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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.

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.

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.

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 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 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.

MUCH work is being directed towards the characterisation of tumour-specific transplantation antigens (TSTA). AL/N strain mouse embryo cells transformed by SV40 virus (SV AL/N cells) are suitable for such studies, because (1) these cells are highly antigenic and are rejected at high dose (>107) by the immunologically competent syngeneic AL/N mouse1,2, (2) TSTA is common to all SV40 virus-transformed cells tested but specific in that only SV40 transformed cells share a common TSTA3, and (3), the SV40 ‘early’ or A gene is relatively small (∼1.8×106 molecular weight), its mapping and the nucleotide sequences of the restriction endonuclease fragments are under intense study, and the various cellular antigens in the transformed cells—the TSTA, the tumour-specific surface antigen (TSSA) (ref. 4), and the nuclear T antigen—probably represent modifications of products of this early gene5–7. Using SV AL/N cells and a detergent technique8 for solubilisation, we demonstrated that immunisation by a partially purified soluble extract containing 100 µg protein effectively protects mice against tumourigenic doses of SV40 transformed cells9. We report here that TSTA activity co-purifies with the T antigen in several steps, that TSTA can be recovered from a DNA column in a fraction which then effectively immunises at the 1-µg level, and that the TSTA, like the T antigen10, binds to double-stranded mammalian DNA.

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.

Spontaneously transformed highly tumorigenic AL/N strain mouse cells and their SV40 virus transformed clonal derivatives were compared. Glycoproteins were labeled by various metabolic precursors (amino acids, glucosamine, mannosamine, etc.). The flow of such labels into various subcellular components was followed. Cell surface membranes were obtained and the components of these membranes were separated by polyacrylamide gel electrophoresis. The biosynthesis of a prominent glycoprotein component with an apparent molecular weight of 180 K dalton was reduced in the SV40 transformed cells, when all the different kinds of cells were grown in their logarithmic phase. Three types of SV40 specific antigens were detected by immunologic assays: (1) T antigens, (2) tumor specific surface antigens (TSSA), and (3) tumor specific transplantation antigens (TSTA). A rapid and sensitive microassay for TSSA guided the development of a mild and efficient detergent (Triton X-100) solubilization technique, and the purification of the antigens that appear to co-purify in various procedures. The antigens expressed on the cell surface that are responsible for immunologic recognition and rejection of the SV40 transformed cells represented a very minor fraction of the cell surface membrane components. The microassay for TSSA allowed partial purification and characterization of the SV40 specific cell surface antigen (s). The(se) SV40 specific cell surface antigen (s) in the AL/N strain mouse functionally opposes cellular tumorigenicity.

Publisher Summary This chapter discusses a study in which the biosynthesis of glycoconjugates in cell culture of a closely related family of AL/N mouse embryo clones were monitored by anion-exchange chromatography of glucosamine-labeled macromolecules. In the study, four clones were studied, the parent clone and three subclones obtained by recloning 1 h after infection by simian virus 40. Of the subclones, two were transformed, as defined by the presence of viral specific nuclear T antigen; the third was T antigen negative. Cell cultures were divided into three compartments: the medium, the trypsinate, and the cells. The compartments were prepared for anion-exchange chromatography by papain digestion, followed by dialysis to equilibrium against the initial column buffer. The ion-exchange profiles of all three compartments of the T antigen negative subclone were similar to those of the parent clone. In each compartment, seven peaks were detected. The first four appeared to be glycopeptides, and the last three glycosaminoglycans.

Publisher Summary This chapter discusses glycolipids in SV40 and polyoma virus-transformed mouse cell lines. In a study described in the chapter, various normal, spontaneously transformed, and DNA tumor virus-transformed mouse cell lines were grown in tissue culture and were analyzed for changes in ganglioside composition. The cell lines included the highly contact inhibited mouse fibroblasts 3T3, its SV40 and polyoma doubly transformed variant SVPY 3T3; an established epithelial-like normal cell line from inbred mouse N AL/N; its spontaneously transformed tumorigenic variant T AL/NT; and its SV40-transformed SVS AL/N and polyoma transformed PY AL/N variants. The decrease in the virus-transformed variants in N-acetylneuraminyl-galactosyl-N-acetylgalactosaminyl-(N-acetylneuranrinyl)-galactosyl-glucosylceramide and galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide occured consistently, irrespective of the growth medium, the substratum, the growth conditions, and the harvesting conditions.

