DNA and RNA from Uninfected Vertebrate Cells Contain Nucleotide Sequences Related to the Putative Transforming Gene of Avian Myelocytomatosis Virus

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0022-538X/79/08-0514/08$02.00/0

DNA

and RNA

from

Uninfected Vertebrate

Cells Contain

Nucleotide Sequences Related to the

Putative

Transforming

Gene of Avian

Myelocytomatosis

Virus

DIANASHEINESS* ANDJ. MICHAELBISHOP

DepartmentofMicrobiology and Immunology, Universityof California,SanFrancisco, California94143

Received forpublication19March1979

The avian carcinoma virus MC29 (MC29V) contains a sequence of

approxi-mately1,500 nucleotides whichmay represent a generesponsiblefor

tumorigen-esisbyMC29V. Wepresent evidence that MC29V hasacquired this nucleotide

sequencefrom the DNAof its host. The hostsequencewhichhasbeen incorpo-rated byMC29V is transcribed into RNA in uninfected chicken cells and thus

probably encodesa cellulargene. We haveprepared radioactive DNA

comple-mentary totheputativeMC29V transforminggene

(cDNAMc2q)

andhave found thatsequences homologousto

cDNAMc2g

arepresentinthe genomesofseveral uninfected vertebratespecies.TheDNAofchicken,the natural host forMC29V, contains at least 90% of the sequences represented by

cDNAMc2q.

DNAs from otheranimals show significant but decreasing amounts of complementarity to

cDNAMc2S in accordance withtheirevolutionary divergencefrom chickens; the

thernal stabilitiesofduplexesformed between

cDNAMc2S

and avian DNAsalso

reflect phylogenetic divergence. Sequences complementary to

cDNAMc2g

are

transcribed into approximately 10 copies per cell of polyadenylated RNA in uninfected chicken fibroblasts. Thus, the vertebrate homolog ofcDNAMc29may

beagenewhich hasbeen conservedthroughoutvertebrateevolution and which served as a progenitor for the putative transforming gene ofMC29V. Recent experimentssuggestthat theputativetransforminggeneofavianerythroblastosis virus, like that of MC29V, may have arisen by incorporation of a host gene

(Stehelinetal., personal communication). These findings for avian

erythroblas-tosis virus andMC29Vclosely parallelpreviousresults, suggestingahost origin forsrc (D. H.Spector, B. Baker, H. E. Varmus, and J. M. Bishop, Cell

13:381-386, 1978; D. H. Spector, K. Smith, T. Padgett, P. McCombe, D. Roulland-Dussoix,C. Moscovici, H. E.Varmus,and J. M.Bishop, Cell 13:371-379, 1978;D.

H. Spector, H. E. Varmus, and J. M. Bishop, Proc. Natl. Acad. Sci. U.S.A. 75:

4102-4106, 1978;D.Stehelin, H. E. Varmus, J.M.Bishop, and P.K.Vogt, Nature

[London] 260:170-173, 1976), the gene responsible for tumorigenesis byavian

sarcoma virus. Avian sarcoma virus, avian erythroblastosis virus, and MC29V, however, induce distinctly different spectra of tumors within their host. The

putative transforming genes of these viruses share no detectable homology,

althoughsequenceshomologoustoall threetypesofputativetransforminggenes

occur and are highly conserved in the genomes of several vertebrate species.

These data suggest that evolution of oncogenic retroviruses has frequently

in-volvedamechanismwherebyincorporationand perhapsmodification of different host genesprovideseachvirus with the ability to induce its characteristic tumors.

Avian retroviruses induceneoplastic transfor- from endogenous sarc are found in

polyriboso-mation in avarietyofcells,includingfibroblasts, mal RNA (23) and aprotein closelyrelated to

epithelialcells,andhematopoieticcells(1). The the product of src has been detected in unin-best-studiedexampleistheaviansarcoma virus fected vertebrate cells (6, 19a).Hence,

endoge-(ASV),which transformsfibroblastsvia asingle nous sarc apparentlyencodes an essential

cellu-viral gene, src(32).Highly conserved nucleotide lar gene that may have served asprogenitorfor sequences homologousto src (denotedendoge- srcof ASV. Thesefindingsraise thepossibility

noussarc) are present in the DNA of chickens that thecapacityof avian retrovirusesto trans-(25, 28) and other vertebrates (25); transcripts form cells other than fibroblastswas also

gen-514

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erated from cellular genetic determinants. We

haveexamined this possibility by analyzingthe

phylogeneticoriginsofMC29 virus (MC29V), an avian retrovirus that induces carcinomas and sarcomasin birdsandtransformsfibroblasts and

macrophages in cellculture, yetlacks thegene src(7, 22, 26).

The genome of MC29V contains a sequence

of approximately1,500nucleotides that may

en-code the oncogenic capacityofthe virus (8, 22). WehavepreparedradioactiveDNA(CDNAMC2S) complementary to thissequence (22) and have used this DNAtodemonstratethat theputative transforming gene of MC29V is unique to

MC29Vand thecloselyrelated virus MH2V (D. Sheiness, L. Fanshier, and J. M. Bishop,

manu-script inpreparation). In thepresent

communi-cation, we show that the genomes of several birds and other vertebrates include a highly conserved homolog of

cDNAMc2S;

this homolog

is transcribed into polyadenylated RNA in

un-infected avian cells. Ourfindings, and those of

Stehelinetal.(D.Stehelin, I. Saule,M.Roussel, A.Sergeant, C. Lagrou, C.Rommens, and M. B.

