Biochemical properties of the 145,000-dalton super-T antigen from simian virus 40-transformed BALB/c 3T3 clone 20 cells

SV3T3 C120 cells contain a 145,000-dalton form of simian virus 40 (SV40) super-T antigen but little if any normal-sized large-T. The subcellular location of super-T, its DNA binding properties, and its interaction with nonviral tumor antigen (NVT) were examined. Immunofluorescence microscopy and subcellular fractionation indicated that super-T is almost exclusively nuclear. Chromatography on double-stranded DNA-cellulose showed that super-T binds to double-stranded DNA and has an elution profile indistinguishable from normal-sized large-T. Super-T also binds specifically to a fragment of SV40 DNA which contains the origin of DNA replication. However, immunoprecipitation of super-T or large-T either with anti-tumor cell serum or with anti-NVT serum from fractions obtained by sucrose density centrifugation of 32P-labeled or [35S]methionine-labeled extracts revealed clear differences in the sedimentation characteristics of these proteins. The bulk of labeled 145,000-dalton super-T sedimented between 4S and 10S, whereas the bulk of 32P-labeled large-T from normal SV40-transformed cells sedimented as two peaks at 23S to 25S and 16S to 18S. By contrast, the sedimentation properties of NVT from the SV3T3 C120 cells were similar to those normally observed with other SV3T3 cell lines. The reason for this apparent difference in complex formation between super-T and NVT and that normally observed with large-T is unclear, but it probably has no deleterious effect on the ability of super-T to maintain transformation.     

Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.

SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.     

Identification of simian virus 40 protein A.

A large simian virus 40 (SV40)-specific protein can be efficiently immunoprecipitated from infected cell extracts with antisera obtained from hamsters bearing SV40-induced tumors. The protein has an apparent molecular weight of 88,000 to 100,000 with respect to markers with known molecular weights, but behaves anomalously on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Cell lines infected by two different strains of SV40 synthesize immunoreactive proteins that differ slightly in mobility during SDS-polyacrylamide gel electrophoresis, evidence that the protein is coded for by the virus. These differences in protein size correlate with differences in the electrophoretic mobility of viral DNA fragments obtained by digestion with HindII and III restriction enzymes. The size of the viral capsid proteins VP2 and VP3 also varies with the strain of virus. dl-1001, a constructed deletion mutant that lacks part of the SV40A gene, directs the synthesis of a 33,000-dalton polypeptide that is not detected in cells infected with wild-type virus. The deletion fragment, like the larger protein, is phosphorylated. Maps of tryptic peptides from the 88,000- to 100,000-dalton protein and the 33,000-dalton fragment show common peptides and provide strong direct evidence that the proteins are products of the SV40 A gene. The deletion fragment reacts with antitumor sera and binds to double-stranded DNA in the presence of the complete A protein.     

An antibody to a synthetic peptide that recognises SV40 small-t antigen.

A peptide Tyr.Arg.Asp.Leu.Lys.Leu corresponding to the carboxy-terminal six amino acids of small-t antigen predicted from the DNA sequence of SV40 was synthesised, coupled to bovine serum albumin and to ovalbumin and used to raise antibody in rabbits. The sera obtained immunoprecipitated [125I]peptide. It also recognised SV40 small-t that was synthesised in vitro from SV40 mRNA or extracted from SV40 infected monkey cells. The immunoprecipitation of small-t was inhibited by added peptide. To demonstrate that the determinant was present at the carboxy-terminal end of the molecule, truncated versions of small-t coded for by 0.54-0.59 deletion mutants were tested. dl 890 small-t, which contains an in-phase deletion removing nine amino acids but leaving the carboxy-terminal sequences intact, was recognised by the antipeptide serum. By contrast dl 885 small-t, which has an out-of-phase deletion leading to an altered carboxy terminus coded in an alternative reading frame, was not recognised. The data confirm the location and specificity of the determinant recognised on small-t by the antipeptide serum.     

