Proc. Nati. Acad. Sci. USAVol. 82, pp. 2354-2358, April 1985Biochemistry

Expression in Escherichia coli of a fusion protein productcontaining a region of the adenovirus DNA polymerase

(synthetic peptide/protein blotting/expression vector/molecular cloning/adenoviral DNA replication)

DAVID REKOSH*t, JEFF LINDENBAUMt, JAMES BREWSTER*, LAWRENCE M. MERTZ*, JERARD HURWITZt,AND LAURA PRESTINE**State University of New York, Department of Biochemistry, Buffalo, NY 14214; and tMemorial Sloan Kettering Institute, Department of Molecular Biology,425 East 68th Street, New York, NY 10021

Contributed by Jerard Hurwitz, December 31, 1984

ABSTRACT The bulk of an open reading frame extendingfrom map coordinates 23.3 to 14.2 in region E2b of theadenoviral genome has been cloned and expressed from achimeric plasmid in Escherichia coli. The cloning strategyused created a fusion protein of 124,000 daltons, whichcontained >98% adenovirus-encoded sequences. Antiserumraised against this protein reacted with the authentic 140,000-dalton adenovirus DNA polymerase. Another serum raisedagainst a synthetic hexapeptide whose sequence correspondedto the predicted carboxyl terminus of adenovirus-encoded DNApolymerase also reacted with the fusion protein and authenticadenovirus DNA polymerase. These results demonstrate thatthe cloned region of DNA encodes the adenovirus DNA poly-merase.

Adenovirus type 2 (Ad2) DNA, isolated from virions, con-tains a 55-kDa terminal protein (TP) covalently attached toeach 5' end (1). This protein is initially synthesized as an80-kDa precursor (pTP), which is processed to the matureform at a late stage in virion assembly (2-4). Efficientreplication of the DNA-protein complex (Ad DNA-pro) invitro requires five proteins. Three of these proteins are viralin origin and include the pTP (5), a DNA polymerase of 140kDa (Ad Pol) (6-8), and a 72-kDa single-strand DNA bindingprotein (Ad DBP) (9, 10). The other two proteins can beisolated from uninfected HeLa cells. Nuclear factor II is atype I topoisomerase required for efficient elongation (11).Nuclear factor I binds to a sequence between nucleotides 17and 48 on the Ad DNA molecule and, in the presence of thethree viral-encoded proteins and Ad DNA-pro, stimulatesthe addition of dCMP to the pTP (12-14).The three viral-encoded proteins have been mapped to

specific regions of the viral genome by using two procedures.The first procedure involved hybrid selection with individualviral DNA fragments ofmRNA from the infected cell. In vitrotranslation systems programed with selected Ad mRNAsyielded products that were approximately the same sizes asthe proteins isolated from Ad-infected cells. For the Ad DBPand pTP, peptide maps provided further evidence for identity(2, 3, 7, 15). The second procedure involved the use oftemperature-sensitive viral mutants defective in DNA repli-cation. Two groups ofAd type S (AdS) mutants (ts36 or ts149;ts125) have been used; they have been mapped by markerrescue or DNA sequencing to specific regions of the viralgenome (16-18). Since extracts made from cells infected atthe restrictive temperature with either group of mutants failto replicate viral DNA, in vitro complementation studies withpurified viral proteins could be carried out (7-10, 19). It wasdemonstrated that the ts36 or ts149 defect was complemented

