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Gene organization of the transforming region of adenovirus type 7 DNA
Gene, 18 (1982) 143-156 Elsevier Biomedical
143
Press
Gene organization of the transforming region of adenovirus type 7 DNA (DNA
sequence;
RNA mapping;
Sl nuclease;
sequence
comparison;
cell transformation)
R. Dijkema, B.M.M. Dekker and H. van Omondt Department of Medical Biochemistv, (The Netherlands) (Received
December
(Revision
received
Sylvius Laboratories,
University of Leiden, Wassenaarseweg
72, 2333 AL Leiden
14th, 1981) and accepted
February
16th. 1982)
SUMMARY
The sequence been determined. be involved sequence
of the leftmost 11% of the weakly oncogenic human adenovirus type 7 (Ad7) DNA has This part of the Ad7 viral genome encompasses early region El which has been shown to
in the process
and determined
of cell transformation
coordinates
in vitro (Dijkema
of the El mRNAs,
et al., 1979). From
we are able to predict
polypeptides encoded by the transforming region of Ad7. The organization of the El region of Ad7 and of other adenovirus
serotypes
the primary
the nucleotide structure
of the
(Bos et al., 1981) leads to
the proposal of a novel mechanism for gene regulation at the translational level in which protein synthesis can initiate at either the first or the second AUG triplet available in mRNA. The differences between the large
Elb-specific
oncogenicity
tumor
antigens
of adenovirus
types
12, 7 and
5 may
explain
the
difference
in
of these viruses.
INTRODUCTION
the criteria
Human adenoviruses have been extensively studied because they can serve as a model for eukaryotic gene expression, and because they in-
types belonging to class A (such as Ad12) are highly oncogenic and induce tumors at high frequency following a short latency period; class B serotypes (e.g. Ad3 and Ad7) induce tumors at low frequency and only after an extended latency
duce tumors in certain
rodents
and transform
non-
This phenomenon
Abbreviations: cDNA,
adenovirus,
complementary
0378-I 119/82/0000-0000/$02.75
can be used as one of
Ad; bp, base pairs;
DNA;
T antigen,
sero-
Transformation by human adenoviruses is a process in which apparently only a small portion of the viral genome is involved; since the introduc-
N, nucleotide;
tumor antigen.
0 1982 Elsevier Biomedical
classification:
period; and serotypes of classes C (e.g. Ad2 and Ad5) to F do not induce tumors in experimental animals (Mackey et al., 1979a, b). However, adenoviruses of all classes can transform rodent cells in vitro (for a review, see Tooze, 1981).
permissive cells in tissue culture. Among the 36 recognised serotypes of human adenovirus, the degree of oncogenicity varies from one serotype to another.
for the following
Press
144
tion of the calcium to transform which
or Ad5 virions
rodent
or intact
sequences
(Tooze,
transformation
is basically
encompasses human Recently,
region
two subregions: Elb
(map position
own
promoter
at least viral
cycle
11% in the DNA sofar (Tooze,
that
1969). Early mRNA
of
was
of the virus
described
(Van
was extracted cycloheximide)
and
viral
from cells at 6 h in the pres-
and late mRNA been
was fractionated
on oligo-(dT)
was used in Sl nuclease
KB
Eb et al..
the cells had
with virus. The RNA
chromatography
der
(or 18 h post infection 24 h after
in
propagated
cellulose
inby
before
it
experiments.
1%) each of which has its
for the synthesis
of a family
of
partially overlapping RNAs (Tooze, 1981). The Ela region corresponds to the smallest DNA fragment able to convert a primary cell into an immortal ‘transformed’ cell line (Ad5 HpaI-E: O-4.3%; Ad7 BgZII-H: O-4.5%). However, cells transformed by region Ela spects from cells transformed
differ in several reby DNA fragments
which, in addition, contain information El b region. Cells transformed by region
from the Ela have
a different morphology, grow to lower saturation densities and have an atypical T antigen distribution pattern. This has led to the conclusion that the Elb region, possibly in conjunction with the Ela region, codes for functions that give rise to the characteristic, transformed transformed cells. We have determined
post infection
fected
and
been
ence of 25 pg/ml was harvested
to consist
Gomen)
the purification
have
of all
O-4.5%)
4.5-l
cells and
198 1).
Ela
position
(strain
DNA
and
El has been shown (map
Ad7
14%
of region El
in the lytic
studied
METHODS
(a) Viral DNA and isolation of cytoplasmic RNA
by Ad2
to the leftmost a function
the leftmost
adenoviruses
the left
1981). This indicates
early
AND
(Van der Eb et al.,
DNA contain
of the genome
MATERIALS
fragments
from
cells transformed
homologous
is expressed
DNA
to originate
of the viral genome
1980); moreover,
which
it has been possible
cells with specific
were all found
terminus
DNA
technique
properties
the nucleotide
of
virus-
sequence
(b) Enzymes Restriction endonucleases HinfI and T4 polynucleotide
HindIII, PstI, HpaII, kinase were isolated
as described earlier (Van Ormondt et al., 1978); endoR. Sin1 was a gift from Mr. M. Lupker. Endonucleases Bgf II, M601, MboII, HhaI, HaeIII, PuuII, XhoI and BumHI were purchased New England Biolabs (Beverly, MA). DNA merase I, Klenow’s fragment A of DNA merase and pancreatic DNase were obtained
from polypolyfrom
Boehringer (Mannheim, F.R.G.), bacterial alkaline phosphatase (BAPF) from Worthington (Freehold, NJ), and Sl nuclease
from Sigma (St. Louis, MO),
T4 DNA ligase was prepared from a lysogen of AT4lig as described previously (Murray et al., 1979).
of
the left-terminal 11% of Ad7 DNA. However, due to the spliced nature of Ad7 El mRNAs (Yoshida and Fujinaga, 1980; Dijkema et al., 1980b; 1981) it is not possible to predict protein sequences from this DNA sequence alone. To that end, we have studied these mRNAs at the nucleotide level using molecular cloning and a modification of the Sl nuclease technique. In the present paper, which is one in a series describing the organization of the El region of Ad7 (Dijkema et al., 1980a, b; 1981) we report the nucleotide sequence of the Ad7 Elb region, and of the mRNAs specified by it. Our results enable us to predict the amino acid sequences of the Ad7 polypeptides involved in cell transformation. A comparison will be drawn between the Elb proteins of adenoviruses of different oncogenicity.
(c) Chemicals [ a-32P]dNTPs were obtained from New England Nuclear (Boston, MA), [ Y-~~P]ATP from Radiochemical Centre (Amersham, UK). Unlabeled dNTPs were purchased from Sigma (St. Louis, MO). The source of the chemicals used for the chemical degradation technique and gel electrophoresis systems have been described (Van Ormondt 1979).
et al., 1978; Maat
and
Van Ormondt,
(d) Sequencing procedures
the
Sequence analysis was performed protocol described by Maxam
according to and Gilbert
145
(1977).
Single-end-labeled
restriction
that served as substrate tion technique
were prepared
with [ Y-~*P]ATP
5’ ends kinase, with
in the chemical
or of recessed Klenow’s
both
cases
A of DNA [a-32P]dNTP
followed
quences
directly
DNA was used for hybridization,
DNA.
adjoining
where in
Nucleotide
se-
enzyme
sites
restriction
These
conditions
scribed in a previous
synthesis
polymerase endonuclease
fragments.
30 min) at 37°C when nickand at
37 and 42°C when the probe was terminally
of
of choice,
by restriction
of the labeled
units/ml,
translated
either by labeling
3’ ends by repair
cleavage
(200 -400
degrada-
and T4 polynucleotide
fragment
and a complementary
fragments
a much
the thermal
termini
of the hybrids, Later
those was
recom-
“breathing”
and thus batches
de-
et al., 1980b)
temperature
to reduce label.
from
paper (Dijkema
lower
mended terminal
differ
labeled
at the
the loss of the
of Sl nuclease
abled us to use higher Sl incubation
en-
temperatures;
homochromatography
(Tu et al., 1976) on thin-
apparently, they did not contain contaminating nucleases. Alkaline agarose gels were as described
layer plates
cellulose
by McDonell
were occasionally
determined
of DEAE
Cell 300 DEAE/HR-2/15). ling of the sequence data programs
described
by two-dimensional (Macherey-Nagel
et al. (1977).
For computer handwe made use of the
by Staden
(1977; 1978). RESULTS
(e) Sl mapping of cytoplasmic RNAs (a) Sequence analysis Poly(A)-containing bridized
overnight
RNA at 52°C
(2-10
pg)
was
to 32P-labeled
hyOur choice
DNA
quencing
fragment (50 ng genome equivalent) in 10 ~1 of 80% formamide (Casey and Davidson, 1977). The RNA-DNA hybrids were treated with Sl nuclease
2col
plzB
5’
~
GU5
-
--M6Z9
map
ob-
U3F4
YlZ3
Ml F2
23 F2
Z5M5
M5Z6
used in se-
tained by the method of Smith and Birnstiel(1976; result not shown). We have previously reported the
R2Z3 _ A5K
F2Y3
enzymes
on a preliminary
2500
R3Z5 BlF2
of restriction
was based
-
LF2
ACY 81
R122
F3 61
Z7A2
H522
~
-Z2Hl
m
FL 22
Y2D - 06Yl
H3M3
pzz2-
BL F3
ACZ3
Ml z5
A6 HI
ZZAB -
ZLYl
-- 63F4
3’
Fl H4
M3 FL
--M7F6
M4D
M685 m
3’
kyF3
Z5R3
w
M5Z5
ZCIFZ F7MF
BSZB MB8
Y3Ml
B
AC Bl MSBl
LHqa
Z5Ml
Z3F3
Z784 ----
Hl z2 a4 z2
F3Z2
HSAl
PlZ2
F3 04
R2 &la BlYl
B
ABZ2 A2F3
Acy 23
PI F6
Zll
H3
YIZL
H5M3 Z2F4
ZLyz
63H3 FL H3
Ml FL
u M3 H3
g7t.J M7Y2
5’ DY2
I
r Fig. 1. Schematic representation of the sequenced tracts of DNA in the I- and r-strands of the region between the BgHI site at position 1564 and the HphI site at 3388. In the fragment names, the first letter denotes the endonuclease used for the primary splitting of this region, the second the endonuclease used for the secondary restriction cut to separate the two end-labeled segments of the primary restriction fragment. The following abbreviations were used: A-AluI; G-BglII;
H-H/WI; Hga-HguI;
K-PstI;
L-EalI;
M-MboI; P-HphI;
AC-Accl; R-EcoRII;
Acy-Acy
I; B-MboII; C-FnuDII; D-PouII; F-HinfI;
U-AsuI; Y-HpoII;
Z-HneIII. At the bottom of the
picture we have indicated with solid lines those parts of both strands (I and r) which have been sequenced.