Raes, Cold Spring Harbor Symp. Quant. Biol.,

in press) with avian erythroblastosisvirus, indi-catethat each of the distinctiveavian retrovirus

transforminggenes mayhavebeen derived from aseparate genetic determinantin the avian ge-nome. The putative progenitors of oncogenic genes appear not to be structurally related to one another, and their function in cellular

growth or metabolism is not presently known. Elucidation of these alleged functionsmight

pro-videuseful insights into the mechanisms of

on-cogenesis and the origins oftargetcell specificity inneoplastic transformation.

MATERIALS AND METHODS Cels andviruses. Fertile chicken eggs were ob-tained from H & N Farms, Redmond, Wash.; quail eggs werefrom Life Sciences, Gainesville, Fla.; duck eggswerefrom Reichard Farms, Petaluma, Calif. A clonal stock of MC29V/MCAV-A was obtained as previously described (22); MC29V/MCAV-A was propagated in C/BE chickcellswhich lacked the avian retrovirus group-specific antigen. XC cells were de-rived froma tumorproduced in rats with the Prague-C strain of ASV (30) and kindly provided by J. A. Levy. TheSchmidt-Ruppin subgroup D strain of ASV

wasobtainedfrom Peter Vogt and propagated in

ran-dom-bredchf-/gs-chicken embryo fibroblasts. Preparation of RNA from uninfected and MC29V-infected chicken cells. Total cellular RNA fromcultures of chicken fibroblasts was purified by a previously published method (33). Some RNA prepa-rationswere passed through acolumn of oligo(dT)-cellulose(T3 grade;CollaborativeResearch Inc., Wal-tham, Mass.) inbuffer containing 0.5 M NaCl-0.003

M EDTA-0.02 M Tris-hydrochloride (pH 7.4)-0.5%

(wt/vol) sodiumdodecylsulfate tobind the polyade-nylated RNA fraction. Bound RNA was eluted with 0.01 M Tris-hydrochloride (pH 7.4)-0.003 M EDTA and thenprecipitatedwith ethanol.

Preparation of cellular DNAs. UnlabeledDNA

wasextracted from the following sources: 10- to

12-day-old chicken, quail, and duck embryos; MC29V-infectedchicken embryo cells grown inculture; rhea lungs; BALB/cmousemammarygland tumors; Esch-erichia coli cells (provided by Herbert Boyer); and XC cells. Rhea tissuewas a kind gift from Lynn A. Griner of the SanDiego Zoological Garden, San Diego, Calif. Homogenized tissuesorcellsweresuspended in buffercontaining0.1 M NaCl, 0.02 M Tris (pH 7.4), 0.003M EDTA, 1% sodiumdodecyl sulfate,and 500

,ug

of Pronase per mlat37°Cfor2 to18hand extracted twice withphenol-chloroform (1:1). NaClwasadded

toafinal concentration of0.3M, and high-molecular-weight DNAwasspooledoutafter the addition oftwo

volumes of ethanol. High-molecular-weight salmon spermand calfthymus DNAswere purchased from Sigma ChemicalCo., St. Louis, Mo., extracted with phenol-chloroform, andspooled out of ethanolas de-scribed above.

The DNAsweresuspended in 20 mM Tris (pH

7.4)-0.01 M EDTA, reducedto anaveragelength of 350 nucleotides by mechanical shearing in an Amicon DNA pressurecellat40,000to50,000lb/in2,extracted withchloroform-isoamyl alcohol (24:1), and precipi-tated withethanol. RNAwasdegradedeitherby diges-tionwith 100,g ofpancreatic RNase (Worthington BiochemicalCorp.,Freehold, N.J.) perml,followed byphenol-chloroform extraction and ethanol precipi-tation,orbytreatinentwith0.1NNaOHat37°Cfor

10minfollowed by neutralization and ethanol precip-itation. Most DNApreparationswerepassedthrough

a column of Sephadex G50 before further use, or

alternatively, werefurtherpurified by being precipi-tated threemoretimeswithethanol.

"C-labeled unique-sequencechicken DNAwas pu-rifiedfrom chicken embryo fibroblasts grown in the presence of ['4C]thymidine (12). After mechanical shearing, RNAse treatment, and passage through Sephadex G50, the DNA was denatured and

rean-nealedto aCotof300in0.6MNaClat68°C;the DNA whichdidnotreassociatewasrecoveredby fractiona-tiononhydroxyapatiteandallowedtoannealto aCot of 600, and thefinal nonreassociatedportionwas iso-lated after another fractionation on hydroxyapatite. Preparationof cDNA's. [3H]cDNAMc2S (2 x 104 cpm/ng),whichwascomplementarytonucleotide se-quences presentinthe genome of MC29V butnotthe genome ofMCAV, waspreparedasdescribed previ-ously(22).

