MC29 virus-coded protein occurs as monomers and dimers in transformed cells.

0022-538X/84/020509-06$02.00/0

CopyrightC 1985,American SocietyforMicrobiology

MC29

Virus-Coded

Protein

Occurs as Monomers and Dimers in

Transformed Cells

JOHN P. BADER* AND DAVID A. RAY

Laboratory of Molecular Oncology, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701

Received 15 August1984/Accepted4October1984

The MC29 virus-coded protein pllG'm-'Yc wasfound exclusively in the nucleus of transformed Japanese quail (Q8) cells, and time courseexperiments indicated that the protein had a half-life of about 30 min. When extracts of either Q8 or chicken embryo cells infected with MC29 virus were prepared with nondenaturing detergents and then sedimented in sucrose gradients, pllO was found in the fractions expected to contain monomers(5.9S), dimers (9.3S), or mixtures of the two. The same extracts treated with denaturing detergent (0.2% sodium dodecyl sulfate) exhibited pllO only in fractions expected for the monomeric protein, but

j8-mercaptoethanol

had noeffecton theoriginal distribution. Gradientsprepared with 0.5 or 1.0 M NaCI failed to dissociate the faster-sedimenting form. No other protein or polyribonucleotide which could increase the sedimentation rate ofpllO wasfound, and neitherRNase norDNasealtered thesedimentationpattern ofpllO innondenatured extracts. A reassociation of monomericpl10into dimers discernibleby gelelectrophoresiswas demonstrated.

A protein encoded by avian MC29 virus, p110ag-mvc, contains both viralgag and cellular mycportionsas ahybrid polypeptide of110kilodaltons (kDa) (5, 17). At least three other independent isolates of avian retroviruses, CMII, MH2, and OK10, contain myc sequences in theirgenomes (6, 7, 15), and they produce either a similarly hybrid viral myc product (CMII and OK10 viruses) (6, 27) or a myc protein translated from a spliced viral mRNA containing cellularsequences(OK10and MH2viruses) (12, 17, 26).The

p1109'9-my'

of MC29 virus is found predominantly in the nucleus (1, 24) and may be associated with chromatin (9). The protein also has been shown to have DNA-binding properties, although sequence specificity for such binding has not beendemonstrated (14, 24). These observations, as well as those on the activation of cellular myc transcription in avariety of circumstances(30), suggest the possibility of a regulatory role formyc protein in transcription or DNA

synthesis.

Anothervirus-codedprotein, the largeTantigen of SV40 (Aprotein), is anuclearprotein (4) and has been shown to have regulatoryactivity for both transcription (29) and the initiation ofDNAreplication (25). The SV40 large Tantigen has been shown to exist in monomeric, dimeric, and tetra-mericforms, butDNA-binding capacity wasobserved only with dimers and tetramers (8). Also, several procaryotic proteins regulate transcriptionorDNA replication as oligo-mers,including the lac repressor (2), theXproteinsCI,Cro, and CAP (10, 31), and the T4 and gene 41 protein (22). These observations prompted us to examine whether p110ag-mYc exists inthecellas anoligomer. Inthe courseof these experiments we also examined the possibility that p1Osam-Ycbecomes associated with a cellularprotein. The resultspresentedin thisreport demonstrate that

pllamg-mc

occursnaturally in both monomeric and dimeric forms. No cellularproteinwasconsistentlyfound to be associated with p110Aag-myc.

* Correspondingauthor.

MATERIALS AND METHODS

Cells andmedia. Cell lineQ8, derived from Japanese quail embryo cells infected by MC29 virus (5), wasused for the analysis of pllar-mYc. These cells produce no infectious virus,but infectious MC29 virus can be rescued from them

by the addition ofRous-associated virus-1 (RAV-1).

