Vol. 38, No. 2JOURNAL OF VIROLOGY, May 1981, p. 430-4370022-538X/81/050430-08$02.00/0

Genome Structure of Avian Sarcoma Virus Y73 and UniqueSequence Coding for Polyprotein p90

MITSUAKI YOSHIDA,`* SADAAKI KAWAI,2 AND KUMAO TOYOSHIMA2

Department of Viral Oncology, Cancer Institute, Toshima-ku,' and Department of Oncology, Institute ofMedical Science, University of Tokyo, Minato-ku,2 Tokyo, Japan

Received 7 November 1980/Accepted 23 January 1981

The genome structure of a newly isolated sarcoma virus, Y73, was studied. Y73is a defective, potent sarcomagenic virus and contains 4.8-kilobase (kb) RNA as

its genome; in contrast, helper virus associated with Y73 had 8.5-kb RNA, similarto other avian leukemia viruses. Fingerprinting analysis of these RNAs demon-strated that the 4.8-kb RNA contains a specific RNA sequence of 2.5 kb, whichrepresents the transforming gene (yas) ofY73. This specific sequence was mappedin the middle of the genome and had at both ends 1- to 1.5-kb sequences incommon with Y73-associated virus RNA. This structure is very similar to thoseof avian and mammalian leukemia viruses. In vitro translation of the 4.8-kb RNAand the immunospecificity of the products directly demonstrated that polyproteinp90, containing p19, is a product translated from capped 4.8-kb RNA and that thespecific peptide portion is coded by the yas sequence. Protein 90, which was alsofound in cells transformed with Y73, was suggested to be a transforming protein.

Among avian retroviruses, Rous sarcoma virus(RSV) has been most thoroughly studied be-cause of its competency in replication and sar-comagenic transformation. The sarcoma gene(src) in RSV codes a protein, p6OBrc (4, 21), whichhas protein kinase activity phosphorylating ty-rosine residues in target proteins (5, 10). Re-cently, we (27) reported that a newly isolatedavian sarcoma virus, Y73 (11), contains a sar-comagenic transforming gene (yas) showing nohomology with the src sequence of RSV. Fur-thermore, a specific protein, p90, was found inY73-transformed cells and shown to have pro-tein kinase activity, which phosphorylated ty-rosine residues in p90 itself and in heavy-chainmolecules of immunoglobulin G complexed withp90 (13). Fujinami sarcoma virus (7) was alsorecently reported to have a distinct class oftransforming gene (8, 16).

Independent isolates of avian sarcoma virusesRSV and B77 are competent in replication andhave virtually the same genome structure: 5' gagpol env src 3' (26). On the other hand, all avianacute leukemia viruses are defective in replica-tion, deleting the pol and env genes and part ofthe gag gene, instead of the region containingthe transforming gene in the middle of the ge-nome (3, 6,9, 18,22). Since Y73 is a sarcomagenicvirus, but is defective in replication (11), itseemed interesting to determine its genomestructure.

In this work, we characterized the Y73 genomeby RNase T1 fingerprinting analysis and in vitro

translation of the genome RNA. Results showedthat the structural features of the Y73 genomeare essentially the same as those of genomes ofother acute leukemia viruses and that polypro-tein p90 is a gene product of the specific se-quence (yas), suggesting that p90 is a transform-ing protein.

MATERLALS AND METHODSCells and viruses. Chicken embryo fibroblasts

were prepared from C/O or C/BE chicken embryosand cultured as described before (28). Y73 viral stockwas grown as described previously (13), and Y73-as-sociated virus (YAV) was isolated by two successivelimiting dilutions on chicken embryo fibroblasts andconfirmed to have no focus-forming ability under thestandard agar layer.

Viral preparations were harvested every 12 h andpurified by precipitation with ammonium sulfate andby successive discontinuous and continuous sucrosegradient centrifugations as described previously (30).Labeling of viral RNA with [32P]phosphate was carriedout exactly as described previously (30).Preparation of viral RNA. Virion RNA was ex-

tracted by proteinase treatment followed by phenolextraction (30). Virions were disrupted by adding 0.1volume of 10% sodium dodecyl sulfate in the presenceof 100 ,Lg of proteinase K per ml. After incubation for30 min at 37°C, RNA was extracted three times withphenol-chloroform (6:4) and then precipitated with 2volumes of ethanol. RNA was dissolved in 0.1% sodiumdodecyl sulfate and heated at 100°C for 1 min andthen fractionated on a gradient of 15 to 30% sucrosecontaining 50 mM Tris-hydrochloride (pH 7.5), 0.1 MNaCl, and 1 mM EDTA by centrifugation at 45,000rpm for 3 h in a Beckmann SW50.1 rotor at 15°C. In

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GENOME STRUCTURE OF Y73 431

some experiments, RNA containing polyadenylic acid[poly(A)] was separated by passing the preparationthrough a column of oligodeoxythymidylic acid-cellu-lose as described before (31).