[32P]cDNAs77(105 cpm/ng)wassynthesized by the endogenous reverse transcriptase of detergent-acti-vated B77ASV and purifiedasdescribed previously (27). cDNA madeby this method consists mostly of a fragment101nucleotideslong which is complementary

tothe5'terminus ofthe B77 genome and also contains

asmall proportion of material transcribed from the

entireASV genome (11).

Nucleicacidhybridization. Stringentconditions for DNA-DNA or DNA-RNA hybridizations were

68°C in 0.6 M NaCl-0.01 M Tris (pH 7.4)-0.01 M

31,

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EDTA. Hybridization of radioactive cDNA with RNA was measured as resistance to hydrolysis by Si

nu-clease (17),andhybridization between radioactive and unlabeled DNA was detectedby fractionationon hy-droxyapatite at60°C. A1- to2-mlamountofpacked hydroxyapatite was used for each200,ugofDNA, and hybridization mixtureswereloaded onto columns in 0.012 M sodium phosphate buffer (pH 6.8); single-stranded DNA waseluted with 0.12 M sodium phos-phate buffer, and double-stranded DNA waseluted with 0.4 M sodiumphosphatebuffer.Radioactivityin eluted fractions was assayed by precipitation with trichloroaceticacid;quenching ofradioactivityin

sam-plescontainingmorethan200Mgofnucleic acidwas

minimizedbysoaking filtersovernightinasolutionof 90%Omnifluor (New England NuclearCorp., Boston, Mass.), 9% Protosol (NewEnglandNuclear),and 1% water (Hetch-Hetchy Reservoir, Yosemite National Park,Calif.)tohydrolyzethe nucleicacids.

Conditions enhancing theformation of stable du-plexes between DNAshaving partially mismatched sequenceswere590C in1.5MNaCl-0.02 M Tris(pH 7.4)-0.003 M EDTA (20). The fraction of radioactive DNA induplex was measuredbyhydroxyapatite chro-matographyat50'C;single-strandedDNAwaseluted with 0.14 M sodium phosphate buffer, and double-stranded DNAwaseluted with 0.4Msodium phos-phate buffer. Radioactivity in eluted fractions was

assayed as described above.

Values forCotandCrtwerecorrected to standard conditions(5).

Thermaldenaturation of DNA-DNAduplexes. Avian DNAswereincubated with[3H]cDNAMc2gfor lengths of time sufficienttoallow the maximal amount ofannealing,then dilutedto afinal volume of5ml of 0.14 M sodium phosphate buffer (pH 6.8). Samples wereloaded onto hydroxyapatite columns in a circu-lating water bath at 50°C. After the columns were washed threetimeswith 1.3 volumes of0.14Msodium phosphatebuffer, an internal standard wasapplied to eachcolumn,consisting ofasampleof XCcellDNA (DNA fromaratcell line transformed by ASV)

an-nealed to [32P]cDNAB77. Columns were thoroughly washed, and then the temperaturewasraised in incre-mentsof4to5°C. After5minateachtemperature, thermallydenatured cDNA waseluted from each col-umnwith 1.3 volumes of 0.14 M sodium phosphate buffer, and radioactivity in each wash was measured asdetailed in the sectiondescribing DNA hybridiza-tion. Tm was defined as the temperatureatwhich 50%

oftheoriginallyhybridized cDNAwas eluted from the

column.

Thermal denaturation of RNA-DNAduplexes.

RNA fromuninfectedorMC29V-infected chicken em-bryoscellswas incubated with [3H]cDNAMc29under conditionsthat ensured maximal hybridization. Por-tions were removed for determination of initial S1 nucleaseresistance, andthen reaction mixtureswere

dilutedto afinal concentration of0.1 M NaCl-0.005

MTris(pH 7.4) and a final volumeof 1.0ml.Aportion of SR-D ASV RNAwhich had hybridized to [32p]_

cDNAB77wasadded to each reaction tubetoserveas an internal standard, and then the mixtures were

covered witha thinlayer of mineraloil.Sampleswere

placedinacirculating water bath at an initial

temper-ature of50°C; after8 min, a 100-ll sample was re-moved to 1 ml ofS1 nuclease buffer containing S1 enzymeandplacedat0°C until the completion of the thermaldenaturation. The temperaturewasraisedat 4 to 5°C intervals, and additional portions were re-moved after 8 minateach temperature. The fraction of cDNA remaining in hybrid at eachtemperature was determined by hydrolysis with S1 nuclease. Tm was definedasthe temperatureatwhich 50% of the radio-activity originally in hybrid became susceptible to S1 nuclease.

RESULTS

Genomes of birds and other vertebrates contain nucleotide sequences related to

cDNAMc29. DNA complementary to the

puta-tive transforming gene ofMC29V

(cDNAMc2q)

annealed under stringent reaction conditions (0.6MNaCl, 68°C)toDNAfromnormal

chick-ens aswellas toDNAfrom several other birds phylogenetically diverged from chickens (Table 1). Annealing of

cDNAMc2g

toDNAfromducks

andrheas,phylogeneticallyremoved from

chick-ensby 80x 106and 100x

10'

years,respectively, wassomewhat lessthan forchicken DNA. When these annealing reactions were repeatedunder nonstringent reaction conditions that facilitate the formation of partially mismatched hybrids (1.5 M NaCl, 590C), the amountof

cDNAMC2J

annealing tovariousavian DNAsincreased

al-most tothevalueseenwith chickenDNA(Table 1). This increaseimplies that the sequencesin duck and rhea DNA that are related to

cDNAMc2g

have undergone divergence relative

tothose inchicken DNA.

