Relationship Among Tau Antigens Isolated from Various Lines of Simian Virus 40-Transformed Cells

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JOURNAL OFVIROLOGY,June1980, p. 650-657 Vol. 34, No. 3 0022-538X/80/06-0650/08$02.00/0

Relationship

Among Tau Antigens Isolated from Various

Lines of

Simian Virus

40-Transformed

Cells

DANIEL T. SIMMONS,`* MALCOLM A.MARTIN,2PETER T. MORA,3 ANDCHUNGMING CHANG3t SchoolofLifeand HealthSciences, University ofDelaware, Newark,Delaware19711';and Recombinant DNAResearch Unit, National InstituteofAllergyandInfectiousDiseases,2andMacromolecular Biology

Section, Immunology Program,National CancerInstitute,3Bethesda,Maryland20205

Inadditiontothevirus-specified tumorantigens,simian virus 40-transforned

cells containatleast oneotherprotein which canbeimmunoprecipitated with

serumfromanimalsbearing simian virus 40-inducedtumors.Thisprotein,which

isdesignated Tauantigen, hasanapparentmolecularweightof 56,000 as

deter-minedbyelectrophoresisonacrylamidegels. Therelationshipamong Tau

anti-gensisolated from different lines of simian virus 40-transformed cellswas

exam-ined bycomparing the methionine-labeled tryptic peptides of theseproteinsby

two-dimensional fingerprintingonthin-layer celluloseplates. Inthisfashion,we

initially determined that the Tau antigens isolated from three different lines of

transformedmouse cellswerevery similar. Second, we found that Tauantigen

isolated fromaline ofrattransformantswascloselyrelated, butnotidentical,to

the mouse cell Tau antigens. Approximately 70% of their methionine peptides

comigrated intwodimensions.Finally,weshowed thatTauantigen isolated from

aline of transformed human cellswasonly partially relatedtothemouseandrat

proteins. About 40%of the methioninepeptides of the humanproteinwere also

contained in the Tauantigens from the othertwospecies. These resultsstrongly

indicate that the Tau antigens isolated from these various simian virus

40-transformed cell lines containcommon amino acid sequences.

Monkey cells infected with simian virus 40

(SV40) synthesize two proteins (94,000 and

20,000 daltons) which can be

immunoprecipi-tated with serum from animals bearing

SV40-inducedtumors (anti-Tserum) (1,4,29). These

proteinsarethelarge and smalltumorantigens

(T-Ag's) andarecoded by the region oftheSV40

genomethat isexpressed earlyinlytic infection

and invirus-transformed cells (22,23).

In addition to the T-Ag's, cells transformed

bySV40 synthesizeoneotherprotein (50,000 to

56,000 daltons) which can be

immunoprecipi-tated withanti-Tserum (5, 9, 10, 16, 17, 19, 21,

28). This protein is probablycoded by the cell

DNA since it contains very few (zero to two)

methionine-labeledtrypticpeptidesincommon

with either large or small T-Ag (5, 16, 28) and

sinceuninfectedmouseembryo carcinoma cells

manufacture a protein that is very similar or

identicalto the 50,000- to 56,000-dalton protein

isolated from SV40-transformed mouse cells

(19). We havepreviouslycalledthis protein Tau

antigentodistinguishitfrom thevirus-coded

T-Ag's (5). Smith et al. (28) have recentlycalled

thisprotein nonviral T-Ag.

tPresent address: Department of Microbiology, National Yang-MingMedical College and Taipei Veteran General Hos-pital, Sheh-Pei, Taipei, Taiwan.

Tau antigens have been detected inall lines

ofSV40-transformed rat, human,hamster,and

mousecells that have been examined(5, 16, 21,

28),although transformed hamstercells appear

tomake smallerquantities of these proteins (28).

In addition, cells transformed by other viruses

(Moloney murine leukemiaor sarcomavirus)or

by chemicals (methylcholanthrene) synthesize

similarly sized proteins whichmaybe Tau

anti-gens(7). Theseproteinsareapparentlynot

pro-ducedinspontaneouslytransformedmousecells

(5), in SV40-transformed cells which have

re-vertedto aT-Ag-negativestate(5),orin normal

untransformedcells (7, 19,28).Aportion of the

Tauantigen in transformed cellsmaybe found

in acomplex withlargeT-Ag (17).

