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Recombinant wild-type protease of human immunodeficiency virus, type 1 (HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/micrograms. Two proteolytically inactive HIV-1 mutant proteases (Arg-87----Lys; Asn-88----Glu) were found to bind to pepstatin A agarose, and and they were purified as the wild-type protease. A third mutant protease Arg-87----Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).

Ac-Lys-Ala-Ser-Gln-Asn-Phe(NO2)-Pro-Val-Val-NH2 (peptide I) and Thr-Phe-Gln-Ala-Phe(NO2)-Pro-Leu-Arg-Glu-Ala (peptide II) undergo hydrolysis between the p-nitrophenylalanyl and prolyl residues catalyzed by the proteases of HIV-1 and AMV, respectively. The specific hydrolyses of peptides I and II are accompanied by a decrease in their uv absorption at 269 nm (delta epsilon = 1000) and an increase at 316 nm (delta epsilon = 600). The use of microspectrophotometric cells allows continuous uv measurements on a volume (60 to 120 microliters) comparable to that required for the HPLC point assay currently used. At the highest substrate concentration possible under the assay conditions, good first-order kinetics were observed with both proteases, and the values of Vmax/Km were obtained.

In an already tumorigenic spontaneously transformed mouse cell, after further transformation by SV40, the virus-specific antigenic function becomes dominant. By transplantation into syngeneic mice SV40 antigen negative revertant tumour cells can be selected out.

A specific cellular protein of molecular weight of 53-55,000 (p53) has been shown to be induced in all SV40 transformed cells. A similar protein has also been shown to be present in embryonal carcinoma cells and in midgestation murine embryo primary cells, which are not infected by SV40. In embryo cell primaries the amount of the protein was shown to decrease with the increase in the stage of embryo development. As differentiation or decrease in cell growth rate can account for this, and since the growth rate of embryo primary cells cannot be measured, we chose to investigate various embryonal carcinoma cells. We report that the p53 is present in a pluripotent embryonal carcinoma cell OTT6050, and in its differentiated parietal endoderm derivative, PYS-2 cells. The amount of p53 is higher in the undifferentiated EC stem cells than in the differentiated PYS-2 (parietal endoderm) cells. The amount of the protein decreases in F9 embryonal carcinoma cells induced to differentiate to a parietal endoderm cell type by treatment with retinoic acid, as it does following spontaneous differentiation of OTT6050 EC cells. To determine if a change in growth rate, rather than differentiation, might account for the diminished levels of this protein, the amount of p53 was measured in growing and in growth arrested cell populations. When the growth rate of F9 cells was reduced by treatment with 8-bromocyclic AMP there was no change in the amount of p53. The half life of the p53 was compared in the undifferentiated and the differentiated cell types to determine if a change in stability might account, in part, for the altered levels of this protein. The p53 is found to be most stable in the SV40 transformed established clonal cells. It is less stable in the fibroblast clonal cells which were not transformed by SV40. The results of these experiments indicate that a decrease in the amount of p53 primarily correlates with differentiation in the embryonal carcinoma cell lines studied and not with cell growth rate. Furthermore, the decrease appears to be related (in part) to the decreased stability of the p53.

Mild detergent treatment of SV40 transformed mouse embryo fibroblasts, followed by (NH4)2SO4 precipitation of solubilized proteins and rate zonal centrifugation in a sucrose gradient, provided direct and rapid determination of the sedimentation coefficient (4S) of tumor specific surface antigen(s) (TSSA), as detected by an antibody dependent, cell lysis assay. A molecular weight range of 50,000–60,000 was estimated for the TSSA. These results agree with those also obtained from Sephadex G-150 exclusion chromatography.

The 297bp gene coding for the HIV-1 protease was chemically synthesized and expressed in E. coli. Single amino acid substitutions (Arg 87 - greater than Lys; Arg 87 - greater than Glu) were introduced in the C-terminally located conserved region GlyArgAsn of the protease gene in the wild-type clone. The products of the mutant and the wild-type clones were expressed at approximately similar levels at 30 minutes of induction but the mutant protease proteins accumulated as a function of time of induction unlike the wild-type protease which declined after 60 minutes. The mutants were completely devoid of proteolytic activity as determined in assays employing as substrates a synthetic nonapeptide and a gag related recombinant polyprotein.

