The nucleotide sequence of the early region of the Tupaia adenovirus DNA corresponding to the oncogenic region E1b of human adenovirus 7

73

Gene, 34 (1985) 73-80 Elsevier GENE 1216

The nucleotide sequence of the early region of the Tupaia adenoviros DNA corresponding to the oncogenic region Elh of human adenovirus 7 (R~ombin~t homologies)

DNA;

cloning vector;

tree shrew; ~~sfo~g

region; restriction

maps;

amino acid

Rolf M. Flligel, Helmut Baanert, Sbdor Suhai* and Gholamreza Darai** Institutefor Virus Research, *Institutefor Documentation, Information and Statistics, German Cancer Research Center, Im Neuenheimer Feld 280,69&J Heidelberg, Tel. (6221)/48461 I, and **Institutefor Medical Virology, Universityof Heidelberg, Im Neuenheimer Feld 324, 6900 Heidelberg (F.R.G.) Tel. (6221~~~63008 (Received October 23rd, 1984) (Revision received November 23rd, 1984) (Accepted December 5th, 1984)

SUMMARY

The nucleotide sequence of the early region Elb of the tree shrew (Tupaia) adenovirus (TAV) DNA has been determined. The sequenced region includes the genes for ~lypeptides of&f, 15000,440OO and 13 400, which are analogous to the small and large Elb proteins and protein IX, respectively, of the three human adenovirus serotypes $7, and 12. The hex~ucleotide consensus signal AATAAA occurs only at the 3’ terminus of the gene for protein IX suggesting that the El region of TAV encompasses one transcription unit. The amino acid sequences of the TAV polypeptides have a higher degree of homology to those of Ad7 and Ad5 than to those of Ad12.

INTRODUCTION

There have been many studies on Elb gene functions of human adenoviruses, since the genes that are located at the left-hand end of all human adenoviral genomes are involved in cell transformation. Based on studies with defined mutants of the human serotypes and on transfection experiments which map in the Elb region, a critical role for the tumor antigens of 19 kDal and of 53 kDal has been postulated to be required in the process of cell transformation and tumorigenicity (Jochemsen et al., Abbreviations: Ad, adenovirus; Ap, ampicillin; bp, base pairs; El, early region 1; ORF, open reading frame; TAV, Tupaia adenovirus; Tc, tetracycline. 0378-I 119/85/%03.30 0 1985 Elsevier Science Publishers

1982; Tooze, 1980). Conclusive evidence has been reported that the small or 19-20-kDal proteins of the Elb region are required for the major transforming activity, whereas the 53-kDal protein seems to be necessary for oncogenic tr~sfo~ation in tbe case of Ad12. The comparison of the deduced amino acid sequences of the Elb polypeptides between the human adenoviruses 5,7, and 12 revealed regions of strong homology (Van Ormondt and Hesper, 1981; Van Ormondt and Galibert, 1984). On the other hand, it was recently shown that the difference in oncogenicity between Ad5 and AdlZ-transformed cells in nude mice is specified by the lb region (Bernards et al., 1982; Van den Elsen, 1983). More knowledge of the Elb gene products is required. One approach

74

to analyze and identify the conserved domains in the Elb polypeptides essential for their physiological function(s) is to carry out comparative sequence studies of an adenovirus of a species phylogenetically separate from Homa ~~p~e~~. We recently reported on the primary structure of the Ela region of the TAV (Beckon et al., 1983a) and postulated a consensus sequence for polar encapsidation of adenoviral DNA. Here we extend the sequence determination to the complete Elb region of the TAV and compare the deduced ammo acid sequences of the TAV Elb polypeptides to those of the three human adenovirus serotypes.

(Brinckmann et al., 1983a). The end-labeled fragments were subjected to sequence analysis according to the method of Maxam and Gilbert (1977). [ cr-32P]dNTPs were purchased from Amersham. Sequence data were handled as described by Van Ormondt and Hesper (1983). A modified program for constructing the phylogenetic tree was used according to Fitch and Margoliash (1967).