Mitochondria were rapidly prepared from rat liver by differential centrifugation. Nucleic acids were extracted by a detergent-phenol method and the DNA and RNA were separated by hydroxyapatite chromatography. Mitochondrial DNA thus prepared contained up to 90% of the un-nicked, twisted, circular form-I, as determined by sedimentation velocity experiments. The mitochondrial DNA was shown to be free of nuclear DNA by its renaturation properties and by its banding pattern in an alkaline cesium chloride equilibrium buoyant-density gradient. Both early-melting and delayed-melting transitions were observed, presumably resulting from thermal denaturation of the intact circular mitochondrial DNA. The form-I mitochondrial DNA was rapidly nicked at pH 12.3 in 3 m -CsCl solution. Upon centrifugation to equilibrium, the two mitochondrial DNA strands separated and banded at 1.740 and 1.771 g/ml. However, a small fraction of the closed-circular form-I was stable enough at 25 °C and pH 12.3 to form a band at a buoyant density of 1.766 g/ml.

The ganglioside pattern of both SV40- and polyoma virus-transformed mouse cell lines differs from that of the parent cell lines or of cell lines that have transformed spontaneously in tissue culture. This is manifested by a dramatic decrease of gangliosides with an oligosaccharide chain larger than sialyllactose. Present investigations indicate that this change probably cannot be attributed to excessive catabolism of gangliosides, but is caused by impaired synthesis of tri- and tetrahexosyl gangliosides in the virus-transformed cell lines. We present evidence for the block of a required step for the biosynthesis of these ganglioside homologs. The block involves the enzyme catalyzing the transfer of N -acetylgalactosamine from uridine diphosphate N -acetylgalactosamine to hematosides ( N -glycolylneuraminyl or N -acetylneuraminylgalactosylglucosyl ceramide). This well-defined enzymatic change opens the way for studies of the biochemical mechanism of the alteration of cell membranes which occurs after transformation by the tumorigenic DNA viruses polyoma and SV40.

Lipids were analyzed from the Rauscher mouse leukemia virus, obtained either from plasma of viremic mice or from virus-infected cells in tissue culture. In both viruses the lipids consisted of 75–80% phospholipids, and the distributions of the various phospholipids were similar. Phosphatidyl choline (lecithin) was in both viruses the main component (68% and 64%) of the phospholipids. The fatty acid compositions of the lipid extracts, however, were different: the virus from plasma contained more polyunsaturated fatty acids, and the virus from tissue culture more shorter chain saturated fatty acids. The lipid from the Moloney mouse leukemia virus obtained from plasma, was similar but not identical in fatty acid composition to the Rauscher plasma virus. Also were compared the fatty acid compositions of phospholipid extracts from two types of tissue-cultured mouse cells: one uninfected with the Rauscher virus, the second similar but infected and producing the tissue culture virus. The fatty acid spectra were alike. Linolenic acid (C 18 , 3 double bonds) was present in considerable amount (about 11%) only in the purified Rauscher tissue culture virus, but not in the Rauscher or Moloney plasma virus or in the uninfected or infected cells. Generally, both the Rauscher and the Moloney plasma viruses contain relatively more fatty acids of shorter chain length, while the tissue culture virus of Rauscher and the cells used to produce this virus contain more polyunsaturated fatty acids.

Charged polyglucose derivatives, such as polyglucose sulfates, with different molecular weights, branching, and degree of substitution precipitated basic proteins, such as lysozyme, ribonuclease, protamine, thymus peptide, polymyxin B, an ACTH preparation, cytochrome c, and serum albumin from aqueous solution at low pH and salt concentration. Enzymic activity of lysozyme, ribonuclease (both on ribonucleic acid and on a cyclic phosphate), and hyaluronidase was inhibited in solution. The inhibition required macromolecular size and polyanionic structure and depended on the type and amount of charged groups on the synthetic polysaccharide derivative. Highly substituted sulfates were the most effective inhibitors, while sulfates with similar molecular size but lower charge density or derivatives with weaker dissociating anionic groups (carboxyls) had less inhibitory activity. The inhibition appears to be due to electrostatic interaction forces between the oppositely charged macromolecules and can be reversed by a polymeric cation stronger than the substrate.