Second-ary cultures of chicken embryo cells were infected with MC29 and RAV-1 and then used when all the cells were morphologically transformed. The cells weregrown at39°C in Eagle minimal essential medium containing 5% fetal

bo-vineserum, 10% tryptose-phosphate broth (Difco

Laborato-ries), added D-glucose (to 11 mM), 0.5% dimethyl sulfoxide, andantibiotics. Cellswere then exposedto [35S]methionine (100 ,uCi/ml) in methionine-free minimal essential medium without serumfor 1 hat 39°C,except thatin initialstudies, longer incubations were performed in the presence of 2%

fetalbovineserum. Whole-cellextracts weremade in stand-ard buffer (10 mM sodium phosphate [pH 7.4], 100 mM sodium chloride, 0.5% Triton X-100, 1 mM phenylmethyl-sulfonyl fluoride, and aprotinin [1 U/ml]). For partially de-naturing conditions, sodiumdodecyl sulfate (SDS)wasadded

to0.1or0.2%. Fractionation intonuclei andcytoplasmwas done with 0.5%Triton X-100 in 20 mM Trishydrochloride (pH 7.4)-2 mM MgSO4-1 mM phenylmethylsulfonyl fluoride-aprotinin (1 U/ml). Nuclei were sedimented and then extracted with standardbuffercontaining 0.1% SDS.

Zonal centrifugation. Standard buffer supplemented with

sucrose was used to prepare 5 to 20% sucrose gradients.

Cellswere scraped froma100-mmpetri dish, centrifugedto

apellet, suspended in standard buffer, and sonicated for 30

s. Theextractswere layeredoverthegradients, whichwere thencentrifugedat38,500rpminaBeckmanSW40rotorfor 22to26hat4°C. Fractionswereremovedfrom thetopwith a meniscus-seeking probe (Densiflo; Buchler Instruments) connected to a 2120 peristaltic pump (LKB Instruments).

Sedimentation coefficients were calculated from the frac-tions(23) byamodification ofacomputerized program(13)

and then confirmed with an internal bovine serum albumin standard.

509

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a b c d e f g h precipitated only myc-related proteins, and the addition of the ZC10

peptide

interfered with the selection of these

proteins.

Immunoprecipitating

proteins

smaller than pllO in the nuclei

presumably

were

degradation

products

of

p110QagmYc,

since a less extensive

labeling

time with

rapid

extraction

produced only

pl110a9mYc*

Itshouldbe noted that $ #e- s9 this anti-mycZC10serumbindsonly a cytoplasmic p90 from chicken or

quail embryo

fibroblasts;

no

p55-58

or other cellular myc

protein

is selected

by

thisantiserum.

Toexaminethe intracellular

stability

of

p110,

cultures of

Q8

cellswere

exposed

to

[35S]methionine

for15 minat

39°C,

rinsed free of label, and exposed to medium containing nonradioactivemethioninefor various

lengths

oftime. Some cells were treated with buffer

containing

Triton X-100 and

RM,

Mn:

separated

into nuclear and

cytoplasmic

fractions. Within the first 15 min of continuous

labeling,

more than 60% of the pllOwasfound in the nucleus. In the next 15 min after the addition of cold

methionine,

pllO was absent from the clearcytoplasmiclocalization of

p1109a0-my?'.

Q8cells cytoplasm (Fig. 2). The prelabeled

pllO

was lost from the ;Simethioninewere fractionated into nuclei and cyto- nucleus and cells with a half-life of about 30

min.

No

pllO

n mixed with the indicated serum.

Immunoprecipitates

was detectable in culture fluids during the experiment (data edandanalyzedon a10%polyacrylamide gel.Lanes:

uclearfractions; e through h, cytoplasmic fractions;a not shown),indicating that the decrease in p110 was due to 7gagserum; b and f, preimmune serum taken before degradation. To obtain a high level of incorporation of 10; c ang g, anti-myc ZC10 serum;d andh, anti-myc [35S]methionine into p1lOand to avoid the accumulation of nixed withimmunizing peptide ZC10. possible

degradation

products,

radioactive

labeling

was

lim-itedtoexposures of1 h in

subsequent

experiments.

and

immunoprecipitation.

