J. gen. Virol. (1983), 64, 2679-2696.
Printed in
Great Britain
2679
Key words: SV40/MoMuSV/viral enhancers/gene transfer
Characterization of Eukaryotic Transcriptional Control Signals by Assay of
Herpes Simplex Virus Type 1 Thymidine Kinase
By J A S C. L A N G , 1'2. N E I L M. W I L K I E 1 AND D E M E T R I O S
SPANDIDOS1 ~
A.
1The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden,
Glasgow G61 1BD and 2Institute o f Virology, Church Street, Glasgow, U.K.
(Accepted 9 September 1983)
SUMMARY
We describe the characteristics of a general assay for eukaryote transcription-control
sequences using the herpes simplex virus (HSV) thymidine kinase (tk) gene. After
transfection of cultured cells with tk-containing recombinant plasmids, two assays
were used to measure gene expression : short term or transient levels of tk m R N A and
TK enzyme activity, and the rate of biochemical transformation from a TK - to a TK ÷
phenotype in selective growth medium (HAT). Deletion of the endogenous tk promoter
results in 500-fold inactivation of gene expression. Replacement with exogenous
transcription-control sequences from the human epsilon globin, mouse fl major globin,
simian virus 40 and Moloney murine sarcoma virus (MoMuSV) genomes results in reactivation of gene expression. The presence of enhancers or activators of gene
expression can also be conveniently measured. The transient expression assay ranged
over two orders of magnitude while the transformation assay was almost two orders of
magnitude more sensitive using the same recombinants. Analysis of the transcriptioncontrol domains in the MoMuSV LTR sequences shows the presence of both an
enhancer and a promoter whose activity equalled that of the tk endogenous promoter.
Insertion of the LTR promoter between the LTR enhancer and the tk promoter had
little effect on modulating gene expression, suggesting no absolute preference for
proximal promoters by this element. The different levels of gene expression obtained
appears to be mediated by transcriptional control of full-length tk mRNA. There was
an apparent correlation between the results obtained with the transient expression and
transformation assays. However, cultured transformed cells all contained roughly the
same levels of tk DNA, tk m R N A and tk enzyme activity. We propose that initial
expression levels have a major effect in determining the transformation efficiency but
that additional genetic controls are superimposed in cells grown in selective HAT
medium.
INTRODUCTION
The genetic control of transcription of eukaryote genes is presently only poorly understood.
Recently, the development of assays for gene expression in vivo and in vitro combined with sitespecific mutagenesis, has made it possible to identify and analyse D N A sequences adjacent to
structural genes that play a role in the transcriptional process. Several distinct regions or
functional domains have been described in DNA located 5' to genes transcribed by RNA
polymerase II (Grosschedl & Birnstiel, 1980, 1982; Benoist & Chambon, 1981; Dierks et al.,
1981, 1983; Moreau et al., 1981; McKnight et al., 1981; McKnight & Kingsbury, 1982;
McKnight, 1982; Everett et al., 1983). These include the cap site, which identifies the sequence
corresponding to the 5' terminus of the mRNA, the so-called TATA consensus sequence located
at approximately - 30 bp from the cap site which appears to position the location of the cap site,
and the so-called CCAAT consensus sequence located at approximately - 80 bp, which appears
t Permanent address: Hellenic Anticancer Institute, Athens, Greece.
0022-1317/83/0000-5830 $02.00
© 1983 SGM
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2680
J. C. LANG, N. M. WILKIE AND D. A. SPANDIDOS
to affect the efficiency of transcription. Other regions described are located up to 120 bases
upstream from the cap site; these contain imperfect repeats of 12 to 15 bp GC-rich sequences
which are also required for efficient transcription (Dierks et al., 1981, 1983; McKnight, 1982;
Everett et al., 1983). It is generally agreed that, in combination, these various regions constitute
a 'promoter' for eukaryote gene transcription.
More distant sequences which influence transcription, termed 'enhancers' (Banerji et al.,
1981 ; De Villiers & Schaffner, 1981 ; Moreau et al., 1981 ; Wasylyk et al., 1983; C a m p o et al.,
1983; Spandidos & Wilkie, 1983) or 'modulators' (Grosschedl & Birnstiel, 1980), have also been
described. Enhancers are short sequences that stimulate transcription of coding sequences from
their own, or other, promoters. They act independent of orientation and at a distance with
respect to the coding sequences. These properties differentiate enhancers from other elements of
eukaryote promoters. Most enhancers so far studied have been derived from eukaryote viruses
such as simian virus 40 (SV40) (Benoist & Chambon, 1981 ; Gruss et al., 1981 ; Banerji et al.,
1981), polyoma virus (De Villiers & Schaffner, 1981, Tyndall et al., 1981), bovine papilloma
virus (Lusky et al., 1982; Campo et al., 1983), herpes simplex virus (J. C. Lang, D. A. Spandidos
& N. M. Wilkie, unpublished results) and retroviruses (Blair e t a / . , 1980; Chang et al., 1980;
H u a n g et al., 1981 ; Levinson et al., 1982; Jolly et al., 1983). However, an enhancer isolated from
the D N A of uninfected cells has also been described (Conrad & Botchan, 1982) and cellular
enhancers may be quite common.
The promoter region of the herpes simplex virus (HSV) thymidine kinase gene (tk) has been
extensively analysed and shown to contain the T A T A and C C A A T consensus signals and two
GC-rich repeated sequences in the 105 bases upstream from the normal cap site ( M c K n i g h t et
al., 1981; McKnight, 1982; McKnight & Kingsbury, 1982). The substitution of this region by
exogenous D N A sequences has allowed the development of a suitable assay system for the
identification and analysis of eukaryote transcription-control elements : the promoter-deleted tk
gene is inactive in gene expression studies but can be reactivated by insertion of D N A sequences
that contain cis-acting regulatory elements. In the present study, we have analysed the
characteristics of this assay using two sensitive and quantitative systems to detect tk gene
expression with transcription-control sequences from the h u m a n epsilon globin gene, the mouse
fl major globin gene, the SV40 early genes and the Moloney murine sarcoma virus (MoMuSV)
long terminal repeat (LTR) as depicted in Fig. l.
METHODS
Construction of recombinant plosmids. Recombinant vectors (Fig. 2 to 4) were constructed by modifying the
parental donor plasmid moleculepTK l, which has been previouslydescribed (Sanders et al., 1982)and contains a
3.5 kb BamHI fragment of HSV-I DNA inserted into the BamHI site of pAT153 (Twigg & Sherratt, 1980). A
detailed description of these recombinants is given in the legends of Fig. 2 to 4. Restriction enzyme-digested
plasmids were treated with calf intestinal alkaline phosphatase (Boehringer Mannheim) to prevent self-ligation
(Ullrich et al., 1977). Ligations were carried out for 5 h at room temperature with T4 DNA ligase (Bethesda
Research Laboratories), with alkaline phosphatase-treated plasmids and the insert DN A fragments at a 1: 5 molar
ratio. Transformations of HB 101 cells were performed as described (Norgard et al., 1978). Molecular linkers were
used as suggested by the suppliers (Bethesda Research Laboratories).