DNAsfrom three vertebrates other thanbirds were tested forreactivitywith

cDNAMc2q.

Calf

thymus DNA showedverylittlereactivity under stringent annealing conditions (Table 1). How-ever, approximately 20% of the cDNA behaved

as duplex by hydroxyapatite chromatography afterannealingtocalfthymus,mouse, orsalmon DNA under nonstringent reaction conditions; chicken DNA annealed to 53% of

cDNAMC2g

under these same reaction conditions. Similar results have been obtained previously with

cDNA corresponding to the src gene of ASV annealed to vertebrate DNAs (25).

cDNAMc2g

annealedonlyslightlytoE. coliDNAanalyzed under identical conditions, thus excluding the

possibilitythat the 20% reactionrepresentedan

artifact ofourassay conditions. Thespecificity

of the reaction of mouse DNA withcDNAMC2S

has been confirmedby thedetectionof aunique fragment of mouse DNA that annealed to

cDNAMc2g

after cleavage with HindlIl restric-tionenzyme (D.Sheiness,S.H. Hughes,and J. Bishop,manuscriptinpreparation).

Copy number of MC29V-specific se-quences in chicken DNA. Thekineticsof the

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CELLULAR ORIGIN FOR MC29V TRANSFORMING GENE 517

TABLE 1. Homology betweencDNAMc2Sand cellularDNAsa

%Homologyunder thefollowing hybridiza-tion condihybridiza-tions:

DNA source Tm AT, Phylogenetic

dis-0.6MNa'; 1.5MNaCi;590C (OC) (OC) tancefromchicken8b 68°C Expt1 Expt 2

MC29-infected chick 74 50 60 - -?

cells

Chicken 62 62 53 77 0 0

Quail - 63 - - - 35-40

Duck 45 54 - 73 4 80

Rhea 39 56 - 72 5 100

Cow 6 - 20 - - 300

Mouse - - 23 - - 300

Salmon - - 21 - - 390

E.coli 0 2 5 - - -600

aDNAs were prepared and hybridization reactions were carried out and assayed on hydroxyapatite as described in thetext. Thermal denaturations were performedasdescribed in the text. Each reaction mixture containedapproximately 1mgof DNA in volumes of100to 200 tleach;final values forCotwere 27 x 104, exceptinthecaseof DNAfromMC29V-infectedcells, where the values forCotwere'104.

bValues indicate megayears;?,

Unknown.

formation ofduplexes between cDNAMc2I and DNA from uninfected chicken embryos are

shown in Fig. 1. For comparison, the rate of reassociation of unique-sequence chicken DNA

wasassayed in thesamereaction mixture. Since 3H-labeled cDNAMc2I annealed with kinetics similarto thoseseen for."C-labeled

unique-se-quence chicken DNA, we concluded that the

homolog ofcDNAMc2S is present as one or, at

the most, a fewcopiesperhaploid chicken ge-nome.

Thermalstabilityofduplexes formed

be-tween

cDNAMC29

andavian DNAs. We used thermal denaturation toevaluate thedegree of

similarity between the

cDNAMc2I

homolog in

chicken DNA and other avian DNAs. Hybridi-zation reactionswereperformedunder

nonstrin-gentconditionstoallowmaximalduplex forma-tion;stabilityof theduplexeswasdeterminedby elution from hydroxyapatite columns with a

thernalgradient (Fig.2and Table 1).Thernal elutionswereintemally standardized by the ad-dition ofduplex formed in aseparate reaction mixture between theASVsequencesinXCcell

DNA and [32P]cDNAB77, a cDNA homologous

to the ASV genome. The duplexes between

cDNAMc2I

and duck and rhea DNAwere

some-whatless stable than those fonned with chicken

DNA; the 4 to5°C depressionofTmindicates a 3 to4%mismatching of bases (18, 31).

Uninfected chicken embryo fibroblasts contain RNA relatedto

cDNAMC29.

RNA

ex-tracted from chicken embryo fibroblasts was

hybridized to cDNAMc2 under stringent

reac-tionconditions. The kinetics of this

hybridiza-tion are illustratedin Fig. 3. In this and other

experiments,

cDNAMc2S

reproducibly reacted

x 5.

0 ~~~~~~~/ O

0.5 /

0~~~~~~

10 102 103 104

Cot(mol.s/l)

FIG. 1. Annealingof

cDNAMc2n

tochickenembryo DNA. DenaturedDNA(0.6to1.0mg)wasincubated in 0.6 MNaClat68°C,asdescribed in the text, with

[3H]cDNAMc29

(0.05ng, 1,000cpm/reaction; 0)and

"C-labeled unique-sequence chicken DNA (1,000

cpm/reaction;0)inreaction volumesof0.1 to 0.2ml fortime periodsrangingfrom3minto5days. For-mationof duplexwasmeasuredbyfractionationon

hydroyapatiteasdescribed in thetext, and valuesfor

thefractionoflabeled DNA induplexwere normal-ized to the finalextent ofreaction foreachprobe.