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2354

by purified Ad Pol and that the ts125 defect was com-plemented by purified Ad DBP. Thus, the genes for the AdDBP and Ad Pol proteins could be positioned on the genomeat the position of the respective mutations.These results, combined with analysis of the viral DNA

sequence (20-23) and R-loop mapping of mRNA from theinfected cell (2), allowed the assignment of the three viralproteins to two early transcription regions termed E2a (forthe Ad DBP) and E2b (for the pTP and Ad Pol) on the viralDNA 1-strand. Early region E2a mRNA is transcribed froma promoter at map coordinate 75.0; it contains small leadersmapping at 75.0 and 68.5 and a main body mapping between66.5 and 61.5. The entire Ad DBP is encoded contiguouslyfrom the main body ofthis sequence (23). At least two distincttranscripts have been identified from E2b (2, 24). Theseappear to initiate at the same promoter as the early E2amRNA since they contain the same first leader. Both alsocontain a leader of about 200 nucleotides that maps atcoordinate 39.0 and main bodies that map between 30.3 and11.2 and between 26.0 and 11.2. The larger ofthe two mRNAsis thought to encode the pTP (from coordinates 28.9 to 23.5)(21), whereas the smaller one encodes Ad Pol (from co-ordinates 24.5 to 14.2) (7). The exact structure of the splicesites in these mRNAs is not known, thereby obscuring theexact nature ofthe amino termini ofthe proteins they encode.

In this report we describe the cloning and expression inEscherichia coli of most of the E2b region of Ad2 DNA thatencodes the Ad Pol protein and the isolation of two antiserareactive against this protein.

MATERIALS AND METHODSConstruction of Chimeric Plasmids and Bacterial Strains.

Pst I-cut Ad2 or AdS DNA was inserted at the unique Pst Isite in plasmid pUC8 by using standard procedures (25).Recombinant plasmids were identified by colony hybridiza-tion with appropriate probes. Plasmids were isolated fromcolonies that appeared positive and were screened further.Initial transformation was into the E. coli strain JM 83 (25).Once identified, the plasmids were isolated and introducedinto strain CSR 603 (26). All experiments were carried out byusing this strain.

In vitro Transcription-Translation of Plasmid DNA. Re-actions were carried out according to the directions providedby the manufacturer, Amersham. In some instances DNAwas fragmented with the restriction enzyme Sal I prior toaddition to the system.

Abbreviations: Ad, adenovirus; Ad DNA-pro, Ad DNA-proteincomplex; Ad DBP, Ad-encoded DNA binding protein; Ad Pol, AdDNA polymerase; pTP, Ad-encoded preterminal protein; bp, basepair(s); HRP, horseradish peroxidase.To whom reprint requests should be addressed.

Proc. Natl. Acad. Sci. USA 82 (1985) 2355

Antisera. 124-kDa serum. Antiserum was obtained againstthe 124-kDa fusion protein by excising the stained proteinband from a polyacrylamide gel, emulsifying the pro-tein-polyacrylamide mixture in an equal volume ofphosphate-buffered saline, and injecting the mixture subcutaneously, atmultiple sites, into a New Zealand White rabbit. The rabbitwas injected at monthly intervals for 3 months before areactive serum was obtained. The fusion protein isolatedfrom -6 ml of bacteria grown to a stationary phase wasinjected at each time.Pol serum. The synthetic peptide was purchased from

Bachem Fine Chemicals (Torrance, CA). Prior to injection,the peptide was conjugated to bovine serum albumin withbis-diazotized benzidine as described elsewhere (27). Onemilligram of conjugate in 1 ml of incomplete Freund adjuvantwas injected into a New Zealand White rabbit at 10 differentintradermal and intramuscular sites, including the footpads,at monthly intervals. Reactive serum was obtained after threeinjections.

Electrophoretic Transfer Blotting. Blotting was performedas described elsewhere (28) following electrophoresis onpolyacrylamide gels. Gelatin was used as a blocking agent.Following incubation with an appropriate dilution of anti-body, in some instances 125I-conjugated protein A was usedto locate the antigen-antibody complexes. In other instances,goat anti-rabbit IgG-horseradish peroxidase (HRP) conju-gate and the Bio-Rad immunoblot assay kit were used asdescribed by the manufacturer. The latter procedure was alsoused in the spot-blot assays performed to determine the titersof the sera.DNA Synthesis and Purified Enzymes. The pTP-Ad Pol

fraction (5), Ad DBP (29), nuclear factor I (12), and factor pL(30) were purified as described. Plasmid pLA1 containing theleft-hand terminal 3.3-kb Bgl II fragment of Ad5 DNA wasprepared as described (31). One unit ofpTP-Ad Pol catalyzedthe incorporation of 1 nmol of [3H]TMP into an acid-insolubleform in 20 min at 30'C.