Fingerprinting analysis of [32PJRNA. Two-di-mensional gel electrophoresis was used for fingerprint-ing RNase Ti-resistant oligonucleotides. The proce-dure was essentially as described by Lee and Wimmer(17). Oligonucleotides separated on gels were extractedwith 0.5 ml of water, lyophilized, and digested withRNase A (0.1 mg/ml). The digests were analyzed byelectrophoresis at pH 3.5 on DEAE-cellulose paper asdescribed by Barrell (1).

In vitro translation of viral RNA. mRNA-de-pendent reticulocyte lysate was prepared as describedby Pelham and Jackson (20) and used to translate Y73RNA as described previously (29). Each reaction mix-ture (12 pl) contained 0.2 jlg of viral RNA and 20 ACiof [3S]methionine in addition to the standard re-agents. Two microliters of the reaction mixture wasused for gel electrophoresis in the buffer described byLaemnmli (15), and the gels were dried for autoradiog-raphy or fluorography. Cells were labeled with [3S]-methionine as described previously (31), and immu-noprecipitation was carried out according to Kessler(14).

RESULTSRNA of Y73. Since the original Y73 stock

contained a large excess of YAV of subgroup Aas helper (11), the transformed cells were clonedin soft agar medium. Viruses released from theselected producer cells were labeled with [32P]-phosphate, and the RNA was analyzed by aga-rose gel electrophoresis. As shown in Fig. la,

a b c

I

8.5kb-

28S -48kb-

0x

Ea

18S-

most of the RNA containing poly(A) migratedas high-molecular-weight aggregates, probably50-70S dimers. After heat treatment at 1000Cfor 1 min, the aggregates were dissociated intotwo RNA species: 4.8-kilobase (kb) and 8.5-kbRNA components (Fig. lb). The 4.8-kb RNAextracted from the gel sedimented as 26S onsucrose gradient centrifugation (Fig. le), and theRNA was smaller than those of known defectiveacute leukemia viruses (3, 6, 12, 18). On theother hand, 8.5-kb RNA sedimented as 35S, andits mobility in the gel was the same as that ofRNA of transformation-defective RSV. Fromthese results, and from the findings that non-transforming YAV isolated by limiting dilutioncontained only 8.5-kb RNA and that the 4.8-kbcomponent has a specific sequence, as shown inthis paper, we concluded that the 4.8-kb RNAspecies is the genome of Y73 carrying the sar-comagenic transforming gene.Analysis of Y73 RNA by fingerprinting.

The relationship between 4.8-kb and 8.5-kbRNAs was analyzed by fingerprinting RNase T1-resistant oligonucleotides. 32P-labeled 4.8-kb and8.5-kb RNAs were separated by agarose gel elec-trophoresis and selected for poly(A)-containingRNA on oligodeoxythymidylic acid-cellulose.The fingerprint of the 8.5-kb RNA (Fig. 2D)showed that the helper 8.5-kb RNA was a singlecomponent, and as expected, the fingerprint of4.8-kb RNA contained the specific oligonucleo-tides that were missing in that of 8.5-kb RNA,

f ract'ion no.

FIG. 1. Analysis of Y73 RNA by agarose gel electrophoresis (a, b, c) and sucrose gradient centrifugation(d, e). [32P]RNA of Y73 virions containing poly(A) before (a) and after (b) heat treatment at 1X0°C for 1 minand [32PJRNA of transformation-defective Prague RSV were subjected to electrophoresis in 2% agarose gelin 40mM Tris-hydrochloride (pH 8.0), 20mM sodium acetate, and 1 mMEDTA. After electrophoresis, RNAswere extracted from the bands corresponding to 4.8-kb and 8.5-kb RNA and centrifuged through 15 to 30%sucrose gradients containing 20mM Tris-hydrochloride (pH 7.5), 0.1 M NaCi, 1 mM EDTA, and 0.2% sodiumdodecyl sulfate in an SW50.1 rotor at 45,000 rpm and 15°C for 180 min. (d) 8.5-kb RNA; (e) 4.8-kb RNA.Dotted lines are [3H]rRNA added as internal markers..