146
of Ad7 region Ela (nucleotides
kema
I- 1564, Dijkema
et al., 1980a) and of the gene of
ported
in the latter
Ad7 polypeptide
IX (nucleotides
tracted
by one because
primary
structures
3350-4010,
Dij-
et al.,
198 1). The nucleotide publication
numbers
re-
have to be sub-
in the meantime
we have
147
I AT s 0 R” R s p I. s Y I( K C’H P E R c N L G I I. N E 0 E GATTGCMCATCAGGTAGGGTGAAGAGTCAGTTGTC~TGAAGA~~CATGTTT~AGAGATGTAATCTTGGCATACTGAATGAAGGTGAAGCAAGGGTCCGCCACTGCGCAGCTACAGA 2778 278% 2790 2800 2810 2820 2830 284% 2858
AR”
R
H
2860
C
A
ATE
2870
2980
TACPILIKGNASYS”N~lCGHSDERPYO*LTCAGGXCNIL
AACniC~~~MATSTTCTAIITAAAGGGAAATGCCAG~~~~AT~~~~~ATC~TGGACATTCGGATGAGAGG~~~~TCAGATGCTAACCTG~ 298%
2910
2948
2950
2970
2990
3000
3110
312%
3230
3240
RVRACECGGRHARPPPVCYDYTEDLRPDHLVLACTGAEFG AAGGGTGCOCGCATGCG4ATGCGGAGGC~GCATGCTAGAT~CAGC~GGTGTGCGTGGAT~G~CTGAAGACCTGAGGCCCGA~ATT~GTGCTTGCC~CACTGGhGCGGAGT~GG 3258 3260 3278 328% 323% 3300 3318 3320 3330 3340 3358
3368
AT”“IVSHARKRWPYPEHNV*T~~T~S~GG~~G~~~~TGC TGCTACCOTGCATATCGTT~ACACACGCAACI\M~CCCTGTATTTGAACATAATGTGA~~C~~GTG~~CCATG~AT~TAGGTGGTCGCAGGGG~ATGTTT*TG~C~~~~AGTG 3810 ,028 383% 3040 3058 3860 3870 3086 3098 31%%
N “NHtlK”M LE PDAFSRVSYTG I BDNN I TAICA-rrATCTGAAGGTAln;TTGGAA~CAGATGCCTTTTCCAGAGT~GCGTAACAGGAATCT~GATA~AATAT~AA~TA~~AAGATCC~AGATA~ATGA~AC~AAACC 313% 314% 3158 3160 3170 3188 3190 328%
PtWK 321%
I
tRY.DDT 3220
R
term 55x S
S
G
E
E
T
D
l
L &iee
1”
TTCTAGTGGTCAAGAAAC~A~GTIAGTAGTGGGGG~~~TGTGGATGGGGACTTTCAGGTTGGTAAGGT~ACAAATTGGGTAAATTTTGTTAA~~~~G~~~TG~~G~~C~n 3378 338% 3390 340% 341% 3426 3430 344% .
eexln
P
P
epzicelrv* ” 3450
3460
3470
3488
358%
3590
3680
3710
372%
*K
GSASPEGGYPSPYtlGRtPPWRGVRPNVMGSTYDGRPVP TGCAAGCGCTTC~~GAGGGGGGAGTATTTA~CC~ATCTUACGGGCAGGC~CCACCA~GGCAGGAGT~GTCAGAA~TCA~GGbTCCACTGT~ATGGGAGACCCGTCC 3498 3588 3518 3520 3538 3548 3559 3160 3570
PANSSTLTYATLSSSPLDAAAANTILGnCYYGS AGCCCGCCMTKCTCIUCGCTGAC~TA~CCAC~TGAGTTCGTCACCAT~GATGCAGCTGCAG~~CGCCGCTAC~CTGCCGCCMCACCATCCTTGGAATGGGCTATTACGGAA 3610 3628 3630 3648 3650 3660 3678 3698 368% 3700
AOLREQTESAVATAKSK’
r---poEg(il1 3 9182
%
3930
3940
m*,pix 3950
3960
TCGCGCGCGGTATGCCCTGGACCATCGGTTTCGATCATTGAGAACTCGGT 3978 398% 3998 4%0% 4010
Fig. 2. The nucleotide sequence of the Z-strand of the region El of Ad7. We have underlined “Goldberg-Hogness boxes” and the sequence AATAAA, and boxed postulated initiation and te~ation codons. Also shown are the polypeptides predicted by the DNA sequence (this paper; Dijkema et al., 198oa; 1981) and RNA mapping data (this paper; Dijkema et al., 1980b; 1981). The splice sites and the messengers in which they occur are indicated by arrowheads and Roman numerals.
deleted one base in the DNA sequence between positions 1564-3350. Fig. 1 is a schematic representation of the tracts sequenced to detertine the primary structure of that interjacent region, i.e. between the BgrII site at position 1564 and the HphI site at position 3388. As is evident from the bottom part of Fig. 1, we succeeded in sequencing both I and r strands virtuaIly completely. Fig. 2 shows the deduced sequence, in combination with the sequence of the adjacent segments determined previously, which together represent the entire Ad7 El region (nucleotides l-4010). We have given the sequence of the 1 strand, since this has the same polarity as the mRNAs transcribed from this region. We have indicated postulated initiation and
termination codons, “Goldberg-Hogness boxes” reported to occur some 30 nucleotides upstream from the nucleotides coding for the 5’ end of mRNAs, and the sequence AATAAA usually associated with the 3’ termini of mRNAs (Proudfoot and Brownlee, 1976). (For all these features see DISCUSSION.) Finally, in Fig. 3 we present the physical map of the region between positions 1564-3350 for a number of restriction endonuclease cleavage sites. (h) Mapping of Elb mRNAs by the Sl nuclease technique In a previous paper we reported that hybridization of the Ad7 BglII-E (positions 1564-3897, i.e.
148
2cm
2500
3Lm
3500
LOO0
boSe I-'""i
L
3
Y- Hpa II
ll
1
1
2
C’CGG
13
Z-Hae
III
3
815 5
U-Asu I
1
74
F-Hinf I
KIJ
12
3
7
L
2
11
1
2
1
1
1
3
I
1
3
6
L
11 2
&318l
GG’CC
6
G’GNCC
161
I
5
G’ANTC
T-Taq I
T’CGA
H-Hha I
1
5
I
I
C-FnuD II A-Alu I
7
M-Mb01
16
B-MboII
8
11~10 121
5
1
5
61
1
1 1
7
3
6
n6n
G CG’C
3x
2
i
3
2"
2
1
I
5
1
1
lslll
2
5
P-HphI
3
2.
C G’CG
1191
3
AG’CT
2
la
‘GATC
i 7
2
2
L
3
GAAGA
8x
GGTGA
ST
R-EcoR 1
‘CC+GG
Dde I
C’TNAG
zoo0 I
25x I
I
,
,
I,,
3an
, , I,
.,
Bx
, I
*,
,
,
Loo0 1
No sites for HindlI,HpaI.EcoRI.Bgl I.BclI.BstEII,ClaI,KpnI.PvuI,RsaI.SmaI.Xma~.SacI.SacII. SalI,MlaI.RruI,XbaI Fig. 3. Physical symbols
maps for a number
in the maps for MboII,
of restriction
Hph I and HgnI
endonuclease
cleavage
shovi the positions
virtually equivalent to region Elb) fragment to polyadenylated RNA, and subsequent nuclease Sl treatment of the RNA-DNA hybrids leads to a number of protected segments: annealing to early mRNA yielded Sl-resistant tracts of 1800 and 440 nucleotides (N), whereas protection with late mRNA resulted in two additional products of 600N and 160N (Dijkema et al., 1981). For a more precise mapping of the transcript segments which comprise the various Elb mRNAs, hybrids were made by annealing late mRNA to internally
sites (arrows)
of the cleavage
in the region between sites relative
positlons
to the recognition
1564-3350.
The
sites.
32P-labeled PuuII (positions 1564-3474) and HguI (positions 1564-2269) subfragments of BgfII-E. Protection of the PvuII subfragment yielded tracts of 1800 N, 600 N and 160 N (Fig. 4A, lane 2), and protection of the HguI subfragment tracts of 700 N and 600N (Fig. 4A, lane 1). Hybridization of the nick-translated BgfII-BumHI fragment (15641910) to early and late mRNA yielded an Sl -resistant product of 335 N in both cases (Fig. 4C) which locates the 5’ terminus of both early and late Elb RNA around position 1575.
B
1910
4
I!%&
3.c7L
2269
BarnHI
i 3897
t
V
1
HgaI
PVUII
mRNA: tsDON
/
l
H
*
P
3m.I
*
____-----
600N
_____..----
600N
___--*___----
__---.._ -----____
__--------____
--__
* ‘.
@ON
---___--__
---_____:
48ON
t%
480N
‘t
180 N
l
Fig.4.
Detection
electrophoresis BglII-E
and approximate gel; nuclease
mapping
Sl-resistant
(1564-3802)
after hybridization
indicating
cleavage
sites for Eg/II, PuuII,
mRNAs;
the broken
showing
the Sl-resistant
line carets portion
late (L) mRNA, next to a Hinfl
denote
of the Elb
mRNAs
tracts of the HgaI to poly(A)RNA BarnHI
of the Ad7 BglII-BarnHI digest of pBR322
and (bottom
sequences. fragment
for siring.
cells. (A) Autoradiograph
lane 1) and PeuII
from Ad7-infected
and HgoI,
the intervening
in Ad’l-infected
(1564-2269,
(1564-3474,
cells. (B) Physical
part) the approximate
(C) Autoradiograph (positions
1564-1910)
of alkaline
lane 2) subfragments
map of the Ad7 &III-E lengths (* 10 nucleotides)
of an alkaline generated
agarose
by protection
agarose of Ad7 fragment
of the EIb
electrophoresis
gel
with early (E) or
150
37O
42O
G
cloned
A
Py
c
the
cDNAs,
nuclease
(1977);
we made use of a modification Sl
instead
lated)
DNA,
template
labeled
and
fragments
SinI-HinfI
Sharp
(nick-trans-
we used 5’- or 3’-terminally
labeled
position
of Berk
(r strand)
3’-labeled 2221;
technique of internally
of
labeled
as probe (Fig. 5). A
fragment
(positions
by incorporation
2119.
of [(Y-~IP]TTP
2119) was hybridized
to late mRNA.
Sin I-Hin f1
nuclease
S 1-resistant
fragment
was then run next to a Maxam-Gilbert
sequence
ladder
The major
portion
at The
of the
of the same fragment
protected
product
(Fig. 5A).
is seen to run as a
band which comigrates with band C2165. This implies that C2164 is the last base in the DNA fragment that is complementary to mRNA, and encodes the donor residue in the RNA splice which joins the 600 N and 440 N transcripts menFig. 5. Sequence Gilbert.
pattern
1977) and
late RNA)
segment
tions 2119-2221.