RESULTS

(a) Determination of nucleotide sequence of TAV Elb MATERIALS AND METHODS

(a) Strains, media and vectors E&u&$& coii K-12 strain C600 (F, hi-, thr-I, Ied36,lacYl, tonA21, supE44, A-) (Ham&an, 1983) was used as recipient cell in all transformation experiments. Bacteria were grown in tryptose phosphate broth medium (Difco 0060-01-06). When needed, 100 pg Ap/ml and 20 pg Tc/ml were added to media or plates. HBV DNA fragment 2685-3188/O-88 bp (Will et al., 1982) cloned in the BarnHI site of pLc24 (KUpper et al., 1981) was used for constructing a vector with the AvaI recognition site (CCCGAG). The HBV plasmid was kindly provided by Heinz Schaller.

The primary structure of TAV region Ela from nucleotides 1-1215 was reported previously (Brinckmann et al., 1983a). To extend the recently determined sequence into the region Elb, the chemical method of Maxam and Gilbert (1977) was used following the strategy outlined in Fig. 1. More than 95% of the sequence was determined from both strands or at least twice when the same strand was used. The nu~leotide sequence shown in Fig. 2 confms the presence or absence of the restriction sites mapped here or as previously described (Beckon

2

Bamtit

I -

AvaI 3

Restriction enzymes, T4 ligase, T4 pol~ucleotide kinase, and the Klenow fragment of the E. edi DNA polymerase I were purchased from Boehringer Marmheim, BRL, Neu-Isenburg or Biolabs, Schwalbath. They were used according to specifications from the manufacturer. The sushi-fr~ent B of TAV was prepared from the recombinant DNA plasmid PTA-BB (Brinckmann et al., 1983b). The construction of the molecular clones PTA-BE-AD has been described previously (Brinckmann et al., 1983a). PnriIication and labeling of recessed 3’-ends of subfra~ents of the BarnHI fragment was pe~ormed as described

TagI

’ 4

Lw3A

1

A-L 2 Q

4

3

--* 3

I 3

7 4

86, f6,

Yl!._G.t-,--t

+ -

e-

4,

1

2

I

Hiifi

2

1

Hpa II

(b) Enzymes, clones and sequences

1

,

8

2

1

3

//7

2

3

3

,l

4

69jC ,, 1

/ 1

/

+_--+t~--. -_ -Ct---l-

-

It

+q--

-+-

c-.-....

I

1

2

3

kb

Fig. 1. Restriction maps of the Elb region of TAV DNA. The cleavage sites shown have been verified by cutting with the appropriate restriction enzyme and separating the resulting DNA fragment on either agarose (t?.8-1.5%) or polyac~lamide geis (7%). Sequences covered in individu~ reactions and gels are shown as arrows pointing from the 5’ to 3’.

--

Fig. 2. Nucleotide sequence of the fragment BumHI-B of TAV DNA. Initiation codons (b) and termination codons (v) are marked by symbols and are underlined. The stop codon at 1003 is postulated to be the terminator for TAV Ela polypeptides. The initiators at 1007, 1360 and 2609 define the start ofthe predicted 15-kDal, 44-kDa1 and IX Elb proteins ofTAV. The corresponding terminators are located at 1418, 2533 and 2975. The hexanucleotide signals AATAAA are underlined with dashed arrows. The TATA promoter for mRNA protein IX is boxed. The sequence starts at nucleotide 1001. For nucleotides l-1000 see Brinckmann et al. (1983a).

et al., 1983a; Matz et al., 1980) with one exception: The TuqI site at position 1455 (TCGATC) is resistant to cleavage under a variety of conditions, due to methylation of the A residue in the cloned DNA (Maniatis et al., 1982).

