Proc. NatL Acad. Sci. USA Vol. 80, pp. 906-910, February 1983 Biochemistry

Purification of biologically active simian virus 40 small tumor (hybrid trp-lac /actin cable effect) ILAN BIKEL*, THOMAS M. ROBERTS*, MARILUCI T. BLADON*, ROBERT GREEN*, EGON AMANNt, AND DAVID M. LIVINGSTON* *The Sidney Farber Cancer Institute and The Departments of Medicine and Pathology, Harvard Medical School, Boston, Massachusetts 02115; and tDepartment of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 Communicated by Morris Friedkin, October 29, 1982 The simian virus 40 small tumor antigen (t antigen) gene has been were grown in L broth containing ampicillin (40 Ag/ml) to an cloned downstream from a hybrid Escherichia coli trp-lac pro- ODW of0.7 and then incubated for 2 hr with 5 mM isopropyl moter anda suitable ribosome binding site. Abacterial clone (865i) f3-D-thiogalactopyranoside. Radiolabeling of with transformed by such a plasmid (pTR865) expresses this gene and, [3S]methionine was performed as described (31). under optimal conditions, can produce .5% ofits total as t Antigen and Protein Assays. t antigen was detected by Na- t antigen. Soluble extracts ofsuch a clone were relatively depleted DodSO4/polyacrylamide slab gel electrophoresis (32) using 4% in t antigen, which was found in the initial pellet fraction. The stacking and 15% separating gels. Marker proteins were bovine protein was recovered from this fraction in a significantly purified serum albumin (Mr 68,000), carbonic anhydrase (29,000), form byextraction with urea-containing buffer. After gel filtration concentrations were deter- of such t antigen-enriched solutions, highly purified protein was and myoglobin (17,000). Protein obtained. When either this fraction (freed ofurea) or NaDodSO4 mined by the method of Lowry et al. (33) with bovine serum gel-purified 865i t antigen (rendered free of detergent) was in- albumin as a standard. jected into untransformed rat cells, dissolution of intracellular Immunoprecipitation. [35S]Methionine-labeled cells were actin cable networks was observed. solubilized and the extracts were immunoprecipitated and elec- trophoresed as described (31). The early region ofsimian virus 40 encodes two products, large Purification of t Antigen. Washed E. coli cells were sus- tumor antigen (T antigen) and small tumor antigen (t antigen), pended (1 g wet weight per 8 ml) in 50 mM Tris-HCI, pH 7.9/ which play an important role in the process of virus-induced 25% sucrose/1% Nonidet P-40/0.5% sodium deoxycholate/2 neoplastic transformation (1-19). t antigen is a 20-kilodalton mM dithiothreitol/5 mM EDTA and sonicated. After centrif- (kDal), nonphosphorylated polypeptide, and most of it can be ugation at 6,000 X g for 30 min at 40C, the pellet, which con- isolated from the cytoplasmic fraction ofinfected cells (20, 21). tained 290% of the cell content oft antigen, was taken as the Its precise biochemical function is unknown, although its pri- starting fraction for further protein purification. This fraction mary structure is characterized by two cysteine clusters which was reextracted twice at 0C by sonicating in 25 mM Tris