Goat antiserum to Zonal sedimentation of

p1O1a09mYc.

Cultures of

Q8

cells )rotein of avian

myeloblastosis

virus was ob- labeled with

[35S]methionine

were

lysed

with the standard ResearchResources,

Biological

Carcinogenesis

buffer

containing nondenaturing detergent

(Triton X-100),

ional Cancer Institute.Anantiserum

specific

for sonicatedto

disrupt

nuclei and other

fragments,

and

layered

ion ofthe MC29

protein

was

developed

with a over alinear5to20%sucrose

gradient.

After

sedimentation,

iignated ZC10) derived from the nucleotide se- thefractionswere mixed with

anti-p279ag

serumand

protein

he myc gene (28). The

synthesis

of

ZC10,

its

A-Sepharose.

The bound

proteins

were eluted

by

denatura-orabbits, and the collection ofantiserum

(anti-

tionand

analyzed by

electrophoresis

in

polyacrylamide

gels.

performed

with the aid and collaboration ofT. Vatson, and S. Showalter, Frederick Cancer

Icility. 1.0

veremixed with 10p.l of antiserum, and

antigen-

.8 oa

C/

\ nplexeswereselected with

protein A-Sepharose

Total Cellular

Nuclear

rmacia Fine

Chemicals)

(11).

Conjugates

were

h a solution

containing

1%

SDS,

1% - 4-

\-tanol, 10%

glycerol,

0.1%

phenol red,

and 10

drochloride (pH

6.8).

The eluates wererun in 8 2 -ontinuous

polyacrylamide gels (19).

The

gels

permeated

with

Enlightening (New

England

R2

^p.), dried, and visualized

by

fluorography (21).

-radioactivity

in

protein

bandsweremadewitha .08

_y\pasi

2955

scanning

densitometer. Individual bands

wA at.i5l nm nntl ni-nL- ars. uuprp intuorhsLtul\

were N;annctu ait JJV 11111, U:IIUPUilKdl1r4l WfZl1 IIIC;dtgIZtU.

RESULTS

Intracellular stability of

plljja9-mYc.

Antisera directed againsttheviralstructural

protein p27"a"

orthe myc

portion

of

p11Oag-mYc

wereusedtoconfirmthe intranuclearlocation of

pllV-a'mYc

(1) andto examinethe time course of locali-zation and stability. After the exposure of

Q8

cells to

[35S]methionine-containing

medium for 6

h,

the cellular extractwasmixed with these

antisera,

and the

immunopre-cipitates

were

analyzed

by

electrophoresis

in

polyacryl-amidegels.

p1109a9-mYc

wasfound

exclusively

in thenucleus (Fig. 1),whereas theidentifiable

p279ag

wasin the

cytoplas-mic fraction. Thep27found in

Q8

extracts in this and later experimentsappeared tobe eithera

degradation product

of pllOor a cellularprotein selected by the

antiserum,

since other gag products (e.g., p76and

p180)

were not found in this nonproductive cell line. The

anti-myc

ZC10 serum

Hours Minutes

FIG. 2. Time course of pllO synthesis and stability. Q8 cells

were exposed to [35S]methionine for 15 min and then rinsed and

incubated in medium containing nonradioactive methionine. At

various intervals, cells were extracted whole or separated into

nuclear and cytoplasmic fractions, and pllO was

immunoprecipi-tated with anti-27 serum and protein A-Sepharose. Solubilized

precipitateswereanalyzed by gelelectrophoresisandfluorography,

and the relativeintensityofpllObands was determined.The dotted

line indicates that no pllOwas detected in cytoplasmic fractions

after theinitial extraction.

1 10

27

-FIG. 1. Nu

exposedto[35 plasmand ther

weresolubilizi

athroughd,ni

and e, anti-p2 injectionofZ( ZC10serum n Antibodies the

p279ag

p tained from.