By means of a direct cytolytic assay, SV40 specific antisera were shown to kill SV40 T antigen positive AL/N mouse cell lines, including lines which were repeatedly passed through the syngeneic mouse as tumors. The sera also killed a polyoma transformed SV40 T antigen negative AL/N cell line, but not untransformed or spontaneously transformed AL/N embryo cell lines. With the antisera tested there was also no lysis observed on SV40 T antigen positive Balb/c mouse, hamster or human cell lines, although by use of a competition assay SV40 specific common antigens were detectable on the surface of these cells.

In most murine cells transformed by the SV40 virus, virtually all of the cellular phosphoprotein p53 is in a complex with the SV40 T antigen. Here, we report that, in SV40-infected T-antigen-positive Balb ST12 mouse cells, most (∼80%) of the p53 is not in complex. Complex formation was determined by measuring the amounts of [ 35 S]methionine-labeled p53 which coprecipitated with T antigen when using monoclonal antibody to T antigen. The amount of complex formation was expressed as a percentage of total p53 present, measured by the amount of p53 precipitated with the monoclonal antibody to the p53. The values were confirmed by Western blotting procedure, in which the steady-state levels of the proteins were measured. In these measurements after complete precipitation with antibody to T antigen, the residual p53 in the supernatant was precipitated by antibody to p53, and this amount was denoted as free p53. There was no significant difference seen between the [ 35 S]methionine-labeled tryptic peptides of complexed and the free p53 (or between complexed and free T antigens) as determined by two-dimensional gel electrophoresis and chromatography. Virus rescue experiments and retransformation by the rescued virus showed that there was no mutation in the SV40 DNA coding for the T antigen which could account for the lack of complex formation. Both p53 and T antigen were underphosphorylated in cells which exhibited reduced complex formation. Tumorigenicity in syngeneic mice and anchorage-independent cell growth in culture of various cloned mouse cells with or without T antigen expression was compared. The changes in the biologic properties were explainable solely on the basis of known or expected effects of expression of the T antigen and were independent of complex formation or of absence of complex formation between p53 and T antigen.

The 297bp HIV-1 protease gene was constructed from five discrete synthetic fragments and expressed in E.coli . A soluble protein product of 11.5 Kd was detected by immunoblotting using protease specific antisera. A quantitative assay system, utilizing a synthetic nonapeptide spanning the cleavage site between p17–p24 in the gag polyprotein, was used to measure the specific protease activity in crude extracts. The protease hydrolyzed tyrosyl-proline bonds with an approximate specific activity of 43 pmoles/min/μg of total protein. The chemical synthesis of the protease gene and it's expression provides a feasible method for rapid mutant analysis, important for structure-function studies and rational design of potential inhibitors.

The cellular tumor antigen p53 was isolated from mouse neuroblastoma cells and was found in a form not complexed to another protein. The p53 in these cells was stable, turning over about every 10 h. Its methionine-labelled tryptic peptides were very similar to those of the p53 isolated from SV40-transformed mouse cells. The labelled protein was purified from neuroblastoma cells by immunoaffinity using specific monoclonal antibodies and was about 80% radiochemically pure. Furthermore, the purified p53 sedimented in sucrose gradients with a sedimentation coefficient of approximately 8S. This correlated with the sedimentation coefficient of p53 prior to purification, showing that the purified protein retained its native size.

A clonal line of highly oncogenic “spontaneously transformed” mouse cells (T AL/N clone 3) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned. The clone of SV40-transformed cells (subclone 1) expressed SV40-specific T (nuclear) and transplantation antigens but was 100 times less tumorigenic than the parent T AL/N clone 3 cells. When large numbers of subclone 1 cells (10 4 -10 5 ) were injected into syngeneic AL/N mice, tumors were produced. From the tumors, cell lines were established in culture, all of which were consistently negative for T antigen. Tumor lines tested were found not to contain SV40-specific transplantation antigen and had again become highly tumorigenic. The original subclone 1 cells contained about one copy of SV40 DNA per diploid amount of cell DNA, as well as RNA complementary to the early region of the SV40 genome. The T antigen-negative cells from tumor line 124 contained approximately 0.5 copy of SV40 DNA per diploid equivalent and did not synthesize any detectable virus-specific RNA. Reassociation kinetic analysis with restriction enzyme fragments of viral DNA demonstrated that the cells from tumor line 124 (and also the clones of this line) had lost DNA sequences predominantly from the early region of the SV40 genome. The results indicate that a set of stably integrated SV40 DNA sequences can be present in a cell without the expression of viral antigens.

