Proc. Nati. Acad. Scd. USAVol. 77, No. 7, pp. 3778-3782, July 1980Biochemistry

Predicted structure of two adenovirus tumor antigens(transformation/splicing/DNA sequence)

MICHEL PERRICAUDET*, JEAN MICHEL LE MOULLEC*, AND ULF PETTERSSONtRecombinaison et Expression G6netique, Institut National de la Sant6 et de la Recherche Medicale U163, Unit6 de G6nie GCnetique, Institut Pasteur, 28 ruedu Docteur Roux, 75015 Paris, France; and tDepartment of Microbiology, University of Uppsala, The Biomedical Center, Box 581, S-751 23 Uppsala, SwedenCommunicated by Hugo Theorell, January 18,1980

ABSTAGCr Early adenovirus type 2 (Ad2)mRNA sequenceshave been cloned by using the pBR322 plasmid as a vector. Twoclones that include sequences from region E1B were identifiedand their DNAs were characterized by hybridization, restrictionenzyme cleavage, and DNA sequence analysis. The resultsshowed that the clones were derived from two different splicedmRNAs. By combining our results with the established DNAsequence for region EIB of the closely related adenovirus type5 [Maat, J., vanBeveren, C. P. & van Ormondt, H. (1980) Gene,in press] it was possible to deduce the structure of a 13S and a22S mRNA. The two mRNAs differ from each other by the sizeof their intervening sequences. If translation starts at the firstAUG following the cap, the 22S mRNA encodes a M, 67,000polypeptide that is terminated by a UGA stop codon locatedimmediately before the splice, whereas the 13S mRNA encodesa Mr 20,000 polypeptide that is translated in different readingframes before and after the splice. The Mr 20,000 and 67,000polypeptides correspond in molecular weight to two proteinsthat invariably are precipitated from infected cell extracts byantisera from animals carrying adenovirus-induced tumors.

Adenovirus type 2 (Ad2) and the closely related adenovirus type5 (AdS) have been extensively used as models to study eukar-yotic gene expression and cell transformation by viruses. Virusmultiplication in permissive cells can be divided in two phasesseparated by the onset of viral DNA replication. A large numberof early and late Ad2 mRNAs have been positioned on the viralgenome (1-3). Most species are spliced, and several overlappinggenes must exist to explain the large number of early poly-peptides that have been identified. Early mRNAs are expressedfrom four regions, designated E1-E4 (4,5). Only a small partof the viral genome is required for in vitro transformation ofrodent cells (6, 7), and the transforming region coincides withthe El region, located at the left-hand end of the linear ade-novirus genome (Fig. 1). Two promoters are present in the Elregion which subdivide it into regions ElA and E1B (8) eachof which is transcribed into several overlapping mRNAs. TheElA region is approximately equivalent to the smallest re-striction endonuclease fragment, Hpa I-E (map coordinates0-4.5), which is sufficient for transformation of cells in vitro(14). However, cells transformed by the Hpa I-E fragmentdiffer in certain aspects from cells transformed by largerfragments; i.e., they have an atypical tumor (T) antigen stainingpattern and grow to lower saturation densities. Consequently,it is thought that the region adjacent to the Hpa I-E fragmentalso plays a role in cell transformation. The complete sequenceof the left-hand 11% of AdS DNA has been established (11, 13,15). However, due to the spliced nature of the early mRNAs,it is not possible to predict protein sequences directly from the

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DNA sequence. A study of the mRNAs from the ElA and E1Bregions at the nucleotide level is obviously necessary to deducethe structure of the polypeptides that are responsible for celltransformation. Therefore we have used a molecular cloningprocedure to amplify the appropriate mRNA sequences. Clonesof mRNAs from the El region have been isolated and charac-terized by restriction enzyme mapping, hybridization, andsequence analysis, and clones corresponding to a 12S and a 13SmRNA from region ElA were described in a recent commu-nication (12). In the present study we have isolated two clonesthat allow the characterization of both a 13S and a 22S mRNAfrom region E1B. By combining our results with those of Maatet al. (11) it is possible to predict the amino acid sequence oftwo partially overlapping polypeptides that may play a key rolein transformation by adenoviruses.