Weinvestigated the relationshipamongTau

antigens isolated from various lines of

SV40-transformed cells to determine whether these

proteins areidentical. For this purpose, we

ex-aminedthemethionine-labeledtrypticpeptides

of Tau antigens isolated from three different

linesofSV40-transformedmousecells,oneline

oftransformedrat cells, and one line of

trans-formed human cells. We found that these

pro-teins were not all identical, and yet they all

contained some peptides in common. In

addi-tion, we noticed that the mouse-derived Tau

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SV40 Tau ANTIGENS 651

antigens were nearly identical to one another,

that the ratprotein was significantly related to,

butclearly distinguishable from, the mouse

pro-teins, and that the human protein was only partially related to the others.

MATERIALS AND METHODS

Cells. SV40-transformed BALB/c and AL/N mousecells(lines 315 and 215, respectively) have been described previously (5). BALB/c (line 11A8) trans-formants were obtained from G. Todaro. Human

transformedcells (line SV80) were isolated originally

byTodaroetal. (30), and rattransformedcells were

isolatedby W. Topp anddescribedbyBotchan et al. (3).

Immunoprecipitation and gel electrophoresis. Transformedcells were grown in150-cm2culture flasks and labeled for3hwith50uCi ofL-['S]methionine

perml,aspreviouslydescribed (5). The cells in each flask were washed three times with ice-cold 0.01 M Tris-0.001 MNa2HPO4-0.137 MNaCl, pH 7.4 (Tris-bufferedsaline),andlysedduring a 15-min period at 00C in the presence of2mlof extraction buffer (5).

Thelysatewascollected andcentrifuged at 150,000x

gfor 40 min at2MC. The supernatantwascarefully removed andincubated at00C for 1hwith 25piof either normal oranti-Thamster serum. The anti-T serum was obtained from hamsters carrying SV40-inducedtumorsandwasprovided by J. Gruber, Na-tional Cancer Institute.

Protein A-bearingStaphylococcus aureus (Cowan Istrain; NCTC 8530) was prepared by the method of Jonsson andKronvall (14). The bacteriawerewashed three timesat00Cwith extractionbuffer, resuspended in thesamebufferto afinal concentration of 10% (wt/ vol), and added (300

pl)

tothe reaction mixture to precipitate the immunecomplexes.After 1hat0°C, the bacteriawerewashedonceinextractionbuffer, six

timesin0.1MTris-0.5MLiCl-1%2-mercaptoethanol,

pH9.0(26), andoncein Tris-bufferedsaline (pH 7.4)

andsuspendedin 150

pl

of 0.075 M Tris-sulfate (pH

8.6)-2% sodium dodecyl sulfate-2%

2-mercaptoetha-nol-0.002%bromophenolblue-15%glycerol (20).

Gel electrophoresis and chromatography of

tryptic peptides. Protein sampleswere heated and

subjectedtoelectrophoresisfor2.5 to 4hat25mA in

gels containing 13% acrylamide and0.26%

bisacryl-amideasdescribedbyTegtmeyeretal.(29).Proteins tobe characterized furtherwere extracted from the

gelanddigestedwithtrypsinaspreviouslydescribed

(27). Trypticpeptideswere separatedintwo dimen-sions onthin-layercelluloseplatesessentially as de-scribed byGibson (11). The peptides in water-pyri-dine-acetic acid (300:10:3,vol/vol), pH5.4 (12),were applied near the corner of a thin-layer plate and

subjectedtoelectrophoresisat40Cinthesamebuffer

for 3 h at 300 V. Chromatography in the second

dimensionwasperformedin butanol-pyridine-water-acetic acid(97:75:60:15,vol/vol) pH5.3(11),for5.5to 6h at roomtemperature.Afterthoroughdryingina

fumehood,theplatesweredippedinmolten(4000)

2-methylnaphthalene containing0.4%

2,5-diphenyloxa-zole,asdescribed byBonner andStedman (2).Kodak

XR2 film was exposed to the plates for 1 to 3 weeks at -70°C before being developed.