Simian virus 40-transformed cells express new species of proteins precipitable by anti-simian virus 40 tumor serum.

In addition to the virus-coded large-T and small-t antigens, two new classes of proteins were immunoprecipitated by anti-simian virus 40 (SV40) tumor serum from extracts of various SV40-transformed cell lines. These were as follows: (i) proteins (termed "super-T proteins") with an Mr higher than that of large-T antigen (86,000), which were found in many SV40-transformed cell lines derived from mouse and rat cells (super-T proteins and large-T antigen appeared to have closely related structures as judged by the Chromobead elution patterns of their methionine-labeled tryptic peptides); (ii) proteins (termed "55K proteins") with an Mr ranging from 50,000 to 60,000, which were present in all SV40-transformed cell lines examined so far, including those obtained by chromosome-mediated gene transfer. The 55K proteins were not structurally related to large-T antigens, as judged by the Chromobead elution patterns of their methionine-labeled tryptic peptides. Our data are compatible with the assumption that the 55K proteins are largely or totally cell coded.     

Identification and partial characterization of new antigens from simian virus 40-transformed mouse cells.

Two new species of antigens were detected in simian virus 40-transformed mouse cells, in addition to the large (94,000 daltons) and small (20,000 daltons) tumor antigens. These antigens were immunoprecipitated from cell extracts by using anti-T serum and not normal, nonimmune serum. One of these was a protein with a molecular weight of approximately 130,000 and was present in some but not all SV40-transformed mouse cells. The other, which we have named Tau antigen, has a molecular weight of 56,000 as estimated by electrophoresis through acrylamide gels and was found in all virus-transformed cells examined. The 13,000-daltons antigen contained about 15 methionine-tryptic peptides which were also present in the large SV40 tumor antigen as determined by ion-exchange chromatography. This strongly suggested that the protein was virus coded. The 56,000-dalton Tau antigen appeared to share only two methionine-tryptic peptides with the large species of SV40 tumor antigen, as determined by ion-exchange and paper chromatographies. Our results are compatible with a cellular origin for Tau antigen. However, our data do not exclude the possibility that this protein contains sequences specified by the virus DNA.     

The influence of fluoro-phenyl-alanine on the synthesis of simian virus 40 DNA and T antigens.

Treatment of Simian Virus 40 (SV40) infected monkey cells with fluorophenylalanine (FPA) resulted in increased uptake of thymidine by the cells, and progressive inhibition of both viral and cellular DNA synthesis. Viral DNA synthesis was more sensitive to inhibition by FPA than cell DNA synthesis. Synthesis of SV40 T antigens was however unaffected by FPA, as judged from immunofluorescence assays. The M.W. of the major polypetides immunoprecipitated from cell extracts by antibodies from tumor bearing hamster sera was similarly unaffected. It is suggested that T antigen synthesized in the presence of FPA is non functional.     

Extraction and fingerprint analysis of simian virus 40 large and small T-antigens.

A study of simian virus 40 (SV40) T-antigens isolated from productively infected CV1 cells using a variety of different extraction procedures showed that under some conditions the highest molecular weight form of T-Ag (large-T) isolated comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with large-T from SV40-transformed H65-90B cells. Other faster-migrating forms of large-T are probably generated during the extraction procedure by a protease which is active at low pH, and such forms are probably experimental artifacts. After extraction under conditions which minimize proteolytic degradation of large-T, a further form of T-antigen was isolated; this has an apparent molecular weight in the range 15,000 to 20,000 and is referred to as small-t. Fingerprint analysis of [35S]methionine-labeled SV40 proteins showed that small-t has 10 to 12 methionine peptides whereas large-T has 15 to 18 methionine peptides. All but two of the methionine tryptic peptides present in small-t are also present in large-T. The fingerprint data also showed that T-antigens have no peptides in common with SV40 VP1. Experiments using reagents which inhibit posttranslational cleavage of encephalomyocarditis virus polyproteins showed that these reagents do not affect the synthesis of small-t and suggest that it is not made by proteolytic cleavage of large-T in vivo. An alternative model, which proposes that large-T and small-t are synthesized independently, is discussed in terms of the fingerprint data and the number of methionine tryptic peptides predicted from the primary sequence of SV40 DNA.     