MATERUILS AND METHODSCells and Viruses. Ad2 was propagated in HeLa cells (16),

and viral DNA was extracted from virions as described byPettersson and Sambrook (17). EarlymRNA was extracted fromcells that had been grown in the presence of cycloheximide at25 gg/ml as described by Persson et al. (16). The RNA wasfractionated by chromatography on oligo(dT)-cellulose beforeit was used in reverse transcription experiments.

Cloning Procedures. Purified poly(A)-containing early Ad2mRNA was used as a template for reverse transcriptase(RNA-dependent DNA polymerase). The second strand wasalso synthesized with reverse transcriptase after alkaline hy-drolysis of the RNA. The double-stranded cDNA was insertedinto the Pst I site of the pBR322 plasmid after tailing withpoly(dG) and poly(dC). All details of our cloning proceduresare described in two recent communications (10, 12). Clonesspecific for region E1B were identified by colony hybridization(1, 18), using fragment HindIII-C of Ad2 DNA (map coordi-nates 8.0-17.0) as a probe.Recombinant DNA Safety Procedures. All cloning exper-

iments were carried out in a P3 facility at the Pasteur Institute.Permission to carry out the cloning experiments and for thesubsequent analysis of the recombinant plasmids was grantedby the French (Delegation G&n6rale a la Recherche Scientifiqueet Technique) and the Swedish recombinant DNA com-mittees.

Sequence Analysis. The protocol of Maxam and Gilbert (19)was followed. The recombinant plasmids were cleaved withendonuclease Sac I, end-labeled with polynucleotide kinase,and recleaved with Pst I. The fragments of interest were iso-lated by electrophoresis on 8% polyacrylamide gels.

Abbreviations: Ad2 and Ad5, adenovirus types 2 and 5; T antigen,tumor antigen; bp, base pair(s); SV40, simian virus 40.

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El

A-

I E2 E4

E3

9S (?) - 1 AAA

B 12S (26,000) ----I-AA -- I

13S (32,000) -.-̂ aW-

w w ~~~.1.5 2.5 3.5 4.5

t tXba I Hpa I

5.5 6.5 7.5 8.5

-c

9.5

:AAA 9S (15,000)___ --_AAA 13S (20,000)

-AAA 22S (67,000)

101W.5

Sac I

11.5

Clone 244

IClone 272

FIG. 1. (A) Illustration of the four easly gene clusters on the Ad2 genome. The arrows indicate the direction of transcription. Region Elcontans two separate promoters (8) and is hence subdivided into regions ElA and ElB. (B) Enlargement of region El, illustrating the structureof mRNAs from this region, which have been detected by electron microscopy (1, 2) and Si nuclease mapping (3). The calibration is in mapunits (1 map unit = 1% of the linear adenovirus genome), and restriction endonuclease cleavage sites are indicated. The common 5' end for the13S and 22S mRNAs of region ElB is located at nucotide 1702 (9). The common poly(A)-addition site for all region ElB mRNAs is at nucleotide4071 (see ref. 10) in the sequence ofMaat et al. (11) for region El ofAd5 DNA. The splice points and the common poly(A)-addition site for regionElA mRNAs have been determined by Perricaudet et al. (12). Sequences that are read from reading frame 1 (empty bar) as well as frame 2 (solidbar) are indicated. The translational reading frames are defmied as described by van Ormondt et al. (13). The position of the gene for the 9SmRNA for polypeptide IX was determined by Alestr6m et al. (10). (C) Sequences present (stippled bar) in clones 244 and 272.

RESULTS

Cloning of mRNA Sequences from Early Region E1B ofAd2. cDNA copies of early Ad2 mRNA, prepared from cellsthat had been grown in the presence of cycloheximide, wereinserted into the Pst I site of plasmid pBR322. Several hundredclones containing Ad2-specific sequences were identified bycolony hybridization, using UP-labeled Ad2 DNA as a probe.In order to identify clones that were specific for region E1B,hybridizations were carried out with fragment HindIII-C asa probe. Two positive clones were found, and they are desig-nated 244 and 272; Plasmid DNA was extracted from the twoclones, labeled in vitro by nick translation, and hybridized toblotted HindIII fragments of Ad2 DNA. The results showedthat DNA from clone 244 hybridized to both fragments Hin-dIII-G (0-8.0) and HindIII-C (8.0-17.0), whereas clone 272hybridized exclusively to fragment Hindfll-C. Previous studies(1-3) using electron microscopy and the S1 nuclease assay haveidentified two spliced mRNAs within region E1B, a 22S anda 13S mRNA species (Fig. 1). In order to correlate our clonesto these two mRNAs, an analysis using restriction endonucleasesPst I, HindIII, Bgl II, Sac I, and Sma I was undertaken. Pst Idigestion of clone 244 excised a 600-base-pair (bp) cDNA insertlaking cleavage sites for restriction endonucleases Hindl and