Branch,

Nati the myc

regi

peptide (des

quence of ti

injection

int(

ZC10)

were

Papas,

D. N Research Fa

Samples

antibody

con

C1-4B

(Phar

eluted

witE

mercaptoeth

mM Tris

hy(

or 10% disc

were

fixed,

Nuclear Cor Estimatesof

Transidyne

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5.9 S 9.3S

4_am ..

110-

-27 *N

1 2 3 4 5 6 7 8 9 10 11 12 13 Top Fraction Number Bot

FIG. 3. Zonal sedimentation ofpllO nondenaturedextracts.Q8 cells exposedto[5S]methionine weretreated withanondenaturing

detergent (Triton X-100), and the extractwaslayered over a 5 to

20% sucrose gradient. After centrifugation, fractions were mixed with anti-p27RaR serum, and immunoprecipitates were solubilized

and analyzed by gel electrophoresis. The fractions calculated to

containpllOmonomers(5.9S) and dimers (9.3S)areindicated.

The most intense bands ofpllO were seen in the fractions expectedtocontainproteins ranging from 110 kDa (5.9S)to about twice that size (9.3S) (Fig. 3). This pattern is best explained by the occurrence of peaks of monomers and dimers ofpllO withoverlappingmixtures inthe intermediate fraction(s). No peak of activity indicative of higher-order oligomers of pllO was observed, although the trailing pat-ternofpllO infractions of high Svedberg values leave this possibility open. In this gradient, a 200-kDa protein of undetermined relevance was found in fractions of 9.3S and 10.3S, which also contained thepresumptive dimer ofp110.

The pellet from the gradient was reextracted with buffer containing SDS and examined for the release of boundp110.

A band equivalent to about 20% of the total pllO in the sample was found (data not shown). Cellular DNA and chromatinsedimented into the pellet; this indicated that only a small portion of the intranuclear pllO was associated directlywith theDNAorchromatin,asreportedby Bunteet al. (9).

When the radioactive cellular sample was treated with a partially denaturing solution (buffer containing 0.2% SDS) before centrifugation, nearly all of the pllO appeared in the monomeric region (Fig. 4A), demonstrating that pllO

mon-omers sediment at the expected rate in thesegradients. In eitherdenaturingornondenaturing conditions, the

gag-spe-cific protein, p27, appeared in the fraction anticipated fora

monomer (Fig. 3).

Therelativelyhighintensityofradioactivityinthedimeric region suggestedaspecificaggregation ofp110. However,to preclude nonspecific ionicinteractions, samples treated sim-ilarly withnondenaturing detergentweresedimentedthrough gradients with higher salt concentrations (0.5 and 1.0 M NaCI).The resultwasessentially thesame:pllO appeared in thefractionsexpected for bothmonomersanddimers and in intermediatefractions, demonstratingthatdissociation could

notbe

accomplished

by merely raising

the ionic strengthof the

gradient (Fig.

4B).

Inother

experiments,

a

possible

role fordisulfidebands in

oligomerization

was examined. The addition of

IP-mercaptoethanol

(1%, vol/vol)

had no

significant

effect on the

rapid

sedimentationof

pllO

innondenatured extracts.

Extracts of chicken

embryo

cellsinfectedwithMC29and RAV-1 were also examined in sucrose

gradients.

Immuno-precipitates obtainedwithanti-p271a9serumshowedthat the

pr769ag

derived from RAV-1 sedimented mainly in the fractions expectedfora76-kDaprotein(Fig. 5A),

indicating

that

pr769aR

exists

predominantly

asamonomerin nondena-turedextracts. In thesame

sample,

pllO

sedimented

consid-erably

further into the

gradient,

withadistribution similarto that noted above. When the extract was treated with 0.1% SDS before sedimentation, pllO wasfound in its

expected

monomeric

position,

sedimenting slightly ahead of

pr76919

(Fig. 5B).