We have explored aspects of a suggested relationship between the expression of simian virus 40 tumor-specific transplantation antigen (TSTA) and tumor antigen (TA). A unique rat embryo cell line transformed by a temperature-sensitive A mutant that loses TA during exposure to the nonpermissive temperature (A28-RE) was found to lose TSTA. On the other hand, a typical control tsA-transformed cell line (A239-MB) expressed both TA and TSTA at the non-permissive temperature. TA in lysates obtained from A239-MB cells was found to be three to four times more thermolabile by covwt-mb) when incubated at either 33 or 40 degrees C. These data complement previous reports using TA from lytic infection and are consistent with the suggestion that TA is virus encoded. In contrast to TA, which even in wild-type-transformed cells was completely destroyed in less than 10 min at 50 degrees C, TSTA, assayed in vivo by tumor rejection, and tumor-specific surface antigen(s) TSSA) defined by an in vitro cytolytic assay, were thermostabile. Even after 24 h of incubation of extracts of 50 degrees C, high levels of TSTA and TSSA activity were present. Since these surface antigens when obtained from cells transformed by tsA mutants were also thermostabile, they could not be distinguished from the wild-type antigens. These results (i) indicate a coordinate expression of TA and TSTA in transformed cells; (ii) confirm that TA is virus encoded; and (iii) confirm that tha antigenic and immunogenic determinants that characterize TA and TSTA activities are distinct. However, the possibility that TSTA, like TA, is of viral rather than cellular origin is not excluded.

The simian virus 40 (sv40) tumor antigen (T-antigen) and tumor-specific transplantation antigen (TSTA) have been partially purified and studied to clarify their relationship. The T-antigen and the TSTA were partially purified from nuclei of SV AL/N cells, and SV40-transformed mouse embryo fibroblast line, by precipitation with ammonium sulfate and chromatography on DEAE- and DNA-cellulose. The T-antigen was assayed by complement fixation, and the TSTA was assayed by its ability to immunize mice against SV40-containing ascites tumor cells. When T-antigen- and TSTA-containing preparations were sedimented through sucrose gradients, each antigen had a major peak of activity at a sedimentation coefficient of 6.7 and minor peaks in other regions. Antiserum against T-antigen (from tumor-bearing hamsters) immunoprecipitated the TSTA activity. A preparation of T-antigen from human SV80 cells, which exhibited only one protein band after sodium dodecylsulfate-polyacrylamide gel electrophoresis, had TSTA activity when as little as 0.6 microgram of protein per mouse was used for immunization. These experiments demonstrate that the T-antigen, the product of the SV40 early A gene is capable of inducing specific immunity against transplantation of SV40-transformed tumor cells in mice.

The phosphoprotein p53 has been investigated mainly because of its relationship with tumorigenic transformation. In this communication, we report that, during the embryonal development of mouse and chicken, there is a decline in the steady-state levels of the p53 protein and an equal decline in p53 mRNA. During the development of the chicken, the relative rates of p53 transcription appear to be constant. p53 mRNA is relatively stable (half-life greater than 12 h) in both chicken and mouse embryos. We conclude that (i) the down-modulation of p53 mRNA (and of protein) during embryonal development has been well conserved during the evolution of the vertebrate, implying that the p53 protein may have a function in embryonal development; and (ii) the mechanism of control is apparently mainly on a post-transcriptional level.

A 55-kilodalton (kDal) protein was detected recently in primary cultures of day 12 mouse embryos by immunoprecipitation with serum from simian virus 40 (SV40) tumor-bearing hamsters (T serum), Preliminary evidence suggested that this protein was similar to a cellular 55-kDal protein induced after SV40 transformation of mouse cells. We now show that specific approximately 55-kDal [35S]methionine-labeled proteins precipitate from primary cultures of midgestation mouse, rat, and hamster embryos on addition of T serum or monoclonal antiserum prepared against the SV40-induced mouse 55-kDal proteins. The two-dimensional maps of the [35S]methionine-labeled tryptic peptides of the mouse, hamster, and rat embryo proteins are similar to the maps of the corresponding proteins from SV40-transformed cells. Primary cells from midgestation mouse, hamster, or rat embryos contain one-third to one-half as much 55-kDal protein as a SV40-transformed mouse fibroblast cell and nearly the same amount as F9 mouse embryonal carcinoma cells. The amount of 55-kDal protein is greatly reduced on replating the mouse, rat, or hamster embryo primary cells. The amount of this protein in mouse embryos is dependent on the stage of the embryo. The embryo proteins are phosphoproteins.