DNA-mediotedgene transJer. The recipient mouse LMTK , LATK (Spandidos & Wilkie, 1983) and Syrian
hamster BHKTK (Spandidos et al., 1982) cells were cultured in SFI2 medium (Flow Laboratories) containing
15% Hycloneserum (Sterile Systems Inc.) Exponentially growing cells were used in each transfection experiment.
Transfections were carried out using the calcium phosphate technique (Graham & van der Eb, 1973) with the
followingmodifications.The DNA calcium phosphate co-precipitate was added to the culture medium at a ratio
of 0.5 ml co-precipitate per 5 ml medium containing I × 10~mouse or 5 × 105 hamster cells exponentially growing
in a 25 cmz flask. After 6 or 24 h, depending on the experiment, the medium was replaced with fresh nonselective medium [SF12 (Flow Laboratories) containing 15% Hyclone serum (Sterile Systems lnc.)] for an
additional 24 h before selective medium containing HAT (Littlefield, 1964) was applied. The medium was
changed every 3 days for up to 2 weeks before colonies were counted. The Methocel assay has been described in
detail elsewhere (Spandidos et al., 1982).
Assay o! thymidine kinase actirity. The assay of HSV-l-specific TK activity in the presence of 0.2 mM-dTTP
(Jamieson & Subak-Sharpe, 1974) has been described previously (Wilkie et al., 1979).
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H S V tk expression assay
2681
Isolation of RNA and DNA and filter hybridizations. Extraction of RNA from cells and blotting onto
nitrocellulose have been described elsewhere (Spandidos & Paul, 1982).
Total high molecular weight cell DNA from recipient and transformed cells was obtained as described by
Gross-Bellard et al. (1973). Extraction of DNA from cell lines was performed as described by Hirt (1967).
Electrophoresis on agarose gels and filter hybridizations were performed in 5 x saline sodium citrate (SSC), 50%
formamide for 24 h at 42 °C with 10 ng/ml probe as described (Wahl et al., 1979) using 2 × Denhardt's solution
(Denhardt, 1966). 3zp-labelled DNA probes with specific activities of 1 x 108 to 3 x 108 ct/min/ktg were made by
nick translation (Rigby et al., 1977). The nitrocellulose sheets were washed in 0-5 x SSC at 60 °C and exposed to
hypersensitized X-ray films at - 70 °C (Laskey & Mills, 1977). DNA and RNA spot hybridization procedures
were carried out as previously described (Spandidos et al., 1981).
RESULTS
Recombinant plasmids
The D N A fragments containing transcription control sequences located 5' to a variety of
eukaryote genes are depicted in Fig. 1, which shows the recognized domains within each
segment. Plasmid pTK1 contains a PvulI site located 195 bp upstream ( - 195) from the cap site
and a BgllI site located 54 bp ( + 54) into the sequence corresponding to the untranslated leader
of the tk m R N A (Wilkie et al., 1980; Preston & McGeoch, 1981). After genetic manipulation
using H i n d l I I molecular linkers, this fragment was inverted, thereby inverting the tk promoter
region (pTK9, Fig. 2). Deletion of the P v u l I - B g l l I fragment, and replacement with a H i n d l I I
site, deletes the promoter (pTK10, Fig. 2). Thus, insertion of D N A fragments into the H i n d l I I
site of p T K 10 places the new exogenous sequences adjacent to the sequences corresponding to
the 5' untranslated leader of the normal tk m R N A . In almost all of the exogenous promoter
fragments shown in Fig. 1, at least one terminus corresponds to a site in the untranslated 5'
leader. Subsequent insertion of these fragments in the 5'-3' orientation relative to the tk gene
results in fusion of 5' untranslated regions of the m R N A of each gene (Fig. 3 and 4). Insertion of
fragments in the opposite orientation results in inversion of the exogenous promoter, in a
manner analogous to pTK9. The one exception shown is the fragment containing the promoter
sequences of the mouse fl major globin gene. In this case, the right-hand end terminates at a
S a u 3 A I site located 15 bp upstream from the cap site (Konkel et al., 1978). In some cases, further
manipulation with molecular linkers was used to separate the enhancer d o m a i n (73 and 72 bp
repeats, Fig. 1) of the MoMuSV fragment from the promoter domain ( C C A A T and T A T A
consensus sequences, Fig. 1) and to insert each domain independently into p T K I 0 (Fig. 3).
Alternatively, the different domains were inserted into the pAT153 moiety of plasmids in which
the tk gene was either under the control of its own promoter, or the human epsilon globin
promoter (Baralle et al., 1980) (Fig. 4). The entire simian virus 40 (SV40) transcriptional control
fragment containing an enhancer (72 bp repeats) and promoter (21 bp repeats and T A T A
consensus) domain (Fig. 1) was also inserted into the p A T 153 moiety of these plasmids (Fig. 4).
The ability of the various reconstructed tk genes to express R N A and active enzyme was then
tested using two distinct techniques. In the first, recombinant D N A was used to transfect
L A T K - or B H K T K - cells in culture, and, 24 to 48 h later, the amount of tk-specific R N A and
T K enzyme activity induced in the cultures was measured. In the second, we determined the
ability of recombinant plasmids to transform L A T K - or B H K T K - cells to a stable T K +
phenotype. We also measured the tk gene copy number, and the levels of tk m R N A and T K
enzyme activity in individual clones of transformed cells.
Short term expression assays
Time course
Mouse L A T K or hamster B H K T K - cells were transfected with p T K M O L T R 1 , a plasmid
in which the 5' untranslated leader sequences of MoMuSV and HSV tk are fused and which
contains the entire L T R transcriptional control signals (Fig. 3). Duplicate cultures were
harvested at various times post-transfection, and duplicates were used to determine T K enzyme
levels and tk-specific m R N A levels by the dot-blot procedure, as described in Methods. The
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2682
J. C. LANG, N. M. WILKIE AND D. A. SPANDIDOS
CGAAT
DS2 I D i l
l
T~TA _ _ . ATG
HSV-1 TK
r
Pv
t
I
E
Bg
CCAAT ATA
I
Human e globin r
I
Pv
B
CCAAT TATA
Mouseflmajor globin I
H
I
Sa
72bp
pBR322
SV40
21 bp
R
R
L3
A
I-----]
E H/Hp
I
H
t
bp 0
I
i
I
I
100
200
300
400
bp R
/\
Mink
73 72 CCAAT TATA
MoMuSV LTR Iv ' V X A / V X / ~ I
v.-... . . . . . . I
I
bp 0
~
Xb
E
Mink
~XAA/N/
Sm
I
I
I
I
I
200
400
600
800
1000
Fig. 1. The promoter regions of HSV-I tk gene (Preston & McGeoch, 1981; Wagner et al., 1981;
McKnight, 1982), human epsilon globin (Baralle et al., 1980), mouse fl major globin (Konkel et al.,
1978), SV40 (Fiers et al., 1978; Reddy et al., 1978) and MoMuSV (Dhar et al., 1980). The structural
sequences are shown as heavy lines. DSI and DS2 on the tk promoter represent the first Distal Signal
and second Distal Signal respectively (McKnight, 1982). The two 72 bp and two 21 bp directly repeated
(R) sequences of SV40 as well as the 73 and 72 bp directly repeated sequences of MoMuSV LTR are
shown as cross-hatched boxes. The horizontal arrow indicates the direction of transcription. TATA,
TATA box homology sequence; restriction sites: E, EcoRl; H, Hindlll; B, BamHI; Pv, PrulI; Bg,
Bglll: Xb, Xbal; Sm, Smal; Hp, HpalI; Sa, Sau3AI.
results are shown in Fig. 5. The o p t i m u m t i m e post-transfection for t k expression was found to
be 24 h for B H K T K - cells and 48 h for L A T K - . In each case, there was a good correlation
b e t w e e n the a m o u n t of tk-specific R N A and e n z y m e activity. A similar t i m e course, but w i t h
l o w e r values, was found for p T K 1 , and the induced e n z y m e activity was specifically i n h i b i t e d by
r a b b i t a n t i - H S V serum (data not shown).