These values were 48%for[3H]cDNAMc29 and 91%

for"C-labeledunique-sequencechicken DNA.

with chicken fibroblast RNAto aplateauvalue

of 90%, normalized to the value to which

cDNAMc2I

reacted with MC29RNA.

cDNAMC2S

hybridized equally well to polyadenylated chicken fibroblast RNA. Chicken fibroblast

RNA did not hybridize appreciably with

cDNAB77 duringthesereactions

(Fig.

3); hence,

thehybridizationofchickenRNA with

cDNAMc2s

cannot be attributed to contamination with VOL. 31,1979

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1.(

Ul w

I-CY

x

0

UB

a I

0

LL

a z a

u

0

z 0

LL 0.!

1.

0.!

1

0

0 60 70 80 90

TEMPERATURE (°C)

FIG. 2. Thermal denaturationof duplexes formed

between cDNAMc29 and avian DNAs. Denatured DNAs (1.0mg/reaction) from chicken embryos (A),

duckembryos (B),and rhealungs (C)wereincubated

with[3H]cDNAMc29(4,500cpm,0.225ng;0)in 1.5M NaCiat59°Casdescribedin thetextfor4to6days

tofinalvaluesof Cot?-7x104. Sampleswereapplied

tohydroxyapatitecolumnsat50°Candelutedwitha

thermalgradient asdescribed in thetext. Thermal elutions were internallystandardizedby the

appli-cationtoeach columnof duplexformedinaseparate

annealing reaction between XC cell DNA and

[32P]cDNAB77 (0).

MC29VRNA, since suchaviral RNA

contami-nant would have annealed with cDNAB77 (22). Thermal stability of duplexes formed

be-tween cDNAMc29 and uninfected chicken

cellRNA. The extent ofcomplementarity be-tween chickenRNA andcDNAMc2gwas

evalu-atedby thermal denaturation. RNA from unin-fected andMC29V-infected chicken embryo

fi-broblastswasreacted withcDNAMc2I tovalues for Crt of3.7x 104and6x 102,respectively;Fig.

3indicates that RNA fromuninfected cellsdoes

not reactappreciablywith

cDNAMc2I

at avalue ofCrt = 6 x 102, whereas the hybridization of

infectedcell RNAwithcDNAMC29wascomplete

atthisvalue for Crt. Consequently, the duplex between RNA from MC29V-infected cells and

cDNAMc2I consisted ofcDNAMC29hybridizedto

RNA transcribed from the integrated MC29V provirus, not ofcDNAMC29 hybridized to RNA

transcribed from theendogenous chicken hom-olog of

cDNAMc2I.

Duplexes between cDNAB77 and ASV RNA were added to each reaction

mixture as an internal standard, and thermal denaturationwas performedandanalyzed with S1 nuclease as described above. Little or no

difference in thermalstabilitywas seenbetween hybrids formed with either uninfected or in-fected chickencellRNA and

cDNAMc2g

(Fig. 4). Similar resultswere obtained when the

experi-ment was repeated with different preparations ofcellular RNAs.Thus,bythe criterion of ther-mal denaturation, thenucleotidesequence

rep-resented by

cDNAMc2g

isindistinguishablefrom

the homologous sequence found in chicken

DNA.

DISCUSSION

Normalvertebrate cells containa homo-log of the nucleotide sequence that may

encode the oncogenic capacity of MC29V. We have found nucleotide sequences

homolo-1.0

x

-z

u

CL 0 z

0

LA 0.

UNINFECTED

CHICK CELL 0

RNA

0

n A

10 102 lo3 104

[image:5.504.81.231.60.449.2]

Crt(mol-s/l)

FIG. 3. Hybridization ofcDNAMc2O withRNAfrom

chicken embryo fibroblasts. RNA extracted from

whole chickenembryo fibroblasts (150to500 ,ug/sam-ple)washybridizedto 13H]cDNAMc2o (1K000cpm,0.05

ng; 0)in 0.6 MNaClat680Casdescribedin the text. Thelength ofincubationranged from 7minto56h.

[32P]cDNAB77 (1,000cpm,0.01ng;0)wasincludedin

each reaction mixture.Hybridizationwasmeasured

by resistance ofthe cDNA tohydrolysis by Sl

nu-clease.

CHICKEN ft

/

5 -.0/

O F;~~

I

oll~~~~~q

DUCK

5

. 9r

I.U c

--RHEA 0 F

.5/

,

I

/

I10

i.