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432 YOSHIDA, KAWAI, AND TOYOSHIMA00: .- fE-; S.-S ffi-. 0'tV,0000000 .. 00.: 00077.?:0

A 00 ;f}0 -u:0- 0-0 SS00: :00 000:E. :fX

FIG. 2. RNase T, fingerprinting analysis of Y73 RNA. [32PJRNA of Y73 was fractionated into 26S (A) and35S (D) fractions, and each fraction was treated with weak alkali to introduce one or two cuts in the RNAmolecule, as described previously (27). Then RNA fragments containingpoly(A) were separated on oligodeox-ythymidylic acid-cellulose and fractionated into six fractions by sucrosegradient centrifugation. Each fractionwas recentrifuged, and the peak fraction was digested with RNase T, and fingerprinted by two-dimensionalgel electrophoresis. (A) 26S RNA; (B) 18-20S RNA from 26S RNA; (C) 5-14S RNA fromt 26S RNA; (D) 35SRNA; (E) 19-25S RNA from 35S RNA; (F) 5-14S RNA from 35S RNA.

indicating that 4.8-kb RNA contained the spe-

cific nucleotide sequences (Fig. 2A). All of theoligonucleotide spots found in the fingerprint of8.5-kb RNA were detected in that of 4.8-kbRNA, but most ofthem were minor components.These minor spots were probably contaminationof the 4.8-kb RNA fraction with fragments of8.5-kb RNA. To verify this explanation, eachspot was cut out from the gels and quantitatedby counting the radioactivity of 32P. Since theaccurate length of each oligonucleotide was notknown, the ratio of the yield of each oligonu-

cleotide from the 4.8-kb RNA fraction to that ofthe corresponding spot from 8.5-kb RNA wasused to estimate the apparent molar yield ofeach oligonucleotide, assuming that pure 8.5-kbRNA gave each oligonucleotide in a molar ratio.In the case of spots specific to 4.8-kb RNA, theyield of oligonucleotides of 8.5-kb RNA whichwere similar in size was used to calculate theratio. The results are summarized in Table 1. Allspots were classified into three groups by theiryield ratio, that is, less than 0.5, between 0.8 and1.2, and more than 1.4. Oligonucleotides showing

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TABLE 1. Yioligonucleotides fSSpots in 26S RNA

fraction

No. Yield(cpm)

1 7752 7363 2954 9855 2916 1267 9788 2309 20110 74511 16812 49413 77914 55615 19216 13917 35318 32019 18523a+b 86724 38925 13127 20128 21629 80630 16431 61632 14238 56439 57540 10141 22742 56843 60744 32045 50046 16447 177

GENOME STRUCTURE OF Y73 433

'ields ofRNase Ti-resistantrom 26S and 35S RNA fractions

Spots in 35S RNAfraction Ratio 26S/

No. Yield 35S(cpm)

(1.03)a(0.98)

51 753 0.3952 643 1.5053 779 0.37

(0.18)54 702 1.3955 502 0.4656 491 0.4157 553 1.3558 441 0.38

(1.12)59 562 1.38

(1.19)60 466 0.4161 379 0.36

(0.91)(0.82)

62 388 0.4765 466 1.86

(1.08)66 358 0.3668 536 0.3769 524 0.4170 542 1.4971 486 0.3472 367 1.6773 486 0.2980 393 1.4381 355 1.6182 234 0.43

(0.97)(1.06)(1.13)(0.83)

77 383 1.3078 415 0.3979 397 0.44

'The ratio of oligonucleotides specific to the 26SRNA fraction was calculated by using the yield (countsper minute; cpm) of oligonucleotides of 358 RNA,which are similar in size to the specific one; this ratiois shown in parentheses.

a ratio of 0.8 to 1.2 were all specific to 4.8-kbRNA, and all other oligonucleotides showing aratio of less than 0.5 or more than 1.4 wereconfirmed by RNase A digestion to have thesame nucleotide composition as the correspond-ing spots obtained from 8.5-kb RNA (data notshown). From these findings it was concludedthat 22 oligonucleotides produced at a ratio ofmore than 1.4 (10 spots) and between 0.8 and 1.2(12 spots) originated from 4.8-kb RNA and thatthe rest of the minor spots produced at a ratioof less than 0.5 represented 8.5-kb RNA frag-

ments contaminating the 4.8-kb RNA fraction.Spot no. 6 showed a very low yield although

it was specific to the 4.8-kb RNA fraction. Thisspot might be explained by a microheterogeneityin the sequence of 4.8-kb RNA, but the reasonis unknown. Thus, spot no. 6 was removed fromthe following considerations.