E f f e c t s o f different transcription-control sequences
T h e effects of replacing the tk p r o m o t e r w i t h different e x o g e n o u s t r a n s c r i p t i o n - c o n t r o l
sequences, or inserting t h e m at various loci relative to genes w i t h a c t i v e p r o m o t e r s is s h o w n in
T a b l e 1. L A T K - or B H K T K - cells were transfected with r e c o m b i n a n t p l a s m i d s and tk-specific
R N A and T K e n z y m e activity m e a s u r e d 48 h ( L A T K - ) or 24 h ( B H K T K - ) later. In all cases,
there was good correlation b e t w e e n the a m o u n t of tk-specific R N A d e t e c t e d and the T K e n z y m e
levels. Deletion of the t k p r o m o t e r results in r e d u c t i o n o f m e a s u r a b l e t k g e n e expression to
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H S V tk expression assay
EH B
'
..Jr-Pv
pTK1
i,
tk~J
~ i ~
,,,.._s~
"/~_.
_E
~ Bg
S
,/
../.~-H
%,.
['/
,
N~H ( B g )
, l l , ~ h l
pWK8
/""
,/
'
,.
~TATA
-~al-'H (Bg)
B
""',,,H(Vv)
~.....
tk I /
(Pv)
I
~,\\\ J
E
.----J--
2683
~
n (ag) ""
E
/_-L-
B
~'~H
~ A T A '///
I
" ~ - t t (Pv)
\
(Pv, Bg)
tki/
'
\",
Fig. 2. Structures of pTK 1 and derivative plasmids pTK8, pTK9 and pTK 10. pTK8 was derived from
pTK 1 after removal of the HindlIl site in the pAT153 moiety, by sequential treatment with Hindlll, the
Klenow fragment of Escherichia cob DNA polymerase I and T4 DNA ligase, followed by conversion of
the PrulI and BglII sites near the 5' end of the tk gene to HindlII sites by using molecular linkers, pTK8
carries the 249 bp HindllI (Pt:ull)-HindIII (Bg/II) fragment containing all the tk promoter functional
domains in 5'-3' orientation and pTK9 inverted in 3'-5' orientation. In pTK 10, the tk promoter region
has been deleted. The broken line indicates pAT 153 DN A, the solid line the 3.58 kb BamHI fragment of
HSV-I. the outer heavy line the structural tk gene, and the inner heavy line the coding sequence. The
arrow indicates the direction of transcription. The maps are not drawn to scale. TATA, TATA box
homology sequence; restriction sites E, EcoRl; H, HindIIl; B, BamHI; Pv, PvuII;,Bg, BglII.
background levels (pTK 10). Replacement of the tk promoter with the promoter of the epsilon
globin gene (pTKel) resulted in reactivation of the tk gene, but to a lower level of gene
expression than the normal tk promoter (pTK 1). Replacement of the tk promoter with the entire
MoMuSV L T R transcription control signals in a 5'-3' orientation ( p T K M O L T R 1 ) resulted in
reactivation to a considerably higher level of gene expression than pTK 1. Inclusion of the entire
SV40 transcription control sequence or the MoMuSV enhancer domain 1 to 4,kb distal from a tk
gene under the control of the epsilon globin promoter resulted in considerably increased gene
expression. This was observed whether these elements were present in 5'-3' orientation
(pTKeSV1 ; pTKeMOE1) or 3'-5' orientation (pTKeSV2; pTKeMOE2) relative to the tk gene.
This strongly suggests that the increased level of gene expression observed with these plasmids is
due to the presence of enhancer elements in the SV40 and MoMuSV L T R sequences. This was
confirmed by the transformation experiments to be described. Fig. 6 shows that inclusion of
enhancer elements increased tk gene expression by regulating the amount of full-length tk
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J. C. LANG, N. M. WILKIE AND D. A. SPANDIDOS
2684
(a)
E _ B
illI~ ~"
'~
H(B)
~
pTKIO
]] , ~
tk~J
B ~ P v
pTKel
"B----~
pTKg2
(d)
E
//f
X ~
t
.....
L,
". _
i/
Pv
E
"~IK
1B(Xb)
ink
H/Hp- t L
• \
~ '
-E ~
TATA
~ ( B g )
I
I_AT n
~
EI
/,,
(b)
///'"
~ATA
',,
(c)
H(Pv)
~ 7 2 bp R
~
'-TATA
~l~E
t
x\
\\ ,
73
tkil/
~']
J ' ¢
\
\
"~,
/
I--Xb
],-TATA_
!j, TATA
~r"-----r pv
H (Sm)
H (Sm)
E "
pTKMOP1 pTKMOLTR 1
Fig. 3. Recombinant plasmids carrying hybrid genes containing the structural thymidine kinase
sequences linked to heterologous eukaryotic promoters. (a) The human epsilon globin promoter. A 197
bp BamHI-PvulI fragment containing the promoter sequences from the human epsilon globin gene
(Baralle et al, 1980) was inserted into the HindllI site of pTK 10 in 5'-3' orientation (pTKel) or in 3'-5'
orientation (pTKe2) relative to the direction oftk transcription. (b) The mouse fl major globin promoter.
The plasmid pTKfll was constructed by replacing the HindlII-BgllI fragment of plasmid pTKI
containing the tk promoter sequences with a 0.4 kb HindlII Sau3AI fragment carrying the promoter
region of the mouse fl major globin gene. The original 2 GT. WesMflG recombinant, from which the
mouse fl globin promoter was derived after subcloning into pBR322, was constructed by Tilghman et al.