RESULTS

Construction of the Chimeric Expression Plasmid. Thestrategy adopted for the cloning and expression of the Ad Polgene is summarized in Fig. 1. When Ad2 or Ad5 DNA is cutwith the restriction enzyme Pst I, the fragments producedinclude a 3432-base-pair (bp) product derived from co-ordinates lying between 23.3 and 13.8 map units; this frag-ment spans most of the open reading frame believed toencode the 140-kDa Ad Pol (Fig. 1 Upper). The fragment wascloned into the expression vector pUC8 at a unique Pst I sitenear the beginning of a small fragment of the E. coli3-galactosidase gene carried on the plasmid (Fig. 1 Upper).The Pst I cloning site occurs 38 nucleotides after the AUGused for the initiation of protein synthesis. Furthermore,analysis of the DNA sequence in both pUC8 and Ad DNAindicated that translation across the Pst I sites was in thesame reading frame (Fig. 1 Lower). However, since the AdDNA fragment was inserted into the vector in two differentorientations, two recombinant plasmids were obtained. Oneplasmid (p26) possessed the sequence encoding the Ad Polprotein in an opposing orientation to the direction of tran-scription. The other plasmid (pPol) had the Ad DNA in thecorrect orientation. The pPol chimera encodes a 124-kDapolypeptide containing 1071 amino acids from the Ad Polopen reading frame and 12 amino acids from the vector.An ethidium bromide-stained agarose gel of restriction

enzyme digests of the plasmids formed from Ad2 DNA isshown in Fig. 2. As expected when pPol and p26 weredigested with Sal I, a single fragment of 6100 bp was obtainedfrom each (lanes 1 and 2), and two fragments of 3400 and 2700

13.8 23.3. 34 Kb Pst I Fragment2

Hin~dm

Hind I Fragments: 2.2Kb3.9 Kb

Hindl Frogments: 4.9Kb1.2Kb

MET. .8.. SER VAL ASP LEU GLN PRO SER LEU. PA.AUG.... TCC GTC GAC CTG CAG CCA AGC TTG,....TAG

24 PstI 249

...128.. GLU VAL ASN LEU GLN GLU LEU PRO.....

........GAG GTA AAC CTG CAG GAG CTC CCG.....TAG384 PstI 3198

MET..8.. SER VAL ASP LEU GLN GLU LEU PROCI.!?AUG..... TCC GTC GAC CTG CAG GAG CTC CCG....- TAG

24 PstI 3198

pUC8

140

FUSION PROTEIN

FIG. 1. Schematic illustration ofthe cloning scheme. (Upper) Theheavy black arrow shows the 140-kDa open reading frame in regionE2b. The 3400-bp Pst I fragment ofAd DNA can be cloned into pUC8in two orientations with respect to lac transcription, yielding pPol (inthe correct orientation) and p26. (Lower) Each line (pUC8, 140 kDa,or FUSION PROTEIN) shows the translation across the Pst I site ofeach DNA. The numbers refer to the number of amino acids ornucleotides not shown in the remainder of each reading frame. Kb,kilobases.

bp were obtained with Pst I (lanes 4 and 5). However,digestion with HindIll (lanes 7 and 8) produced different pairs