HCI, also occur in a group ofglycopeptide tropic hormones (22). Fur- pH 8.4/2 mM dithiothreitol (buffer A) containing 2 M NaCl and thermore, repeated, specific co-immunoprecipitation of papo- 2 M urea. After centrifugation, each of these supernatant frac- vavirus t antigen and two cell proteins (56 and 32 kDal) from tions was discarded. Finally, the pellet was suspended in buffer crude cell extracts has been observed (23-25). These results A containing 10 M urea, sonicated, and then centrifuged at 18°C suggest that the antigen can form an intracellular complex with for 30 min at 6,000 x g. This extraction was repeated twice, and these two cell gene products. Finally, there is evidence linking the supernatants were combined. Subsequent purification the synthesis of intact t antigen to the acute disappearance of steps were performed on this fraction (extract A). The yield of intracellular actin cable networks (26, 27), the new appearance t antigen in extract A was found to be independent of the use ofcentriolar structures (28), and, in the presence of T antigen, of lysozyme prior to detergent treatment and sonication. For the promotion ofcell DNA synthesis in untransformed, resting further purification of t antigen, 1 ml of extract A, containing cells (3, 27, 29). How these phenomena result from the func- 0.5-2 mg of protein, was applied to a 2.6 X 40 cm column of tion(s) oft antigen is unknown. Sephacryl S-200 (Pharmacia) equilibrated at 32°C in buffer A This report describes the generation of a rich, prokaryotic containing 10 M urea; 0.8-ml fractions were collected. Fractions source of intact simian virus 40 t antigen and its extensive pu- containing t antigen and 14-kDal t antigen (see Results) were rification in a biologically active form. The availability of such pooled and dialyzed against three changes of 500 vol of buffer a preparation ofthe antigen hopefully will facilitate future anal- A. Both proteins were stored in a soluble form at 0°C in buffer yses of its functions. A at 60-100 ,ug/ml. Purification of tAntigen byNaDodSO4Gel Electrophoresis. MATERIALS AND METHODS t antigen and 14-kDal t antigen (see Results) were purified to simian homogeneity from extract A by preparative NaDodSO4/poly- Growth of Cells and Radiolabeling of Proteins. A Each was eluted- virus 40-transformed cell line, SV80 (30), was grown as de- acrylamide gel electrophoresis (32). protein scribed (31). Escherichia coli W3110 (rK-, mK+, lac iq) a high- from crushed, unfixed, unstained gel strips into 0.2 M ammo- level lac repressor-producing strain, was transformed with nium bicarbonate, pH 8.0/0.4% NaDodSO4 at 320C for 48 hr. pTR865 (see Results). W3110iq/865 (865i) transformed colonies Eluates were lyophilized, and the solid residue was redissolved in water. Each solution was dialyzed at 40C for two 16-hr pe- The publication costs ofthis article were defrayed in part by page charge payment.. This article must therefore be hereby marked "advertise- Abbreviations: t antigen, small tumor antigen; T antigen, large tumor ment" in accordance with 18 U. S.. C. §1734 solely to indicate this fact. antigen; kDal, kilodalton.