(1977). The Sau3AI site is located 15 bp upstream from the cap site of the fl globin gene (Konkel et al.,
1978). (c) The SV40 early region promoter. Insertion of the EcoRI-HindlIl fragment from pSO59
containing the SV40 early and late gene promoter sequences into an EcoRI-HindllI deletion of pTK10
places tk transcription under control of the SV40 early promoter (pTKSV1). (d) The Moloney murine
sarcoma virus LTR promoter. Insertion of an EcoRI HindlII fragment from pmlspl (not shown)
containing 473 bp from the MoMuSV LTR and 5' flanking mink DNA sequences (McClements et al.,
1980; Dhar et al., 1980) places tk transcription under control of the promoter regulating MoMuSV viral
gene expression (pTKMOLTRI). The CCAAT and TATA consensus sequences within the MoMuSV
LTR lie 3' to the XbaI and 5' to the Smal sites (Fig. 1; Dhar et al., 1980). Conversion of the XbaI site to a
BamHI site and subsequent BamHI cleavage allows their separate recloning within a BamHI fragment
linked to the structural tk gene. Religation to BamHl-cleaved pAT153 produces pTKMOPI.
m R N A present shortly after transfection. W e found little difference in the total amount of
transfected plasmid D N A in short term cultures whether or not enhancers were p r e s e n t ( d a t a
not shown), suggesting that the effect on transcription was independent o f gene copy number.
Transformation of TK- cells
The second way of measuring gene expression of tk plasmids, measuring the efficiency of
biochemical transformation, was carried out by transfecting mouse L M T K - , L A T K - cells or
hamster B H K T K - cells with recombinant plasmid D N A in the presence of carrier D N A ,
transferring the cultures to selective H A T medium, and counting the n u m b e r of T K ÷
transformed colonies after 10 to 14 ( L M T K - , L A T K - cells), or 5 to 7 ( B H K T K - cells) days
respectively. In each case, a dose-response curve using increasing amounts of r e c o m b i n a n t
plasmid D N A was constructed, and the results are expressed as the ratio o f the n u m b e r o f
colonies obtained per Ixg of recombinant D N A over the number of colonies obtained per Ixg o f
p T K 1 D N A (containing the standard tk gene). W e routinely obtain 2000 to 4000 colonies per ~tg
of pTK1 D N A when measuring in this way.
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2685
H S V t k expression assay
/ ....
B
"~--l~" ¢21 bp R
B
" ~ J T A T A Pv':DS1
H
(B)~ , H ~ [
-TATA
H (Pv)
pS059
WB
.
tk
,"
//
I
(b)
tk
-H (Pv)
-H (B)
B
B
B
pTK1SV1
pTKeSV ]
pTKt~SV2
EH
J-L
///
/
:
B (E)
\t\
I?
°'K'
',
B (Sm)
xu
'e°°
~73>bP
. . . . B(Sm)
B (E)
pTK 1MOLTR 1 pTK l MOLTR2
E
E
/,_~,73.7bp
R B
B
B
B/Bg
?~D.SA• [.ATA i
'lAimj ~_H(p,)I
I
pMOEI
I ~ tk
"-.
/
/
It
. ';
"" ....
"
R 1.72
\ I''ATA
J'
t} ~ v
~'S
TA
//
"x x
(c)
B
~
\
t
B
II
tktk
"1
\
B
B
-H (Pv,Bg)
tk
~-H (Pv)
B
B
B
pTK]MOEllpTK,MOE21pTKMOE2
pTKeMOE1 pTKMOE]
Fig. 4. Construction of recombinants carrying enhancers at distances from the tk and tk hybrid genes.
(a) The SV40 enhancer upstream from the tk gene. BamHl cleavage of pSO59 leaves a single cloning site
3' to the SV40 sequences. Subsequent insertion of the BamH I tk fragment from pTK 1 places the tk gene
5'-3' relative to the direction of transcription of the SV40 early genes (pTK 1SV 1). Similar insertion of
the BamHI tk fragment from pTKs1 5'-3' gives pTK~SVI, and 3'-5' gives pTKeSV2. (b) The MoMuSV
enhancer and promoter upstream from the tk gene. The Sinai site within the MoMuSV LTR and the
EcoRI site in the mink sequences were converted to BamHl sites by using molecular linkers. Subsequent
insertion of this BamHl (EcoRl) BamHl (Smal) LTR fragment into the BamHl site upstream of the tk
gene in pTKI 5'--3' gives pTKIMOLTR1, and 3' 5' pTK1MOLTR2. (c) The MoMuSV enhancer
inserted upstream or downstream of tk genes and into promoter-negative recombinants. Levinson et al.
(1982) have shown that the Sau3AI Xbal fragment from the MoMuSV LTR can functionally substitute
for the SV40 enhancer, suggesting that sequences 3' from the Xbal site are non-essential for enhancer
function. Conversion of the XbaI site to BamHI using molecular linkers allows separate recloning of the
enhancer-containing moiety (pMOEI). Religation to the BamHl tk fragment of pTKI in 5'-3'
orientation gives pTKIMOEI. Similar religation to the BamH] tk fragment of pTKel in 5' 3'
orientation gives pTK~MOEI, and 3' 5' pTKeMOE2. Ligation to the BgllI-BamHI tk fragment of
pTKI in 5'-3' orientation gives pTKMOEI. In the same way, ligation to the BamHI tk fragment of
pTK10 in 5'-3" orientation gives pTKMOE2.
T h e effects o f r e p l a c e m e n t or i n c l u s i o n of e x o g e n o u s e u k a r y o t e t r a n s c r i p t i o n - c o n t r o l
s e q u e n c e s o n t k g e n e a c t i v i t y is s h o w n in T a b l e s 2 a n d 3. T h e m e a s u r e d t r a n s f o r m a t i o n r a t e s
r a n g e o v e r a b o u t 4 log10 units. T h e r e f o r e , t h e t r a n s f o r m a t i o n a s s a y is at l e a s t o n e h u n d r e d t i m e s
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2686
J. C. L A N G ,
l0
-~
~,
.g
N . M. W I L K I E
i
I
12
24
A N D D . A. S P A N D I D O S
I
I
I
Ca)
5
.,o
:2
(b)
~
z
~
20
10
>
:2
t
0
I
48
Time (h)
I
72
96
Fig. 5. (a) HSV TK activity in lysates of LATK- (O) or BHKTK- (O) cells transfected with
pTKMOLTR1. Transfection of cells is described in Methods. The DNA-calcium phosphate coprecipitate was removed after 6 h and the medium replaced with fresh non-selective medium. At
various time intervals the cells were harvested, resuspended at a concentration of 2 x 107 cells/ml in 50
mM-Tris-HCl pH 7.5, 5 mM-fl-mercaptoethanol and 5 gM-deoxythymidine and the sonicated lysates
were stored at - 70 °C until assayed. (b) HSV tk RNA from LATK- or BHKTK - cells transfected with
pTKMOLTRI as above analysed by spot hybridization (Spandidos et aL, 1981). Cells harvested from
the experiment described above were used for the isolation of RNA as described in Methods. Twenty gg
of each RNA was spotted in duplicates onto nitrocellulose filters and the filters hybridized with nicktranslated 32p-labelled0.6 kb BgIII-SstI fragment tk DNA. The autoradiographs were scanned and the
average is given for each time point.
more sensitive than the short term assay (which has a range of only 2 log units, see Table 1) and is
capable of the detection of low-level activity not possible with the short term assay.
Deletion of the tk promoter (pTK10) results in a gene inactivation of 500-fold (Table 2).