Possibleassociation of cellularmacromolecules with p110. In theexperiments described above,inwhichproteinswere

immunoprecipitated from nondenaturing extracts, several polypeptide bands unrecognized as either gag-related or

myc-related proteins were observed. The occurrence of these

bands,

especiallyinfractions containingthe presump-tivepllOdimer,raised thepossibility that pllO isassociated with cellular protein under natural conditions. The same

fractions shown in Fig. 1 were treated with partially dena-turing detergent (0.2% SDS) to release any polypeptide associated with pllO before immunoprecipitation. Under

A

4

3_

2-o B

2-B

0

0~~~~~-,4_ + +~/

2_ 0o

2 4 6 8 10 12 14

SW20

FIG. 4. (A)SedimentationofSDS-treatedextract.The sedimen-tation properties of a nondenatured extract of [35S]methionine-labeledQ8cells(0)werecompared with those ofthesameextract treated with 0.2% SDS (0). Both were run in sucrose gradients containing0.1 MNaCl, with 0.1% SDS addedtothegradientforthe SDS-treatedextract.(B)Sedimentation inhigh-ionic-strength gradi-ents. The same nondenatured extract used in the 0.1 M NaCl gradientwas runingradientscontaining0.5(-)or1.0(O)MNaCl. Arrows indicate the predicted location of pllO monomers and dimers.

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these conditions, only gag-related proteins were precipi-tated, and several bands disappeared from the pattern (data not shown).

Toexaminethispossibility further, an antiseruminduced against myc-specific peptidesequences(anti-myc ZC10)was used to immunoprecipitate pllO in the absence of any affinity for viral structural proteins. This antiserum had a common affinity for p110, but presumably differed in its ability to recognize and immunoprecipitate cross-reacting cellular proteins or degradation products of p110. When anti-ZC10 serum was used with fractions from sedimented Q8extracts, pllOprecipitated inbothmonomeric and oligo-meric regions (Fig. 6). Although other polypeptides ap-peared in thesefractions, nosingle polypeptide was promi-nent in the gradient fractions containing the presumptive pllOdimer.Asingle lightbandofapproximately71 kDa was common to anti-p27 and anti-myc antisera, but its distribu-tion in the gradient failed to support the possibility of a special association whichwouldincrease the sedimentation ofp110.

In several experiments with direct Q8 cellular extracts (without zonal centrifugation), the anti-gag and anti-myc sera precipitated only pllO in common. It seems unlikely, therefore, that a cellular protein forms a natural aggregate with p110. The heterologous polypeptides found in pllO fractions canprobably be attributed toinadvertent degrada-tionofpllOin thegradient beforeimmunoprecipitation, with subsequent differential recognition by the antisera.

Possible association of RNA withp110. The possible asso-ciation of a cellular RNA species with pllO was also examined. After 4 h of exposure to

[14C]uridine,

Q8 cells were treated with nondenaturing detergent, sonicated, and sedimented in a sucrose gradient. Portions ofthefractions were analyzed for radioactivity both directly and after immunoprecipitation with anti-p27.Inthis gradient,5SRNA, tRNAs, and smallerspecieswould be found in the firstfew fractionsatthe topofthegradient, and 18S rRNA andlarger species would befoundin thebottomfourfractions (Fig.7). After immunoprecipitation, a small but distinct peak of radioactivity was found associated with the 9.OS fraction, the expected peak fraction for the pllO dimer. When the experiment was repeated, a similar radioactive peak was seen inthe9Sregion. The immunoprecipitableradioactivity

6.oS

A

9.5S

110-

76-B

110-

76-e

4, 12...

2 3 4 5 6 7 8 9 10 11 12

[image:4.612.342.536.69.278.2]

Fraction Number

FIG. 5. Chicken embryocells infected with MC29 and RAV-1

werelabeledwith [35S]methionine and lysed, and thenextractswere

sedimentedinsucrosegradients.The fractionswere immunoprecipi-tated with anti-p27gag serum. (A) Nondenaturing Triton X-100

extract. (B) Treatedwith0.1% SDS beforecentrifugation.