(b) Three ORFs

The nucleotides from 1007 to 1418 of the established sequence represent an ORF assuming that the first AUG is used as initiation codon. The deduced

amino acid sequence of a 15kDal TAV protein that corresponds to the 21-19-kDal Elb proteins of the human serotypes 5, 7, and 12 is shown in Fig. 3A. The overall homology of the 15-kDal TAV protein is about 60 % when compared to any one of the corresponding human Elb proteins and it is by 39 and 41 amino acids shorter than the Ad5 or Ad7 proteins (see Table I and Fig. 3). The amino acid composition of the 15-kDal polypeptide is similar to that of Ad5, but it lacks histidine residues. A second long ORF starts at ATG-1360 and runs up to the stop codon TAA-2533. The deduced amino acid sequence of this TAV protein, which is 391 amino acids long and corresponds to the 54-kDal E 1b of the human adenoviruses (Van Ormondt and Hesper, 1983) is shown in Fig. 3B. It reveals a high degree of homology of 63 y0 to the human adenovirus Elb proteins (see Fig. 5 and Table I). The TAV 44-kDal protein is about 100 amino acids shorter than the large Elb proteins of the three human serotypes. The NH,-tern&al part of the TAV 44-kDal protein differs from the human Ad sequence, whereas the central part has a relatively high degree of homology (Fig. 3B). It is noteworthy that there is a small gap of 76 bp between the termination codons of the 44-kDal polypeptide and the start codon for polypeptide IX at nucleotide 2609. The stop codon of the 44-kDal TAV protein falls within a consensus splice donor site. In contrast, in human Ad Elb region the corresponding splice donor sites are a few bp downstream from the Elb terminators (Van Ormondt and Galibert, 1983; Anderson et al., 1984). The size of the gap is quite similar to the distances reported for the

Fig. 3. Amino acid sequence of the TAV (DNA) Elb polypeptides 15 kDa1 (A), 44 kDal (B) and IX (C), as deduced from the nucleotide sequence of TAV DNA (Fig. 2).

corresponding intergenic regions of Ad5, 7, and 12 which are 103,97, and 81 bp long. There is a TATA box for protein IX at nucleotides 2567-2574, The stop codon of TAV protein IX is located at nucleotide 2975 generating a polypeptide IX of 122 amino acid residues (Fig. 3C), Again, this TAV polypeptide is shorter than its human analogs. The 3’ terminus of the Elb transcription unit is characterized by the occurrence of the consensus signal sequence AATAAA at 2976. The only other poly(A)-addition signal within the first 3210 bp of the 1 strand of TAV DNA occurs further downstream at nucleotide 3 117 (Fig. 2). It is interesting to note that the corresponding hexanucleotide MTAM for the transcripts of protein IVa2 and region E2b that is located on the r strand of the Ad genomes falls within the reading frame of the TAV protein IX at twelve nucleotides before the stop codon (Fig. 4). Another poly(A)-addition signal on the I strand precedes the putative stop codon of the polypeptide IVa2 by 12 bp and is therefore an unlikely candidate for the corresponding transcriptional signal. The carboxy-ter-

TABLE I Degree of amino acid homologies between Elb polypeptides of TAV and human Ad 12, 7, and 5. Protein homologies to TAV Virus

12 I 5

15 kDa1 Elba

44 kDa1 Elba

Polypeptide IXB

Amino acids Identical (%)

Similar (%)

Amino acids Identical (% )

Similar (% )

Amino acids Identical (%)

Similar (%)

33 (24.1) 30 (21.9) 33 (24.1)

83 (60.6) 81 (59.1) 84 (61.3)

114 (29.2) 119 (30.4) 117 (29.9)

243 (62.2) 250 (63.9) 246 (62.9)

34 (27.9) 37 (30.3) 35 (28.7)

85 (69.7) 90 (73.8) 92 (75.4)

a Percentage values were obtained by using 137 and 391 as lengths for the small and large TAV EIb polypeptides and 122 for protein IX. (Length of corresponding sequences in Ad12: 163 and 483; Ad7: 178 and 493; Ad5: 176 and 497.)