906 Downloaded by guest on September 29, 2021 Biochemistry: Bikel et al. Proc. Natl. Acad. Sci. USA 80 (1983) 907

riods, once against 500 vol ofbuffer B (25 mM Tris HCI, pH 7.4/ a relevant segment ofthe E. coli trp operon is cloned in the Cla 0.14 M NaCi) containing 1% cholic acid and once against 500 I site of pBR322. In one of the resulting plasmids, pEA108, vol ofbuffer B containing 0.1% cholic acid. After three further there is a unique Cla I site adjacent to the -35 region of the 16-hr dialyses against 500 vol ofbuffer B containing 0.1% cholic trp promoter. acid, dialysis was performed against three changes of 500 vol In the actual construction (Fig. 1), an Hpa II-EcoRI fragment of buffer B. from pTR436C, bearing the small t antigen gene, ribosome Extraction with Solutions Containing Organic Solvent. Var- binding site, lac operator, and lac promoter through base -21, ious amounts ofextract A (0.3-0.6 mg/ml) were dialyzed against was removed from pTR436C (a modification of pTR436 kindly two changes of 100 vol of (i) 40% 1-propanol/0.25 M urea/0.6 provided by Kai Zinn in which the first Hha I site distal to the M Na acetate, pH 8.1, (ii) 75% 1-propanol/0.25 M Na acetate, t antigen encoding region of pTR436 has been converted to an pH 8.1, or (iii) chloroform/methanol, 1.8:1 (vol/vol), for 16 hr EcoRI site by using a synthetic EcoRI linker) and cloned be- at room temperature. A precipitate formed in each case and was tween the unique Cla I and EcoRI sites of pEA108. In the re- removed by centrifugation at-the end of the dialysis period. sulting plasmid, pTR865, the -35 consensus sequence of ptrp Preparation of Monospecific Anti-t/T Antigen. Two New (cross-hatched area) is joined to the pTR436C sequence. Thus, Zealand White rabbits were each injected, on days 1, 8, and 28, plac is converted to ptac with no modification ofthe messenger with NaDodSO4 gel bands containing 200 pAg of 865i t antigen encoding regions ofthe hybrid gene. This plasmid was used to emulsified in Freund adjuvant. Sera were assayed for [35S]- transform E. coli W31JJOi, a strain that constitutively produces methionine-labeled t/T antigen binding by immunoprecipita- high levels of the lac repressor. A typical colony (865i) grew tion and were absorbed with a sonicated extract ofuntransformed efficiently and synthesized easily detectable amounts of a 20- E. coli W3110iq cells (10 mg of protein per ml). kDal protein (Fig. 2a; compare lanes B and G) which migrated Manual Injection of Various Solutions into Rat-1 Cells and with pTR436c [35S]methionine-labeled t antigen. In addition, Visualization of Actin-Containing Cable Structures. Microin- when a culture of 865i was incubated in the presence of iso- jection of Rat-1 cells growing on glass coverslips and immu- propyl thiogalactoside, the concentration ofthe 20-kDal protein nofluorescent screening of these cells for actin cable networks increased dramatically. were performed as described (19, 26). In all experiments, scor- To extract the 20-kDal protein, isopropyl thiogalactoside-in- ing ofinjected cells was performed under conditions such that duced 865i cells were lysed by sonication in the presence of the analyst was not aware of what had been injected into the dithiothreitol, Nonidet P-40, and deoxycholate. Under these cells under consideration. conditions, most of the 20-kDal band was found in the initial pellet fraction (Fig. 2b). This band contained t/T antigen com- RESULTS mon determinants because it was specifically immunoprecipi- tated by rabbit anti-NaDodSO4 gel-purified T antigen (Fig. 2c). Generation of a Bacterial Clone That Overproduces t An- Given these observations and the fact that pTR865 and pTR436 tigen. Previous results showed that it was possible to construct have identical t antigen messenger encoding sequences, it was a plasmid carrying the t antigen coding sequence, a relatively assumed that the 20-kDal bands in cells transformed by each strong (UV5) E. coli lac promoter, and a functional hybrid ri- plasmid were identical. Prior results indicated that the pTR436 bosome binding site (31, 34). Bacteria carrying such a plasmid 20-kDal immunoreactive protein was t antigen lacking only an synthesized intact t antigen, but the amounts present were NH2-terminal acetyl group (31). lower than those needed for purifying biochemical quantities In one test of this point, antibody was raised against the of this protein. In an effort to increase the yield of t antigen, NaDodSO4 gel-purified 20-kDal band. This serum specifically we elected to construct a more efficient version of the lac pro- immunoprecipitated T antigen, t antigen, and an 8- to 12-kDal moter recently described by DeBoer et aL (35). Amann et al. band from SV80 cells (Fig. 2d). The 8-kDal band is composed (36) have constructed plasmids in which a fragment containing largely oft/T antigen shared sequences which are encoded by (-35) (-10) MACA TATAAT ATG UAAI pTR436C A_~~~~~~~~~...... I promoterlac operatorlac SDl1c t ontigen

HpaII R I \ \ \ RI~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

(-35) ClCa I TTG(CAI- - -I pEA108 4 pl- -mq I promoterTrI I%

ft..~~~ ~ ~ __

oft*-.'..''.%.1ftS 1 ...... R 1 (-35) 1 (-10) TTGACA TATAAT%Irr pTR865 F03_ xrBr_A _~ promoterTac operatorlac SDlc t antigen

FIG. 1. Construction of pTR865. Downloaded by guest on September 29, 2021 908 Biochemistry: Bikel et al. Proc. Natl. Acad. Sci. USA 80 (1983) (q) (h) (C.) (d.) A B C D ARB c A BC D _m*- 68K-