Insertion of HindIII molecular linkers at the PvuII ( - 1 9 5 ) and BglII ( + 54) sites (pTK8) has
very little effect on transformation ability. Surprisingly, inversion of the tk promoter (pTK9)
resulted in retention of activity at a level some 20- to 50-fold lower than p T K 1 or p T K 8 (Table 2).
The same effect was observed when the tk promoter was replaced by the h u m a n epsilon globin
promoter. When inserted into the HindIII site of p T K 1 0 in a 5'-3' orientation ( p T K e l ) , an
activity two- to fivefold lower than pTK1 was obtained. W h e n the epsilon fragment was
inverted (pTKe2), a further 50-fold reduction in transformation rate was observed (Table 2).
Replacement of the tk promoter with the mouse fl major globin promoter ( p T K fl 1) (which does
not include the normal cap site) in 5'-3' orientation also reactivated tk to about the same level as
the human epsilon promoter. Exogenous transcription-control sequences that contain an
enhancer were able to reactivate the tk gene to give markedly elevated transformation
efficiencies. Thus replacement of the tk promoter by the entire SV40 control region ( P T K S V I )
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H S V tk expression assay
2687
T a b l e 1. Short term expression o f H S V tk-specific R N A and T K activity in L A T K
B H K T K - cells transfected with H S V tk recombinants
tk-specific R N A t
Donor DNA*
HSV TK activity +
A
t"
pTK 1
pTK 10
pTK~I
pTKeSV 1
pTK~SV2
pTK~MOE1
pTKeMOE2
pTK MOLTR 1
Salmon
and
LATK
3.7
0.2
1.0
11
7.3
9.3
6.7
17
<0.1
BHKTK ~
3.0
0.2
1.0
9.3
7.3
13
9.3
16
<0.1
LATKC
BHKTK
3-4
0. l
1"0
7-2
4.1
9.3
5-1
18
<0.1
4.4
0-2
1.0
13
5.0
15
6.3
17
<0-1
* Donor DNA (10 gg) was added to the recipient cells as a calcium phosphate co-precipitate (in the absence of
carrier) as described in Methods. Six h later, the medium was replaced with fresh medium and incubation
continued at 37 °C. Cells were trypsinized and harvested at 48 h after addition of DNA for LATK -, and 24 h for
BHKTK cells.
t RNA was located as described in Methods and tk-specific RNA was assayed using the 0.6 kb Bglll SstI tk
DNA fragment as a probe for spot hybridization analysis (Spandidos et al., 1981). The autoradiographs were
scanned and tk-specific RNA is given in arbitrary units.
:[: HSV TK activity was assayed in the presence of 0.2 mM-dTTP as described (Wilkie et al., 1979) and the ratios
found when compared to TK activity induced by pTK~I are given. HSV TK activity for pTK~l-transfected
LATK- cells was 0-31 pmol/h/105 cells and BHKTK- was 0.47 pmol/h/105 cells.
T a b l e 2. Transformation o f mouse L M T K Donor DNA*
pTK 1
pTK8
pTK9
pTKI0
pTK~I
pTKE2
pTKfll
pTKMOP1
pTKMOLTR1
pTKSVl
Promoter orientation
5'-3'
5'-3'
3'-5'
5'-3'
3'-5'
5'-3'
5'-3'
5'-3'
5'-3'
cells with HSV-1 tk recombinants
Transformation ratios#
1.0
1.1 2.0
0-024).06
0.002
0.1-0.6
0.002-0.009
0-1 0-5
0-7-1.1
29 47
15 36
* Donor DNAs (1 ng to 1 gg) were mixed with carrier salmon sperm DNA at the final concentration of 20 ~tg/ml
in the calcium phosphate precipitate and transformation was carried out using the calcium phosphate technique
(Graham & van der Eb, 1973) as described in Methods.
~ Only flasks or plates containing between 10 and 100 colonies were considered at the above concentrations of
DNA, since values outside these ranges, both for colony counts and donor DNA concentrations, were not in the
linear part of the transformation curve. Transformation frequencies were within the range of 1000 to 4000
colonies/lag pTK 1 DNA. The averages of three to six experiments are given.
o r t h e e n t i r e M o M u S V c o n t r o l r e g i o n ( p T K M O L T R 1 ) g a v e a 15- to 50-fold i n c r e a s e i n
t r a n s f o r m a t i o n efficiency r e l a t i v e to p T K 1 . I n t e r e s t i n g l y , t h e e n h a n c e r d o m a i n o f t h e L T R
s e q u e n c e could b e deleted, to leave a r e g i o n c o n t a i n i n g t h e r e c o g n i z e d p r o m o t e r c o n s e n s u s
signals ( p T K M O P 1 ) w h i c h w a s a b o u t as a c t i v e as t h e n o r m a l tk p r o m o t e r . T h u s , t h e M o M u S V
L T R s e q u e n c e c o n t a i n s s e p a r a t e a n d well-defined e n h a n c e r a n d p r o m o t e r d o m a i n s . (See also
T a b l e 1, a b o v e , a n d T a b l e 3, below.)
T a b l e 3 s h o w s a m o r e d e t a i l e d a n a l y s i s o f t h e effect o f i n c l u d i n g e n h a n c e r s e q u e n c e s o n t h e
t r a n s f o r m a t i o n efficiencies o f t k - c o n t a i n i n g p l a s m i d D N A s . T h e d e t a i l e d o r g a n i z a t i o n o f t h e
r e l e v a n t p l a s m i d s c a n b e f o u n d in Fig. 2 to 4. R e s u l t s are e x p r e s s e d as r a t i o s c o m p a r e d to e i t h e r
t h e s t a n d a r d tk g e n e ( p T K 1 ) or t h e tk g e n e c o n t r o l l e d b y t h e h u m a n e p s i l o n g l o b i n p r o m o t e r
( p T K e l ) . T h e results o b t a i n e d u s i n g t h e l i q u i d or semi-solid a s s a y s a n d L M T K - , L A T K - or
B H K T K - cells were f o u n d to b e c o m p a r a b l e . I n c l u s i o n o f t h e e n t i r e S V 4 0 o r M o M u S V L T R
e n h a n c e r c o n t r o l signals only, in e i t h e r o r i e n t a t i o n , i n t o t h e p A T 15 3 m o i e t y o f tk p l a s m i d s , 1 to 4
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2688
J.C.