SI-resistant
of 3’4abeled
labeled
(by protection
SinI-Hinfl
at Slnl
is shown,
end of the intron
fragment
The deduced
while in the complementary
and the donor
nucleotide
in the previous
paragraph
(mRNA
V in
Fig. 6; see DISCUSSION).
with (posi-
site). The temperatures
the lanes are those of the Sl treatment. (r-strand)
tioned
G, A. Py.
(lanes
nuclease
over
sequence
strand
the 5’ DISCUSSION
(2164) are indicated.
(a) Mapping of Ad7 Elb mRNAs To
determine
the
above-mentioned insofar
map
transcripts
coordinates at the nucleotide
as they were not determined
5po
lop0
t’
BamHI
’
of
c”
the level,
In this paper
by sequencing
‘500 Hlndrn
*v
*opo
‘1
‘t’
quence
T
Barn HI
BglI
we describe
for the BglII
t
390
the Ad7
site at 4.5% (position
3YJ
t
se-
1564)
LOP0
11’
Earn HI XhoI
Hlnd!J
DNA
Bgl II
FRAME ‘7 2 3 75% nn I
512
1155?\1219 AiAn’,
I
28 K proten
1062I <,,‘\.~ ~-12‘9
II x__---
_s_._-___
t 3 term,nus ptza2 rnRM
naunnn
12‘9 H
2LK
,.
63K
,.
,577 216‘ _____-------
P
_____------
TZI
Fig.
6. The
stop
frames.
codons
(TAA, TAG, TGA;
In the long termination
indicated
codonfree
--.-------______._
are represented
as solid black lines. In the bottom
paper;
and Fujinaga,
Yoshida
as tick marks) in the /-strand
stretches
the presumed
.^.3‘V
initiation
l&o
21 K f 55K
”
+
21K+
9K
”
~ 1LK
IpIg
1938 v114
of the Ad7 region triplets (ATG)
part the results of the Ad7 RNA mapping
1980) are summarized.
+
---------“‘-___________~~,~
H
reading
aao.nn 0 iL;fL
3386,‘. 1‘75
El arranged
are indicated.
data (Dijkema
+
pmtetns
according
to their
The coding
regions
et al., 19gOb; 1981; this
up to the HphI site at position 3388. Tracts surrounding this sequence have already been described in two previous publications (Dijkema et al., 1980a; 1981) so that the Ad7 (subgroup B) DNA sequence now determined stretches from the left terminus of the viral genome to nucleotide 4010. As will become evident this sequence encompasses the entire early region 1 (El). The primary aim of our sequencing effort is to predict which polypeptides are encoded by this transforming region, and to compare these with the proteins specified by the corresponding regions of subgroup A and C adenoviruses. However, a prediction of polyp~tides is not possible on the basis of the DNA sequence alone but also requires knowledge about the coordinates of the mRNAs specified by the investigated DNA region. Such information we obtained by the nuclease Sl technique of Berk and Sharp (1977), and by sequencing cloned cDNAs derived from Elb mRNAs. Initial mapping was performed by hybridizing Elb mRNA to the BglII-E fragment (positions 15643897) and to shorter subfragments thereof: BglIIPuuII (1564-3474) and Bg/II-HguI (1564-2269) and degrading the non-hybridized stretches of nucleic acid with nuclease Sl. The BglII-E fragment is protected by four transcripts of 1800N, 600 N, 440 N and 160 N, when late mRNA is used for hyb~~zation. In the case of the PvuII subfragment three Sl-resistant products of 1800 N, 600 N and 160 N were generated; consequently, the 440 N transcript observed by hybridization to the &$11-E fragment maps to the right of the PuuII site at position 3474. Hybridization of the HguI subfragment to late mRNA generates two Sl-resistant products of 700 N and 600 N. Of these, the 600 N tract is the same as the one seen in the previous two hybridization experiments, whereas the 700N tract represents the stretch of DNA that is protected by the 18OON transcript. This interpretation is based on the fact that the 1800 N and 600N transcripts have the same 5’ terminus (see below: 5’ terminus of Elb mRNAs). From the absence of the 160N tract we conclude that the 160 N transcript maps between the HguI and PuuII sites at positions 2269 and 3474. In a previous paper we reported that the 1800 N and 440N tracts are covalently linked, as shown by sequencing cloned cDNA and by running a
nuclease Sl-resistant RNA . DNA hybrid in a neutral agarose electrophoresis gel (Dijkema et al., 1981). In the autoradiograph of this gel we detected bands representing material of 2250 and 450 bp long (the latter is generated by the quasi-late mRNA for viral polypeptide IX which was found to coincide to a large extent with the 440N segment). Unfortunately, the same autoradiograph did not allow us to demonstrate the presence of hybrids 1050 and 1200 bp long, because of the high background in this portion of the gel. The latter two bands were expected to arise by hybridization of the DNA probe to mRNAs consisting of the 6OON+~ON and 6OON+ 160N+440N segments, respectively. This expectation is based on the fact that the similarly organized Elb regions of Ad2 and Ad12 (Tooze, 1981) both encode an mRNA species of the 600 N -t- 440 N type. Moreover, Yoshida and Fujinaga (1980) have recently demonstrated this mRNA as a late species in Ad7-infected cells. Finally, the coordinates of the 160 N segment were inferred from those of a minor Ad2 El b mRNA observed by L.T. Chow (personal co~unication). The map positions of the Elb mRNAs as discussed above are given in Fig. 4B. (b) The 5’ terminus of Elb mRNAs
The nuclease Sl experiment shown in Fig. 4C located the 5’ end of Elb mRNA around position 1575. By hybridization to late Elb mRNA of the HindHI-Hue111 fragment situated between positions 1384 and 1678 we obtained a major Sl-resistant tract of 100 -t 3 N (results not shown). The Hind111 site is well within the Ela region so that the RNA-protected portion of this fragment must be complementary to the 5’-terminal 100 nucleotides of Elb mRNAs and the 5’ cap template, of all El b mRNAs, has to be at Al577 -t 3. This is in excellent agreement with the presence of a “Goldberg-Hogness box”, TATATAA, at positions 1548-1554. (c) The 3’ terminus of Elb mRNAs
In a previous publication (Dijkema et al., 1981) we reported that Ad7 Elb mRNA IV and the quasi-late messenger for polypeptide IX (mRNA
152
VII) have a common 3’-poly(A) attachment site encoded by nucleotides 3938-3941. This informa-
various
tion
of Fig. 6.
was obtained
derived
by sequencing
from the said mRNAs.
cloned Nuclease
cDNA
adenylation
(Proudfoot
curs
times
three
mRNAs
in
associated and
the
with 3’-poly-
Brownlee, 3’-terminal
of the Ela
sequence)
mRNAs
are given
and in the bottom
part
Sl data
(not shown) suggest that Elb mRNAs V and VI share this 3’ terminus as well, in spite of the fact that the signal AAUAAA
coordinates
in Fig. 2 (DNA
(f) mRNA VII Messenger
RNA
VII is, in spite of its location
1976) oc-
in early region I, not an early mRNA,
region
synthesized
predominantly
late stages
of infection
and
independent
promoter
(Tooze,
of
IV-VII.
(d) Splice sites in Elb mRNAs IV, V and VI
to the vast majority mRNAs,
since it is
at the intermediate transcribed
from
an
1981). In contrast
of characterized
it is not spliced
and
eukaryotic
and, in all probability,
The initial RNA mapping studies indicate that the Ad7 EIb region specifies three differently spliced mRNAs with identical 5’ and 3’ termini
specifies
(Fig. 4B) and with a common 480 N long 3’ segment (the length of this segment is measured from the splice point to the BglII site at position 3897,
since it must contain
plate for mRNA VII. Curiously enough, the gene for Ad7 polypeptide IX, in contrast to the corre-
and thence
sponding
to the polyadenylation
site at position
viral polypeptide
IX. It should
be noted
that the common part of the introns of mRNAs IV, V, and VI is an important regulatory region the promoter
and cap tem-
gene of Ad5 (Van Ormondt
et al., 1980)
3938). The 5’ end of this 480N segment (acceptor residue) is the same in all three El b mRNAs
and Ad12 (Bos et al., 1981), does not feature a “Goldberg-Hogness box”. The coordinates of
(Dijkema
mRNA
hybrid cDNA
et al., 1981). By sequence
plasmid constructed derived from mRNA
analysis
of a
from pBR322 and IV it was established
that this acceptor nucleotide corresponds to C3475 in Ad7 DNA and is covalently linked to a donor nucleotide corresponding to A3386 (Dijkema et al., 1981). In the present paper (Fig. 5) we designate position 2164 as specifying the 3’-terminal residue of the 600N transcript in mRNAs V and VI; this point was determined by the nuclease Sl technique (Fig. 5). The location of the 160N tract
VII are given in Figs. 2 and 6.
(g) Coding capacity To interpret the nucleotide sequence in terms of its coding capacity we have arranged the nonsense codons present in the Ad7 El region according to the three possible reading frames (Fig. 6). The El a region contains two stretches open for protein synthesis which are separated by an AT-rich region containing nonsense codons in all three read-
was inferred by analogy from the position of the 180N segment observed in a minor Ad2 Elb
ing frames. We have shown (Dijkema
mRNA species (L.T. Chow, personal communication) and of a similar tract in Ad12 El b ‘mRNA
the three Ad7 Ela
(Virtanen et al., 1982). The various splice points have been indicated in the DNA sequence of Fig. 2, and in the mRNAs in the bottom part of Fig. 6. (e) Ela mRNAs I, II and III In a recent paper (Dijkema et al., 1980b) have described the organization of Ad7 early gion Ela. It was found to encode three 5’3’-coterminal mRNAs differing in the amount internal sequences removed by RNA splicing.