Analysis of the nucleotide composition of 22oligonucleotides originating from 4.8-kb RNAsuggested that 11 oligonucleotides were specificand produced in a ratio of 0.8 to 1.2 and that 10oligonucleotides were shared with YAV 8.5-kbRNA and thus were produced at a ratio of morethan 1.4 (Table 2). Assuming a random distri-bution of the oligonucleotides along the genome,the results suggest that about half the 4.8-kbsequence is specific to 4.8-kb RNA.For determination of the location of the oli-

gonucleotides specific to 4.8-kb RNA within thegenome, RNA fragments containing poly(A)were fractionated and purified by sucrose gra-dient centrifugation, followed by recentrifuga-tion of each fraction and fingerprinting (Fig. 2Band C). Although the 4.8-kb RNA fraction wascontaminated with fragments of 8.5-kb RNA,only 22 spots, which were concluded to be pro-duced from 4.8-kb RNA (Table 2), were fol-lowed, and the other spots were ignored. Thus,a tentative oligonucleotide map was constructed(Fig. 3). YAV 8.5-kb RNA was analyzed similarly(Fig. 2E and F), and the two oligonucleotidemaps were compared. Ten oligonucleotides werecommon to both RNAs, and 4 of these 10 were

TABLE 2. Qualitative analysis ofRNase T,oligonucleotides of Y73 26S RNA and

YAV 35S RNA

Sptn.Spot no.

of Y73 oSfYA RNase A digestion products26S RNA RNA

1 U,C,G2 U, C, AC, G4 52 U, C, AC, AU, AAG6 U, C, AC, AU, AAAU,G7 54 U,C,AC,AU,AAC,AAG

10 57 U, C, AAG, AAAU12 U, C, AC, AG, AAAU13 59 U, C, AU, AG, AAC, AAAC14 U,C,AC,AU,AAC,G17 U, C, AC, AU, AAC, AAU, G18 C, AC, AU, AAC, AAU, AAAC, G23 a + b 65? U, C, AC, AU, AG, AAC, AAAG24 C, AC, AG, AAAC29 70 U, C, AC, AU, G31 72 U,C,AC,AU,AAC,G38 80 U, C, AU, AAU, G39 81 U, C, AU, G41 U, C, AU, AG42 U, C, AC, AU, AAU, AAAN,a G43 U, C, AC, AU, AG, AAC, AAU44 U, C, AC, AU, AG45 77 C, AU, AAU, AAAC, Ga N, Not identified.

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434 YOSHIDA, KAWAI, AND TOYOSHIMA

mapped within about 1 kb adjacent to poly(A)at the 3' end; the other 6 were detected only inthe poly(A)-containing RNA fragments largerthan 20S for the Y73 and 30S for YAV, and werethus mapped within about 1 to 1.5 kb in the 5'regions of the genomes. The remaining oligonu-cleotides specific to 4.8-kb RNA were mapped inthe middle portion of the 4.8-kb genome RNA.These results suggest that the 4.8-kb specificRNA sequence is composed of a continuous se-quence of about 2.5 kb of RNA and is flanked atthe two ends with 1- to 1.5-kb RNA sequencescommon with those of helper YAV RNA.In vitro translation ofY73 RNA. To study

YAV / O X 00t1- J o 1f CNiYAV ( Ln UINr) , U" 0 00 i.0 D P.,1 r-... N- to LODto

the gene products coded by the specific se-quence, poly(A)-containing RNA isolated fromY73 virions was fractionated by size (Fig. 4A)and translated in an mRNA-dependent reticu-locyte lysate (20). Polyacrylamide gel analysis(Fig. 4B) of the translation products showed thatPr76 was the main product from RNA fractionslarger than 28S, as found in previous experi-ments on translation of avian erythroblastosisvirus RNA (29). This result was also expected,because the higher-molecular-weight fraction ofRNA contained mainly helper YAV RNA inwhich the gag gene is located near the 5' ter-minus and the protein-synthesizing system from

L1 1. _-. He_CA NDioxo.1. U iS sto r-I s sP. to W to )>A

5' I I I I C0Ci8.5kb 8 7 6 5 4 3 2 1 0

4.8kbY73 CaPE2ZE= I I 71;7yrovKzj :!