In mouse cell lines transformed by SV40 virus, a decrease was observed in the higher ganglioside homologues disialo-ceramidetetrahexoside and monosialo-ceramidetetrahexoside. Such change was observed only in cells which carry the virus genome, and it correlated with increased saturation density in tissue culture and with rejection in immunologically competent syngeneic host. This indicates that the change is one of the virus-regulated functions, and it is postulated that it relates to the rejection of the virus-transformed cells.

A rapid in vitro assay for SV40 virus-associated tumor-specific transplantation antigen (TSTA) has been developed using SV40-transformed cell lines from inbred mice. This assay, which is based on antibody-complement-mediated cytolysis, permits quantitative measurement of the new antigen on the surface of living cells and in subcellular fractions. The procedure can be done in 5–10 μl of solution in 3 hr using microtest tissue culture plates, with cytolysis indicated either by trypan blue dye uptake or by the release of 51 Cr from labeled target cells. Mouse antiserum kills only those mouse cells which have been transformed by SV40 virus. It also kills hamster cells which carry SV40 T antigen and TSTA; however, it does not kill hamster cells which carry the “S” antigen but neither the T antigen nor the TSTA. The assay is suitable for monitoring analytical scale biochemical work on solubilized TSTA by the method of cytolysis-inhibition. The cytolytic activity of the mouse antiserum elutes from Sephadex G-200 with the immunoglobulin G-containing fractions. Incubation of SV40-transformed cells with the specific antiserum, without complement, enhances the tumorigenicity of these cells when they are injected into syngeneic animals. This assay has permitted the direct measurement of TSTA on the surfaces of cells infected, abortively transformed, or stably transformed by SV40 virus.

A general procedure is presented for the preparation of diethylaminohydroxylpropyl ethers of polysaccharides. This involves reaction between various ratios of 1-diethylamino-2,3-epoxypropane and the polysaccharide (amylose, amylopectin, dextran, and a chemically synthesized polygalactose) and concentrated aqueous solution of sodium carbonate. These ratios control the degree of substitution, and give as high as 1.4 cationic groups per monosaccharide unit in the case of certain polysaccharides. The cationic polysaccharides possess high hemagglutinating activity which is proportional to the degree of substitution. Other biological applications are reviewed.

An ideal solute for density gradient ultracentrifugation of polymers in aqueous solution should be inert and readily soluble in water to form an extended range of solution densities of low viscosity. High molecular weight is an added attraction because osmotic effects are minimized. Highly branched spherical synthetic polysaccharides fulfill these requirements. High degree of branching is a consequence of the condensation of polyfunctional monomers. Density and relative viscosity of solutions of polyglucose, sucrose, and of a natural sucrose polymer, Ficoll, are compared. The behavior of various polyelectrolytes was studied in low viscosity polyglucose density gradients in equilibrium buoyant density measurement in the ultra-centrifuge. Macromolecules or macromolecular complexes attain low apparent equilibrium buoyant density, probably because of an excluded volume effect of the solute. This allows sedimentation to isopycnic position of complex biopolymers in inert polyglucose solutions, which ot...

Inactivation of bacteriophages T 1 −T 7 by polyglucose sulfuric acid was studied under controlled conditions. Release of DNA indicating rupture of the phage head was prevented. Full inactivation of T 1 , T 3 and T 7 was achieved at pH 4.7, of T 2 at pH 4.2, of T 5 at pH 4.3 and of T 4 , T 6 and T 4−01 at pH 4.0. The inactivation curves showing the effects of temperature, pH and concentration of polyglucose sulfuric acid indicate that the odd members are closely related among themselves and the even members among themselves. Attempts to reverse inactivation were not successful. Incubation of the inactivated phage with putrescine and spermidine, or with bacteria at pH 5.6, failed to restore viability. The inactivated phages release their DNA in 0.2 M lysine at pH 8.8 and lose their ability to kill or lyse the bacteria, but retain normal attachment to Escherichia coli B. Ghosts treated with polyglucose sulfuric acid also lose bacterial-killing and -lysing abilities. It is suggested, therefore, that during the process of inactivation the proximal tail structure is damaged.