5.9S 9.3S

11Q0

--71

--f_

3 4 5 6 7 8 9 10 11 12

Top Fraction Number Bol

FIG. 6. Immunoprecipitation of gradient fractions with anti-myc sera.Anti-ZC10 antiserum directedagainst myc-specific peptidewas used to precipitate pllO and associated proteins from gradient fractions. The fractions expected to contain pllO monomers or dimersareindicatedbyarrows.

represented about 1% ofthe '4C in the fraction. When the eluted immunoprecipitate was analyzed by acrylamide gel electrophoresis, no polynucleotide band was found; all of the radioactivity was found at the migrationfront. We are currently examiningthepossibility that thepllOdimer was nucleotide-bindingability.

Inanotherexperiment, DNase I(10 ,ug/ml) and RNase A (10,ug/ml) were added to extractsof [35S]methionine-labeled cells before centrifugation through a sucrose gradient con-taining 0.5 M NaCl. No differences in the sedimentation pattern of pllO were observed compared with that of un-treated extract. Bands of immunoprecipitable pllO were found in both monomeric(6.OS) and dimeric(9.5S) regions. Theaddition ofSDS tonuclease-treatedextractsresulted in theappearance ofpllOonlyin the monomeric region.

Preliminary attempts to reassociate monomers into dimers. Weattemptedtoreconstitute dimers frompllO monomers in cellular extracts prepared with a partially denaturing solu-tion(0.2% SDS). On severaloccasions,weobservedaband estimatedtobe twice the size ofpllOafter

immunoprecipi-tationandelutionandcooling oftheeluate in the presenceof

3-mercaptoethanol

for several hours beforeresolutionby

gel

electrophoresis (Fig. 8). The use of anti-ZC10 serum di-rected against the myc peptide avoided confusion of this p200-220 band with the

pl80gag-PoI

precursor of reverse transcriptase, and the addition of the free myc ZC10peptide successfully decreased the amount of

radioactivity

in the band. This dimeric form has been observedonly inextracts of MC29-infected cells, includingthe

nonproducing

Q8and Q5 quail lines, and of chicken embryo cells infected with MC29virus.Attempts toreconstitutedimersbyothermeans are in progress.

DISCUSSION

The data presented here suggest that the virus-coded proteinof avian MC29 virus

(p1199ag-mYc)

occurs asboth free monomers and as dimers in infected cells. The higher-mo-lecular-weightformwasfound notonlyin sucrose

gradients

ofmoderate saltconcentration(0.1 M

NaCI)

but alsoin salt

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concentrations (0.5 and 1.0 M NaCI) that are expected to dissociate superficialaggregations.Inseveralgradients,some pllO was found in fractions estimated to contain proteins considerably in excess of220 kDa. However, no peak of activity was found in any single fraction, and the relative intensities of the bands decreased with increasing relative Svedberg values. It seems unlikely, therefore, that pllO occurs as natural oligomers larger than dimers.

Asignificant portion of the intracellularpllO sedimented into the pellet, presumably in association with DNA (24), chromatin (9), or the nuclear matrix (17). We have not yet been able to ascertain whetherthis association involves the monomeric ordimeric form orbothofp110.

Atleasttwootherpossibilities of dimerization of retrovi-ral proteins have been reported. (i) A 120-kDa form of pp60srcisproducedbyaRous sarcomavirus-transformedrat cell line (18). This protein appears to be a linear tandem polypeptide translated from a large (28S) form of viral mRNA and is not dissociable by SDS. (ii) Murine pr65sar occurs, in atleasttwocases, asintravirion 130-kDadimers, which arenondissociable bySDS butreducedto65 kDa by ,-mercaptoethanol (32). Since p1109a9-mYc dimers were dis-sociated by SDS but not by

P-mercaptoethanol,

the aggre-gation of pllO appeared to be different from that ofthese other proteins. In fact, the intracellular pr76 of RAV-1 occurred as amonomerwithorwithout ,-mercaptoethanol. Therefore,theaggregation ofpllOapparentlyoccursthrough domains inthe myc region thatareindependent of disulfide bands.