we reand of The
mRNAs
et al., 1980b) that in (Fig.6;
mRNAs
I, II
and III) this intervening sequence is removed by RNA splicing. As a result, mRNAs I and II give rise to completely overlapping polypeptides (28000 M, and 24000 M,) initiating at AUG 576 and terminating at UGA1452, their only difference being 3 1 internal amino acids absent in the protein specified by mRNA II. The polypeptide encoded by mRNA III shares the N-terminal part but is translated in a different reading frame beyond the splice point; consequently, it terminates at an earlier stop codon (UAA 1348) and has an M,-value of 6300. In the region of the EIb mRNAs (1577-3938)
153
Ad7 DNA
contains
tein synthesis frame
III,
three stretches
between
TGA1482
between
TAAl904
frame II, and between frame DNA
mRNAs
stretch
(frame
as a probable peptides
(Kozak,
mRNAs
in
would have an &&-value of 55000. The splicing
in
mRNAV
(1602) available
between protein
codon
1978). The
splices
IV, V and VI all eliminate
situated
downstream terminates
the first open
frame
III. If protein
synthesis
poly-
present
in
tracts that are
from stop codon
which
serves
for the Elb
DNA
of 20600
that in adenovirus
Elb
mRNAs
must exist in which protein initiated at AUG1907 available methionine
exclu-
we propose situation
can also be
by the deletion
of one of Ad5
of an adenovirusElb-specific
(iii) The synthesis in vitro of both a 19,210OO M, and 55-65000 M, polypeptide from Elb mRNA IV, the presence of the 55-65000 M, protein being dependent on the cell-free translation system used (Spector et al., 1980; Lupker et al., 1981; Esche et al., 1980). (iv) The absence of any apparent peptide relatedness between the Ad5 Elb-specific 19000 it4, and 65000 M, proteins (Van der Eb et al., 1980; Bos et al., 1981). When, as proposed, protein synthesis is indeed
which eliminates
by nucleotides
J4, protein,
product
the N-terminal
1907-2164)
II) of mRNA IV it and the RNA splice
the tract between nucleotides
this
alternative
3386
mRNA
should code for a polypeptide An alternative explanation protein
synthesis
at
C terminus
mRNA
IV has its 5’ terminus
the splice
protein
of 9000 M,. for the initiation
AUG1907
will part
of the 55000
but it will have a different
might
be
downstream
thus of that from
AUG1602. Our evidence indicates that this is not the case (Fig. 4C) but we are searching for other 5’ termini for this messenger. Downstream
from the tract
encoding
the Elb
polypeptides, the third stretch available for translation (AUG3480 to UAA3894) is entirely within the confines of the Elb mRNAs IV, V and VI, where apparently this potential information is silent, but also falls within the quasi-late mRNA VII. Between the said translation signals, mRNA VII can code for a protein of 14000 M,. In view of the sequence
homologies
at the nucleic
acid and
protein levels with the corresponding genes of Ad2 and Ad5 (Dijkema et al., 1981) which have been shown
to encode
serotypes, mRNA tion for Ad7.
55-65 000 M, polypeptide both in vitro and in vivo (Van der Eb et al., 1980; Tooze, 1981).
initiated at AUG1907 (frame would terminate at UAA3383,
with
V
base, as a result of which the organization Elb is identical to that of Ad7 and Ad12. (ii) The synthesis
(specified
the second
acids
beyond
which would be the second triplet if all Elb mRNAs
has been amended
86 amino
not only at AUG1602
point;
in
have their 5’ terminus at position 1577. Evidence supporting the idea that in adenovirusElb mRNAs protein synthesis can also be initiated from the second available AUG triplet is based on the following observations. (i) In Ad5 and Ad12 Elb DNA the same distribution of initiation and termination signals can be observed (Bos et al., 1981). It should be noted that recently (Bos et al., 1981) the Ad5 Elb DNA sequence
2164 and 3475. If in mRNA V
is initiated
also at AUG1907,
share
of
of the region
specified by two codons (leu-pro)
another
synthesis
nucleotides synthesis
deletion
stretch
is initiated
M,. However,
in the
at AUG1907
UAG2136
sively from the first available AUG1602, mRNAs IV, V and VI all would give rise to an identical polypeptide
but
results
initiated
sequence.
TAA3383
in the first open
IV protein
the coding
and TAA3894
III) and consequently
initiator
mRNA
fall outside
The
triplet
is situated
and 3475 would
in
and
TAA3315
III. The first AUG
in the Elb
open for pro-
and TAG2136
viral
polypeptide
IX of these
VII should have the same func-
(h) Comparison of Ad7 and Ad5 Elb polypeptides A comparison of the postulated structures of the 55000 i%4,and 19000 M, polypeptides of Ad7 and Ad5 is given in Fig. 7. The computer program AAFIT (Staden, 1978) which compares amino acid sequences, assigns each amino acid to one of four classes: basic (R, K, H), acidic (D, E),’ hydrophobic (V, A, L, I, P, F, Y, W, M) and polar (uncharged) (S, T, Q, N, C, G). Using the AAFIT program we have aligned the amino acid sequences as to give an optimum fit. Lines have been drawn above and below strictly identical amino acids and asterisks indicate the residues belonging to the same class as defined by the AAFIT program. As
_ ^
-
*
-
-
*
***
*
***
***
xRF?%~sssF ZNMEGSBDE
*
***
GGYLLDF~~~ H~A~RK
*********
*
**
PATWF-RPP
**
DNLRLLASAA
N~LL~LSS~
*** ***
*
***
***
QE~QQ~DNPR
GDNT~GAAA
SGSSRD-TET t **
AGtDPRE*-
AGLDPPIJEE***t* *
****
****
* *****
** XLVNIRNCCY -LRPDCKYKIS ---
-LRPDKQ?R'~T FKINIRNACY
*****if**+
A~CA~TE CGGMHV&SV
TA?~WJZGN * #I******
_
of the postulated
.
a
-
.
.
_
.I
-
by asterisks
Elh polypeptides
1978)amina acids are indicated
acid sequences
of the amino
with similar (Staden,
Positions
Fig. 7. Comparison
proteins.
400
I
,^ , . -. _ - -
and those with identical
of Ad7 (upper-sequence)
-
(A) the 21000
M, proteins:
I
_ .-
.a_
..,_
acids by lines above and below the sequences.
and Ad5 (lower sequence): amino
7 5
5
A~~IV~A~~~V I?;K?TMiiIzG HSDE~PY~AG~H~NI~ ***t* *** ** * l ** **if**** l * ******** ********** KTIHVASHSR KAWPVFENNI LTRCSLHLGN NCED_RASQML TCSDGNCHLr; AVIKHNMVCG __."w-.._w-__
-~SVKSNMXCG l ********
%%ACECGGK NA~FPPV~~???-~?%DLRPDHLVLACTGZZZZGZE?~ ~M~M~MN~~~~DAP~R~s~ FI~DMNIQL~Z?IZZ%?TKP $I********* **c* l **** ** ******* ******* ** ********* * t******* *** *** ** ********** ***** **** ** **+* RCRPCECGGK Hl~N~~L~ ~EELRPDHL VLACTRAEFG SSDEDLD RRGVFL~LS~TKIL~ESM~K~LN ~V~TMKIW -- KVLRYDK~RT __ ---
396
ASIKKCLFER --- CTLGILSEGN SWNVASD 30; -
200
--TCVEAWGQ% VRGCSFYACW IATSGiiVEQ ~sGNGAEVXIQT~KAZ~?! cZMGXZ~V ZZEAITLZII ~~-RZDGYNZI ~M~K~~X%Z%GFNN *** ** ******* ******* * **** x* ** ******** * ** ******* ***+*a**** *** * **** ** * **** ****a* ***** VFLANTNLIL HGVSFYGFNN TCVEAWTD_m VRGCAFYCCW ISGNGAEVEI RTEDRVAFRC SbJINmVL EDGWIt.J$V LFTGPNFSGT KGWCRPKSR --_-w-_ ---
195
100
************* *
LDFSTPGRTA AAVAFLTEL ******** * l ******** LDFSTPGRAA AAVAFLSSK
PTDHASGSXG ZAGGQSESR ~GPsG---GG * * * ** l t* ***** * AAGGSQAASA $#ZPMEPESR ~PSG~NWQ VAELYPELRR ILTITEDGQC -_-
-EAIIPTEEQ QQQQ~EARRR RQ~S~NPR
**
QFLGVAGI-L RHP~TMPA~
--w-wNRzImNP SGNNSRTE-L ALSLMSRRRP ET'JWWHEVQS EGRD!?VSI-iQEKYSLEQLKT CxE;E?%W: VAiiiNGIS ******* * ** * l t** *** **** * *********t *** * * ********* * t * ** ** LKGVKRERGA CEATEEARNI..AFSLMTRHRP ECITFQQXKD NCANgI.DL_LAQKYSIUTJJ YWWLQPGDDFE EAXRWAKVA _--*-
97
**
K45v?.B1?iA A?.Z&WKARR M~TI~~~Pv
)jERRzPSERG VPAG_FSGHAS ESGCET@S 1
**
??DPP%LQ@
1
B
101
*
****
~SESTHLS
*t*
~IRQ~F~
I.01
1 -W*AXX!Z~~I. RQTS~L~LENA SDGVSGLZZ? WFF;GDLXRE FRIXQ~%EE FEXLLDDIPG EEALNLGHQ AH%~%?LSV *** ****** t * **** **** **** * l *** * * * **** **** **** *** *** l ** ** MEAWEC~F SA~N~QS sNsT~WF~?~? L~$SSQ~KE,,~cRIKEDYKW~E~KSCGE ~D~LNLGHo AL~Q~IKT -1
A
(B) the 55Oaa M,
155
TABLE
1
Amino
acid
polypeptides
of transformed composition
of the 21000
M, and
55000
M,
addition (4.5-6%)
of Ad7 and Ad5
almost Proteins Amino
2looo
55000 M,
M,
Ad5
Ad1
Ad5
encodes
that
transformed
tumors
specified
Ad-l
functions
complete
to induce
acids
cells (Van der Eb et al., 1980). In
to region Ela, the first part of region Elb
in suitable
by the intact
give rise to an
phenotype animals.
early
Functions
region I (O-l 1.5%)
give rise to a transformed
phenotype cells.
similar
phe
10
11
18
19
leu
23
24
31
30
transformed
ile
5
8
21
27
genie
met
2
3
15
18
val
I
11
39
31
ser
16
5
31
38
(Jochemsen,
pro
6
10
22
18
oncogenic
result of the predicted differences of their Elbspecific 55000 M, polypeptides or whether, in
5
8
2-i
26
ala
14
16
36
33
tyr
2
3
10
9
his
4
4
13
15
gin
11
8
12
16
asn
5
3
21
25
lys
9
7
21
21
asp
7
12
22
22
glu
18
13
36
32
cys
3
27
21
trp
8
I
6
9
arg
14
14
37
31
gly
I
11
39
45
176
178
496
492
Total
can be seen, the Ad5 and Ad7 are tion of a variable 23-67 (Ad5) and equal lengths and
differences in the highly related not known.
with
the
55000 iL4, polypeptide
Whether
the
difference
in
of Ad5, Ad7 and Ad12 is the
are required,
re-
The authors are indebted to Dr. A. de Waard for the generous supply of T4 DNA ligase, and also express their gratitude for his stimulating interest
or to
region, or both, is
to Dr. A.J. van der Eb in this work.
This work was supported by the Netherlands Organization for the Advancement of Pure Research (ZWO) cal Research
through
the Foundation
in the Netherlands
of Chemi-
(SON).
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1979. ribosomes
A simple
(1979) 437-445.
and mes-
genes
types 5 and
do eucaryotic
in messenger
D.J., Halbert,
adenoviruses. Staden,
of wild-type
Nature
M.L.:
2398. Spector,
IX of human
region
mRNA.
site mapping.
Expression
polypeptide
adenovirus
sis, Univ. of Leiden, M.:
organization
J. Mol. Biol. 192 (1980) 399-417.
of human
m eukaryotic
H.O. and Birnstiel.
restriction
13 (1981) 375-385.
of the transforming
H.: Studies
products
H.. De Waard,
B.M. and Lewis, J.B.: Proteins
adenoviruses.