Bgd I, whereas Sac I cleavage gave two fragments, 150 and 450

bp. These results, together with hybridization data, indicatedthat clone 244 corresponded to the 13S mRNA of the regionE1B (Fig. 1). Clone 272 contained a 500-bp cDNA insert thatcontained cleavage sites for endonucleases Bgl and Sac I. Thepresence of a Bgl II cleavage site suggested that the insertedcDNA was different from that of clone 244 and might be re-lated to the 22SmRNA (Fig. 1). To esablish whether the cDNAof clone 272 included the spliced region of the 22S mRNA,DNAs from this clone as well as from another clone containing

fragment HindI-C of Ad2 DNA inserted into the HindmI siteof pBR322 were digested by a mixture of endonucleases Sac Iand Bgl II. The analysis of the digestion products was facilitatedby the absence of cleavage sites for Sac I and Bgl II in thepBR322 plasmid. The sizes of the excised Sac I/Bgl II fragmentsfrom the two plasmids were compared and found to differ byapproximately 100 bp, which would be expected if the re-

combinant plasmid contained a spliced sequence (Fig. 1). Thuswe concluded that clone 272 contains sequences from thespliced region of the 22S mRNA. Neither of the clones con-

tained a Sma I cleavage site, which indicated that in both cases

the poly(A) junctions are missing, because the 3' end of regionE1B mRNAs is located in fragment Sma I-M (10). Alestrom etal. (10) have, however, determined the position of the common3' end for region E1B mRNAs by determining the sequence ofa cloned cDNA copy of the quasi-late polypeptide IX mRNAfrom E1B.

Partial Sequence Analysis of Clones 244 and 272. In orderto study the structure of the cloned mRNAs at the nucleotidelevel, sequence analysis of selected regions of the recombinantplasmids was performed according to the Maxam and Gilbertprocedure (19). To locate the splice junctions that were expectedto be present in the two clones, we made use of the Sac Icleavage site at map position 10.5 (Fig. 1). In both cases se-

quence analysis from this cleavage site in the leftwards directionidentified splice points (Fig. 2). Ad2 and Ad5 belong tosubgroup C adenovirus, and the sequences of their transformingregions are nearly identical (12, 21). Therefore it is very likelythat RNA splicing in Ad5 follows the same lines, and it shouldbe possible to combine our results with the DNA sequence thathas been established for the leftmost 11% of Ad5 I)NA (11, 13,15) to deduce the structure of the 13S and the 22S mRNAs. Asis shown in Fig. 3, the splice in the 1SS mRNA results in a de-

A

C

! :::Z=I

Biochemistry: Perricaudet et al.

t - > - - 1

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3780 Biochemistry: Perricaudet et al.

/A

.--G G

I-

C_

*_ ~~ C

B

IG T

-ftf

--a

--wg-1f- ..... I-f

ftf.a,,'11

C

*>..

asX%

"'l..ksi.... %

.", N

,,W\N'k

FIG. 2. Sequencing gels showing the splice points in clones 244and 272. DNA from the clones was cleaved with endonuclease Sac I,end-labeled with polynucleotide kinase, and recleaved with Pst I. Thefragments of interest were isolated from an 8% polyacrylamide gel andtheir nucleotide sequences were determined according to the Maxamand Gilbert (19) procedure. Shown are 20% sequencing gels, illus-trating the splice point for clones 244 (A) and 272 (B). The arrow in-dicates the position ofthe splices when Chambon's rule (20) is applied.The sequence starts with nucleotide 3606 (bottom of the gel) andcorresponds to the r strand; i.e., the sequence is complementary tothat of the E1B mRNAs.

letion of sequences between nucleotides 2257 and 3595, andthe splice in the 22S mRNA deletes a region between nucleo-tides 3512 and 3595.