1 2 3 4 5 6 7 8 9 MORIGIN

12.2 Kb

-8 . 1~~~~~~~~~~~~.5 _ - 1 6 . 1~~~~~~~~~~.

- ~~~~~~~~~~~~~5.1-4.1

3.1

- _ ~~~~~~~~2.0-1.6

FIG. 2. Restriction enzyme analysis of pPol, p26, and pUC8. Theplasmids were derived from Ad2 DNA. Each plasmid was cut withthe indicated enzyme, subjected to electrophoresis on a 0.7% agarosegel, and stained with ethidium bromide. Sal I: lane 1, pPol; lane 2,p26; lane 3, pUC8. Pst I: lane 4, pPol; lane 5, p26; lane 6, pUC8.HindIII: lane 7, pPol; lane 8, p26; lane 9, pUC 8. Lane M containeda 1-kilobase ladder as the size marker.

Biochemistry: Rekosh et al.

Proc. Natl. Acad. Sci. USA 82 (1985)

of fragments from each, indicating that the Ad DNA wascloned in a different orientation in each plasmid. pUC8 wasalso digested and analyzed on this gel as a control (lanes 3,6, and 9). A corresponding pair of plasmids was also made byusing Ad 5 DNA as starting material (data not shown).

Expression of the Ad Pol Chimeric Protein. To examine theproduction of the Ad Pol fusion protein, cultures of E. colicontaining Ad2-derived pPol or p26, and pUC8, were grownovernight to stationary phase in L-broth, harvested, andboiled in gel sample buffer containing 1% NaDodSO4. Anappropriate amount of each sample, together with molecularmass markers, was loaded onto a NaDodSO4/15% polyacryl-amide gel; after electrophoresis the gel was stained withCoomassie brilliant blue (Fig. 3A). A dark band at theexpected position corresponding to 124 kDa was visible in thesample containing pPol (lane 1) and absent in the twocontrols. In another experiment (Fig. 3B), the mobilities ofthe polypeptides derived from cells carrying the Ad2 versionof pPol (lane 3) were compared with the polypeptides derivedfrom cells carrying the Ad5 counterpart (lane 2). The stainedgel showed that the two polypeptides have approximately thesame apparent molecular mass but that the AdS-derivedpolypeptide migrated slightly faster than the Ad2 protein.Although the reason for this reproducible difference inmigration is not known, this result suggests that the proteinsare derived from plasmid-encoded sequences and not fromthe E. coli host.A more direct experiment was performed to confirm that

the 124-kDa protein observed in the culture containing pPolwas actually encoded by the plasmid. In this experiment theAd2 pPol was added to an in vitro system that efficientlytranscribed and translated added DNA into [35S]methionine-labeled protein. As controls, p26 and pUC8 were alsosubjected to the same procedure in separate reaction mix-tures. In additional reactions, all three plasmids were alsodigested with the restriction enzyme Sal I prior to addition tothe transcription-translation system. Sal I cuts just after thef3-galactosidase ribosome binding sequence, thus separatingthe inserted Ad DNA from an active promoter. The proteinsproduced were then analyzed by NaDodSO4/polyacrylamidegel electrophoresis followed by fluorography. In addition tothe background bands present in all of the samples, those

A

205 - ..123

96 -tt68- _.;

43-

29 -

B1 2 3

116 -

96 -

68 -

45 -

FIG. 3. Protein analysis of bacteria transformed with pPol, p26,and pUC8. Bacterial cultures containing each plasmid were grown tostationary phase. One-hundred microliters of each was analyzeddirectly on NaDodSO4/polyacrylamide gels that were stained withCoomassie brilliant blue. Positions of stained molecular mass mark-ers (given in kDa) are shown. (A) A 15.5% polyacrylamide gel. Theplasmids were derived from Ad2 DNA. Lane 1, pPol; lane 2, p26;lane 3, pUC8. (B) A 10% polyacrylamide gel. Lane 1, pUC8; lane 2,AdS-derived pPol; lane 3, Ad2-derived pPol.

A B C1 2 3 1 2 3 l 2

-116- -

-96 _

-68 -