71

DISCUSSION 14*

P&k

t-strand PI5

r-strand

--Tz= 4

6

10

8 I

1

2

3

mu. kb

Fig. 4. Arrangement ofthe early region El b of TAV genome. The bottom line gives the nucleotide position (in kb) and the map units (mu.) of TAV DNA. The three upper lines represent the three reading phases. The ORFs are indicated by solid bars, and the predicted M, of the encoded polypeptides (in kDa1). Vertical lines denote stop codons. The ORF of the COOH-terminal part of protein IVa2 is also included.

minal70 amino acids sequence of TAV polypeptide IVa2 were compared to the corresponding sequences of Ad5 and 7 (Van Beveren, 198 1; Engler and Van Bree, 1982) (not shown). The high degree of homology of 74-76x found is consistent with the interserotypic homology of the human Ad sequences. Likewise, the gene of IVa2 of bovine Ad7 also showed high homology to human Ad2 DNA (Hu et al., 1984). These data indicate that the carboxy end of the IVa2 gene has been highly conserved during the evolution of adenovimses. (c) Evolutionary relationships To determine the evolutionary relatedness of TAV to each of the human adenoviruses, the amino acid sequences of four proteins of Ad5, Ad7, Ad12 and TAV served to construct phylogenetic trees by two difIerent methods (Fitch and Margoliash, 1967; Dayhoff and Barker, 1978). For example, the evolutionary distances between the largest Elb proteins as judged by the number of accepted point mutations per 100 amino acid residues are approximately twice as small among the human Ad’s than between TAV and the human Ad’s Apparently, TAV has a distinct phylogenetic placement different from the three human Ad’s. TAV is phylogenetically more closely related to Ad7 and Ad5 than to Ad12. A similar result was obtained for the other three Ad proteins and when the method of Fitch and M~go~ash (1967) was used.

There are a number of diierences as well as common features between the El region of TAV when compared to that of the human Ad serotypes which deserve comment. Starting with the differences it is obvious that the intra- and intergenic distances are considerably shorter in TAV genome than in human Ad DNAs. The initiation codons of the three major TAV polypeptides start at nucleotide positions which are closer to the internal end of the inverted terminal repeats and are followed at relatively short distances by stop codons (see Fig. 2). The intergenic region between Ela and Elb of 150-171 bp of the human Ad serotypes is compressed to a single bp in the TAV El region (Fig. 2). This raises the interesting question whether the tr~s~~ption signals of region Ela in TAV DNA are used for the synthesis of Elb mRNAs. Obviously, S 1 mapping experiments should provide an answer whether it is meaningful to subdivide the early region of TAV or not. Common features of the four Ad’s are more obvious in the amino acid sequence of the predicted polypeptides. The largest TAV Ela polypeptide possesses a central domain with a long run of a conserved sequence characterized by equidistantly located cysteine residues (Beckon et al., 1983b). The small TAV Elb polypeptide of 15 kDal shares 30 amino acids with the corresponding proteins of all three human Ad’s, most of which are located around the aspartic acid residue 78 (LDF-“;SXGR where X is an undefined amino acid). This aspartic acid residue has been shown to be essential for transforming activity of the 19-kDal protein of Ad2, since its replacement by tyrosine leads to a loss of the transforming ability (Chinnadurai, 1983). In line with human Elb 20-kDal pol~eptides which lack any homology in the c~~xy-te~~~ part of their molecules, the TAV IS-kDal polypeptide has virtually no matches at its COOH-terminus and is even shorter by 26-41 amino acids. However, in view of its essential role in cell transformation (Jochemsen et al., 1982; Chirmadurai, 1983; Fukui, 1984) it is intriguing to fmd a relatively low homology even in its NH,-terminal part. Those parts of the El region with greatest variations between the three human serotypes and even between Ad5 and Ad2, namely the COOH-te~~ moiety of the 20-kDal Elb polypeptides and the NH*-te~~~ portion of the large

78 10

30

30

50

A0

MDFLKPFCC3YKVLKSVS~SSWSRF-FGPKITKLIYSTKVSNRWFLDSLP--RG *z 2 1 . 5s 3**23 l 5 l * ‘“7 s14*56 *

3

60

**2*

3

l

l

MEVWAILEDLRQTRLLLENASDGVSGLWRFWFGGDLARLVFRIKCYREEFEKLLDDIPG

YLDGLASU)FSKI-DCLV~QLDFTSAGRLTASLSLLVLWEEWSARSCVSFDILTELLCV 21r3 ’ $4 2 553 ***,I,** 1’151 * 552 * 2

13525

LFEALNL~HFKfKVLSVLDFSTPGRT~VAFLTFILDKWlRQTHFSKGWLDFtAA

~A~_______)(,,A~A~~~A--____-~---______~_______-----~PS~~V. 1

/*3*

**

3*1

t

2

ALWRTW((ARRMRTILDYWPMPLGVAGlLRHPPTMPAVLQEECX)EDFIPRAGLDPPVfE*

B

,

~~s~o____________VEGA5PAK-_____--_____~~~~~__~~~~~~~~~~____ 1C 20 30 10 50 80 *333**