29K- -29K

20K- 17K- -17K -14K t

FIG. 2. Induction, extraction, and characterization of 865i t antigen. (a) Cultures (1 ml) of 865i cells were grown and induced without (lane B) or with isopropyl thiogalactoside at 1 mM (lane C), 5 mM (lane D), or 10 mM (lane E). After centrifuging and washing, the bacterial pellets were lysed with NaDodSO4 gel sample buffer (32) and the extracts were boiled for 3 min and electrophoresed. Lane F, extract of am maxicell preparation (37) ofE. coli RBI13 which contains and expresses the t antigen gene of pTR436c. Lane G, NaDodSO4 extract of pBR322-containing W311jiq cells. Lane A, marker proteins (sizes shown in kDal). The migration position of pTR436c [35Shmethionine-labeled t antigen (20 kDal) identified in an autoradiogram of lane F is indicated. The gel was stained with Coomassie brilliant blue. (b) Coomassie brilliant blue-stained NaDodSO4 gel elec- tropherogram of a whole-cell NaDodSO4 extract of 865i'prepared as in a (lane B), as well as the supernatant (lane C) and pellet (lane D) of such cells extracted by sonication in Nonidet P-40/sodium deoxycholate-containing buffer. Lane A, marker proteins. The positions of t antigen and 14- kDal t antigen are noted. (c) Autoradiogram of a NaDodSO4 gel electropherogram containing -immunoprecipitated from 865i. Lanes: A, preimmune rabbit serum immunoprecipitate of an extract of 865i; B; rabbit anti-gel-band-purified T antigen immunoprecipitate of this extract; C, [(Slmethionine-labeled maxicell extract from pTR436c/RB113. (d) Anti-865i t antigen immunoprecipitation of SV80'cells and characterization of extract A from isopropyl thiogalactoside-induced, [35S]methionine-labeled 865i cells. Lanes: A, rabbit antibody raised against NaDodSO4' gel- purified 865i 20-kDal t antigen was used to immunoprecipitate an extract of [35Slmethionine-labeled SV80 cells; B, a preimmune rabbit serum precipitate of the same extract; C, an aliquot of 865i extract A. Lane D contained an aliquot of, a NaDodSO4 whole-cell extract of these labeled bacteria. An autoradiogram of this gel is presented. the 0.655-0.59 map unit segment ofthe viral (21). An anti-t/T antigen immunoreactive 14-kDal. band was also de- tected in 865i extracts, and it migrated with a pTR436c 14-kDal I (b) in a maxicell extract (31) (Fig. 2c). Previously, A B C band observed T N T-N this polypeptide was shown to share methionine-labeled tryptic I§ peptides with t antigen (34, 38), and our preliminary sequence results suggest that it is composed of the COOH-terminal 123 -68K residues oft antigen (unpublished data). At nucleotides 5,026- 5,023, there is a typical Shine-Dalgarno sequence followed by an ATG (nucleotides 5,010-5,008) 13 base pairs downstream, -29K which may constitute an effective ribosome binding site. Measurements of the t antigen concentration in extracts of isopropyl thiogalactoside-induced 865i revealed that it consti- -1 tuted .5% of total cell protein or at least 400,000 monomers i-17K per cell. In some induced 865i cultures, t antigen constituted -14K t -10% of total cell protein. Purification of t Antigen from 865i Cells. Logarithmically growing 865i cells were induced with 5 mM isopropyl thioga- 14K14.t lactoside and extracted in the presence ofdetergents. After cen- trifugation, the pellet, fraction -was washed in the same lysis mixture and then in buffer containing 2 M urea and 2 M NaCl. ELUT ION with buffer containing 10 M urea. The It was finally extracted FIG. 3. (a) Autoradiogram of a NaDodSO4 gel electropherogram final soluble fraction~contained nearly all ofthe t antigen present of Sephacryl S-200 gel filtration column fractions. [3"S]Methionine- in the original pellet (Fig. 2d, lane C) and it was chromato- labeled extract A was chromatographed in a column of Sephacryl S- graphed in a column of-Sephacryl S-200 equilibrated in buffer 200, and'every otherfraction was precipitated with trichloroacetic acid containing 10 M urea. The antigen eluted in a number of frac- and electrophoresed. Elution was from right to left. (b) Immunopre- tions including the void volume (Fig. 3a). However, highly pu- cipitation9fpurified35S-labeled t and 14-kDal t antigens. The 14-kDal with a small amount of 14- t antigen (lane A) and 20-kDaI t antigen (lane B) bands were excised rified t antigen (contaminated only from NaDodSO4 gels loaded with a NaDodSO4 extract of 865i, the pro- kDal t antigen) was detected in the distal portion of the chro- teins were,eluted, and the detergent was removed. Lane C, Sephacryl matogram. More distal fractions sometimes contained highly S-200-purified t and 14-kDal t antigen-containing fractions were di- purified 14-kDal t antigen, which was largely depleted in the alyzedfee of urea. Halfof eachfraction was-then allowed to react with 20-kDal band (data not shown). The yield of purified t antigen rabbit antiserum raised against gel band-purified t antigen (lanes I) was =150 pAg from a 70-ml culture of induced 865i grown to an or with preimmune rabbit serum (lanes-N). Downloaded by guest on September 29, 2021 Biochemistry: Bikel et al. Proc. Natl. Acad. Sci. USA 80 (1983) 909