FIG. 4. In vitro coupled transcription-translation of pPol, p26,and pUC8. Plasmids (Ad2 derived) were added to the in vitro systemsupplemented with [35S]methionine. The resulting proteins weresubjected to NaDodSO4/polyacrylamide gel electrophoresis.Fluorograms of the gels are shown, together with the positions ofstained molecular mass markers (given in kDa). (A) Uncut plasmidswere used as templates. Lane 1, pPol; lane 2, p26; lane 3, pUC8. (B)Plasmids were digested with Sal I prior to use as templates. Lane 1,pPol; lane 2, p26; lane 3, pUC8. (C) Uncut plasmids were used astemplates. Lane 1, pPol; lane 2, pUC8. The results presented inA andB were from a single experiment; those described in C were obtainedwith a different extract.

containing uncut pPol (Fig. 4A, lane 1) showed severaldistinct bands that were absent in the uncut p26 or pUC8samples (Fig. 4A, lanes 2 and 3) and in the samples digestedwith Sal I (Fig. 4B). The largest pPol-specific band migratedas a 124-kDa protein and had an identical mobility as theprotein detected in pPol-transformed E. coli. Surprisingly,this band was not the predominant product (Fig. 4A). In otherexperiments, such as the one shown in Fig. 4C, it was asubstantial part of the product, although a large array of otherbands was still detected. The variation in these experimentsis perplexing, in part because the origin of the smallermolecular mass bands is not clear. However, Fig. 4B (lane 1)clearly demonstrated that only background bands werepresent in the pPol sample cut with Sal I prior to its use as atemplate for transcription and translation. Thus, it is reason-able to assume that the additional bands detected in the uncutpPol samples were derived from mRNA transcribed from thep8-galactosidase promoter. The smaller molecular mass bandsmay be proteolytic breakdown products of the larger 124-kDapolypeptide, premature termination products caused by non-optimal in vitro translation or transcription, or the result ofinternal initiations on the long mRNA produced, as observedpreviously with other eukaryotic genes expressed in E. coli(32, 33).

Production and Characterization of Antisera DirectedAgainst Ad Pol. Two types of antisera were raised in rabbits.The first serum, called 124-kDa serum, was obtained from arabbit that was injected with the Ad2 Pol fusion polypeptidethat had been excised from a Coomassie blue-stained,NaDodSO4/polyacrylamide gel. The second serum, calledPol serum, was obtained from a rabbit injected with asynthetic peptide conjugated to bovine serum albumin. Thesequence of the peptide was Tyr-Trp-Ile-Glu-Met-Pro. Thelast five amino acids of this peptide comprise the sequencepredicted from the Ad DNA sequence to be the carboxylterminus of the Ad Pol protein. This assumes that the end ofthe large open reading frame in the Ad DNA encodes theterminus of Ad Pol. The amino-terminal tyrosine was addedto facilitate coupling to bovine serum albumin.Both sera were tested for reactivity against the Ad Pol

fusion protein from pPol-transformed bacteria. For thispurpose, extracts prepared from cultures of bacteria trans-formed with either pPol or pUC8 were subjected to electro-phoresis on a NaDodSO4/polyacrylamide gel. The proteinswere then transferred to nitrocellulose by electroblotting.

2356 Biochemistry: Rekosh et al.

Proc. Natl. Acad. Sci. USA 82 (1985) 2357

The nitrocellulose replicas were incubated with appropriatesera, washed, and probed with 125I-labeled protein A. Fig. 5Ashows a Coomassie blue-stained portion of the gel that wasnot blotted; Fig. 5B shows an autoradiogram of the blots inwhich either preimmune (lanes 1 and 2) or 124-kDa immune(lanes 3 and 4) serum was used at a dilution of 1:100. Fig. 5Cshows the resultant autoradiogram when preimmune (lanes 1and 2) or Pol immune (lanes 3 and 4) serum was used at adilution of 1:1000. With each immune serum, a predominantreactive band with a mobility corresponding to the 124-kDaAd Pol fusion polypeptide was detected. Less reactivesmaller molecular mass bands were also observed in theselanes at positions similar to those seen in the in vitrotranslation experiment described above.The reaction of the Pol and 124-kDa.sera with authentic Ad