Jochemsen,
Gene
B.M.M., for
type 7. Gene
Esche, M., Mathews, senger
sequences
12 (1980b) 287-299.
R., Maat, J., Dekker,
adenovirus
Lupker,
DNA.
Van Ormondt.
region of weakly oncogenic
the Ela region.
Kozak,
type7
Smith,
R., Dekker,
Dijkema,
adenovirus
141-156.
are
region
of highly-
and non-
Nature
295 (1982) 705-707.
K.: Unique region
P. and
structures
species of mRNA
1 in cells transformed
fragment.
J. Virology
tran-
from by
36 (1980)
143
Press
Gene organization of the transforming region of adenovirus type 7 DNA (DNA
sequence;
RNA mapping;
Sl nuclease;
sequence
comparison;
cell transformation)
R. Dijkema, B.M.M. Dekker and H. van Omondt Department of Medical Biochemistv, (The Netherlands) (Received
December
(Revision
received
Sylvius Laboratories,
University of Leiden, Wassenaarseweg
72, 2333 AL Leiden
14th, 1981) and accepted
February
16th. 1982)
SUMMARY
The sequence been determined. be involved sequence
of the leftmost 11% of the weakly oncogenic human adenovirus type 7 (Ad7) DNA has This part of the Ad7 viral genome encompasses early region El which has been shown to
in the process
and determined
of cell transformation
coordinates
in vitro (Dijkema
of the El mRNAs,
et al., 1979). From
we are able to predict
polypeptides encoded by the transforming region of Ad7. The organization of the El region of Ad7 and of other adenovirus
serotypes
the primary
the nucleotide structure
of the
(Bos et al., 1981) leads to
the proposal of a novel mechanism for gene regulation at the translational level in which protein synthesis can initiate at either the first or the second AUG triplet available in mRNA. The differences between the large
Elb-specific
oncogenicity
tumor
antigens
of adenovirus
types
12, 7 and
5 may
explain
the
difference
in
of these viruses.
INTRODUCTION
the criteria
Human adenoviruses have been extensively studied because they can serve as a model for eukaryotic gene expression, and because they in-
types belonging to class A (such as Ad12) are highly oncogenic and induce tumors at high frequency following a short latency period; class B serotypes (e.g. Ad3 and Ad7) induce tumors at low frequency and only after an extended latency
duce tumors in certain
rodents
and transform
non-
This phenomenon
Abbreviations: cDNA,
adenovirus,
complementary
0378-I 119/82/0000-0000/$02.75
can be used as one of
Ad; bp, base pairs;
DNA;
T antigen,
sero-
Transformation by human adenoviruses is a process in which apparently only a small portion of the viral genome is involved; since the introduc-
N, nucleotide;
tumor antigen.
0 1982 Elsevier Biomedical
classification:
period; and serotypes of classes C (e.g. Ad2 and Ad5) to F do not induce tumors in experimental animals (Mackey et al., 1979a, b). However, adenoviruses of all classes can transform rodent cells in vitro (for a review, see Tooze, 1981).
permissive cells in tissue culture. Among the 36 recognised serotypes of human adenovirus, the degree of oncogenicity varies from one serotype to another.
for the following
Press
144
tion of the calcium to transform which
or Ad5 virions
rodent
or intact
sequences
(Tooze,
transformation
is basically
encompasses human Recently,
region
two subregions: Elb
(map position
own
promoter
at least viral
cycle
11% in the DNA sofar (Tooze,
that
1969). Early mRNA
of
was
of the virus
described
(Van
was extracted cycloheximide)
and
viral
from cells at 6 h in the pres-
and late mRNA been
was fractionated
on oligo-(dT)
was used in Sl nuclease
KB
Eb et al..
the cells had
with virus. The RNA
chromatography
der
(or 18 h post infection 24 h after
in
propagated
cellulose
inby
before
it
experiments.
1%) each of which has its
for the synthesis
of a family
of
partially overlapping RNAs (Tooze, 1981). The Ela region corresponds to the smallest DNA fragment able to convert a primary cell into an immortal ‘transformed’ cell line (Ad5 HpaI-E: O-4.3%; Ad7 BgZII-H: O-4.5%). However, cells transformed by region Ela spects from cells transformed
differ in several reby DNA fragments
which, in addition, contain information El b region. Cells transformed by region
from the Ela have
a different morphology, grow to lower saturation densities and have an atypical T antigen distribution pattern. This has led to the conclusion that the Elb region, possibly in conjunction with the Ela region, codes for functions that give rise to the characteristic, transformed transformed cells. We have determined
post infection
fected
and
been
ence of 25 pg/ml was harvested
to consist
Gomen)
the purification
have
of all
O-4.5%)
4.5-l
cells and
198 1).
Ela
position
(strain
DNA
and
El has been shown (map
Ad7
14%
of region El
in the lytic
studied
METHODS
(a) Viral DNA and isolation of cytoplasmic RNA
by Ad2
to the leftmost a function
the leftmost
adenoviruses
the left
1981). This indicates
early
AND
(Van der Eb et al.,
DNA contain
of the genome
MATERIALS
fragments
from
cells transformed
homologous
is expressed
DNA
to originate
of the viral genome
1980); moreover,
which
it has been possible
cells with specific
were all found
terminus
DNA
technique
properties
the nucleotide
of
virus-
sequence
(b) Enzymes Restriction endonucleases HinfI and T4 polynucleotide
HindIII, PstI, HpaII, kinase were isolated
as described earlier (Van Ormondt et al., 1978); endoR. Sin1 was a gift from Mr. M. Lupker. Endonucleases Bgf II, M601, MboII, HhaI, HaeIII, PuuII, XhoI and BumHI were purchased New England Biolabs (Beverly, MA). DNA merase I, Klenow’s fragment A of DNA merase and pancreatic DNase were obtained
from polypolyfrom
Boehringer (Mannheim, F.R.G.), bacterial alkaline phosphatase (BAPF) from Worthington (Freehold, NJ), and Sl nuclease
from Sigma (St. Louis, MO),
T4 DNA ligase was prepared from a lysogen of AT4lig as described previously (Murray et al., 1979).
of
the left-terminal 11% of Ad7 DNA. However, due to the spliced nature of Ad7 El mRNAs (Yoshida and Fujinaga, 1980; Dijkema et al., 1980b; 1981) it is not possible to predict protein sequences from this DNA sequence alone. To that end, we have studied these mRNAs at the nucleotide level using molecular cloning and a modification of the Sl nuclease technique. In the present paper, which is one in a series describing the organization of the El region of Ad7 (Dijkema et al., 1980a, b; 1981) we report the nucleotide sequence of the Ad7 Elb region, and of the mRNAs specified by it. Our results enable us to predict the amino acid sequences of the Ad7 polypeptides involved in cell transformation. A comparison will be drawn between the Elb proteins of adenoviruses of different oncogenicity.
(c) Chemicals [ a-32P]dNTPs were obtained from New England Nuclear (Boston, MA), [ Y-~~P]ATP from Radiochemical Centre (Amersham, UK). Unlabeled dNTPs were purchased from Sigma (St. Louis, MO). The source of the chemicals used for the chemical degradation technique and gel electrophoresis systems have been described (Van Ormondt 1979).
et al., 1978; Maat
and
Van Ormondt,
(d) Sequencing procedures
the
Sequence analysis was performed protocol described by Maxam
according to and Gilbert
145
(1977).
Single-end-labeled
restriction
that served as substrate tion technique
were prepared
with [ Y-~*P]ATP
5’ ends kinase, with
in the chemical
or of recessed Klenow’s
both
cases
A of DNA [a-32P]dNTP
followed
quences
directly
DNA was used for hybridization,
DNA.
adjoining
where in
Nucleotide
se-
enzyme
sites
restriction
These
conditions
scribed in a previous
synthesis
polymerase endonuclease
fragments.
30 min) at 37°C when nickand at
37 and 42°C when the probe was terminally
of
of choice,
by restriction
of the labeled
units/ml,
translated
either by labeling
3’ ends by repair
cleavage
(200 -400
degrada-
and T4 polynucleotide
fragment
and a complementary
fragments
a much
the thermal
termini
of the hybrids, Later
those was
recom-
“breathing”
and thus batches
de-
et al., 1980b)
temperature
to reduce label.
from
paper (Dijkema
lower
mended terminal
differ
labeled
at the
the loss of the
of Sl nuclease
abled us to use higher Sl incubation
en-
temperatures;
homochromatography
(Tu et al., 1976) on thin-
apparently, they did not contain contaminating nucleases. Alkaline agarose gels were as described
layer plates
cellulose
by McDonell
were occasionally
determined
of DEAE
Cell 300 DEAE/HR-2/15). ling of the sequence data programs
described
by two-dimensional (Macherey-Nagel
et al. (1977).
For computer handwe made use of the
by Staden
(1977; 1978). RESULTS
(e) Sl mapping of cytoplasmic RNAs (a) Sequence analysis Poly(A)-containing bridized
overnight
RNA at 52°C
(2-10
pg)
was
to 32P-labeled
hyOur choice
DNA
quencing
fragment (50 ng genome equivalent) in 10 ~1 of 80% formamide (Casey and Davidson, 1977). The RNA-DNA hybrids were treated with Sl nuclease
2col
plzB
5’
~
GU5
-
--M6Z9
map
ob-
U3F4
YlZ3
Ml F2
23 F2
Z5M5
M5Z6
used in se-
tained by the method of Smith and Birnstiel(1976; result not shown). We have previously reported the
R2Z3 _ A5K
F2Y3
enzymes
on a preliminary
2500
R3Z5 BlF2
of restriction
was based
-
LF2
ACY 81
R122
F3 61
Z7A2
H522
~
-Z2Hl
m
FL 22
Y2D - 06Yl
H3M3
pzz2-
BL F3
ACZ3
Ml z5
A6 HI
ZZAB -
ZLYl
-- 63F4
3’
Fl H4
M3 FL
--M7F6
M4D
M685 m
3’
kyF3
Z5R3
w
M5Z5
ZCIFZ F7MF
BSZB MB8
Y3Ml
B
AC Bl MSBl
LHqa
Z5Ml
Z3F3
Z784 ----
Hl z2 a4 z2
F3Z2
HSAl
PlZ2
F3 04
R2 &la BlYl
B
ABZ2 A2F3
Acy 23
PI F6
Zll
H3
YIZL
H5M3 Z2F4
ZLyz
63H3 FL H3
Ml FL
u M3 H3
g7t.J M7Y2
5’ DY2
I
r Fig. 1. Schematic representation of the sequenced tracts of DNA in the I- and r-strands of the region between the BgHI site at position 1564 and the HphI site at 3388. In the fragment names, the first letter denotes the endonuclease used for the primary splitting of this region, the second the endonuclease used for the secondary restriction cut to separate the two end-labeled segments of the primary restriction fragment. The following abbreviations were used: A-AluI; G-BglII;
H-H/WI; Hga-HguI;
K-PstI;
L-EalI;
M-MboI; P-HphI;
AC-Accl; R-EcoRII;
Acy-Acy
I; B-MboII; C-FnuDII; D-PouII; F-HinfI;
U-AsuI; Y-HpoII;
Z-HneIII. At the bottom of the
picture we have indicated with solid lines those parts of both strands (I and r) which have been sequenced.