Following morphological transformation of mouse cell lines in culture by a murine sarcoma virus, a decrease occurs in a single glycolipid glycosyltransferase. This same enzyme change has been observed previously in DNA-virus-transformed cells, but is not seen after infection with a non-transforming murine leukaemia virus.

Chromatin and DNA from a minimal deviation rat hepatoma and from rat liver were prepared and various physicochemical and template properties studied. More RNA and total protein but similar amounts of histone were associated with hepatoma chromatin than with liver chromatin. The spectral and thermal denaturation properties of the two chromatins are nearly the same, as is the case for DNA from the two sources. The kinetics of incorporation of labeled nucleoside triphosphates and the divalent ion requirements of DNA-directed RNA polymerase with hepatoma chromatin template are similar to those published for liver chromatin. The base compositions of the in vitro transcribed RNA from hepatoma and liver chromatins were similar to each other and (A + U)-rich. The RNA products from the two DNA templates also had similar compositions but were more (A + U)-rich than chromatin products. The relation of this work to the study of RNA populations in liver and hepatoma is briefly discussed.

Analysis of mouse cell lines revealed that m all the cell lines investigated which were transformed in tissue culture by the tumorigenic DNA viruses, the highest ganglioside homolog N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GD1a) is decreased or is nearly absent, and concomitant with this N-acetylneuraminylgalactosylglucosylceramide (GM3) becomes the principal ganglioside. In the untransformed control or parent cell lines, or in cells transformed “spontaneously” in culture, GD1a occurs in well quantifiable amounts. Incorporation studies with the radioactive precursors N-acetyl- d -[ 3 H]mannosamine and N-acetyl- d -[ 3 H]glucosamine confirmed these results. We postulate that these changes in the membrane glycolipids are expressions of a common virus-regulated biochemical function in SV40- and polyoma virus-transformed mouse cells.

The activities of four glycosyltransferases involved in ganglioside biosynthesis were measured in two different established mouse cell lines and in the SV40 and polyoma transformed variants of these lines. The only consistent change observed was the very reduced activity of UDP-GalNAc: hematoside N-acetyl-galactosaminyltransferase in all of the transformed cell lines (15–20% of normal cells). There was no significant differences in any of the glycosyltransferases with increased cell density and cell-to-cell contact.

Transformation of mouse cells in culture by oncogenic DNA and RNA viruses is associated with an altered ganglioside metabolism in these cells. The change is specific: a drastic reduction in hematoside: N -acetylgalactosaminyltransferase. Though the underlying molecular basis of this change is unknown, it is clearly related to viral transformation per se and not to abnormal morphology in culture. Although a correlation between transformation in culture and malignancy in vivo has not been conclusively established (48), the possibility of a relationship between the functioning of N -acetylgalactosaminyltransferase and neoplastic processes in vivo is being examined.

A series of synthetic polyglucose carboxyl derivatives with different carboxyl content has been used to inhibit ribonuclease depolymerizing activity on ribonucleic acid as measured by the KUNITZ assay. The enzyme inhibitory activity increased greatly with the charge density of the polyglucose carboxyl derivatives. From this and similar experiments it is concluded, that COULOMBIC attracting forces have an important role in bringing the enzyme and the substrate together. Eine Reihe synthetischer Polyglukose-carboxylderivate mit verschiedenem Carboxylgehalt wurde zur Hemmung der Ribonuklease-entpolymerisierenden Wirkung an Ribonukleinsaure, die nach der KUNITZ-Probe gemessen wurde, verwandt. Die enzymhemmende Wirkung nahm mit der Ladungsdichte der Polyglukose-carboxylderivate stark zu. Durch diese und ahnliche Experimente ergibt sich, das COULOMB-Anziehungskrafte beim Zusammenfuhren von Enzym und Substrat eine wichtige Rolle spielen.

The slow reduction of cytochrome c by certain synthetic polycations, which we reported previously, has been further investigated in an attempt to clarify the role of the charged groups in the reaction. We were unable, however, to eliminate the possibility that trace quantities of reducing structure may be present in the synthetic polyglucose derivatives employed. For this reason, we now favor this simpler interpretation. The effects of alcohols, urea, guanidine, and elevated temperature on the reduction have also been investigated during the course of these studies.