Polypeptide IX mRNA

DISCUSSIONThe proposed structures of the 22S and the 13S mRNAs areshown in Fig. 3. The DNA sequence (11, 15) shows that the 5'and 3' parts of the 13S mRNA are read from different readingframes, whereas the 22S mRNA is read in a single readingframe, identical to that used in the 5' part of the 13S mRNA.From our studies it is not possible to locate the common 5' endfor the region EIB mRNAs, which has been mapped at position4.5 by electron microscopy (1, 2) and the SI nuclease assay (3).However, by sequence analysis of the capped Ti oligonucleo-tide from region EIB mRNAs, Baker and Ziff (9) have posi-tioned the 5' end to nucleotide 1702 in the sequence of Maatand van Ormondt (15). If we assume that both polypeptides areinitiated at the first ATG that follows the cap at position 1714,the 13S and 22S mRNAs code for 186- and 598-amino-acid-longpolypeptides with molecular weights of 20,000 and 67,000,respectively. Halbert et al. (22) have selected mRNAs fromregion EIB by hybridization and identified their proteinproducts by in vitro translation. They have observed Mr 53,000and 15,000 polypeptides that probably correspond to thepolypeptides that can be predicted from our study. The dis-crepancy in size is most likely due to the use of inaccurate sizemarkers. Van der Eb and his colleagues (23) have used markerproteins with molecular weights known from their primarysequences and, in their hands, the immunoprecipitated poly-peptides have molecular weights of 65,000 and 19,000, whichis in close agreement with our predicted values.There are, obviously, many ways in which the splicing re-

action can be exploited by a virus to utilize its genetic infor-mation in the most efficient way. Several interesting exampleshave emerged from the analysis of early and late adenovirusmRNAs:

(i) Late during adenovirus infection, splicing is used to addthe common tripartite leader to many different mRNA bodies(24, 25), presumably to make certain that the viral mRNAs cansuccessfully compete with cellular mRNAs for ribosomes andfactors required for translation.

(if) During transcription of region EIA, splicing makes itpossible to eliminate a region from the mRNAs that is blockedin all three translational reading frames by numerous stop co-dons (12). The splice in the 12S and 1SS mRNAs of region EIA

Cap Stop

AUCUGUUUUGCAG CAGCCGCCGCCGCCAUGAGC # JAA--//-CAAAA3610 4030 4071

Frame 1

Cap AUG Stop'I .113S '// GUACAG GUGGCUGA. UUUUGUAUCUGUUUUGCAG ---C-C----U-//- AAAA

1702 1714 2257 3595 3611 4071

1I fi.I

Frame 1 Frame 2

Cap AUG.,

Stop

'I// AUUGAGIGUAM UUUUGUAUCUGUUUUG(3508 3512

CAG CAGCC /- AAAAA4

593595 4071

Frame l

FIG. 3. Proposed structures ofthe 13S and 22S mRNAs from region E1B. The sequence is indexed according to Maat et al. (11). The positionof the cap is from Baker and Ziff (9), and the position ofthe poly(A) junction for region E1B was determined by Alestr6m et al. (10). The locationsof the initiator AUG and of the stop codons, which terminate translation, are shown. The translational reading frames as defined by Maat etal. (11) are also indicated. The exact positions ofthe splice points cannot be established due to the presence oftandem repeats. The boundariesof the intervening sequence have been positioned according to Chambon's rule (20). The cap and the initiation AUG in the polypeptide IX mRNA(10) are also indicated.

22S4 4

1702 1714

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Proc. Natl. Acad. Sci. USA 77 (1980) 3781

MET GLU ALA TRY GLU CYS LEU GLU ASP PHE SER ALA VAL ARG ASN LEU LEU GLU GLN SER

SER ASN SER THR SER TRY PHE TRY ARG PHE LEU TRY GLY SER SER GLN ALA LYS LEU VALCYS ARG ILE LYS GLU ASP TYR LYS TRY GLU PRE GLU GLU LEU LEU LYS SER CYS GLY GLULEU PHE ASP SER LEU ASN LEU GLY HIS GLN ALA LEU PHE GLN GLU LYS VAL ILE LYS THRLEU ASP PHE SER THR PRO GLY ARC ALA ALA ALA ALA VAL ALA PHE LEU SER PHE ILE LYSASP LYS TRY SER GLU GLU THR HIS LEU SER GLY GLY TYR LEU LEU ASP PHE LEU ALA MET