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).     

Association of a murine 53,000-dalton phosphoprotein with simian virus 40 large-T antigen in transformed cells.

Serum raised against a mouse 53,000-dalton (53K) phosphoprotein precipitates both the 53K immunogen and simian virus 40 large-T from lysates of simian virus 40-transformed 3T3 cells. This serum, designated F5, does not recognize antigenic determinants on native or denatured large-T and precipitates large-T because the 53K phosphoprotein forms a stable complex with large-T. This complex sediments at 23S on sucrose density gradients, corresponding to a molecular weight of 600K to 1,000K, and appears to contain only 53K and large-T as major components. It is held together by noncovalent bonds and is located in the cell nucleus. All the 53K immunoprecipitated from cell lysates by F5 is present in the high-molecular-weight complex, but large-T can be separated into a complexed and a free form on sucrose density gradients. The complexed form of large-T is more readily phosphorylated than the free form. We have been unable to detect an association of large-T with comparable host cell proteins during productive infections with simian virus 40.     

Covalent affinity labeling by periodate-oxidized [alpha-32P]ATP of the large-T proteins of polyoma and SV40 viruses

Incubation of crude extracts from cells lytically infected with polyoma virus in the presence of periodate-oxidized [alpha-32P]ATP led to the radioactive labeling of one main polypeptide immunoprecipitated by anti-T antigen antibodies. It was absent from extracts of mock-infected cells and exhibited an apparent Mr value of 105,000, identical with that of the large-T viral protein. This polypeptide was unambiguously identified as large-T on the basis of the heat sensitivity of the in vitro labeling in extracts from cells infected with a tsa mutant. The amount of incorporated radioactivity was found to increase linearly with that of infected cell extract and with the incubation time. Labeling resulted from the specific binding of an oxidized ATP molecule to a nucleotide binding site of the large-T protein, since it was efficiently completed by unlabeled ATP. Study of the dependence on the concentration of oxidized ATP evidenced a first order kinetic for the labeling reaction. Similar results were obtained for the large-T protein of SV40 virus.     

Characterization of different tumor antigens present in cells transformed by simian virus 40.

In addition to large T and small t antigens, cells transformed by simian virus 40 (SV40) commonly contain other proteins which specifically immunoprecipitate with SV40 anti-T serum and which are not detected in untransformed cells. The additional tumor antigens (T-Ags) fall into two groups: those having a close structural relationship with normal SV40 T-Ags, and those unrelated to large T and small t. The latter are probably nonviral T-Ags (NVT-Ags). The NVT-Ags comprise a family of proteins of molecular weight 50,000-55,000. Fingerprint analysis shows that NVT-Ags have few if any peptides in common with large T or small t, and that they lack the amino terminal tryptic peptide and the peptides unique to small t. NVT-Ags from different species have different fingerprints, but those isolated from different transformants of the same cell line are identical. The size of NVT is unaltered in cells transformed by mutants of SV40 with deletions in the region 0.60-0.55 map units. The mRNA for NVT does not hybridize to SV40 DNA. The other forms of T-Ag isolated from transformed cells fall into three classes: shortened forms of large T (truncated large T); multiple species of T-Ag with molecular weights very similar to, but distinct from, those of normal large T (large T doublets and triplets); and elongated forms of large T (super T). These proteins all contain the normal amino terminus of SV40 T-Ags, and the truncated forms of large T lack peptides from the carboxy terminal half of large T. One species of super T (molecular weight 130,000) contains only those methionine tryptic peptides present in normal large T, although it may contain some peptides in more than one copy.     