0d

ct .o *

¢ l

I

_-i

r%

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519

gous to the putative transforming gene of

MC29V in the genomes of several birds and

other vertebrates (Table 1). Homologywas de-tected byhybridization withcDNAMc2z and was

virtually complete in the case of the chicken

genome; 90% of

cDNAMc2g

annealed to RNA from uninfected chicken cells, and thermal de-naturation ofduplexes consisting ofcDNAMC2S and chicken RNA didnotreveal thepresenceof

any mismatched base pairs. The reaction

be-tween

cDNAMc2I

and chicken DNA was less extensive (approximately 60%);weattributethe

failure of this reaction to reach completionto

stoichiometric limitations that can impede

re-actions in solution between single-stranded cDNAsand denaturedcellularDNAs (4, 18).

The extent of homology with

cDNAMc2I

de-creases in the DNA of various vertebrates in rough accord with their phylogenetic distance from chickens (Table 1). These resultsare

rem-iniscent of previous results indicating that a

homolog of the ASVsrcgeneiswidely distrib-uted in the DNAof vertebrate species (25). Our thermal denaturation studies, however,suggest

thatthe avianhomolog of

cDNAMc2I

isslightly

moreconservedthanis the avianhomolog ofsrc

(25).

We observed that approximately 20% of

cDNAMc2g

formed duplexes with DNAs of

mam-malsand fish(Table 1). Comparableamountsof

hybridizationwereseenwhen cDNA

represent-ing thesrcgene wasannealedtoDNA of

verte-brates other than birds (25). Detailed investiga-tions revealed that most of the src sequences wereactually present in mammalian DNA, but thatmismatched sequencesin the duplexes

be-tween mammalian DNA and cDNA

represent-ingsrcprecludedmoreextensiveduplex

forma-tion (25). The fact that annealing of

cDNAMC2I

tocalf DNA occurred only under nonstringent

annealingconditions(Table 1)suggeststhat

du-plexes between

cDNAMc2S

and its endogenous

homolog in mammalian DNA likewise contain

mismatchedsequences.Accordingly,wesuspect

that more than 20% of the sequences in

cDNAMc2I

may be present in DNA of

verte-brates other than birds. Fossil records indicate that theseparationofbirds,mammals, and fish occurredapproximately400x

10'

years ago;the detection ofhomologywith

cDNAMc2I

through-out these groups suggests that the vertebrate

homolog of

cDNAMc2I

must have existed as a cellulargenebeforethedivergence ofthe various vertebrates and that this sequence has been

highly conservedthroughout vertebrate

evolu-tion.

Endogenous homolog of

cDNAMC2I

may

encode an essential cellular function. The

U 1.0 UNINFECTED

NMC29-NFrED

CHICKCELLO4IICKCELL

RNA MA *

o .

<

Z

0.5 h'

I

0 50 70 90 0 50 70 90

TEMPERATURE(°)

FIG. 4. Thermal denaturation of hybrid formed between cDNAMc2 and RNAfrom uninfected and MC29V-infectedchickencells. RNAsextractedfrom culturesof (A)uninfected(2mgof RNA perreaction) andfrom (B)MC29V/MCAV-infectedchicken fibro-blasts (60 pgofRNA perreaction) werehybridized

with[3H]cDNAMc29(5,000 cpm,0.25ng;0) in 0.6 M

NaCiat680Cas described in thetext. Incubations werefor24 to 48htovaluesofC,tthat allowed the maximum amounts of hybrid to form. Hybridized samples wereplaced in a waterbath at50°Cand denatured withathermalgradient, and the extentof denaturation was assayed with Sl nucleaseas de-scribed in thetext.Denaturation wasstandardized internally by adding hybridforned in a separate reaction between SR-D ASVRNAand

[32PJcDNAB77

(0).

widespread occurrence ofsequences related to

cDNAMc2g

in vertebrategenomes suggests that thesesequencesencodesomevital cellular func-tion which has ensured theirevolutionary

con-servation.Ourfindingthatsequencesrelatedto

cDNAMc2g

aretranscribed into polyadenylated

RNA (Fig. 3) substantiates this proposal.

Fur-thermore,theendogenoushomolog of

cDNAMC2I

exists inchicken DNA as asingle- orlow-copy

sequence (Fig. 1); codingsequencesfor

proteins

areusuallyrepresented byone or afewcopiesin

thegenomic DNA(2,9, 13, 16,29).

The endogenous homolog of

cDNAMc2g

is

transcribed into only a few copies

(approxi-mately 3 to 10) of RNA percell (Fig. 3). This low value is nevertheless similartothe intracel-lularconcentration oftranscriptsfrom the

en-dogenous homolog of src (24), which we now know toencodeaproteinsimilartothatencoded

by the ASV src gene (6, 19a). A low level of

generalized transcriptionis notresponsible for

the presence inchickenRNA ofsequences hy-bridizing to

cDNAMc2I,

since globin or

ovalbu-min RNA could not previously be detected in

chicken fibroblasts (24). Thus, despite the low VOL. 31,1979

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520 SHEINESS AND BISHOP

intracellular concentration of transcripts, we

conclude that transcription of the endogenous homologueofcDNAMc2S probably has functional significance.