**a

311

1 M)PPNSLQQGIRFGFHSSSFVE~GSMfDNLRLLASAASGSSRDIETP ,7

___1_______________________--___-____L__________________-_

61

~~SESAPGRSGGGVADLFPELR~LTRSTTS~RGIKREANPSGNNSRTELALSLM

17 121 78 178

-RRRLDQVTYSE1UU3FRSGSFSDFFMEKYNFAY~G~~l~IELDP f** 23s 8 ‘6 12 *33 3 l **3 3% 1

52*2***3

*

2

l **3*

l

SRRRPE~EVQSEGRDEV--SILQEKYSLEQLKTCULEPEDDWEVAIRNYAI(ISLRP TKf~lLS~~lQSNCYlf~iVlVGEffil~KVLTKSF~Vl7~AVSFT~FQ 3*2* l 5 * 33*** l l* DKQYRiYKKlNiR~CYlS~EVilDT~~F

S!i*

123

l *4 1 + 5 3’ *51 RCMYXIYPGWGMEAITLlrHIRF-

13’8

RRDSYN~VFTCASQVLFH~FFVGFTGTClTST~LNRGMFLACYRPlMFLMFDLT l l **** ** 3, 55 l 3* l l *33**53, 135,****3* **a

238

RGDGYNGIVFMNTKLILHGCSFFGFNNTCVEAWGG)VSVRGCSFYACwiATSGRVKSQLS

,

IS6

VKHCVFDKCVIAISTECDFEISSNLCTDSCCFLSAAGTGIFSYNSIVNPFTLQDSAEFSM

288

**4*5*24* Sli 3**2 5 121 *ifi **3 ‘31 VKKCMFERCNLGlLNEGEAHC~lETACFILlKGNASVKHNJICW---SDERPY(X(

256

VTCADAKVQLLHTIHIHSNPKLWPQFM-INVLLRAKLFVGRRRGGFHPHFCSLKYSLLTL

355

5**+314 251 *s** * 14 61 l l **5 1 5 51 *** LTCAGGHCNILATVnIVSHARKKWPVFEHNVITKCThMIGGRR~MPYQC~H

316

AKGSERKVNLSTCYPDGLKWKVLNRNPNRLFTRLCECDAS~T~IVLGEVGLPATADP

415

EPDAFSRVSVTGIFDM,lQLWKiLRYDDTKPRVRACEC~

376

TLDSVDCLEF-SSDEfW 513 XI **3**

175

‘“LACTGAEFGSSGffTD

3

C 1

31

4X3511

10

63

35

58*5*

30

2334

30

l

***a,

*3

x

2

2

24.1

3*3

*

l

l

l 35

2’1

383s

51

3

1*

QPVCVDVFEDCRPDH

40

50

MSSNDN-SGIVNTCFLTTRLPAWAGVRQDEVG~DVNGLP~IPSNSMOIA---------SR l *,333 3* c 1 6**,***,****“*2 s**3*2* “5 *,x” 3s

60

3

1 MSGSASFEGGVFSPYLTGRLPPWAGVRQNVMCSTVDGRPVQPANSSTLTYATLSSSPLDA

Sl

~TTDIU\TEPSTR~LI~LLRSVTELNESI~E----LQOKMTELEKRLKlMffK,ffIKLA

61

**1i3*11313* l 53 +5 3 33* 22 22x5 * * 532453*5 AI\AAAATAAANTILGM~YYGSIVANSSSSNEIPSTLAEDKLLVLLAQLEALTORLGELSKO

107 121

3

LAN--PL IENPHDGNF ,“I 512 3 ‘31 313 l VAQLREQTESAVATAK%*

5. Comparison of the 15-k&l (A), 44-kDal (B) and IX (C) TAV Elb proteins with the co~espon~ng proteins of human Ad7. Asterisks indicate identity of amino acids. Numbers stand for the following amino acid homologies: 1 = STPAG (hydroxyl/smali aliphatic),2 = NDEQ (acid/acid amide), 3 = 1+ 2 ~hydrophilic), 4 = HRK (basic), 5 = MILV (aliphatic), and 6 = FYW (aromatic). Each upper row (TAV); each lower row (Ad7).

Fig.

Elb 55-kDal protein are also found to have virtually no homology in TAV. The amino terminal part of the 44-kDal TAV polypeptide which again is shorter than its human counterparts, is read’in an overlapping ORF with the 15-kDal pol~eptide and is predicted to start from an internal AUG in virtually complete analogy to the