146
of Ad7 region Ela (nucleotides
kema
I- 1564, Dijkema
et al., 1980a) and of the gene of
ported
in the latter
Ad7 polypeptide
IX (nucleotides
tracted
by one because
primary
structures
3350-4010,
Dij-
et al.,
198 1). The nucleotide publication
numbers
re-
have to be sub-
in the meantime
we have
147
I AT s 0 R” R s p I. s Y I( K C’H P E R c N L G I I. N E 0 E GATTGCMCATCAGGTAGGGTGAAGAGTCAGTTGTC~TGAAGA~~CATGTTT~AGAGATGTAATCTTGGCATACTGAATGAAGGTGAAGCAAGGGTCCGCCACTGCGCAGCTACAGA 2778 278% 2790 2800 2810 2820 2830 284% 2858
AR”
R
H
2860
C
A
ATE
2870
2980
TACPILIKGNASYS”N~lCGHSDERPYO*LTCAGGXCNIL
AACniC~~~MATSTTCTAIITAAAGGGAAATGCCAG~~~~AT~~~~~ATC~TGGACATTCGGATGAGAGG~~~~TCAGATGCTAACCTG~ 298%
2910
2948
2950
2970
2990
3000
3110
312%
3230
3240
RVRACECGGRHARPPPVCYDYTEDLRPDHLVLACTGAEFG AAGGGTGCOCGCATGCG4ATGCGGAGGC~GCATGCTAGAT~CAGC~GGTGTGCGTGGAT~G~CTGAAGACCTGAGGCCCGA~ATT~GTGCTTGCC~CACTGGhGCGGAGT~GG 3258 3260 3278 328% 323% 3300 3318 3320 3330 3340 3358
3368
AT”“IVSHARKRWPYPEHNV*T~~T~S~GG~~G~~~~TGC TGCTACCOTGCATATCGTT~ACACACGCAACI\M~CCCTGTATTTGAACATAATGTGA~~C~~GTG~~CCATG~AT~TAGGTGGTCGCAGGGG~ATGTTT*TG~C~~~~AGTG 3810 ,028 383% 3040 3058 3860 3870 3086 3098 31%%
N “NHtlK”M LE PDAFSRVSYTG I BDNN I TAICA-rrATCTGAAGGTAln;TTGGAA~CAGATGCCTTTTCCAGAGT~GCGTAACAGGAATCT~GATA~AATAT~AA~TA~~AAGATCC~AGATA~ATGA~AC~AAACC 313% 314% 3158 3160 3170 3188 3190 328%
PtWK 321%
I
tRY.DDT 3220
R
term 55x S
S
G
E
E
T
D
l
L &iee
1”
TTCTAGTGGTCAAGAAAC~A~GTIAGTAGTGGGGG~~~TGTGGATGGGGACTTTCAGGTTGGTAAGGT~ACAAATTGGGTAAATTTTGTTAA~~~~G~~~TG~~G~~C~n 3378 338% 3390 340% 341% 3426 3430 344% .
eexln
P
P
epzicelrv* ” 3450
3460
3470
3488
358%
3590
3680
3710
372%
*K
GSASPEGGYPSPYtlGRtPPWRGVRPNVMGSTYDGRPVP TGCAAGCGCTTC~~GAGGGGGGAGTATTTA~CC~ATCTUACGGGCAGGC~CCACCA~GGCAGGAGT~GTCAGAA~TCA~GGbTCCACTGT~ATGGGAGACCCGTCC 3498 3588 3518 3520 3538 3548 3559 3160 3570
PANSSTLTYATLSSSPLDAAAANTILGnCYYGS AGCCCGCCMTKCTCIUCGCTGAC~TA~CCAC~TGAGTTCGTCACCAT~GATGCAGCTGCAG~~CGCCGCTAC~CTGCCGCCMCACCATCCTTGGAATGGGCTATTACGGAA 3610 3628 3630 3648 3650 3660 3678 3698 368% 3700
AOLREQTESAVATAKSK’
r---poEg(il1 3 9182
%
3930
3940
m*,pix 3950
3960
TCGCGCGCGGTATGCCCTGGACCATCGGTTTCGATCATTGAGAACTCGGT 3978 398% 3998 4%0% 4010
Fig. 2. The nucleotide sequence of the Z-strand of the region El of Ad7. We have underlined “Goldberg-Hogness boxes” and the sequence AATAAA, and boxed postulated initiation and te~ation codons. Also shown are the polypeptides predicted by the DNA sequence (this paper; Dijkema et al., 198oa; 1981) and RNA mapping data (this paper; Dijkema et al., 1980b; 1981). The splice sites and the messengers in which they occur are indicated by arrowheads and Roman numerals.
deleted one base in the DNA sequence between positions 1564-3350. Fig. 1 is a schematic representation of the tracts sequenced to detertine the primary structure of that interjacent region, i.e. between the BgrII site at position 1564 and the HphI site at position 3388. As is evident from the bottom part of Fig. 1, we succeeded in sequencing both I and r strands virtuaIly completely. Fig. 2 shows the deduced sequence, in combination with the sequence of the adjacent segments determined previously, which together represent the entire Ad7 El region (nucleotides l-4010). We have given the sequence of the 1 strand, since this has the same polarity as the mRNAs transcribed from this region. We have indicated postulated initiation and
termination codons, “Goldberg-Hogness boxes” reported to occur some 30 nucleotides upstream from the nucleotides coding for the 5’ end of mRNAs, and the sequence AATAAA usually associated with the 3’ termini of mRNAs (Proudfoot and Brownlee, 1976). (For all these features see DISCUSSION.) Finally, in Fig. 3 we present the physical map of the region between positions 1564-3350 for a number of restriction endonuclease cleavage sites. (h) Mapping of Elb mRNAs by the Sl nuclease technique In a previous paper we reported that hybridization of the Ad7 BglII-E (positions 1564-3897, i.e.
148
2cm
2500
3Lm
3500
LOO0
boSe I-'""i
L
3
Y- Hpa II
ll
1
1
2
C’CGG
13
Z-Hae
III
3
815 5
U-Asu I
1
74
F-Hinf I
KIJ
12
3
7
L
2
11
1
2
1
1
1
3
I
1
3
6
L
11 2
&318l
GG’CC
6
G’GNCC
161
I
5
G’ANTC
T-Taq I
T’CGA
H-Hha I
1
5
I
I
C-FnuD II A-Alu I
7
M-Mb01
16
B-MboII
8
11~10 121
5
1
5
61
1
1 1
7
3
6
n6n
G CG’C
3x
2
i
3
2"
2
1
I
5
1
1
lslll
2
5
P-HphI
3
2.
C G’CG
1191
3
AG’CT
2
la
‘GATC
i 7
2
2
L
3
GAAGA
8x
GGTGA
ST
R-EcoR 1
‘CC+GG
Dde I
C’TNAG
zoo0 I
25x I
I
,
,
I,,
3an
, , I,
.,
Bx
, I
*,
,
,
Loo0 1
No sites for HindlI,HpaI.EcoRI.Bgl I.BclI.BstEII,ClaI,KpnI.PvuI,RsaI.SmaI.Xma~.SacI.SacII. SalI,MlaI.RruI,XbaI Fig. 3. Physical symbols
maps for a number
in the maps for MboII,
of restriction
Hph I and HgnI
endonuclease
cleavage
shovi the positions
virtually equivalent to region Elb) fragment to polyadenylated RNA, and subsequent nuclease Sl treatment of the RNA-DNA hybrids leads to a number of protected segments: annealing to early mRNA yielded Sl-resistant tracts of 1800 and 440 nucleotides (N), whereas protection with late mRNA resulted in two additional products of 600N and 160N (Dijkema et al., 1981). For a more precise mapping of the transcript segments which comprise the various Elb mRNAs, hybrids were made by annealing late mRNA to internally
sites (arrows)
of the cleavage
in the region between sites relative
positlons
to the recognition
1564-3350.
The
sites.
32P-labeled PuuII (positions 1564-3474) and HguI (positions 1564-2269) subfragments of BgfII-E. Protection of the PvuII subfragment yielded tracts of 1800 N, 600 N and 160 N (Fig. 4A, lane 2), and protection of the HguI subfragment tracts of 700 N and 600N (Fig. 4A, lane 1). Hybridization of the nick-translated BgfII-BumHI fragment (15641910) to early and late mRNA yielded an Sl -resistant product of 335 N in both cases (Fig. 4C) which locates the 5’ terminus of both early and late Elb RNA around position 1575.
B
1910
4
I!%&
3.c7L
2269
BarnHI
i 3897
t
V
1
HgaI
PVUII
mRNA: tsDON
/
l
H
*
P
3m.I
*
____-----
600N
_____..----
600N
___--*___----
__---.._ -----____
__--------____
--__
* ‘.
@ON
---___--__
---_____:
48ON
t%
480N
‘t
180 N
l
Fig.4.
Detection
electrophoresis BglII-E
and approximate gel; nuclease
mapping
Sl-resistant
(1564-3802)
after hybridization
indicating
cleavage
sites for Eg/II, PuuII,
mRNAs;
the broken
showing
the Sl-resistant
line carets portion
late (L) mRNA, next to a Hinfl
denote
of the Elb
mRNAs
tracts of the HgaI to poly(A)RNA BarnHI
of the Ad7 BglII-BarnHI digest of pBR322
and (bottom
sequences. fragment
for siring.
cells. (A) Autoradiograph
lane 1) and PeuII
from Ad7-infected
and HgoI,
the intervening
in Ad’l-infected
(1564-2269,
(1564-3474,
cells. (B) Physical
part) the approximate
(C) Autoradiograph (positions
1564-1910)
of alkaline
lane 2) subfragments
map of the Ad7 &III-E lengths (* 10 nucleotides)
of an alkaline generated
agarose
by protection
agarose of Ad7 fragment
of the EIb
electrophoresis
gel
with early (E) or
150
37O
42O
G
cloned
A
Py
c
the
cDNAs,
nuclease
(1977);
we made use of a modification Sl
instead
lated)
DNA,
template
labeled
and
fragments
SinI-HinfI
Sharp
(nick-trans-
we used 5’- or 3’-terminally
labeled
position
of Berk
(r strand)
3’-labeled 2221;
technique of internally
of
labeled
as probe (Fig. 5). A
fragment
(positions
by incorporation
2119.
of [(Y-~IP]TTP
2119) was hybridized
to late mRNA.