In mouse cell lines after transformation with SV40 or polyoma virus there was shown to be a reduction in the content of higher ganglioside homologs and in the activity of an enzyme (uridine diphosphate N -acetylgalactosamine: hematoside 2 N -acetylgalactosaminyltransferase) necessary for the synthesis of the higher gangliosides. These changes were found to be reversibly coupled to the changes in growth properties in tissue culture: in the flat derivatives of the virus transformed cell lines which reverted to normal phenotypic growth properties in culture there was a trend of reversion in ganglioside pattern and in enzymatic activity near to that of the parent 3T3 line, in spite of the continued presence of the viral DNA. Mouse cells productively infected with polyoma virus did not show decrease in the aminosugar transferase activity. Cocultivation of virally transformed cells with enzymatically active cells did not result in reduced enzyme activity in the enzymatically active cells, or in the restoration of enzyme activity in the virally transformed cells. Similarly, no change in enzyme activity occurred when admixing homogenates of the two type of cells. These biochemical findings are consistent with a similar repressor-like mechanism in both SV40 and polyoma virus induced cell transformation .

In addition to the virus-specified tumor antigens, simian virus 40-transformed cells contain at least one other protein which can be immunoprecipitated with serum from animals bearing simian virus 40-induced tumors. This protein, which is designated Tau antigen, has an apparent molecular weight of 56,000 as determined by electrophoresis on acrylamide gels. The relationship among Tau antigens isolated from different lines of simian virus 40-transformed cells was examined by comparing the methionine-labeled tryptic peptides of these proteins by two-dimensional fingerprinting on thin-layer cellulose plates. In this fashion, we initially determined that the Tau antigens isolated from three different lines of transformed mouse cells were very similar. Second, we found that Tau antigen isolated from a line of rat transformants was closely related, but not identical, to the mouse cell Tau antigens. Approximately 70% of their methionine peptides comigrated in two dimensions. Finally, we showed that Tau antigen isolated from a line of transformed human cells was only partially related to the mouse and rat proteins. About 40% of the methionine peptides of the human protein were also contained in the Tau antigens from the other two species. These results strongly indicate that the Tau antigens isolated from these various simian virus 40-transformed cell lines contain common amino acid sequences.

A specific protein of molecular weight (MW) approximately 55,000 (55K) was found recently by immunoprecipitation in all SV40 virus-transformed mammalian cells, in addition to the SV40 large T antigen (appoximately 94K) and small antigen (approximately 17K), which are the only proteins coded by the 'early half' of the SV40 genome. The 55K protein is encoded by cellular DNA; its peptide pattern is different from that of the SV40 antigens and it is species specific in mouse, rat, hamster, monkey and human SV40-transformed (or infected) cells. A 55K protein with a similar peptide pattern was found in mouse embryonal carcinoma cells not exposed to SV40. Similar proteins were reported in mouse sarcomas and leukaemias induced by a great variety of aetiological agents and also in a spontaneously transformed mouse fibroblast cell line, and it has been suggested that the protein may be a general correlated of cellular tumorigenicity. We now report that the approximately 55K protein is present in primary cell cultures from 12-14 day old mouse embryos, but not in 16-day old mouse embryos. The embryo protein has a peptide pattern virtually indistinguishable from that of the SV40-induced protein. We also show by comparing closely related cell families that spontaneously transformed highly tumorigenic mouse cells do not possess the 55K protein.

A family of mouse fibroblast cell lines in exponential phase of growth were compared in protein constitution of their cell membranes. In preparations from these cells enriched in cell-surface membrane we observed one protein component (apparent molecular weight about 250 000) consistently to be reduced or absent in an SV40 virus transformed cell line, when compared with the normal cell line. No such compositional difference was observed in a spontaneously transformed tumorigenic clonal derivative cell line, or in subclones of such a derivative cell line, with or without SV40 virus infection. However, in metabolic labeling experiments with 14C-labeled mixed amino acids, a consistent decrease also was demonstrated in the biosynthesis of the same protein in the SV40 virus infected subclone, as compared to an uninfected sister subclone, during exponential growth. This specific difference in biosynthesis is apparently related to the presence and functioning of the SV40 gene, and correlates with the ability of these cells to grow in viscous medium, but not with cellular tumorigenicity.