M P S

68K -

FIG. 4. Extraction oft and 14-kDal t antigens 29K - into an organic solvent-containing solution. 865i extract A was dialyzed against buffer containing 40% 1-propanol and 0.25 M urea. The ensuing t precipitate (lane P) and a trichloroacetic acid pre- t " ~ cipitate of an aliquot of the supernatant (lane S) 17K - were separately dissolved in electrophoresis sam- 4 Kt - plebuffer and electrophoresed; the NaDodSO4 gel was stained with Coomassie brilliant blue. Lane M, marker proteins. ODsw of 1.2 or 10-20% of the t antigen present in the starting material. When pooled and dialyzed free ofurea, both t antigen and the 14-kDal t antigen were immunoreactive, as were the t antigen and 14-kDal t antigen eluted directly from NaDodSO4/ polyacrylamide gels and freed of detergent (Fig. 3b). In addition, when a partially purified preparation of20-kDal and 14-kDal t antigens was dialyzed into either chloroform/ methanol, 1.8:1 (data not shown), or a buffer containing 40% 1-propanol and 0.25 M urea (Fig. 4) or 75% 1-propanol without urea (data not shown), 60-80% of the t and 14-kDal antigens remained soluble although, in the first case, continuing precip- itation was noted with time. This suggests that these proteins display unusual hydrophobicity, a property consistent with ear- lier findings by others (39). Assay of Purified t Antigen for Biological Activity. A known effect of the presence of intact t antigen in certain cells is the acute disappearance of actin cable structures (26, 27). Hence, to test whether purified 865i t could induce such an effect, Rat- 1 cells were injected with aliquots of Sephacryl S-200-purified t and 14-kDal t antigens (freed of urea) or with NaDodSO4 gel band-purified t and 14-kDal t antigens (freed ofdetergent). The buffer in which each of these proteins was dissolved also was injected in parallel. All injections included rhodamine-conju- gated human IgG in order to identify injected cells (19). Injec- tion oft antigen from both sources led to significant actin cable dissolution, whereas no such effect was noted after injecting buffer or comparable amounts of the 14-kDal t antigen (Table 1; Fig. 5). DISCUSSION FIG. 5. Effect of injecting purified 865i t antigens into Rat-1 cells. Progress in understanding t antigen function has been limited At 22 hr after injection of test solutions into subconfluent Rat-1 cells, by a lack ofpurified, biologically active protein. In this report, the cells were fixed, treated with specific antibody, and examined for the generation of an enriched bacterial source oft antigen and actin cable fluorescence. (A, C, and E) Fluorescein fluorescence. (B, a method for it have been D, and F) Rhodamine fluorescence. Solutions injected were: A and B. purifying described. Overproduction buffer; C and D, Sephacryl S-200-purified 865i t antigen dialyzed free of urea (-0.1 mg/sud); E and F, NaDodSO4 gel band-purified 865i t Table 1. Effect of various solutions upon actin cable structures of antigen dialyzed free of detergent (-0.3 ,ug/,ul). All solutions were Rat-1 cells made 6 mg/ml in rhodamine-conjugated human IgGprior to injection. % cells Actin Actin of the antigen in E. coli was achieved by reconstructing the Injection deficient positive promoter governing t antigen in an existing Buffer 9 91 plasmid (pTR436c) (31). Specifically, the introduction of a -35- Gel-band t antigen 41 56 base-pair consensus sequence from a promoter ofconsiderable Gel-band 14-kDal t antigen 19 81 strength (ptrp) was accompanied by a 10- to 20-fold increment Sephacryl S-200 t antigen 61 39 in t antigen concentration, suggesting that a major increase in