Evidence for Simian Virus 40 (SV40) Coding of SV40 T-Antigen and the SV40-Specific Proteins in HeLa Cells Infected with Nondefective Adenovirus Type 2-SV40 Hybrid Viruses

HeLa cells infected with the nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses (Ad2(+)ND1, Ad2(+)ND2, Ad2(+)ND4, and Ad2(+)ND5) synthesize SV40-specific proteins ranging in size from 28,000 to 100,000 daltons. By analysis of their methionine-containing tryptic peptides, we demonstrated that all these proteins shared common amino acid sequences. Most methionine-containing tryptic peptides derived from proteins of smaller size were contained within the proteins of larger size. Seventeen of the 21 methionine-containing tryptic peptides of the largest SV40-specific protein (100,000 daltons) from Ad2(+)ND4-infected cells were identical to methionine-containing peptides of SV40 T-antigen immunoprecipitated from extracts of SV40-infected cells. All of the methionine-containing tryptic peptides of the Ad2(+)ND4 100,000-dalton protein were found in SV40 T-antigen immunoprecipitated from SV40-transformed cells. All SV40-specific proteins observed in vivo could be synthesized in vitro using the wheat germ cell-free system and SV40-specific RNA from hybrid virus-infected cells that was purified by hybridization to SV40 DNA. As proof of identity, the in vitro products were shown to have methionine-containing tryptic peptides identical to those of their in vivo counterparts. Based on the extensive overlap in amino acid sequence between the SV40-specific proteins from hybrid virus-infected cells and SV40 T-antigen from SV40-infected and -transformed cells, we conclude that at least the major portion of the SV40-specific proteins cannot be Ad2 coded. From the in vitro synthesis experiments with SV40-selected RNA, we further conclude that the SV40-specific proteins must be SV40 coded and not host coded. Since SV40 T-antigen is related to the SV40-specific proteins, it must also be SV40 coded.     

Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells.

Mouse cells transformed by simian virus 40 (SV40) have been shown to contain a complex of the virus-coded large-T antigen with a host 53,000-molecular-weight (53K) protein. Initial attempts to detect a similar complex in lytically infected cells were unsuccessful, and it therefore seemed that the complex might be peculiar to transformed or abortively transformed nonpermissive cells. Immunoprecipitation of [32P]phosphate-labeled extracts of SV40-infected CV-1 African green monkey kidney cells with antibodies specific for large-T or the 53K protein revealed that the large-T-53K protein complex was formed during lytic infections. Only a minor fraction of the large-T present was associated with 53K protein, and large-T and the 53K host protein cosedimented during centrifugation through sucrose gradients. We used monospecific sera and monoclonal antibodies to study the rate of synthesis and phosphorylation of the 53K protein during lytic infections. Infection of CV-1 cells with SV40 increased the rate of synthesis of the 53K protein fivefold over that in mock-infected cells. At the same time, the rate of phosphorylation of the 53K protein increased more than 30-fold compared with control cultures. Monkey cells transformed by UV-irradiated SV40 (Gluzman et al., J. Virol. 22:256-266, 1977) also contained the large-T-53K protein complex. The formation of the complex is therefore not a peculiarity of SV40-transformed rodent cells but is a common feature of SV40 infections.     

Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen.

Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.     

Relationship between the methionine tryptic peptides of simian virus 40 and BK virus tumor antigens.

The monomer form of BK virus (BKV) tumor antigen (T Ag) was immunoprecipitated from extracts of BKV-transformed cells and had a molecular weight of approximately 113,000. This compared with 97,000 for the molecular weight of either BKV or simian virus 40 (SV40) T Ag from lytically infected cells. The SV40 and BKV T Ag's from productively infected cells were compared by examining their methionine-labeled tryptic peptides. Out of a total of 20 SV40-and 21 BKV-specific peptides, there were seven pairs of similar peptides on the basis of ion-exchange chromatography, These coeluting peptides contained approximately 25 to 30% of the total methionine radioactivity. Similar results were obtained when the tryptic peptides of SV40 T Ag from lytically infected cells were compared with those of BKV T Ag from virally transformed cells.     