Putative transforming gene of MC29V

apparently originated from a highly

con-served sequence found in vertebrate

ge-nomes. The ubiquitous distribution in

verte-brate genomesofnucleotide sequencesrelated

to cDNAMc2S indicates ahigh degree of

evolu-tionary conservation for these sequences. The similarity between the chicken homolog of

cDNAMc2Sand thecorresponding viralsequence

suggeststhat MC29V acquired these 1,500

nu-cleotides byincorporationof this conserved cel-lular sequence. Although the genetic element responsible for transformation by MC29V has

not yet been identified, the host sequence

ac-quired by the virus isalikelycandidate for the transforminggeneofMC29V. Recent investiga-tionssuggestthat thesequencecorrespondingto

cDNAMc2g

encodesaportion ofa110,000-dalton protein which isspecified by the MC29Vgenome (16a,19).Sincenootherprotein encodedbythe MC29Vgenomehas beendetected, the

110,000-daltonproteinor someportion thereofmaybe responsible for the morphologicalchangesseen

in MC29V-infected cells. Thus, the genetic in-formationacquiredfromacellularsequence may

encode the unique oncogenic capacity of

MC29V.

The incorporation of host sequencesmaybe

a general mechanism for the acquisition of

on-cogenic capacities by retroviruses. A probable host origin has been previously demonstrated for thesrcgeneofASV; nucleotidesequencesof avian erythroblastosis virus which may be

re-sponsibleforits tumorigenicityarealso foundto

sharehomology with host and other avianDNAs

(Stehelin et al., Cold Spring Harbor Symp.

Quant. Biol., in press). Several murine leukemia viruses simultaneously acquired sarcomagenic

capacities and assimilated host nucleotide

se-quences into their own genomes as a result of

passage through laboratory animals (10, 21).

More recently, transformation-defective mu-tantsofASVfromwhichthemajority of the src gene wasdeletedwerefoundtocause sarcomas when injected into chickens; ASV recovered from these tumors contained a functional src geneapparently acquiredbyassimilationof the hosthomolog ofsrc(14, 15). Theseobservations

suggestthattheevolution of an oncovirus may

frequently involve the acquisitionand possible modificationofanormalhostgene which

there-afterprovides eachviruswithits distinct

onco-genic capacity.

ACKNOWLEDGMENTS

We thankDeborahSpector for usefuldiscussionsand

Ber-thaCook for stenographic assistance.

Thiswork wassupported by Public Health Service grants CA12705,CA 19287,and traininggrant1T32 CA 09043 from theNationalCancer Institute, and by American Cancer So-ciety grant VC-70E.

LUERATURE CITED

1. Bishop, J. M. 1978. Retroviruses. Annu. Rev. Biochem. 47:3548.

2. Bishop, J. O., R. E.Pemberton,and C.Baglioni.1972. Reiteration frequency of haemoglobin genes in the duck. Nature(London)NewBiol. 235:231-234. 3. Bister, K., M. J.Hayman,and P.K.Vogt.1977.

De-fectiveness of avianmyelocytomatosisvirusMC29: iso-lationoflong-termnonproducer cultures and analysis ofvirus-specificpolypeptide synthesis. Virology 82:431-448.

4. Bonner,T. I., D. J. Brenner, B. R.Neufeld, andR. J. Britten. 1973. Reduction in the rate of DNA reassocia-tion by sequence divergence. J. Mol. Biol. 81:123-125. 5. Britten, R.J., D. E.Graham,and B. R.Neufeld.1974. Analysis ofrepeatingDNA sequencesbyreassociation. MethodsEnzymol.29:363-418.

6. Collett,M.S.,J.S.Brugge, and R. LErikson.1978. Characterizationof anormalavialcell proteinrelated tothe avian sarcoma virustransforming geneproduct. Cell15:1363-1370.

7. Duesberg,P.H.,K.Bister,and P. K.Vogt.1977.The RNA of avianacuteleukemia virus MC29. Proc.Natl. Acad.Sci.U.S.A.74:4320-4324.

8. Duesberg,P.H.,P. K.Vogt,K.Bister,D.Troxler, andE. M. Scolnick. 1978.Oncogenic (onc) genes of sarcoma,leukemia,and carcinomaviruses,p.95-111.In Avian RNAtumorviruses,PiccinEditore,Padua.

9. Firtel,R.A.,A. Jacobson, and H.F.Lodish. 1972. Isolationandhybridizationkinetics of messengerRNA fromDictyosteliumdiscoideum. Nature(London)New Biol. 239:225-228.

10.Frankel,A.E.,R.L.Neubauer, andP. J.Fischinger. 1976.Fractionation of DNAnucleotide transcriptsfrom Moloneysarcoma virusandisolationof sarcoma

virus-specificcomplementaryDNA. J. Virol. 18:481-490. 11. Friedrich,R., H. J.Kung,B.Baker,H. E.Varmus,

H. M.Goodman, andJ.M.Bishop.1977. Character-ization of DNA complementary to a nucleotide se-quenceatthe 5' terminusof the avian sarcoma virus genome.Virology79:198-215.

12. Fujita,D.J., J.Tal,H. E.Varmus,and J. M.Bishop. 1978.env geneof chicken RNA tumor viruses: extent of conservationincellularand viral genomes. J. Virol. 27:

465-474.

13. Greenberg,J.R.,and R. P.Perry.1971.Hybridization propertiesof DNA sequencesdirectingthesynthesis of messenger RNA and heterogeneous nuclear RNA. J. CellBiol. 50:774-786.