situation in the Elb region of all three human adenovirus serotypes (Bos et al., 1981). When the amino acid sequence of this TAV polypeptide is compared to any of the human 55-kDal polypeptides or to all of them (in a four-match comparison), a s~~s~~y high degree of homolo~ of 62-63.4% (Table I) is found. It is remarkable that only a few

79

gaps had to be introduced to maximize homology among the large polypeptides of different species. The matches found are located in the central domain of the polypeptide. Furthermore, the DNA sequence allows the prediction of a smaller, coterminal TAV Elb polypeptide analogous to the Elb 18-kDal protein of Ad2 shown to be derived from an alternatively spliced Elb mRNA by Anderson et al. (1984). The greatest differences between TAV El region and that of the three human serotypes is obviously the presence of an SO-171-bp intergenic region in human Ad’s in contrast to that of TAV. This raises the possibility that Ela-Elb co-transcripts are synthesized in the case of TAV. In support of this, it was recently reported that in vitro synthesized Ad2 mRNA contains Ela-Elb co-transcripts which in vitro direct the Ela-related polypeptides (Hashimoto et al., 1984). If TAV El region mRNA is also synthesized as one long transcript, the AUG initiator at 1007 might alternatively be used as an internal start signal for translation. Alternatively promoters for Elb transcript might coincide with the coding region for the E 1a proteins. Further experiments are necessary to decide between these alternative pathways. As to the relatedness of TAV to the three human Ad’s, it is of particular interest that, independent of the method or the viral proteins used, the phylogenetic arrangement indicates that Ad7 and Ad5 are more closely related to TAV than Ad12. In contrast, the sizes of the inverted terminal repeats and of the intergenic regions of the El region of Ad12 resemble more those of TAV than those of the other human serotypes.

REFERENCES Anderson, C.W., Schmitt, R.C., Smart, J.E. and Lewis, J.B.: Early region lb of adenovirus 2 encodes two coterminal proteins of495 and 155 amino acids. J. Virol. 50 (1984) 387-396. Bernards, R., Houweling, A., Schrier, P.I., Bos, J.L. and Van der Eb, A.J.: Characterization of cells transformed by Ad5/Ad12 hybrid early region 1 plasmids. Virology 120 (1982) 422-432. Bos, J.L., Polder, L.J., Bernards, R., Schrier, PI., Van den Elsen, P.J., Van der Eb, A.J. and Van Ormondt, H.: The 2.2 kb Elb mRNA of human Ad12 and Ad5 codes for two tumor antigens starting at different AUG triplets. Cell 27 (1981) 121-131.