RESULTS

Immunoprecipitation of Tau antigens.

Tau antigensarecellularproteins that are

syn-thesized inavariety ofSV40-transformed(5, 16,

17, 19, 28) and non-SV40-transformed (7, 19)

cells. However, they havenotbeen detected in

normal, untransformed cells (7, 19, 28). Since

the Tau antigens isolated from various

trans-formedcellshave thesameapproximate

molec-ularweight (50,000to 56,000),we were interested

in determining whether the same protein was

made in all cells transforned by SV40 or

whethertheproteins variedamong different cell

lines.Furthermore, if differenceswere found,we

wanted to determine the relationship among

Tauantigensisolated fromvarious lines of cells

of thesamespecies and ofdifferent species. We

therefore examined the peptides of theTau

an-tigens isolated from three lines of mouse

trans-formants(lines 1lA8,315,and215;derivedfrom

BALB/c, BALB/c, and AL/N mouse strains,

respectively), one line oftransformed rat cells

(line 14B), and one line oftransforned human

cells(line SV80). Theseproteinswere

immuno-precipitated from extractsofcells labeled with

L-[3S]methionine and subjectedto

electropho-resis (Fig. 1) in acrylamide gels. Figure1 shows

that each of these cell lines contained one or

more forms of Tau antigen (54,000 to 56,000

daltons) that were immunoprecipitated with

anti-T serum but not with normal serum. In

particular, lines 215, 315, and SV80 containedat

leasttwoforms ofimmunoprecipitable Tau

an-tigens (Fig. lb through d). All cell lines also

containedalargeT-Agwithanapparent

molec-ularweightof 94,000.Largerforms ofT-Agwere

alsoapparentin lines 11A8(100,000daltons)and

315(130,000daltons) (Fig. laandc).These and

other high-molecular-weight forms oflarge

T-Ag have been detected previously in various

linesofSV40-transformedcells(5, 15, 16, 18, 24,

28). The small T-Ag of SV40was not readily

detectedin theseimmunoprecipitates, partially

because the anti-Tserumused waschosen for

maximumprecipitationof Tauantigensand

par-tiallybecause theamountsofanti-Tserumused

weretoosmalltoprecipitate small

T-Ag

quan-titatively (6).

Two-dimensional

fingerprinting

of

me-thionine-labeled tryptic

peptides.

To

com-pare thepeptidesof Tauantigensisolatedfrom

various lines ofSV40-transformedcells,labeled

immunoprecipitated

proteins

were

applied

to

preparative acrylamide gels. The bands

corre-sponding to the

slower-migrating

Tau

antigen

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652 SIMMONS ET AL.

a

b

c

d

e

N

T

N

T

N

T

N

T

N

T

130K

100 K

94K

0

56 K

54K

'-FIG. 1. Acrylamide gelelectrophoresis ofproteins immunoprecipitated fromvariouslinesof

SV40-trans-formedcells: labeledproteinsprecipitatedwith eithernormal(N) oranti-T(T)hamsterserumfrom

SV40-transformedline 11A8 (a),215(b), 315(c), SV80(d), and 14B(e)cells. SV40-transformedmousecells (lines

11A8, 315, and215), ratcells (line 14B), and human cells (lineSV80) were labeledfor3h with L-[35S]-methionine. Extractswerepreparedandincubated with either normaloranti- T hamster serum,followedby incubation withproteinA-bearingS.aureus.Proteins in the washedprecipitatesweresubjectedtoacrylamide

gelelectrophoresisfor2.5hat25mA,and the labeledproteinsin thegelweredetectedbyexposuretoX-ray

film. Molecularweights wereestimated bythe relativeratesof migration ofmarkerproteinswithknown molecularweights(5). 130K=130,000 daltons.

species of each cell line (Fig. 1) were excised

from thegels,and theproteinwasextracted and

treated withtrypsin.Theresulting peptideswere

separated intwodimensionsbyelectrophoresis

and chromatography (fingerprinting) on

thin-layer cellulose plates (11). Methionine-labeled

peptidesweredetectedontheplates by

fluorog-raphy,using2-methylnaphthalene (2).To

com-pare any two proteins, peptide samples were

analyzedseparatelyondifferentplatesandas a

mixture on a third plate. Figure 2 shows the

fingerprintsof themethionine-containingtryptic

peptides of the Tau antigens isolated from the

mousetransformants 11A8 (Fig. 2A), 315 (Fig.