Althoughotherpolypeptideswerefound in immunoprecipi-tates containing p110, association ofcellular proteins was not responsible forthe increased sedimentationrate ofthe presumptivedimer. Acellularprotein(s)inassociation with

9.0 $

4

.-4

9

3

I

2

E

01 9

2 4 6 8 10 12 P

Fraction Number

FIG. 7. Sedimentation of 14C-labeled extracts. Q8 cells were exposedto [14C]uridine for4h, treated with nondenaturing deter-gent, sonicated,and centrifugedin a sucrosegradient asdescribed for [35S]methionine-labeled extracts. Portions of fractions were takendirectly for theassay ofradioactivity(0),and the amount of

14C bound toanti-p27 immunoprecipitates wasdetermined (0). P, Definition.

a

200-

m-1 m-10

_6

b --..

[image:5.612.387.495.73.196.2]

-S

FIG. 8. Reassociation of pllO monomers into dimers. A Q8 extract prepared with 0.2% SDS was immunoprecipitated with anti-mycZC10. Thesolubilized eluatewascooledovernightat 5°C and then analyzed directly by gel electrophoresis without further treatment. Lanes: Q8 extract; b, Q8extract mixed with the same

peptideused toinduceanti-mycZC10 antiserum.

pllO would be expected to immunoprecipitate with pl0 regardless of the specificity of the antiserum. Noprominent protein except pllO was selected in common by antisera against p279ag and against a peptide derived from a major exon of the myc gene. Heterologous polypeptides immuno-precipitated from p110-containing fractions were probably derived from pllO degraded during sedimentation despite

our efforts to inhibit proteolysis. Different-sized fragments would be recognized by antisera of differing specificity, producing the observed result. In any case, an association similar to that of the SV40 large T antigen with cellularp53 (20) appears to be unlikely. We also were unable to find a

polyribonucleotide associated withpllO that couldincrease its sedimentation rate in sucrose gradients, and neither RNase norDNaseaffected thesedimentationpattern ofpllO fromnondenatured extracts.

The large T antigen of SV40 has been shown to exist in monomeric, dimeric, and tetrameric forms (8). Only the aggregated forms exhibited two recognized activities of T antigen, ATPase and DNA binding, suggesting that T-anti-gen oligomers are the active forms of this virus-coded protein. No special biochemical activity of p11lagg-mYc asso-ciated with its biological function has yet beendiscovered, although the reported general affinityofpllOfor DNA(14) maybe useful indistinguishingthecapabilitiesof the mono-meric anddimeric forms. The activities of several procary-oticregulators oftranscription or of DNAreplicationrequire oligomerization (2, 10, 22), and our observation that pllO occurs as adimer is atleast consistentwith thenotionthat it has aregulatoryfunction in nucleic acid synthesis.

Transcription of cellular myc mRNA occurs at least at a low level in mostgrowingavian and mammalian cells, and presumablymyc mRNAis translatedintoactivecellularmyc protein. The cellular myc protein has been described as a polypeptideofapproximately45 to 60 kDa(3, 16, 17) and as a 55-kDa protein (p 55) in our own unpublished observa-tions. The findingof pllO dimers raises the possibility that pllO in excess combines withp55c-mYc to formheterologous p11Oam-'Yc-p55c-mY" dimers. Withoutexpandingon this pos-sibility, we can state that we observed no association of a cellular 55-kDa protein with pllO with any of the antisera describedabove.

LITERATURE CITED

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2. Adler, K., K. Beyreuther, E. Fanning, E. Gersten, B.

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enborn, A. Kelmm, B. Mueller-Hill, M. Pfahl, and A. Schmitz. 1972. How lac repressor binds to DNA. Nature (London) 237:322-327.

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