HIS LEU TRY ARG ALA VAL VAL ARG HIS LYS ASN PRO PRO ALA THR VAL VAL PHE ARG PRO

PRO GLY ASP ASN THR ASP GLY GLY ALA ALA ALA ALA ALA GLY GLY SER GLN ALA ALA ALA

ALA GLY ALA GLU PRO MET GLU PRO GLU SER ARG PRO GLY PRO SER GLY MET ASN VAL VAL

GLN

VAL ALA GLU LEU TYR PRO GLU LEU ARC ARG ILE LEU THR ILE THR GLU ASP GLY GLN GLYLEU LYS GLY VAL LYS ARG GLU ARG GLY ALA CYS GLU ALA THR GCU GLU ALA ARG ASN LEU

ALA PHE SER LEU MET THR ARG HIS ARG PRO GLU CYS ILE THR PHE

ASN CYS ALA ASN GLU LEU ASP LEU LEU ALA GLN LYS TYR SER ILE

TYR TRY LEU GLN PRO GLY ASP ASP PHE GLU GLU ALA ILE ARG VAL

LEU ARG PRO ASP CYS LYS TYR LYS ILE SER LYS LEU VAL ASN ILE

ILE SER GLY ASN GLY ALA GLU VAL GLU ILE AS P THR GLU AS P ARGSER MET ILE ASN MET TRY PRO GLY VAL LEU GLY MET ASP GLY VAL

ARG PHE THR GLY PRO ASN PHE SER GLY THR VAL PHE LFU ALA ASN

GLN GLN ILE LYS AS P

GLU GLN LEU THR THR

TYR ALA LYS VAL ALA

ARG ASN CYS CYS TYR

VAL ALA PHE ARG CYS

VAL ILE MET ASN VAL

THR ASN LEU ILE LEUHIS GLY VAL SER PHE TYR GLY PHE ASN ASN THR CYS VAL GLU ALA TRY THR ASP VAL ARC

VAL ARC GLY CYS ALA PHE TYR CYS CYS TRY LYS GLY VAL VAL CYS ARC PRO LYS SER ARC

ALA SER I LE LYS LYS CYS LEU PHE GLU ARC CYS THR LEU GLY I LE LEU SER GWU GLY ASNSER ARC VAL ARC HIS ASN VAL ALA SER ASP CYS GLY CYS PHE MET LEU VAL LYS SER VAL

ALA VAL ILE LYS HIS ASN MET VAL CYS CLY ASN CYS GWu ASP ARC ALA SER GLN MET LEU

THR CYS SER ASP GLY ASN CYS HIS LEU LEU LYS THR ILE HIS VAL ALA SER HIS SER ARC

LYS ALA TRY PRO VAL PHE GLU HIS ASN ILE LEU THR ARC CYS SER LEU HIS LEU GLY ASNARC ARC GLY VAL PHE LEU PRO TYR GLN CYS ASN LEU SER HIS DIR LYS ILE LEU LEU GLUPRO GLU SER MET SER LYS VAL ASN LEU ASN GLY VAL PHE ASP MET THRLYS VAL LEU ARG TYR ASP GLU DIR ARG DIR ARG CYS ARG PRO CYS GLUHIS ILE ARG ASN GLN PRO VAL MET LEU ASP VAL THR GWu Gu LEU ARGVAL LEU ALA CYS DIR ARG ALA GLU PHE GLY SER SER ASP GLU ASP DIR

GLN PRO PRO PRO PRO

also results in a frame shift whereby the coding capacity of thisregion is most efficiently put to use (12). Moreover, splicing inthis region gives rise to two mRNAs encoding completelyoverlapping polypeptides, the only difference being that 46internal amino acids are absent from the polypeptide specifiedby the shorter mRNA (12).

(iii) For the mRNAs in region E1B yet another situation isapparent. The splice in the 22S mRNA eliminates only 84 nu-cleotides and occurs after the stop codon that terminates theonly open translational reading frame in this mRNA. The splicedoes not alter the coding potential of the 22S mRNA, and in thisrespect it resembles the mRNA for the small tumor antigen ofsimian virus 40 (SV40) (26, 27). In both cases it is difficult to seeany obvious advantage of the splicing reaction. It has, however,recently been proposed that splicing may be a mandatory stepfor maturation and transport of certain mRNAs, because mu-tants of SV40 that lack the intervening sequence of the VPI genefail to make a functional mRNA from this gene (28). The splicein the 13S mRNA of region E1B has different consequences.If translation starts at the first AUG in the sequence, the NH2terminus of the protein specified by this mRNA is identical tothe NH2 terminus of the Mr 67,000 protein encoded by the 22SmRNA, but 417 amino acids in the Mr 67,000 protein are absentin the Mr 20,000 polypeptide (Figs. 3 and 4). It is noteworthythat 26 out of 29 half-cystine residues are eliminated by thesplice, which might result in drastic structural alterations.