2B), and 215 (Fig.

20).

The fingerprint of a

mixture of the Tau antigen peptides derived

fromcelllines 11A8and 215is shown inFig. 2D.

Themethionine-labeled tryptic peptides of the

Tau antigens fromtwoofthesecell lines (lines

11A8and 215) were indistinguishable (Fig. 2A,

C,andD). Those derivedfromthe third mouse

cell line (line315) (Fig.2B) were slightly

differ-entinthat one peptide (peptide a, Fig. 2A and

C) wasabsent. The appropriate mixing

experi-mentshowedthat theremaining peptides of that

protein corresponded to the peptides derived

fromlinesllA8and215(data not shown). Since

lines 11A8 and 315originatedfrom theBALB/

cstrain ofmice, whereas line215originatedfrom

theAL/N strain, the observeddifferences can-not be due simply to a difference in strains.

Rather, they suggestthatmouse Tau antigens

mightvaryslightly from oneanother,

irrespec-tive of the strains from which the transformed

celllineswerederived. The similarities of these

fingerprints do indicate that the Tau antigens

isolated from mouse transformants are very

closely related proteins. This is in agreement

with the data of Linzer and Levine (19), Smith

etal. (28), andChangetal.(5).Therelationship

among the peptides of the three mouse Tau

antigensis shownbelow(see Fig. 5a).

In asimilarmanner,the Tauantigens isolated

from the rat and human transformants were

comparedwitheach other and withthose of the

mouse cell lines. Figures 3A through C show

that the rat(line 14B) Tau antigen contained16

methionine peptides (Fig. 3B) that were also

present in mouse (line 11A8) Tauantigen (Fig.

3A)and 7peptidesthat weredifferent.The Tau

antigen from human line SV80, on the other

hand,containedonly eightmethionine peptides

(Fig. 3D) that were also present in the mouse

protein (peptides 1, 3, 4, 6, 12, 13, 14, and 16)

(Fig. 3A, D, and E). These eightpeptideswere

alsopresent in the rat Tauantigen (Fig. 3B, D,

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*':.

ts...

4*~~~~~~~~~41

*

FIG. 2. Fingerprintsof methionine-labeledtrypticpeptidesofmouse Tau antigens isolatedfromline11A8

(A),315(B),215(C),andl1A8plus 215(D) cells. SV40-transformed mouse cell lines11A8,315,and 215 were labeledwith L-[3SJmethionine,andthe extracts of these cells were incubated with hamster anti-T serum in separate immunoprecipitation reactions. Tau antigensfrom each of these cell lines were isolated from preparativeacrylamide gels and treated withtrypsin,and the resulting peptides were applied near the corner ofathin-layer cellulose plate (11). Electrophoresis was carried out at 300 V for 3 h at4°C. Thedirection of electrophoresis wasfromleft to right near the bottom of eachfingerprint. This was followed by ascending thin-layerchromatography (frombottom to top in thefingerprints). Plates were dipped in 2-methylnaphthalene andexposedtoX-ray filmat-70°C for about 3weeks.

and F) and were therefore a subset of the 16

peptides common to the mouse and rat Tau

antigens. These dataaresummarized below(see

Fig. 5b).

The relationship betweenthe different

elec-trophoretic forms (56,000and 54,000daltons) of

theTauantigens isolated fromtransformed

hu-mancells (Fig. ld)wasinvestigatedby

compar-ing their methionine-labeled tryptic peptides.