14. Halpern, C.C., W. S. Hayward, and H. Hanafusa. 1979. Characterization of some isolates ofnewly

re-covered avian sarcoma virus. J. Virol.29:91-101.

15.Hanafusa,H.,C. C.Halpern,D.L.Buchhagen,and S. Kawai.1977.Recovery of aviansarcomavirusfrom tumorsinduced by transformation-defective mutants.

J.Ezp.Med. 146:1735-1747.

16.Harrison,P.R., A.Hell, G.D.Birnie,and J. Paul. 1972.Evidence forsingle copies of globin genesinthe

mousegenome. Nature(London)239:219-221.

16a.Hu, S. S.F.,M. M.C.Lai,and P. K.Vogt.1979.The genome of avianmyelocytomatosisvirus MC29

ana-lyzed byheteroduplexmapping.Proc. Natl. Acad.Sci. U.S.A. 76:1265-1268.

17.Leong, J.A.,A. C.Garapin,N.Jackson,LFanshier, W.Levinson, and J. M.Bishop.1972.Virus-specific ribonucleicacidincellsproducingRoussarcomavirus: detection and characterization.J. Virol. 9:891-902.

http://jvi.asm.org/

(8)

18.McCarthy,B.J., and M. N. Farquhar. 1972. The rate of change of DNA in evolution. Brookhaven Symp. Biol. 23:1-43.

19.Mellon, P.,A.Pawson, K.Bister,G. S.Martin,and P. H.Duesberg. 1978. Proc. Natl. Acad. Sci. U.S.A. 75: 5874-5878.

19a.Oppermann, H. D., A. Levinson, H. E. Varmus, L Levintow, and J. M. Bishop. 1979. Uninfected ver-tebratecells contain a protein that is closely related to the product of the avian sarcomavirustransforming gene(src). Proc.Natl. Acad. Sci. U.S.A. 76:1804-1808. 20. Rice,N., and P. Paul. 1972. Reassociation of single-copy DNAsequences.CarnegieInst.Washington Yearb. 71: 262-264.

21. Scolnick, E., J. M. Maryak, and W. P. Parks. 1974. Levels of ratcellularRNA homologous toeither Kirsten sarcomavirus or rat typeC virus incelllines derived fromOsborn-Mendelrats. J. Virol.14:1435-1444. 22. Sheiness, D., LFanshier, and J. M. Bishop. 1978.

Identification of nucleotide sequences which may en-code theoncogenic capacity of avian retrovirus MC29. J.Virol. 28:600-610.

23. Spector, D. H.,B. Baker,H. E. Varmus, and J. M. Bishop. 1978.Characteristicsofcellular RNA related to thetransforming gene of avian sarcoma viruses. Cell 13:381-386.

24. Spector,D.H.,K.Smith, T.Padgett, P.McCombe,D. Roulland-Dussoix, C. Moscovici, H. E. Varmus, andJ. M.Bishop.1978.Uninfected avian cells contain RNA related to thetransforming gene of avian sarcoma viruses.Cell13:371-379.

25. Spector, D.H., H. E. Varmus, and J. M. Bishop. 1978. Nucleotide sequencesrelated to the transforming gene

of avian sarcoma virus are present in DNA of uninfected vertebrates. Proc.Natl. Acad.Sci.U.S.A.75:4102-4106. 26. Stehelin,D.,andT. Graf. 1978. MC29andAEVlack the

transforminggene srcof ASVs. Cell13:745-750. 27. Stehelin,D., R. V.Guntaka, H. E. Varmus, and J. M.

Bishop. 1976. Purification of cDNAcomplementaryto nucleotidesequences required forneoplastic transfor-mation of fibroblasts by aviansarcoma viruses. J. Mol. Biol.101:349-365.

28. Stehelin,D., H. E. Varmus, J. M.Bishop,and P. K. Vogt. 1976. DNArelatedtothetransforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature(London) 260:170-173.

29. Suzuki, Y., L. P.Gage,and D. D. Brown. 1972.The genes for silk fibroin inBombyx mori. J. Mol. Biol.70:

637-649.

30. Svoboda, J.,P. P.Chyle,D.Simkovic,and I.Hilgert. 1963.Demonstration of the absence of infectious Rous virus in rat tumorXC, whosestructurallyintact cells produce Rous sarcoma whentransferredtochicks.Folia Biologica9:77-81.

31. Ullman, J.S.,and B. T.McCarthy.1973.The relation-ship betweenmismatched base pairsand the thermal stabilityof DNAduplexe.Biochim.Biophys.Acta 294: 405-424.

32. Vogt, P. K.1977.Genetics of RNAtumorviruses,p.

341-430.In H. Fraenkel-Conrat and R. P.Wagner (ed.), Comprehensive virology, vol. 9. Plenum Press, New York.

33. Weiss, S.R., H. E.Varmus,and J. M.Bishop.1977.

The sizeand geneticcomposition ofvirus-specificRNAs inthecytoplasm of cellsproducing avian sarcoma-leu-kosis viruses. Cell 12:983-992.

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