Brinckmann, U., Darai, G. and Flilgel, R.M.: Tupaia (tree shrew) adenovirus DNA: sequence of the left-hand fragment corresponding to the transforming early region of human adenoviruses. EMBO J. 2 (1983a) 2185-2188. Brinckmann, U., Darai, G. and Fltlgel, R.M.: The nucleotide sequence of the inverted terminal repetition of the tree shrew adenovirus DNA. Gene 24 (1983b) 131-135. Chinnadurai, G.: Adenovirus 2 Ip+ locus codes for a 19 kd tumor antigen that plays an essential role in cell. transformation. Cell 33 (1983) 759-766. Dayhoff, M.O. and Barker, W.C.: Atlas of Protein Sequence and Structure, Vol. 5, National Biomedical Research Foundation, Washington DC, 1978. Engler, J.A. and Van Bree, M.P.: The nucleotide sequence of the gene encoding protein IVa2 in human adenovirus type 7. Gene 19 (1982) 71-80. Fitch, W.M. and Margoliash, E.: Construction of phylogenetic trees. Science 155 (1967) 279-284. Fukui, Y., Saito, I., Shiroki, K. and Shimojo, H.: Isolation of transformation-defective, replication-nondefective early region lb mutants of adenovirus 12. J. Virol. 49 (1984) 154-161. Hanahan, D.: Studies of transformation ofE. coli with plasmids. J. Mol. Biol. 166 (1983) 557-580. Hashimoto, S., Symington, J. and Matsuo, T.: Cell-free translation of adenovirus 2 Ela- and Elb-specific mRNAs and evidence that Ela-related polypeptides are produced from Ela-Elb overlapping mRNA. J. Biol. Chem. 259 (1984) 7016-7023. Hu, S.-L., Battles, J.K. and Potts, D.: Restriction analysis and homology studies ofthe bovine adenovirus 7 genome. J. Virol. 51 (1984) 880-883. Jochemsen, H., Daniels, G.S.G., Hertoghs, J.J.L., Schrier, PI., Van den Elsen, P.J. and Van der Eb, A.J.: Identification of adenovirus-type 12 gene products involved in transformation and oncogenesis. Virology 122 (1982) 15-28. Kiipper, H., Keller, W., Kerz, C., Forss, S., Schaller, H., Franze, R., Strohmaier, K., Marquardt, O., Zaslovsky, V.G. and Hofschneider, P.-H.: Cloning of cDNA of major antigen of foot-and-mouth disease virus and expression in E. coli. Nature 289 (1981) 555. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982, pp. 102-103. Matz, B., Delius, H., Fliigel, R.M. and Darai, G.: Physical map of Tupaia adenovirus DNA by cleavage with restriction endonucleases and partial denaturation. J. Gen. Virol. 51 (1980) 421-423. Maxam, A.M. and Gilbert, W.: A new method for sequencing DNA. Proc. Natl. Acad. Sci. USA 74 (1977) 560-564. Tooze, J.: Molecular Biology ofTumor Viruses (2nd ed.), Part 2, DNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1980, pp. 932-949. Van Beveren, C.P., Maat, J., Dekker, B.M.M. and Van Ormondt, H.: The nucleotide sequence of the gene for protein IVa2 and of the 5’ leader segment of the major late mRNAs of adenovirus type 5. Gene 16 (1981) 179-189. Van den Elsen, P.J., De Pater, S., Houweling, A., Van der Veer,

80

J. and Van der Eb, A.J.: The relationship between region Ela and E 1b of human adenoviruses in cell transformation. Gene 18 (1982) 175-185. Van den Elsen, P., Houweling. A. and Van der Eb, A.J.: Expression of region E 1b of human adenoviruses in the absence of region Ela is not sufficient for complete transfo~ation. Virology 128 (1983) 377-390. Van Ormondt, H. and Hesper, B.: Compa~son of the nudeotide sequences of early region Elb DNA of human adenovirus types 12, 7 and 5 (subgroups A, B and C). Gene 21 (1983) 211-226.

Van Ormondt, H. and Galibert, F.: Nucleotide sequences of adenovirus DNAs, in Doerfler, W. (Ed.), The Molecular Biology of Adenovirus 2. Springer-Verlag, Berlin, 1984, pp. 73-142. Will, H., Kuhn, C., Cattaneo, R. and Schaller, H.: Replication of hepatitis B virus, in Miwa, M., Nishimura, S., Rich, A., SBB, D.G. and Sugimura, T. (Eds.), Primary and Tertiary Structure of Nucleic Acids and Cancer Research. Japan Sci. Sot. Press, Tokyo, 1982, pp. 237-247. Communicated by H. van Ormondt.