For preparative purposes these proteins were

separated from one another by subjecting the

samplestoelectrophoresisforalonger periodof

time than for thegelshown inFig.1.Figures4A

and B show thefingerprints of the

methionine-containingtrypticpeptidesof the slower (56,000

daltons) and faster (54,000 daltons) species of

lineSV80 Tauantigen,respectively.The results

ofthemixingexperiment areshown in Fig.4C.

These two proteinscontained thesame 18

pep-tides. The 56,000-dalton form of the protein

containedanadditionalpeptide(Fig.4AandC,

peptideb).Thisresultissummarized below(see

Fig. 5c).

DISCUSSION

Ananalysis ofmethionine-labeled tryptic

pep-tidesshowed that the Tauantigens isolated from

three different linesoftransformedmousecells

were very similar. This is in agreement with

previous observations madebyus(5),by Linzer

andLevine (19),and mostrecently by Smithet

al. (28). We noticedin this study, however, as

did Smithetal.(28),thatallmouseTauantigens

were not identical. The Tau antigen isolated

fromline315lackedoneofthepeptides (Fig. 1,

peptide a)presentintheproteinsfrom the other

two mouselines(lines llA8and215).

Bycomparingthepeptidesofmousecell Tau

antigen (line 11A8) with those ofrat(line 14B)

and human (line SV80) transformed cells, we

found that the three proteins apparently

con-tained thesameeightmethionine-labeledtryptic

peptides (Fig. 5b). Furthermore,themouseand

rat Tau antigens appeared to be more closely

related, sharing an additional eight peptides.

The moststraightforwardinterpretationof these

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654 SIMMONS ET AL.

'A 16

*

~~~~~1

*14

~13

12

*;::'sV;0 ....

~~~24 $~~~~~~

-'~~~~~~-g

16

*14

13

12

3

..

:

*

1 a:?. .16

#16

#14

t12

3 :

FIG. 3. Comparison ofmethionine-labeledtryptic peptides ofTauantigensisolatedfrommouse,rat,and humantransformants: fingerprints ofmethionine-labeledpeptides ofTauantigensisolatedfromlines 11A8 (A), 14B (B), 11A8plus14B (C),SV80(D), SV80plus 11A8(E), andSV80plus14B(F). Tauantigenswere

isolatedfrommethionine-labeledtransformedmouse(lineMMA8),rat(line 14B),andhuman(lineSV80)cells by immunoprecipitation and gel electrophoresis. Tryptic peptides wereprepared and separated in two dimensionsonthin-layercelluloseplates.Thenumberedpeptidesin(A),(B),and(C) refertopeptidescommon

tomouseandratTauantigens. Those in(D), (E),and(F) refertopeptidespresent inmouse,rat,and human

Tauantigens.

results is that these Tau antigens are

descend-antsofasingle proteinwhich has evolved

differ-ently in mice,rats,and humans. Thissituation

would be analogous to the evolution of

cyto-chrome cor hemoglobin (8), in which proteins

from closely related species are more similar

than those fromdistantly related species. This

wouldexplain the observedsimilarity in the Tau

antigens of mouse and rat transformants and

their more distant relationship to the human

protein. The eight peptides thatwerefound in

all oftheTauantigens thatweexaminedmight

correspond to regions in the proteins that are

conserved to maintain proper structure and

function.

Some lines of SV40-transformed celLs

con-tained more than one electrophoretic form of

Tauantigen(Fig. 1). This observation has been

madepreviously byus (5) and others (9, 16, 21,

28). Fingerprint analysis of the

methionine-la-beledtryptic peptides of the 56,000- and

54,000-daltonforms of theTauantigens isolated from

SV40-transformed human cells showed that

these proteins were closely related (Fig. 4 and

5c). Therefore theremaybeaprecursor-product

relationship between these two forms of Tau

C

..

D

.:I

E

..

::.;...ft 16

.:...

F

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SV40 Tau ANTIGENS

A

0

b

*:

E*'!

%.:w

,:::

g.:..

i .M-

w:.''...

.S x fUN...

.?,,::::

g£ 4.:' ': se, :te .b

,,l_-;. .': ::N.:'.:::

?: ..:,

4>a

W...

::§:. ,:^ .:

:,X.'.'";.: :. ssMe,

, .:

t%'*

:..:t,@::.