A small subclass of SV40 T antigen binds to the viral origin of replication

We examined the affinities of SV40 large T antigen for unique viral DNA sequences by binding SV40 Bst NI DNA fragments in extracts of infected or transformed cells, and then immunoprecipitating the T antigen-DNA complex. The G fragment, which spans the viral origin of replication (ori) was quantitatively bound to T antigen. A T-antigen-specific monoclonal antibody (McI 7), which recognized only 5%-10% of the T antigen from infected or transformed cells, immunoprecipitated the majority of the ori-binding activity. This suggests that only a minor subclass of wild-type T antigen is active in binding to the origin. C6 cells contain a replication-defective mutant T antigen that when tested in the DNA-binding immunoassay, showed no affinity for the ori fragment. McI 7 not only failed to immunoprecipitate ori binding in C6 cells, but also did not detect any labeled C6 T antigen whatever. Thus McI 7 recognizes an immunologically distinct subset of wild-type 7 antigen that comprises the origin-binding form of the viral protein, which is absent in the C6 T antigen population. McI 122, which recognizes a 53 kilodalton host protein that complexes with T antigen, immunoprecipitated ori-binding activity from extracts of infected or transformed cells, but not from C6 cells. Thus wild-type T antigen can bind ori sequences even when complexed to the host protein. These data suggest that T antigen consists of different subpopulations with different functions.     

Subclasses of simian virus 40 large T antigen: differential binding of two subclasses of T antigen from productively infected cells to viral and cellular DNA

Two major subclasses of simian virus 40 (SV40) large T antigen were separated by zone velocity sedimentation of crude extracts from productively infected cells. These subclasses, which have been shown to differ biologically and biochemically ( Fanning et al., 1981), sedimented at 5-6S and 14-16S. The amount of T antigen in each form was estimated by complement fixation and by immunoprecipitation of T antigen from extracts of cells chronically labeled with [35S]methionine. Each form of T antigen was tested for specific binding to end-labeled restriction fragments of SV40 DNA using an immunoprecipitation assay. The 5-6S and 14-16S forms of T antigen both bound specifically to DNA sequences in the SV40 HindIII C fragment. The sequences required for binding both forms were localized in the same 35-bp region of the origin. However, significant differences in binding activity and affinity for specific and nonspecific DNA were demonstrated. These properties suggest that T antigen subclasses may serve different functions in the lytically infected cell.     

Cell surface location of simian virus 40-specific proteins on HeLa cells infected with adenovirus type 2-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2.

HeLa cells infected with the nondefective adenovirus type 2-simian virus 40 hybrid viruses Ad2+ND1 or Ad2+ND2 were analyzed for cell surface location of the SV40-specific hybrid virus proteins by indirect immunofluorescence microscopy. Two different batches of sera from SV40 tumor-bearing hamsters, serum from SV40 tumor-bearing mice, or two different antisera prepared against purified sodium dodecyl sulfate-denatured SV40 T-antigen, respectively, were used. All sera were shown to exhibit comparable T- and U-antibody titers and to specifically immunoprecipitate the SV40-specific proteins from cell extracts of Ad2+ND2-infected cells. Whereas analysis of living, hybrid virus-infected HeLa cells did not yield conclusive results, analysis of Formalin-fixed cells resulted in positive cell surface fluorescence with both Ad2+ND1- and Ad2+ND2-infected HeLa cells when antisera prepared against sodium dodecyl sulfate-denatured SV40 T-antigen were used as first antibody. In contrast, sera from SV40 tumor-bearing animals were not or only very weakly able to stain the surfaces of these cells. The fact that the tumor sera had comparable or even higher T- and U-antibody titers than the antisera against sodium dodecyl sulfate-denatured T-antigen but were not able to recognize SV40-specific proteins on the cell surface suggests that SV40 tumor-specific transplantation antigen may be an antigenic entity different from T- or U-antigen.