LANG,
N . M. W I L K I E
(a)
AND
D. A. SPANDIDOS
LATK ¢-,I
o
;~
(b)
©
--2
5
J
2J''
Fig. 6. Northern blot tk RNA analysis in transfected cells. Twenty gg of total cell RNAs l¥om cells
transfected with the recombinants indicated on the figure were fractionated on 1~ agaroseformaldehyde-containing gels, blotted onto nitrocellulose and probed with 3-'P-labelled 0.6 kb BgllISstI tk DNA. LAT6 is an LATK + stable cell line obtained after transformation of LATK - cells with
pTKI. (a) The au~oradiograph. (b) Scan across the 1.3 kb region.
kb distal from the t k coding sequences resulted in a substantial (5- to 20-fold) increase in
transformation rate (pTKeSV1, pTKeSV2, pTK1SV1, p T K e M O E 1 ,
pTK~MOE2,
p T K 1 M O L T R 1 , p T K 1 M O L T R 2 , p T K 1 M O E 1 ) . These results are comparable to those
obtained using the same constructions in the short term expression assay shown in Table l. This
is a clear demonstration of the 'enhancer' effect. It should be noted that the adjacent presence of
the MoMuSV promoter domain has little effect on the M o M u S V enhancer activity (compare
p T K 1 M O L T R 1 and p T K 1 M O E 1 ) when these elements are inserted upstream in 5'-3'
orientation relative to the normal t k gene. Surprisingly, we also found that the enhancer domain
of the L T R could activate the t k gene when fused to the 5' untranslated leader sequence and also
when inserted about 1 kb upstream from the tk gene from which the promoter had been deleted
( p T K M O E 1 , p T K M O E 2 , Table 3). The reasons why an enhancer can activate an apparently
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H S V tk expression assay
2689
Table 3. Transformation o f L M T K - or L A T K - mouse or B H K T K - hamster cells with HSV-1
tk reeombinants using liquM or semi-solid Methoeel assays
Transformation ratios
A
Liquid assay'j"
LMTK- cells
'
Donor DNA*
pTK1
pTK 10
pTK~I
pTKeSVI
pTKeSV2
pTKeMOEI
pTKeMOE2
pTKSV1
pTKMOLTRI
pTKISVI
pTK 1MOLTR 1
pTK 1MOLTR2
pTKIMOE1
pTKMOE1
pTKMOE2
Enhancer
orientation
5' 3'
3'-5'
5'-3'
3' 5'
5'-3'
5' 3'
5' 3'
5'-3'
3'-5'
5' 3'
5' 3'
5' 3'
r
Semi-solid assay:~
J'
LATK
BHKTK-
X
X
X
X
pTK 1
1-0
0.002
pTK~I
6-13
0-01
1.0
19-29
8 15
12-14
7 12
pTK~I
9.4 + 2.8
0.01
1-0 ± 0.2
22 ± 4-0
11 ± 4.5
18 ± 9.4
12 _+ 5.6
pTKe,1
6.2 + 1.6
0-01
1.0 ± 0.1
18 _ 5.7
11 ± 4.7
19 ± 9-7
I1 ± 7.7
405 ± 119
213 _+ 59
15-36
29-47
9-10
11-13
17-26
11--20
0-9-1.4
0-1-0.2
* and t, see Table 2.
~: Cells were plated in Methocel medium containing HAT at concentrations of 102 to 10° cells per 100 mm plate
as described by Spandidos et al. (1982). Only plates containing between 10 and 100 colonies per plate were
considered at the above concentrations of DNA, since values outside these ranges both for colony counts and
donor D N A concentrations were not in the linear part of the transformation curve. Plating efficiency of recipient
cells in Methocel medium was 80%. The average and standard deviations of transformation frequencies expressed
as colonies/~tg DNA were derived from the counts of 4 to 12 plates per donor DNA from two independent
experiments. Transformation frequencies were in the range of 2000 to 4000 colonies/~tg pTKI DNA.
promoterless gene are presently unclear, but we h a v e also o b s e r v e d this p h e n o m e n o n w i t h an
e n h a n c e r from b o v i n e p a p i l l o m a v i r u s ( C a m p o et al., 1983).
O n e final point to note in this section is that although the range and sensitivity o f the short
term assay and the t r a n s f o r m a t i o n assay are different, the trend o f the results is the same.
T r a n s c r i p t i o n a l control sequences that h a v e low activity in the short t e r m assay h a v e low
t r a n s f o r m a t i o n activities, while those with high short t e r m activities h a v e high t r a n s f o r m a t i o n
activities.
Gene expression in stably transJormed cells
Several i n d e p e n d e n t colonies from t r a n s f o r m a t i o n e x p e r i m e n t s i n v o l v i n g different r e c o m b i n ant plasmids were isolated and p r o p a g a t e d in H A T m e d i u m . Cultures were h a r v e s t e d 2 to 3
weeks after isolation and used to d e t e r m i n e the tk-specific D N A and R N A levels using the dotblot h y b r i d i z a t i o n t e c h n i q u e and the levels o f T K e n z y m e activity as described. Results are
given in T a b l e 4. Despite the large differences b e t w e e n different p l a s m i d s o b s e r v e d in the short
term gene expression assay and the t r a n s f o r m a t i o n assay (see T a b l e s 1, 2 and 3), there was
r e m a r k a b l y little variation in the tk R N A or T K e n z y m e levels a m o n g the resulting stably
t r a n s f o r m e d cell lines. Fig. 7 shows that an HSV-specific tk m R N A of a p p r o x i m a t e l y 1.3 kb was
present at roughly the same c o n c e n t r a t i o n in a n u m b e r of d i f f e r e n t t r a n s f o r m a n t s p r o d u c e d by
plasmids in w h i c h the tk gene was u n d e r the c o n t r o l of either the tk or epsilon globin promoters.
This strongly suggests that the tk gene is being transcribed from the tk and substitute p r o m o t e r s
in the transformed cell lines, since the normal tk m R N A is k n o w n to h a v e a size of 1-3 kb and
initiation from the epsilon globin cap site in the epsilon tk hybrid gene ( p T K e l ) and t e r m i n a t i o n
at the n o r m a l tk polyadenylation signal would result in an R N A of similar size. Thus, a l t h o u g h
m a r k e d l y different levels of tk gene expression (as m e a s u r e d by the short t e r m and
t r a n s f o r m a t i o n assays) result from altered control by different t r a n s c r i p t i o n control sequences,
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2690
J. C . L A N G ,
N . M. W I L K I E
AND
D. A. SPANDIDOS
T a b l e 4. HSV-1 T K activity and tk copy numbers in L A T K + and B H K T K ÷ transformed cells
with tk recombinants
Cell line*
LAT- 1
LAT-2
BT-1
BT-2
LAT~,I-I
LATe, I-2
BT~,I-1
BTe,1-2
LATr, SV 1- 1
LATc,SV 1-2
BTr,SV 1- 1
BTcSVI-2
LATe,SV2-1
LATc,SV2-2
BTcSV2-1
BTcSV2-2
LAT~MOE 1-1
LAT~:MOE 1-2
BTe,MOE 1- 1
BTc,MOEI-2
LATc,MOE2-1
LATr, MOE2-2
BTcMOE2-1
BT~:MOE2-2
LATMOLTRI-I
LATMOLTRI-2
BTMOLTRI-1
Bq-MOLTR 1-2
Donor D N A t
pTK 1
pTK~I
pTKeSV 1
pTKr, SV2
pTK~MOE 1
pTKeMOE2
pTKMOLTRI
HSV-TK activity:~
2.8
4-5
4-4
4.0
3.7
3.5
4-4
2-6
2.6
2-0
4-4
4.7
5.3
5.3
3-7
5,7
4.7
2.2
2.1
2.7
5.0
4,8
4-1
2.2
2.3
2.6
2.9
3.2
tk DNA copies/cell§
2
1
5
2
2
3
4
2
4
1
1
1
4
3
3
6
4
1
2
5
4
2
4
5
3
3
5
5
RatiosU
1.4
4.5
0.9
2.0
1.9
1.2
1.1
1.3
0.7
2.0
4.4
4.7
1.3
1.8
1.2
0-9
1.2
2.2
1.0
0.5
1.2
2.4
1.2
0.4
0.8
0-9
0.6
0.6
* Each transformed cell line (LAT- for LATK and BT- for B H K T K - transformants) had been grown in HAT
for 2 to 3 weeks after the original isolation.
t Donor DNA was added to the cells as a calcium phosphate precipitate in the absence of carrier as described in
Methods.