*.. h.i:;:. |; ...

':

.+,. ,.

': :: ^+!

[image:6.504.110.398.69.333.2]

..

FIG. 4. Comparison ofthemethionine-labeledtrypticpeptidesoftwo different forms ofhuman Tau antigen:

fingerprints ofthemethionine-labeledpeptides of the56,O0-dalton (A),54,000-dalton (B), and a mixture of

the 56,000- and54,000-dalton(C) Tauantigens isolated fromlineSV80 cells. Human transformed cells (line SV80)werelabeled withL-[3S]methionine, and extracts of these cells were incubated with anti-T serum in an

immunoprecipitationreaction. The 56,000- and 54,000-dalton forms of line SV80 Tau antigen (Fig. 1) were

separated bygel electrophoresisfor4hat 25mA. The trypticpeptides of these proteins were subjected to

electrophoresisandchromatography on thin-layer cellulose plates.

a IIAS

b

IIAS

315

C

svo 56K

sv8o 54K

FIG. 5. Relationships among the methioni beled trypticpeptides ofmouse Tau antigens (a), mouse, rat,and human Tauantigens(b),and human

56,000-dalton(56K)and54,000-dalton(54K)Tau

an-tigens (c). The numbers in the areas where three circlesoverlapin(a) and(b) refertothe numbersof

peptidescommon tothethreespecies ofTauantigen

indicated. The numbersofpeptidescommontoonly twoofthe threeproteinsare indicated inthe other

antigen, or the proteins may be products of

nearly identicalgenes. Pulse-chase experiments

or labeling experimentsperformed in the

pres-ence of protease inhibitors might distinguish

between these

possibilities.

)

Recent datafromourlaboratory (manuscript

in

preparation)

indicatethataTau

antigen

pro-SV80 tein can be immunoprecipitated from monkey

cells after infection with SV40. These results

imply that Tau antigens are induced in either

permissive or nonpermissive (i.e., mouse [19])

cells after infection with SV40. An analysis of

the methionine-labeled tryptic peptides of the

Tauantigen isolatedfrom infected monkeycells

showed that thisprotein isveryclosely related

to the Tau antigens from transformed human

ne-la-

cells

(line SV80). Thisobservation supports our

regions where the circles overlap. The numbers of peptides uniquetoindividualproteinsareindicated outsidetheoverlappingregions. In(c),inwhichonly twoproteins are compared, the number within the region ofoverlap refers to the number ofpeptides commontothesetwoproteins.

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656 SIMMONS ET AL.

contention thatTauantigensareevolutionarily

conservedproteins.

Theinformationpresented in thispaperand

the evidence cited aboveallow us to speculate

about the role of Tau antigens in infected or

transformed cells. Given that Tauantigenscan

beisolatedfromtransformed cells in theformof

acomplex withT-Ag(17),that thesynthesisof

Tauantigens is induced after infection of

mon-keycells withSV40, and that Tauantigensare

conserved proteins, we can hypothesize that

theseproteinsareinvolvedinafunctionusually

attributed toT-Ag (forexample, the induction

ofhost DNA synthesis). It is well known that

theactivation ofhost DNAsynthesis isoneof

the early events that occur in permissive or

nonpermissive cells after infection with SV40

(13, 25). The agent that is responsible for this

process would have to be made in these celLs

early after infection and presumably in

trans-formedcellsaswell. Inadditiontomeeting this

requirement, Tau antigens appear tohave the

abilitytobindtoT-Ag,atleast intransformed

cells (17). We can speculate, therefore, that a

complex ofT-Ag andTauantigen isresponsible

forthis induction.Fromthismodel,it iseasyto

explain the observation that Tau antigens are

conserved proteins since they would have to

interact with SV40 T-Ag, as well as with the

DNA-synthesizingmachinery ofmanydifferent

typesofcells.

ACKNOWLEDGMENTS

WeacknowledgeMargaret Sexauer for her expert technical assistance.

This workwassupportedin partbyPublic Health Service grant CA25942toD.T.S.fromtheNational Cancer Institute.

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