(..oow-.o.oo~~~~~~~~~~~~~cNc%JX N-o0.0-.0~~~~~~~~~~~~~-r!.X- C4__ ____( 4- tn X--_-CN M-

Wx "- ;-) a

.5o90Protein r

9 + -ppl9 + ? specific polypeptide

FIG. 3. Physical maps of oligonucleotides and polypeptide coded by Y73. Spot numbers with a single lineare oligonucleotides shared with 4.8-kb and 8.5-kb RNAs, and those with a double line are specific to 4.8-kbRNA. The orders of the oligonucleotides within the respective parentheses are uncertain.

A

(I) /) (I)

04CI N oo

B b 3 4 5 b 7 8 2<

90-Pr76-

fraction no.

FIG. 4. Polyacrylamide gel electrophoresis of the translation products of Y73 RNA and helper YAVRNA.Y73 RNA containing poly(A) was fractionated on a sucrose gradient (A) as described in Fig. 1, and about 0.2pg ofRNA in each fraction was translated in an mRNA-dependent rabbit reticulocyte lysate, containing 20pCi of [35S]methionine, and analyzed on a 7 to 15%o polyacrylamide gradient gel (B). Track b had no addedRNA; other tracks are labeled with numbers corresponding to the fractions ofRNA from the sucrose gradientin (A). Track YAV is 0.5 pg of heat-treated 70S RNA ofhelper YA V.

OKIOP"& I

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eucaryotic cells translates only one gene nearthe 5' end of polycistronic viral mRNA (19, 21,25).

In contrast to Pr76, a product with a molecularweight of about 90,000 (p90) was synthesizedmost efficiently from slightly smaller RNA than28S (Fig. 4b). Since fractions of similar sizedRNA ofYAV did not support detectable synthe-sis of p90, the results indicate that p90 was codedby the 4.8-kb RNA of Y73. The effect of 7-methyl-GTP, a cap analog, on the synthesis ofp90 was tested to know whether p90 was synthe-sized by intact capped RNA or fragmented RNA(2). As seen in Fig. 5, the syntheses of p90 andPr76 were both strongly inhibited by the addi-tion of 7-methyl-GTP. On the other hand, theamount of smaller minor products was not re-duced by addition of 7-methyl-GTP. These find-ings strongly support the idea that p90 is syn-thesized by intact 4.8-kb RNA which has a capstructure at the 5' terminus and also that smallerproducts are synthesized by fragmented RNA.The in vitro product p90 was found to be

immunoprecipitated with serum against dis-rupted virions of B77 (Fig. 5). Since this antise-rum mainly contained antibody to gag proteins,p90 was suggested to contain amino acid se-quences related to gag proteins. This anti-B77antiserum precipitated p90 from an extract ofcells transformed with Y73, and, as seen in Fig.5, no detectable difference was observed in themolecular sizes of the in vivo and in vitro prod-ucts. This strongly suggests that these two p90proteins are identical. When another antiserumthat had no antibody activity against p19 wasused, Pr76 and p27 were precipitated from trans-formed cells, but p90 was not (Fig. 5). This resultdemonstrated that p90 contained the amino acidsequence of p19, but not that of p27. However,this experiment does not exclude the possiblepresence of a partial sequence of p27 in the p90molecule.

DISCUSSIONIn this paper, we characterized the genome

structure of Y73, a newly isolated avian sarcomavirus which, like RSV, induces fibrosarcoma inchickens (11). The transforming gene (yas) ofY73 was previously characterized as a new typeof sarcoma gene with no homology with src ofRSV or fps of Fujinami sarcoma virus (27). Inthe present study, the yas sequence, which wascharacterized by specific oligonucleotides, wasmapped in the middle portion of the genome,with portions of 1 to 1.5 kb common to the 3'and 5' ends of helper YAV RNA at the two ends.This structural feature of the genome makes itvery similar to the genomes of avian acute leu-