Sephacryl S-200 14-kDal t antigen 12 88 t antigen synthesis resulted from a stimulation of t antigen cis- tron transcription. The concentrations of the various proteins were: NaDodSO4 gel The initial band-purifiedt and 14-kDal t antigens, -0.15 ig/uLI; Sephacryl S-200- problem encountered in the purification of t an- purified t and 14-kDal t antigens, -0.05 ,ug/,l. Approximately 200 tigen was its insolubility. This was overcome in part by using cells were injected with each solution. All solutions contained rhoda- graded urea extraction of the initial pellet fraction which is de- mine-conjugated human IgG (19). void of most of the cell proteins. The dissolved antigen, now Downloaded by guest on September 29, 2021 910 Biochemistry: Bikel et al. Proc. Natl. Acad. Sci. USA 80 (1983) highly purified, could then be separated from other detectable 9. Feunteun, J., Kress, M., Gardes, M. & Monier, R. (1978) Proc. E. coli proteins by gel filtration in urea, the only visible con- Natl Acad. Sci. USA 75, 4455-4459. taminant being 14-kDal t antigen. After being freed ofurea, the 10. Rassoulzadegan, M., Perbal, B. & Cuzin, F. (1978) J. Virol 28, 1-5. product remained soluble at relatively low protein concentra- 11. Fluck, M. M. & Benjamin, T. L. (1979) Virology 96, 205-228. tions (<60-150 ,ug/ml) and reacted with specific antibody. 12. Seif, R. & Martin, R. (1979) J. Virol 31, 350-359. Although substantial in an absolute sense, the yield ofpurified 13. Seif, R. & Martin, R. (1979)J. Virol. 32, 979-988. t antigen was limited by the fact that a significant fraction ofthe 14. Martin, R. G., Setlow, V. P., Edwards, C. A. F. & Vembu, D. antigen, from either animal cells or bacteria, remained aggre- (1979) Cell 17, 635-643. gated in isotonic medium, as noted previously (34, 39), or in 15. Martin, R. G., Setlow, V. P., Chepelinsky, A. B., Seif, R., Lewis, A. M., Jr., Scher, C. D., Stiles, C. D. & Avila, J. (1979) high concentrations of urea. This resulted in its comigrating Cold Spring Harbor Symp. Quant. Biol 44, 311-324. with a number of cell proteins in the void volume and high 16. Steinberg, B. M. & Pollack, R. (1979) Virology 99, 302-311. molecular weight fractions of Sephacryl S-200 columns. 17. Frisque, R. J., Rifkin, D. B. & Topp, W. C. (1979) Cold Spring Of major initial concern was the question of whether puri- Harbor Symp. Quant. Biol 44, 325-331. fied 865i t antigen would have detectable biological activity af- 18. Sugano, S., Yamaguchi, N. & Shimojo, H. (1982) J. Virol 41, ter exposure to potentially solvents. Because both 1073-1075. denaturing 19. Rubin, H., Figge, J., Bladon, M. T., Chen, L. B., Ellman, M., Sephacryl S-200- and gel band-purified t antigens were active Bikel, I., Farrell, M. P. & Livingston, D. M. (1982) Cell 30, 469- in the actin cable dissolution assay, it appears that at least some 480. of the purified t antigen molecules possess this activity. The 20. Prives, C., Gilboa, E., Revel, M. & Winocour, E. (1977) Proc. proportion might be small because 6 x 107 molecules of t Nati Acad. Sci. USA 74, 457-461. antigen were injected per cell but only incomplete effects were 21. Spangler, G. J., Griffin, J. D., Rubin, H. & Livingston, D. M. of 14-kDal t in (1980) J. Virol. 36, 488-498. observed. However, the lack ofactivity antigen 22. Friedmann, T., Doolittle, R. F. & Walter, G. (1978) Nature this assay strongly suggests that the actin cable effect was spe- (London) 274, 291-293. cific. The 14-kDal t antigen was the onlydetectable contaminant 23. Yang, Y.