MET LYS ILE TRY

CYS (LY GLY LYS

PRO ASP HIS LEU

ASP

67,000

FIG. 4. Predicted amino acid se-

quences of the Mr 67,000 and 20,000polypeptides from Ad5. The sequence isbased on the assumption that the firstAUG in the sequence is used for initiation.The two polypeptides are partially over-

lapping. The last five amino acids are

unique for the Mr 20,000 polypeptide.The sequence of the two proteins was

deduced by combining our sequence in-formation for the splice points with theknown sequence for the left-hand 11% of

20,000 the Ad5 DNA (11, 13, 15).

Unlike the 22S mRNA, the 13S mRNA would be translated afterthe splice even though only five residues are added before theUGA stop codon at position 3611 is encountered. (In Ad2 DNAone of the CCG codons after the splice has been deleted, andthus only three prolines and one glutamine are added at theCOOH terminus of the Mr 20,000 polypeptide from Ad2; seeref. 10.) The common part of the intervening sequence for the22S and 13S mRNA is an important regulatory region becauseit includes the promoter as well as the cap site for the poly-peptide IX mRNA (10). The common acceptor site for splicingin region E1B at position 3595 is located within the gene forpolypeptide IX (10). At the termination site for the Mr 20,000polypeptide the sequence is ... AUGA ... (Fig. 3), and it isinteresting to note that the AUG in this sequence serves as theintiator codon for polypeptide IX (10).The E1B region shows similarities as well as clear differences

when compared to the early region of SV40. In both cases alarge and a small protein with related sequences are formed bysplicing (26, 27). In the adenovirus case, however, the large Mr67,000 protein is coded by the large mRNA in a contiguoussequence like the small SV40 tumor antigen. An interestingsimilarity is that both the Mr 20,000 polypeptide of region E1Band the large T antigen of SV40 are equipped with proline-richCOOH termini because of the splice. A direct comparison be-tween the amino acid sequences for the region E1B proteins andthe SV40 T antigens reveals no regions of homology longer thanfour amino acids.

20,000and67,000

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There is as yet no clear answer as to which protein from re-gion El plays the key role in cell transformation. Immu-noprecipitation of extracts from infected cells with sera fromtumor-bearing animals precipitates peptides with molecularweights of 53,000 and 15,000 consistently, and sometimes otherminor polypeptides also (28, 29). For reasons given above theMr 53,000 and 15,000 polypeptides probably correspond to theElB polypeptides with predicted molecular weights of 67,000and 20,000. Thus if we define T antigens as proteins that in-variably are precipitated by sera from tumor-bearing animals,the Mr 67,000 and 20,000 polypeptides described in this studyshould qualify as adenovirus T antigens. The problem is,however, complicated by the observation of van der Eb et al.(14) that cells can be "transformed" by fragment Hpa I-E ofAd5, which does not include any part of region E1B. These"transformed" cells are obtained with an exceptionally lowfrequency and do not have the same growth potential as cellstransformed by larger fragments or virions. Recently it has beenshown that a gene product from region ElA regulates theproduction of mRNAs from the other early regions on the ad-enovirus genome (30, 31). An alternative interpretation of therole of region ElA may be that one or more of its gene productsact primarily by switching on the expression of region E1B.

In cells transformed by the Hpa I-E fragment, the viral geneproduct(s) could function by turning on critical cellular func-tions that would induce phenotypic alterations typical for thesecells.