Pol protein, isolated from infected HeLa cells, was exam-ined. Initially, spot-blot tests were performed with 0.003 unitof purified pTP-Ad Pol complex bound to nitroceilu ose.Subsequently, an appropriate dilution of a commercial oatanti-rabbit serum conjugated to HRP was incubated with theblots. After washing, the Bio-Rad HRP color reagent wasused to develop the blots. Both sera gave a significant signalabove background. The lowest serum dilution that gave adetectable signal was 1:1000 for the Pol serum and 1:100 forthe 124-kDa serum (data not shown).To furthen characterize the 124-kDa serum, a highly

purified fraktion from infected cells containing the pTP-AdPol complex was loaded onto a glycerol gradient and sub-jected to centrifugation as described. The gradient wasassayed for DNA polymerase activity by using an assay thatis specific for Ad Pol (Fig. 6 Upper). Fractions were thenpooled or taken individually as indicated and subjected toelectrophoresis on a NaDodSO4/polyacrylamide gel. Theproteins were then transferred to nitrocellulose, probed witha 1:50 dilution of immune 124-kDa or nonimmune rabbitserum, and developed with HRP-conjugated goat anti-rabbitantibody and Bio-Rad HRP color reagent. The pooled frac-tion contained an immunoreactive band at the positioncorresponding to authentic Ad Pol (140 kDa) (Fig. 6 Lower),as do adjacent fractions of the gradient, indicated by + in Fig.6 Upper.

A B

1 2 1 2 3 4

116

96-

68

43-

29-

C

1 2 3 4

a

FIG. 5. Immunoblots of bacteria transformed with pPol or pUC8.Bacterial cultures containing each plasmid were subjected to elec-trophoresis as described in the legend to Fig. 3. (A) Part of the gel wasstained with Coomassie brilliant blue. Lane 1, pPol; lane 2, pUC8.The remainder of the gel was transferred to nitrocellulose byelectroblotting and probed with various antisera. 251I-conjugatedprotein A was used to detect the bound antibody. Fluorograms of theblots are shown. (B) A 1:100 dilution of serum was used in each case.

Lane 1, pPol with preimmune 124-kDa serum; lane 2, pUC8 withpreimmune 124-kDa serum; lane 3, pPol with immune 124-kDaserum; lane 4, pUC8 with immune 124-kDa serum. (C) A 1:1000dilution of serum was used in each case. Lane 1, pPol withpreimmune Pol peptide serum; lane 2, pUC8 with preimmune Polpeptide serum; lane 3, pPol with immune Pol peptide serum; lane 4,pUC8 with immune Pol peptide serum.

4

00

co3

CD

E0coT

I-2:

- _-++ ++

i

30 Fraction- 140-kDa band

2

200 -

97-

68-

FIG. 6. Reaction of 124-kDa antiserum with Ad Pol. (Upper)Glycerol gradient centrifugation of pTP-Ad Pol complementingactivity. Aliquots (1.5 ul) of indicated fractions were assayed forcomplementing activity with 0.075 jig of EcoRI-linearized pLA1DNA as template. Indicated fractions were subjected to electropho-resis on a NaDodSO4/7.5% polyacrylamide gel that was electroblot-ted to nitrocellulose and prdbed with a 1:50 dilution of 124-kDaantiserum. Fractions marked with a + contained a detectable bandat 140 kDa. (Lower) Immunoblot of pTP-Ad Pol. pTP-Ad Pol (0.12unit) from pooled fractions of the gradient in Upper indicated withthe |-, were run on a gel and electroblotted to nitrocellulose. The blotwas probed with antiserum at a dilution of 1:50 and developed withHRP-conjugated second antibody and Bio-Rad color reagent. Al-though it does not photograph well, the 140-kDa band was clearlyvisible on the original. Positions of molecular mass markers (given inkDa) are shown. Lane 1, immune 124-kDa serum; lane 2, nonimmunerabbit serum.