-C., Hearing, P. & Rundell, K. (1979)J. Virol 32, 147- in Sephacryl S-200-purified fractions. Because it failed to dis- 154. solve actin cables at approximately equimolar concentration, it 24. Rundell, K., Major, E. 0. & Lampert, M. (1981) J. Virol 37, probably was not responsible for the actin cable effect induced 1090-1093. from this 25. Rundell, K. (1982)J. Virol 42, 1135-1137. by the Sephacryl S-200-purified material. However, 26. Graessmann, A., Graessmann, M., Tjian, R. & Topp, W. C. result, it should not be argued that 14-kDal t antigen is biolog- (1980)J. Virol 33, 1182-1191. ically inert; when isolated differently or injected under other 27. Hiscott, J. B. & Defendi, V. (1981) J. Virol. 37, 802-812. conditions or at higher concentration, it may be active. Fur- 28. Shyamala, M., Atcheson, C. L. & Kasamatsu, H. (1982)J. Virol thermore, the existence ofimmunologic and biological activity 43, 721-729. in the NaDodSO4 gel-purified t antigen fractions suggests that 29. Tjian, R., Fey, G. & Graessmann, A. (1978) Proc. Natl Acad. Sci. to a dena- USA 75, 1279-1283. the antigen can be renatured after exposure strong 30. Todarao, G., Green, H. & Swift, M. R. (1966) Science 153, 1252- turant. This is nota surprising finding because biological activity 1254. has been detected in other NaDodSO4 gel-purified growth-af- 31. Roberts, T., Bikel, I., Yocum, R., Livingston, D. M. & Ptashne, fecting polypeptides such as a and y interferons, platelet-de- M. (1979) Proc. Natl. Acad. Sci. USA 76, 5596-5600. rived growth factor, and murine T-cell suppressor factor (40- 32. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 42). 33. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. The availability of biochemical quantities of purified t anti- (1951)J. Biol Chem. 193, 265-275. 34. Thummel, C. S., Burgess, T. L. & Tjian, R. (1981) J. Viroi 37, gen may make new structural, cytochemical localization, and 683-697. functional studies of this protein possible. 35. DeBoer, H. A., Comstock, L. J., Jansura, D. G. & Heyneker, H. C. (1982) in Promoter Structure and Function, eds. Chamberlin, The authors thank Drs. Lan Bo Chen, Geoffrey Cooper, and Charles M. 0. & Rodriguez, R. (Praeger Scientific, Eastbourne, E. Sus- Stiles for invaluable discussions and advice during the course of these sex, U.K.). studies. We are also grateful to Ann Desai for her expert assistance 36. Amann, E., Brosius, J. & Ptashne, M. (1982) Proc. Natl Acad. during the preparation ofthis manuscript. This work was supported by Sci. USA, in press. Grants CA21082 and CA30002 from the National Cancer Institute. 37. Sancar, A., Hack, A. M. & Rupp, W. D. (1979)J. Bacteriol. 137, 692-693. 1. Tooze, J., ed. (1981) DNA Tumor Viruses, Molecular Biology of 38. Derom, C., Gheysen, D. & Fiers, W. (1982) Gene 17, 45-54. Tumor Viruses (Cold Spring Harbor Laboratory, Cold Spring 39. Tegtmeyer, P., Spillman, T. & Schuetz, F. R. (1979) Cold Spring Harbor, NY), 2nd Ed. Harbor Symp. Quant. Biol. 44, 159-164. 2. Brugge, J. S. & Butel, J. S. (1975) J. Virol. 15, 619-635. 40. Yip, Y. K., Barrowclough, B. S., Urban, C. & Vilcek, J. (1982) 3. Chou, J. Y. & Martin, R. G. (1975)J. Viroi 15, 145-150. Proc. Natl Acad. Sci. USA 79, 1820-1824. 4. Osborn, M. & Weber, K. (1975)J. Virol. 15, 636-644. 41. Antoniades, H. N. (1981) Proc. Natl. Acad. Sci. USA 78, 7314- 5. Tegtmeyer, P. (1975)J. Virol 15, 613-618. 7317. 6. Shenk, T. E., Carbon, J. & Berg, P. (1976)J. Virol. 18, 664-671. 42. Fresno, M., McVay-Boudreau, L. & Cantor, H. (1982) J. Exp. 7. Bouck, N., Beales, N., Shenk, T., Berg, P. & Di Mayorca, G. Med. 155, 981-993. (1978) Proc. Natl Acad. Sci. USA 75, 2473-2477. 8. Sleigh, M. J., Topp, W. C., Hanick, R. & Sambrook, J. F. (1978) Cell 14, 79-88. Downloaded by guest on September 29, 2021