Sin I-Hin f1
nuclease
S 1-resistant
fragment
was then run next to a Maxam-Gilbert
sequence
ladder
The major
portion
at The
of the
of the same fragment
protected
product
(Fig. 5A).
is seen to run as a
band which comigrates with band C2165. This implies that C2164 is the last base in the DNA fragment that is complementary to mRNA, and encodes the donor residue in the RNA splice which joins the 600 N and 440 N transcripts menFig. 5. Sequence Gilbert.
pattern
1977) and
late RNA)
segment
tions 2119-2221.
SI-resistant
of 3’4abeled
labeled
(by protection
SinI-Hinfl
at Slnl
is shown,
end of the intron
fragment
The deduced
while in the complementary
and the donor
nucleotide
in the previous
paragraph
(mRNA
V in
Fig. 6; see DISCUSSION).
with (posi-
site). The temperatures
the lanes are those of the Sl treatment. (r-strand)
tioned
G, A. Py.
(lanes
nuclease
over
sequence
strand
the 5’ DISCUSSION
(2164) are indicated.
(a) Mapping of Ad7 Elb mRNAs To
determine
the
above-mentioned insofar
map
transcripts
coordinates at the nucleotide
as they were not determined
5po
lop0
t’
BamHI
’
of
c”
the level,
In this paper
by sequencing
‘500 Hlndrn
*v
*opo
‘1
‘t’
quence
T
Barn HI
BglI
we describe
for the BglII
t
390
the Ad7
site at 4.5% (position
3YJ
t
se-
1564)
LOP0
11’
Earn HI XhoI
Hlnd!J
DNA
Bgl II
FRAME ‘7 2 3 75% nn I
512
1155?\1219 AiAn’,
I
28 K proten
1062I <,,‘\.~ ~-12‘9
II x__---
_s_._-___
t 3 term,nus ptza2 rnRM
naunnn
12‘9 H
2LK
,.
63K
,.
,577 216‘ _____-------
P
_____------
TZI
Fig.
6. The
stop
frames.
codons
(TAA, TAG, TGA;
In the long termination
indicated
codonfree
--.-------______._
are represented
as solid black lines. In the bottom
paper;
and Fujinaga,
Yoshida
as tick marks) in the /-strand
stretches
the presumed
.^.3‘V
initiation
l&o
21 K f 55K
”
+
21K+
9K
”
~ 1LK
IpIg
1938 v114
of the Ad7 region triplets (ATG)
part the results of the Ad7 RNA mapping
1980) are summarized.
+
---------“‘-___________~~,~
H
reading
aao.nn 0 iL;fL
3386,‘. 1‘75
El arranged
are indicated.
data (Dijkema
+
pmtetns
according
to their
The coding
regions
et al., 19gOb; 1981; this
up to the HphI site at position 3388. Tracts surrounding this sequence have already been described in two previous publications (Dijkema et al., 1980a; 1981) so that the Ad7 (subgroup B) DNA sequence now determined stretches from the left terminus of the viral genome to nucleotide 4010. As will become evident this sequence encompasses the entire early region 1 (El). The primary aim of our sequencing effort is to predict which polypeptides are encoded by this transforming region, and to compare these with the proteins specified by the corresponding regions of subgroup A and C adenoviruses. However, a prediction of polyp~tides is not possible on the basis of the DNA sequence alone but also requires knowledge about the coordinates of the mRNAs specified by the investigated DNA region. Such information we obtained by the nuclease Sl technique of Berk and Sharp (1977), and by sequencing cloned cDNAs derived from Elb mRNAs. Initial mapping was performed by hybridizing Elb mRNA to the BglII-E fragment (positions 15643897) and to shorter subfragments thereof: BglIIPuuII (1564-3474) and Bg/II-HguI (1564-2269) and degrading the non-hybridized stretches of nucleic acid with nuclease Sl. The BglII-E fragment is protected by four transcripts of 1800N, 600 N, 440 N and 160 N, when late mRNA is used for hyb~~zation. In the case of the PvuII subfragment three Sl-resistant products of 1800 N, 600 N and 160 N were generated; consequently, the 440 N transcript observed by hybridization to the &$11-E fragment maps to the right of the PuuII site at position 3474. Hybridization of the HguI subfragment to late mRNA generates two Sl-resistant products of 700 N and 600 N. Of these, the 600 N tract is the same as the one seen in the previous two hybridization experiments, whereas the 700N tract represents the stretch of DNA that is protected by the 18OON transcript. This interpretation is based on the fact that the 1800 N and 600N transcripts have the same 5’ terminus (see below: 5’ terminus of Elb mRNAs). From the absence of the 160N tract we conclude that the 160 N transcript maps between the HguI and PuuII sites at positions 2269 and 3474. In a previous paper we reported that the 1800 N and 440N tracts are covalently linked, as shown by sequencing cloned cDNA and by running a
nuclease Sl-resistant RNA . DNA hybrid in a neutral agarose electrophoresis gel (Dijkema et al., 1981). In the autoradiograph of this gel we detected bands representing material of 2250 and 450 bp long (the latter is generated by the quasi-late mRNA for viral polypeptide IX which was found to coincide to a large extent with the 440N segment). Unfortunately, the same autoradiograph did not allow us to demonstrate the presence of hybrids 1050 and 1200 bp long, because of the high background in this portion of the gel. The latter two bands were expected to arise by hybridization of the DNA probe to mRNAs consisting of the 6OON+~ON and 6OON+ 160N+440N segments, respectively. This expectation is based on the fact that the similarly organized Elb regions of Ad2 and Ad12 (Tooze, 1981) both encode an mRNA species of the 600 N -t- 440 N type. Moreover, Yoshida and Fujinaga (1980) have recently demonstrated this mRNA as a late species in Ad7-infected cells. Finally, the coordinates of the 160 N segment were inferred from those of a minor Ad2 El b mRNA observed by L.T. Chow (personal co~unication). The map positions of the Elb mRNAs as discussed above are given in Fig. 4B. (b) The 5’ terminus of Elb mRNAs
The nuclease Sl experiment shown in Fig. 4C located the 5’ end of Elb mRNA around position 1575. By hybridization to late Elb mRNA of the HindHI-Hue111 fragment situated between positions 1384 and 1678 we obtained a major Sl-resistant tract of 100 -t 3 N (results not shown). The Hind111 site is well within the Ela region so that the RNA-protected portion of this fragment must be complementary to the 5’-terminal 100 nucleotides of Elb mRNAs and the 5’ cap template, of all El b mRNAs, has to be at Al577 -t 3. This is in excellent agreement with the presence of a “Goldberg-Hogness box”, TATATAA, at positions 1548-1554. (c) The 3’ terminus of Elb mRNAs
In a previous publication (Dijkema et al., 1981) we reported that Ad7 Elb mRNA IV and the quasi-late messenger for polypeptide IX (mRNA
152
VII) have a common 3’-poly(A) attachment site encoded by nucleotides 3938-3941. This informa-
various
tion
of Fig. 6.
was obtained
derived
by sequencing
from the said mRNAs.
cloned Nuclease
cDNA
adenylation
(Proudfoot
curs
times
three
mRNAs
in
associated and
the
with 3’-poly-
Brownlee, 3’-terminal
of the Ela
sequence)
mRNAs
are given
and in the bottom
part
Sl data
(not shown) suggest that Elb mRNAs V and VI share this 3’ terminus as well, in spite of the fact that the signal AAUAAA
coordinates
in Fig. 2 (DNA
(f) mRNA VII Messenger
RNA
VII is, in spite of its location
1976) oc-
in early region I, not an early mRNA,
region
synthesized
predominantly
late stages
of infection
and
independent
promoter
(Tooze,
of
IV-VII.
(d) Splice sites in Elb mRNAs IV, V and VI
to the vast majority mRNAs,
since it is
at the intermediate transcribed
from
an
1981). In contrast
of characterized
it is not spliced
and
eukaryotic
and, in all probability,
The initial RNA mapping studies indicate that the Ad7 EIb region specifies three differently spliced mRNAs with identical 5’ and 3’ termini
specifies
(Fig. 4B) and with a common 480 N long 3’ segment (the length of this segment is measured from the splice point to the BglII site at position 3897,
since it must contain
plate for mRNA VII. Curiously enough, the gene for Ad7 polypeptide IX, in contrast to the corre-
and thence
sponding
to the polyadenylation
site at position
viral polypeptide
IX. It should
be noted
that the common part of the introns of mRNAs IV, V, and VI is an important regulatory region the promoter
and cap tem-
gene of Ad5 (Van Ormondt
et al., 1980)
3938). The 5’ end of this 480N segment (acceptor residue) is the same in all three El b mRNAs
and Ad12 (Bos et al., 1981), does not feature a “Goldberg-Hogness box”. The coordinates of
(Dijkema
mRNA
hybrid cDNA
et al., 1981). By sequence
plasmid constructed derived from mRNA
analysis
of a
from pBR322 and IV it was established
that this acceptor nucleotide corresponds to C3475 in Ad7 DNA and is covalently linked to a donor nucleotide corresponding to A3386 (Dijkema et al., 1981). In the present paper (Fig. 5) we designate position 2164 as specifying the 3’-terminal residue of the 600N transcript in mRNAs V and VI; this point was determined by the nuclease Sl technique (Fig. 5). The location of the 160N tract
VII are given in Figs. 2 and 6.
(g) Coding capacity To interpret the nucleotide sequence in terms of its coding capacity we have arranged the nonsense codons present in the Ad7 El region according to the three possible reading frames (Fig. 6). The El a region contains two stretches open for protein synthesis which are separated by an AT-rich region containing nonsense codons in all three read-
was inferred by analogy from the position of the 180N segment observed in a minor Ad2 Elb
ing frames. We have shown (Dijkema
mRNA species (L.T. Chow, personal communication) and of a similar tract in Ad12 El b ‘mRNA
the three Ad7 Ela
(Virtanen et al., 1982). The various splice points have been indicated in the DNA sequence of Fig. 2, and in the mRNAs in the bottom part of Fig. 6. (e) Ela mRNAs I, II and III In a recent paper (Dijkema et al., 1980b) have described the organization of Ad7 early gion Ela. It was found to encode three 5’3’-coterminal mRNAs differing in the amount internal sequences removed by RNA splicing.
