-
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
-41-
~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 ~ `~
42-
~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-
--48--
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
-48A-
~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
-49-
~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.
-50-
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,
-52-
~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
-53-
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
-57-
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
- -57A-
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
- 58
~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
-59-
~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 ,
-60-
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
-61-
~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
-62-
~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
-63-
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
-64-
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.
L5
~0
-66-
~%~ 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
--74--
~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
-77-
~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.
L0
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.
--78--
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
-81-
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-
-85- -
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
-86-
:~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
-88-
~ 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~ ~ ~
--89
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.
I5
--91--
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
9 2--
~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.
--96 -
89
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
-97-
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
- 99 -
~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~
2S
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~
--100--
~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) .
-101--
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
-102-
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.
--1 0 3--
~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
36--480
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).
-104-
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.
-105-
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
-106-
~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~