++Pmol/h/10 5 cells.
§ Determined from spot hybridization assays after scanning the autoradiogram by densitometer. A Bglll SstI
0.6 kb tk DNA fragment was used as probe.
II HSV-TK activity:tk DNA copies/cell.
long t e r m culture in selective H A T m e d i u m selects cells in w h i c h t h e level o f tk g e n e e x p r e s s i o n
is f u r t h e r c o n t r o l l e d to a ' s u i t a b l e ' level. T h e m e c h a n i s m s i n v o l v e d i n t h i s a d d i t i o n a l c o n t r o l a r e
n o t yet k n o w n .
A f t e r f u r t h e r p a s s a g e of t r a n s f o r m e d cells, c h a n g e s in t h e copy n u m b e r o f d o n o r tk g e n e s h a v e
b e e n d e s c r i b e d ( S p a n d i d o s & Paul, 1982). W e h a v e d e t e r m i n e d t h e c o p y n u m b e r a n d s t a t e o f tk
g e n e s in several lines o f cells t r a n s f o r m e d w i t h o u r h y b r i d genes. Fig. 8 s h o w s a S o u t h e r n b l o t
a n a l y s i s ( S o u t h e r n , 1975) o f a B H K T K + cell line t r a n s f o r m e d w i t h p T K e l , a p l a s m i d w i t h
m o d e r a t e l y low levels o f gene e x p r e s s i o n in t h e s h o r t t e r m a n d t r a n s f o r m a t i o n assays. T h e d o n o r
D N A was p r e s e n t in five to t e n c o p i e s o f t a n d e m l y r e p e a t e d u n i t s in h i g h m o l e c u l a r w e i g h t
D N A. It is n o t a p p a r e n t w h e t h e r t h e r e p e a t e d c o p i e s are i n t e g r a t e d i n t o c a r r i e r D N A or h o s t cell
D N A. A s i m i l a r result h a s b e e n o b s e r v e d for o t h e r p l a s m i d c o n s t r u c t i o n s w i t h m o d e r a t e or p o o r
levels o f g e n e e x p r e s s i o n . In c o n t r a s t , t h e result o b t a i n e d w i t h B H K T K + a n d L A T K + cell l i n e s
t r a n s f o r m e d w i t h t h e highly efficient p T K M O L T R 1 ( T a b l e s 2 a n d 3) is q u i t e d i f f e r e n t (Fig. 9).
T h e d o n o r D N A is p r e s e n t in o n e to five c o p i e s s e p a r a t e l y i n t e g r a t e d i n t o h o s t or c a r r i e r D N A
a n d does n o t a p p e a r to be amplified.
DISCUSSION
T h e results p r e s e n t e d a b o v e d e m o n s t r a t e t h e usefulness o f t h e HSV-1 tk g e n e as a n a s s a y for
e u k a r y o t e t r a n s c r i p t i o n - c o n t r o l sequences. D e l e t i o n o f t h e n o r m a l p r o m o t e r for tk results in a
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HSV
tk e x p r e s s i o n a s s a y
2691
"7
"7
"7
"7,
[...,
<
[-.,
[...,
<
,.d
<
t-.,
<
t-.,
<
[..,
,<
[.
<
~
<
,-.1
¢~
,.-1
[..,
Origin
1.3 kb
Fig. 7. Northern blot hybridization analysis of tk RNA present in long term transformants. Twenty ~tg
of total cell RNAs from each transformed cell line indicated on the figure and grown under selective
conditions (medium containing HAT)were fractionated on l~agarose formaldehyde-containinggels,
blotted onto nitrocellulose and probed with 3-~P-labelledtk DNA as in Fig. 6.
500-fold inactivation of gene expression. The reason for the slight amount of residual activity is
not known, but could be due either to very weak cryptic transcription-control sequences present
in p T K 1, or to integration of the t k coding sequences adjacent to transcription control sequences
in the carrier D N A or host cell D N A , or both. Activity can then be recovered by insertion of
exogenous transcription-control D N A fragments. The system also provides a useful assay for
enhancers or activators which increase gene expression at a distance.
Using the t k system, gene expression can be measured in two ways: after transfecting cells
with recombinant plasmids in short term or transient assays for tk-specific m R N A and T K
enzyme levels, or the rate or efficiency of transformation of T K - cells to a T K + phenotype.
Although less sensitive than the transformation assay, the transient assay has several advantages
over other systems for the assay o f gene expression : it can be used in almost any cell type and is
quantitative, highly specific, rapid and relatively inexpensive. Our results obtained using it
show that in cells transfected with different recombinant plasmids the amount of tk-specific
m R N A correlated very well with the amount of enzyme present, although there was little
difference in the total amount o f t k D N A present. We have previously demonstrated that at 48 h
post-transfection of an SV40 recombinant in H e L a cells, the donor D N A is assembled into
regularly spaced nucleosomes and it is present in a high copy number per cell (Gilmour et al.,
1982). In the present study, although we cannot rule out effects on D N A replication or
recombination in a minor fraction of the cells within the transfected population, the results
strongly suggest that the exogenous D N A sequences mediate their effect on t k gene expression
by regulating the amount of t k m R N A transcribed. Enhancers probably increase transcription
from the promoter controlling the t k gene, as they increase the amount of the full length t k specific 1-3 kb m R N A present (Spandidos & Wilkie, 1983; J. C. Lang, D. A. Spandidos & N. M..
Wilkie, unpublished results).
Comparison of the data shown in Tables l, 2 and 3 shows that the results obtained using the
transformation assay correlate very well with the levels of t k m R N A and T K enzyme activity
detected in the transient assay. Transcription control sequences that gave low transient assay
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J. C. LANG, N. M. WILKIE AND D. A. SPANDIDOS
2692
BTe 1-l
I
pTKel
Pvull
HpaI
Xbal
EcoRI
KpnI
1
pTKel
I
kl~
kb
0
OC
L7.1
.~----- CC
.~-----3-7
•~---- 3.4
.
.
.
.
.