DISCUSSIONWe have shown that a 3400-bp fragment ofAd DNA, mappingbetween coordinates 23.3 to 13.8, is expressed efficientlyfrom a chimeric plasrhid in E. coli. When inserted in thecorrect orientation downstream from transcription and trans-lation initiation signals a 124-kDa polypeptide is formed.Antibody raised against this polypeptide strongly reactedwith the 140-kDa Ad Pol extracted from Ad-infected HeLacells. Thus, our data provide direct evidence that at least partof the Ad Pol is encoded by this fragment of viral DNA. Inaddition, we have shown that an antiserum raised against asynthetic peptide reacts with the purified protein fractioncontaining Ad Pol activity. Since the sequence of the syn-thetic peptide was derived from the sequence of the DNA atthe 3' end of a large open reading frame contained within thepiece of viral DNA, our. data also position this sequencewithin the Ad Pol. Thus, this report both confirms andextends the existing body of data that suggest that the bulk of

Bio'chemistry: Rekosh et al.

Proc. Natl. Acad. Sci. USA 82 (1985)

the Ad Pol is encoded contiguously within this region of theviral genome.Although both antisera are reactive against the Ad Pol

polypeptide when it is bound to nitrocellulose, these antiserado not inhibit the in vitro DNA polymerase activity or theprotein-priming reaction that leads to the formation of thepTP-dCMP complex (data not shown). It may be that theantibodies were raised against denatured forms of the poly-peptide since a small peptide as well as the denaturedbacterial fusion protein were used as immunogens. If thiswere the case, the value of these sera as affinity reagents forprotein purification would be limited. However, an alterna-tive possibility is that antibody binds to the native proteinwithout affecting its activity.

Expression of the viral DNA from the chimeric pPolplasmid leads to the synthesis both of the intact 124-kDapolypeptide and smaller fragments in in vitro tran-scription-translation assays as well as in the transformedbacterium. We know that most of the fragments isolated fromtransformed bacteria contained the carboxyl-terminal se-quence since they reacted with the antipeptide antiserum.Although it has not been rigorously demonstrated that thefragments seen in vitro are identical to those found in vivo,they have the same apparent molecular mass. Initiation oftranslation on eukaryotic mRNA sequences can occur inbacteria at internal sites ordinarily not used by eukaryotes(32, 33); thus, it is reasonable to assume that a similarmisreading has occurred here. If this were the case it wouldexplain why most of the observed fragments appeared tocontain the carboxyl-terminal sequence.Attempts have been made to purify the 124-kDa protein

from transformed E. coli by using conventional methods.Efforts to isolate the protein by using nondenaturing lysisprocedures failed to solubilize the 124-kDa polypeptide. Theprotein was solubilized by incubation in 8 M urea for at least12 hr and by incubation in the presence of 1% NaDodSO4.Incubation with lower concentrations of urea as well as with2 M potassium isothiocyanate did not solubilize the protein.Others have experienced similar difficulties with the expres-sion of the Ad-encoded Ela protein in E. coli. In this system,however, the protein was solubilized with 2 M potassiumisothiocyanate and was shown to be active after renaturation(34). To assess the biological activity of the 124-kDa protein,we must develop conditions that lead to the solubilization ofnative protein.The ability to produce large amounts of the Ad Pol as well

as other enzymes involved in Ad replication is essential. Therelatively small amounts of these proteins isolated fromAd-infected cells have severely limited studies of this proc-ess. For this reason, additional cloning and purificationstrategies are needed.

We thank Siu-Pok Yee and Phil Branton of McMaster Universityfor help with the anti-peptide antiserum. This work was supported byGrant 5 R01 CA 25674 from the National Cancer Institute (to D.R.),National Institutes of Health Training Grant T32 GM 7288 fromNational Institute of General Medical Sciences (to J.L.), and Grant5 R01 GM 34559 (to J.H.).

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