Cells transformed by the HindIII-G fragment of Ad5 exhibita more transformed phenotype than cells transformed by theHpa I-E fragment, although the T antigen distribution in thesecells often is atypical as compared to cells transformed by virusor fragments that contain the entire El region (23). From thepredicted structure of the 22S mRNA (Fig. 4) it is obvious thatthe Mr 67,000 polypeptide would be truncated and lack abouthalf of its amino acids if it is at all expressed as a stable proteinin these cells. In contrast, the sequence for the Mr 20,000polypeptide would be present in HindIII-G-transformed cellsexcept the five last amino acids, which are encoded beyond thesplice. A more serious consequence might be that the acceptorsite for splicing of the 13S mRNA is absent in the HindIII-Gfragment. Because transformation with the HindIII-G frag-ment occurs with a low frequency, it is conceivable that in therare cases when transformation is established an acceptor sitefor splicing as well as a suitable stop codon are found within thehost sequences at the site of integration. Because most of thesequences for the Mr 20,000 polypeptide are present in cellstransformed by fragment HindIII-G and because more prop-erties characteristic of transformation are expressed in thesecells, we suggest that the Mr 20,000 polypeptide may play a keyrole in adenovirus transformation in vttro.The present study, together with two recently published

reports, outlines the structure at the nucleotide level of all majorspecies of early mRNA from the transforming region of Ad2that so far have been described (1-3, 22). The discrepancy be-tween previously described mRNA species from this region canalso be resolved through our results. By electron microscopy (2)the small splice in the 22S mRNA from region E1B was notobserved, whereas the SI nuclease assay failed to reveal the 13SmRNA with the large splice. It is obvious from the present re-sults that both types of spliced mRNAs exist in infected cells.One additional mRNA has been described from the trans-forming region, although no polypeptide has yet been corre-lated with its appearance (22). This so-called 9S mRNA fromregion ElA is predominantly synthesized late after infection.We have so far been unable to clone this mRNA in Escherichiacoli.

We are grateful to Hans van Ormondt and Peter Alestrbm forcommunicating their results prior to publication. We thank PierreTiollais for laboratory facilities and discussions, Marianne Gustafsonfor excellent secretarial help, and Margaret Bywater and LennartPhilipson for careful reading of the manuscript. This study was sup-ported by grants from the Swedish Cancer Society, The SwedishMedical Research Council, and Institut National de la Sante et de laRecherche MWdicale. During part of this investigation U.P. was sup-ported by a fellowship from the European Molecular Biology Orga-nization.

1. Kitchingman, G. R., Lai, S. P. & Westphal, H. (1977) Proc. Natl.Acad. Sci. USA 74,4392-4396.

2. Chow, L. T., Broker, T. & Lewis, J. B. (1979) J. Mol. Biol. 134,265-303.

3. Berk, A. J. & Sharp, P. A. (1978) Cell 14, 695-711.4. Sharp, P. A., Gallimore, P. & Flint, S. J. (1974) Cold Spring

Harbor Sympt. Quant. Biol. 39,457-474.5. Pettersson, U., Tibbetts, C. & Philipson, L. (1976) J. Mol. Biol.

101,479-501.6. Graham, F. L., Abrahams, P. J., Mulder, C., Heinjneker, H. L.,

Warnaar, S. O., De Vries, F. A. J., Fiers, W. & van der Eb, A. J.(1974) Cold Spring Harbor Symp. Quant. Biol. 39,637-650.

7. Gallimore, P., Sharp, P. A. & Sambrook, J. (1974) J. Mol. Biol.89,49-72.

8. Wilson, M. C., Fraser, N. W. & Darnell, J. E. (1979) Virology 94,175-184.

9. Baker, C. & Ziff, E. (1979) Cold Spring Harbor Symp. Quant.Biol., in press.

10. Alestr6m, P., Akusjarvi, G., Perricaudet, M., Mathews, M. B.,Klessig, D. & Pettersson, U. (1980) Cell 19, 671-681.

11. Maat, J., van Beveren, C. P. & van Ormondt, H. (1980) Gene, inpress.

12. Perricaudet, M., Akusjarvi, G., Virtanen, A. & Pettersson, U.(1979) Nature (London) 281, 694-696.

13. van Ormondt, H., Maat, J., de Waard, A. & van der Eb, A. J.(1978) Gene 4,309-328.

14. Van der Eb, A. J., Mulder, C., Graham, F. L. & Houweling, A.(1977) Gene 2, 115-132.

15. Maat, J. & van Ormondt, H. (1979) Gene 6, 75-90.16. Persson, H., Pettersson, U. & Mathews, M. B. (1978) Virology 90,

67-79.17. Pettersson, U. & Sambrook, J. (1973) J. Mol. Biol. 73, 125-

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