GENOME STRUCTURE OF Y73 435

Bh

-p9O~Pr76

FIG. 5. Comparison of in vitro and in vivo trans-lation product p90. (A) Effect of 7-methyl-GTP on invitro synthesis ofp90; 35S RNA (a, b) and 26S RNA(c, d) were translated as described in Fig. 4 in theabsence (a, c) or presence (b, d) of200mM 7-methyl-GTP. The translation products in the absence of 7-methyl-GTP were immunoprecipitated with rabbitantiserum against disrupted virions, andprecipitateswere analyzed on a 9% gel; (e) and (t) are immuno-precipitated translation products from 35S RNA and26S RNA, respectively. (B) Extracts of cells infectedwith YAV (g) or with Y73 (h, i) were mixed with anti-gag serum, which had anti-p19 activity (g, h) or noanti-p19 activity (i), and the precipitates were ana-lyzed on the gel.

kemia viruses such as avian erythroblastosis vi-rus (3) or MC29 (18), to those of Fujinami sar-coma virus (8, 16), and to those of mammaliansarcoma-acute leukemia viruses (23, 24). Be-cause of the close similarity of the genome struc-ture of Y73 to that of avian acute leukemiaviruses, we have tested its leukemogenic capac-ity in chickens. One-week-old chickens were in-oculated intravenously with Y73 (1.5 x 104 focus-forming units) or with avian erythroblastosisvirus pseudotyped with Rous-associated virustype 1 (104 focus-forming units) as control. Allchickens inoculated with avian erythroblastosisvirus developed erythroblastosis within 8 days;however, no sign of leukosis was detected inchickens inoculated with Y73 by examination of

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436 YOSHIDA, KAWAI, AND TOYOSHIMA

peripheral blood at 3- to 5-day intervals up to 1month. Most of these birds developed subcuta-neous sarcoma at the site of intravenous inoc-ulation within 2 to 3 weeks. Eight survivingchickens were autopsied at 1 month; six hadsarcomas in one or multiple sites or internalorgans such as spleen, lung, liver, or muscle, inaddition to subcutaneous tumors. These obser-vations unequivocally confirmed Y73 as a defec-tive sarcoma virus. Thus, the replication-com-petent avian sarcoma viruses RSV and B77 areexceptional in genome structure, and generallythe features of sarcoma-acute leukemia viralgenomes are defective, with the transforminggene located in the middle of the genome.

In vitro translation of 4.8-kb RNA has directlyshown that a polyprotein of 90,000 daltons (p90)containing p19 but not p27 is a specific transla-tion product. The p90 was translated from agene near the 5' end of the intact genome RNAcontaining the cap structure. The presence of anapproximately 1.5-kb sequence homologous withthe 5' region of YAV RNA suggested that p19was the N-terminal polypeptide component inp90, because even if a long untranslated regionsuch as 0.5 kb were present in the 5' region, theRNA sequence coding for p19, an N-terminalpeptide in Pr76, could be included in the 5'region homologous with YAV RNA. Thus, apolypeptide of about 71,000 daltons should becoded by a specific nucleotide sequence of about2.5 kb and therefore would be a specific polypep-tide. This conclusion was supported by the find-ings that anti-gag serum that had no anti-p19activity did not precipitate p90 and that rabbitantiserum against p6Oarc coded by Schmidt-Rup-pin strain RSV did not cross-react with p90 (13).

Since most of the specific sequence in the 4.8-kb genome would be used for p90, p90 seems tobe the only polypeptide coded by the Y73 ge-nome. This possibility is consistent with thefinding that 4.8-kb RNA is the only speciesdetectable with specific complementary DNA(cDNAya,) in cells transformed with Y73. Theseconsiderations strongly suggest that p90 is in-volved in the mechanism of sarcomagenic trans-formation of infected cells. This prediction issupported by the finding that p90 detected incells transformed with Y73 had protein kinaseactivity for phosphorylation of tyrosine residuesin p90 itself and in heavy-chain immunoglobulinG complexed with p90 (13). However, we did notexclude the possible formation of smaller prod-ucts coded by the Y73 and undetectable by ourmethods.

ACKNOWLEDGMENTSWe thank R. Ishizaki for providing rabbit antiserum, and

Y. Hirayama for excellent technical assistance.This work was partly supported by a Grant-in-Aid for

Cancer Research from the Ministry of Education, Sciencesand Culture of Japan.

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