+
Fig. 8. Southern blot hybridization analysis ofa BHKTK + transformant, BT~I-1 containing multiple
copies of the pTK~I recombinant. 3-'P-labellednick-translated pTKel plasmid was used as a probe. 500
pg of uncut (U) or Pvull (Pv)-linearized pTKel plasmid was used as a marker. P, Hirt precipitate; S,
Hirt supernatant. HpaI, XbaI and KpnI do not cut pTK~I DNA, whereas PvuIl cuts once (7.1 kb linear
pTK~I) and EcoRI cuts twice (3.7 and 3-4 kb fragments), OC, open circles; L, linear molecules; CC,
closed circles.
values gave low transformation efficiencies, while those that gave high transient values resulted
in high transformation efficiencies. There was a tendency towards slightly higher values using
the transformation assay; this may be due either to its greater sensitivity and range or to
additional events occurring during the selection and growth of stably transformed T K +
colonies. Despite the correlation between the transient gene expression values and the
transformation ratios, there was very little difference in the tk D N A , tk m R N A or T K enzyme
levels in low passage stably transformed cell lines. Thus, although initially an altered control of
expression of the tk gene is obtained using different transcription control sequences, culture in
H A T medium selects cells in which the level of tk gene expression is further controlled. The
mechanisms involved in this additional control are not yet known. Nonetheless, taking these
results together, we propose that the initial rate of expression of tk is a major determining factor
in the subsequent rate of biochemical transformation, and that this may be a general
phenomenon. Although early after transfection (2 or 3 weeks after isolation of transformed
clones) donor D N A copies per cell are relatively low (Table 4) further growth (3 to 6 months)
under selective conditions alters the copy n u m b e r depending on the transforming r e c o m b i n a n t
used.
The state of tk D N A in stably transformed cell lines depends on the D N A sequence
controlling tk gene expression. Using sequences which give a high transient expression and
transformation rate when introduced into recipient cells in the form of covalently closed
supercoiled D N A (i.e. the MoMuSV LTR), the transformed cells contain 1 to 5 copies of the
transforming gene, each independently integrated into either carrier D N A or (in the cases where
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H S V tk expression assay
2693
0.2 ng
1 ng
pTK
BT
BT
LAT
LAT
pTK
MOLTR 1
MOLTR 1-1
MOLTR 1-2
MOLTR 1- 1
MOLTR 1-2
MOLTR 1
U
B II B
E
K II B
E
K I[ B
E K ]1 B
E
K "1[ B
U i
kb
OC
L
6.6
CC
3.7
2.9
4h
exposure
40 h exposure
4h
exposure
Fig. 9. Southern blot hybridization analysis of BHKTK + and LATK + transformants obtained after
transformation with pTKMOLTR1. Cell lines transformed with pTKMOLTRI are described in Table
4. BTMOLTRI-I and LATMOLTRI-I were obtained by transfection in the absence of carrier and
BTMOLTR1-2 and LATMOLTRI-2 in a 1000-fold excess of salmon sperm DNA as carrier, s_,p_
labelled nick-translated pTKMOLTR1 DNA was used as a probe. OC, open circles; L, linear
molecules; CC, closed circles; B, BamHI; E, EcoRI; K, KpnI. Kpnl does not cut pTKMOLTRI,
whereas BamH1 cuts once (6.6 kb linear) and EcoRI cuts twice (3.7 and 2.9 kb fragments).
no carrier was used) into recipient cell D N A . There is evidence, as others have noted (Reyes et
al., 1982) for some limited duplication of some of the integrated transforming genes. In contrast,
when weak control sequences which result in low transformation ratios are used, transformed
cells normally contain 5 to 20 copies of tandemly duplicated donor plasmid D N A , with little
evidence of sequence rearrangement. The presence or not of carrier D N A does not affect the
result. In most cases, it is difficult to determine whether or not the duplicated array is integrated
into carrier or host D N A , but at least in some cases, evidence for extrachromosomal copies of
the tk gene can be obtained (Kretchmer et al., 1981 ; Spandidos & Paul, 1982; Spandidos et al.,
1982). The reasons for these consistent differences in the fate of transforming D N A are not yet
known. It could" be argued that L T R sequences affect the recombination properties of the
introduced sequences, but it seems more likely that the results reflect the different ways in which
cells can respond to obtain an 'average' level of expression (for which growth in H A T m e d i u m
selects). In transformed cells, it seems most likely that T K translation is from m R N A
transcribed from the tk-proximal promoter. Thus, a 1.3 kb tk-specific m R N A was present at
roughly the same concentration in a number of different transformants induced by different
plasmids in which tk was under control of either the endogenous tk promoter, or the exogenous
human epsilon globin promoter, whether or not enhancers were present. Initiation at the epsilon
globin cap site, and termination at the tk polyadenylation site would result in an R N A very close
to the normal tk 1.3 kb m R N A . This interpretation remains to be confirmed by S 1 mapping. In
the case of plasmids in which the tk gene is under control of the mouse/7 major globin promoter
fragment, which lacks a cap site, S1 mapping has revealed several different 5' ends located both
within the mouse sequences and the HSV untranslated leader sequences (R. S. Gilmour & D. A.
Spandidos, unpublished observations).
We have used the tk assay for transcription-control sequences to analyse several different
genetic systems. In the present study, we have separated different domains of the M o M u S V
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2694
J. C. L A N G , N. M. W I L K I E A N D D. A. S P A N D I D O S
LTR, and shown them to contain a promoter with an efficiency similar to that of the tk
promoter, and an enhancer, in confirmation of the suggestion by Gruss et al. (1981).
Interestingly, when the LTR enhancer is placed upstream of tk under control of its own
promoter, insertion of the LTR promoter between the enhancer and the tk promoter has no
effect in modulating gene expression. In contrast to the recent report by Wasylyk et al. (1983)
this strongly suggests that however enhancers work, there is no absolute preference for the
proximal promoter. This difference in results is not understood but may reflect differences in the
techniques used to analyse gene expression, or in behaviour between different promoters. We
also observe that enhancers activate gene expression when placed proximal or distal, to the
promoter-deleted tk gene (this study, and Campo et al., 1983). The reasons for this are not clearly
understood but may reflect activation of cryptic promoter sequences. One such sequence may be
located within the normal tk coding sequence and would be predicted to initiate transcription of
a 0-9 kb m R N A with the potential to encode an active TK polypeptide fragment. Interestingly,
in some recent experiments using a variety of enhancers we have observed induction of both a
1-3 kb, and a 0.9 kb tk-specific m R N A (Spandidos & Wilkie, 1983).
In other recent experiments we have used the approach described herein to detect and analyse
the properties of promoters and enhancers in the genomes of bovine papillomavirus and herpes
simplex virus (Campo et al., 1983; J. C. Lang, D. A. Spandidos & N. M. Wilkie, unpublished
results). Additionally, the same techniques have been used to identify a D N A sequence
proximal on the mouse genome to the fl major globin promoter fragment which prevents tk gene
activation by this promoter in fibroblasts (R. S. Gilmour, D. A. Spandidos & J. Paul,
unpublished results).
W e would like to t h a n k Mrs M a r y F r e s h n e y and R o s e m a r y Balfour for help with the Southern analysis, D r J o h n
Paul for critical reading of the m a n u s c r i p t and s t i m u l a t i n g discussion, Drs Peter R i g b y and G e o r g e V a n d e W o u d e
for cloned M o M u S V and SV40 sequences and the C a n c e r R e s e a r c h C a m p a i g n of G rear B r i t a i n for support. J. C. L.
was the r e c i p i e n t of an M R . C . research studentship.
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(Received 24 June 1983)
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