we reand of The
mRNAs
et al., 1980b) that in (Fig.6;
mRNAs
I, II
and III) this intervening sequence is removed by RNA splicing. As a result, mRNAs I and II give rise to completely overlapping polypeptides (28000 M, and 24000 M,) initiating at AUG 576 and terminating at UGA1452, their only difference being 3 1 internal amino acids absent in the protein specified by mRNA II. The polypeptide encoded by mRNA III shares the N-terminal part but is translated in a different reading frame beyond the splice point; consequently, it terminates at an earlier stop codon (UAA 1348) and has an M,-value of 6300. In the region of the EIb mRNAs (1577-3938)
153
Ad7 DNA
contains
tein synthesis frame
III,
three stretches
between
TGA1482
between
TAAl904
frame II, and between frame DNA
mRNAs
stretch
(frame
as a probable peptides
(Kozak,
mRNAs
in
would have an &&-value of 55000. The splicing
in
mRNAV
(1602) available
between protein
codon
1978). The
splices
IV, V and VI all eliminate
situated
downstream terminates
the first open
frame
III. If protein
synthesis
poly-
present
in
tracts that are
from stop codon
which
serves
for the Elb
DNA
of 20600
that in adenovirus
Elb
mRNAs
must exist in which protein initiated at AUG1907 available methionine
exclu-
we propose situation
can also be
by the deletion
of one of Ad5
of an adenovirusElb-specific
(iii) The synthesis in vitro of both a 19,210OO M, and 55-65000 M, polypeptide from Elb mRNA IV, the presence of the 55-65000 M, protein being dependent on the cell-free translation system used (Spector et al., 1980; Lupker et al., 1981; Esche et al., 1980). (iv) The absence of any apparent peptide relatedness between the Ad5 Elb-specific 19000 it4, and 65000 M, proteins (Van der Eb et al., 1980; Bos et al., 1981). When, as proposed, protein synthesis is indeed
which eliminates
by nucleotides
J4, protein,
product
the N-terminal
1907-2164)
II) of mRNA IV it and the RNA splice
the tract between nucleotides
this
alternative
3386
mRNA
should code for a polypeptide An alternative explanation protein
synthesis
at
C terminus
mRNA
IV has its 5’ terminus
the splice
protein
of 9000 M,. for the initiation
AUG1907
will part
of the 55000
but it will have a different
might
be
downstream
thus of that from
AUG1602. Our evidence indicates that this is not the case (Fig. 4C) but we are searching for other 5’ termini for this messenger. Downstream
from the tract
encoding
the Elb
polypeptides, the third stretch available for translation (AUG3480 to UAA3894) is entirely within the confines of the Elb mRNAs IV, V and VI, where apparently this potential information is silent, but also falls within the quasi-late mRNA VII. Between the said translation signals, mRNA VII can code for a protein of 14000 M,. In view of the sequence
homologies
at the nucleic
acid and
protein levels with the corresponding genes of Ad2 and Ad5 (Dijkema et al., 1981) which have been shown
to encode
serotypes, mRNA tion for Ad7.
55-65 000 M, polypeptide both in vitro and in vivo (Van der Eb et al., 1980; Tooze, 1981).
initiated at AUG1907 (frame would terminate at UAA3383,
with
V
base, as a result of which the organization Elb is identical to that of Ad7 and Ad12. (ii) The synthesis
(specified
the second
acids
beyond
which would be the second triplet if all Elb mRNAs
has been amended
86 amino
not only at AUG1602
point;
in
have their 5’ terminus at position 1577. Evidence supporting the idea that in adenovirusElb mRNAs protein synthesis can also be initiated from the second available AUG triplet is based on the following observations. (i) In Ad5 and Ad12 Elb DNA the same distribution of initiation and termination signals can be observed (Bos et al., 1981). It should be noted that recently (Bos et al., 1981) the Ad5 Elb DNA sequence
2164 and 3475. If in mRNA V
is initiated
also at AUG1907,
share
of
of the region
specified by two codons (leu-pro)
another
synthesis
nucleotides synthesis
deletion
stretch
is initiated
M,. However,
in the
at AUG1907
UAG2136
sively from the first available AUG1602, mRNAs IV, V and VI all would give rise to an identical polypeptide
but
results
initiated
sequence.
TAA3383
in the first open
IV protein
the coding
and TAA3894
III) and consequently
initiator
mRNA
fall outside
The
triplet
is situated
and 3475 would
in
and
TAA3315
III. The first AUG
in the Elb
open for pro-
and TAG2136
viral
polypeptide
IX of these
VII should have the same func-
(h) Comparison of Ad7 and Ad5 Elb polypeptides A comparison of the postulated structures of the 55000 i%4,and 19000 M, polypeptides of Ad7 and Ad5 is given in Fig. 7. The computer program AAFIT (Staden, 1978) which compares amino acid sequences, assigns each amino acid to one of four classes: basic (R, K, H), acidic (D, E),’ hydrophobic (V, A, L, I, P, F, Y, W, M) and polar (uncharged) (S, T, Q, N, C, G). Using the AAFIT program we have aligned the amino acid sequences as to give an optimum fit. Lines have been drawn above and below strictly identical amino acids and asterisks indicate the residues belonging to the same class as defined by the AAFIT program. As
_ ^
-
*
-
-
*
***
*
***
***
xRF?%~sssF ZNMEGSBDE
*
***
GGYLLDF~~~ H~A~RK
*********
*
**
PATWF-RPP
**
DNLRLLASAA
N~LL~LSS~
*** ***
*
***
***
QE~QQ~DNPR
GDNT~GAAA
SGSSRD-TET t **
AGtDPRE*-
AGLDPPIJEE***t* *
****
****
* *****
** XLVNIRNCCY -LRPDCKYKIS ---
-LRPDKQ?R'~T FKINIRNACY
*****if**+
A~CA~TE CGGMHV&SV
TA?~WJZGN * #I******
_
of the postulated
.
a
-
.
.
_
.I
-
by asterisks
Elh polypeptides
1978)amina acids are indicated
acid sequences
of the amino
with similar (Staden,
Positions
Fig. 7. Comparison
proteins.
400
I
,^ , . -. _ - -
and those with identical
of Ad7 (upper-sequence)
-
(A) the 21000
M, proteins:
I
_ .-
.a_
..,_
acids by lines above and below the sequences.
and Ad5 (lower sequence): amino
7 5
5
A~~IV~A~~~V I?;K?TMiiIzG HSDE~PY~AG~H~NI~ ***t* *** ** * l ** **if**** l * ******** ********** KTIHVASHSR KAWPVFENNI LTRCSLHLGN NCED_RASQML TCSDGNCHLr; AVIKHNMVCG __."w-.._w-__
-~SVKSNMXCG l ********
%%ACECGGK NA~FPPV~~???-~?%DLRPDHLVLACTGZZZZGZE?~ ~M~M~MN~~~~DAP~R~s~ FI~DMNIQL~Z?IZZ%?TKP $I********* **c* l **** ** ******* ******* ** ********* * t******* *** *** ** ********** ***** **** ** **+* RCRPCECGGK Hl~N~~L~ ~EELRPDHL VLACTRAEFG SSDEDLD RRGVFL~LS~TKIL~ESM~K~LN ~V~TMKIW -- KVLRYDK~RT __ ---
396
ASIKKCLFER --- CTLGILSEGN SWNVASD 30; -
200
--TCVEAWGQ% VRGCSFYACW IATSGiiVEQ ~sGNGAEVXIQT~KAZ~?! cZMGXZ~V ZZEAITLZII ~~-RZDGYNZI ~M~K~~X%Z%GFNN *** ** ******* ******* * **** x* ** ******** * ** ******* ***+*a**** *** * **** ** * **** ****a* ***** VFLANTNLIL HGVSFYGFNN TCVEAWTD_m VRGCAFYCCW ISGNGAEVEI RTEDRVAFRC SbJINmVL EDGWIt.J$V LFTGPNFSGT KGWCRPKSR --_-w-_ ---
195
100
************* *
LDFSTPGRTA AAVAFLTEL ******** * l ******** LDFSTPGRAA AAVAFLSSK
PTDHASGSXG ZAGGQSESR ~GPsG---GG * * * ** l t* ***** * AAGGSQAASA $#ZPMEPESR ~PSG~NWQ VAELYPELRR ILTITEDGQC -_-
-EAIIPTEEQ QQQQ~EARRR RQ~S~NPR
**
QFLGVAGI-L RHP~TMPA~
--w-wNRzImNP SGNNSRTE-L ALSLMSRRRP ET'JWWHEVQS EGRD!?VSI-iQEKYSLEQLKT CxE;E?%W: VAiiiNGIS ******* * ** * l t** *** **** * *********t *** * * ********* * t * ** ** LKGVKRERGA CEATEEARNI..AFSLMTRHRP ECITFQQXKD NCANgI.DL_LAQKYSIUTJJ YWWLQPGDDFE EAXRWAKVA _--*-
97
**
K45v?.B1?iA A?.Z&WKARR M~TI~~~Pv
)jERRzPSERG VPAG_FSGHAS ESGCET@S 1
**
??DPP%LQ@
1
B
101
*
****
~SESTHLS
*t*
~IRQ~F~
I.01
1 -W*AXX!Z~~I. RQTS~L~LENA SDGVSGLZZ? WFF;GDLXRE FRIXQ~%EE FEXLLDDIPG EEALNLGHQ AH%~%?LSV *** ****** t * **** **** **** * l *** * * * **** **** **** *** *** l ** ** MEAWEC~F SA~N~QS sNsT~WF~?~? L~$SSQ~KE,,~cRIKEDYKW~E~KSCGE ~D~LNLGHo AL~Q~IKT -1
A
(B) the 55Oaa M,
155
TABLE
1
Amino
acid
polypeptides
of transformed composition
of the 21000
M, and
55000
M,
addition (4.5-6%)
of Ad7 and Ad5
almost Proteins Amino
2looo
55000 M,
M,
Ad5
Ad1
Ad5
encodes
that
transformed
tumors
specified
Ad-l
functions
complete
to induce
acids
cells (Van der Eb et al., 1980). In
to region Ela, the first part of region Elb
in suitable
by the intact
give rise to an
phenotype animals.
early
Functions
region I (O-l 1.5%)
give rise to a transformed
phenotype cells.
similar
phe
10
11
18
19
leu
23
24
31
30
transformed
ile
5
8
21
27
genie
met
2
3
15
18
val
I
11
39
31
ser
16
5
31
38
(Jochemsen,
pro
6
10
22
18
oncogenic
result of the predicted differences of their Elbspecific 55000 M, polypeptides or whether, in
5
8
2-i
26
ala
14
16
36
33
tyr
2
3
10
9
his
4
4
13
15
gin
11
8
12
16
asn
5
3
21
25
lys
9
7
21
21
asp
7
12
22
22
glu
18
13
36
32
cys
3
27
21
trp
8
I
6
9
arg
14
14
37
31
gly
I
11
39
45
176
178
496
492
Total
can be seen, the Ad5 and Ad7 are tion of a variable 23-67 (Ad5) and equal lengths and
differences in the highly related not known.
with
the
55000 iL4, polypeptide
Whether
the
difference
in
of Ad5, Ad7 and Ad12 is the
are required,
re-
The authors are indebted to Dr. A. de Waard for the generous supply of T4 DNA ligase, and also express their gratitude for his stimulating interest
or to
region, or both, is
to Dr. A.J. van der Eb in this work.
This work was supported by the Netherlands Organization for the Advancement of Pure Research (ZWO) cal Research
through
the Foundation
in the Netherlands
of Chemi-
(SON).
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