DNA binding properties of simian virus 40 T-antigens synthesized in vivo and in vitro.

Simian virus 40 large T- and small t-antigens have been shown previously to share immunological determinants and common sequences and to have roles in virus-induced cell transformation. However, only large T-antigen is a DNA binding protein. Under all conditions tested, small t-antigen did not interact with DNA. Large T-antigen synthesized in infected cells bound to both native calf thymus and simian virus 40 DNAs. As its binding efficiency was less than 100%, it is likely that there are different forms of T-antigen which vary in their affinity for DNA. Large T-antigen synthesized in cell-free protein-synthesizing systems primed by simian virus 40 mRNA also bound to DNA-cellulose, whereas small t-antigen similarly synthesized in vitro did not. An 82,000-molecular-weight T-antigen polypeptide synthesized in cell-free protein-synthesizing systems primed by simian virus 40 complementary RNA transcribed in vitro from simian virus 40 DNA by Escherichia coli RNA polymerase bound efficiently to simian virus 40 DNA. As this product did not share sequences with the small t-antigen, it can be concluded that the amino-terminal portion of the T-antigen is not required for some of its specific DNA binding properties.     

Insensitivity of a ricin-resistant mutant of Chinese hamster ovary cells to fusion induced by Newcastle disease virus.

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.     

Serological Relatedness of Bacterial Deoxyribonucleic Acid Polymerases

A number of bacterial species have been surveyed for serological activities with antiserum to Escherichia coli B deoxyribonucleic acid (DNA) polymerase I (EC 2.7.7.7.). The degree of serological cross-reaction is taken as a measure of relatedness of both the enzyme molecules from various species and the bacterial species themselves. Extracts were assayed by complement fixation only after treatment with deoxyribonuclease, since DNA bound to DNA polymerase alters the serological activity of the enzyme. Antiserum to E. coli DNA polymerase I did not react with either purified E. coli DNA polymerase II or the phage T4-induced DNA polymerase.     

Immunochemical measurement of conformational heterogeneity of poly(inosinic acid).

Several pure poly(I) preparations differed in: (a) their complement fixation reactivity with anti-poly(I) antiserum; (b) their ability to bind to a solid-phase anti-poly(I) antibody-Sepharose column; (c) their ability to inactivate serum complement; and (d) their reactivity with purified antibodies to double-stranded RNA. In particular, poly(I) samples that could induce interferon production differed from non-inducer poly(I)s; the inducers reacted weakly with anti-poly(I) antiserum and were the only ones that reacted with antibodies to double-stranded RNA. One inducer poly(I) did not inactivate complement, and differed from non-inducer poly(I) in quantitative aspects of poly(I) . poly(C) formation with varying amounts of poly(C). An additional type of poly(I) preparation reacted poorly with anti-poly(I) antiserum, did not react with anti-double-stranded-RNA antibodies and failed to induce interferon production. The varying forms of poly(I) were not interconvertible by boiling and rapid chilling. These results indicate that several different stable structural forms of poly(I) may result from a standardized synthetic procedure.     

Relationship between RNA polymerase I activity and ornithine decarboxylase in rat liver tissues.

In order to examine the relationship between RNA polymerase I and ornithine decarboxylase (ODC), three lines of experiments were performed, with the following results. The glucocorticoid-induced increase of RNA polymerase I in rat liver nuclei was not abolished by administration of inhibitors of ODC synthesis and activity, namely 1,3-diaminopropane and 2-difluoromethylornithine respectively. Anti-ODC antibody did not cross-react with RNA polymerase I solubilized from rat liver nucleoli, indicating the absence of a common protein sequence in these enzymes. The ODC preparation which was treated with transglutaminase in the presence of putrescine could not stimulate the activity of RNA polymerase I in nuclei of liver and prostate. All these results suggest that the increases in ODC protein or activity are not a prerequisite to the increase in RNA polymerase I after hormonal or physiological stimuli, but rather that the increases in both enzymes are separate responses to the primary stimuli.     

Antibodies against the subunits of E. coli RNA polymerase as probes for subunit-specific binding of DNA and other ligands.

Antibodies against the isolated subunits alpha, beta, and beta' of DNA-dependent RNA polymerase from E. coli have been prepared. They have been used to compare the extent of antibody-binding, as measured by complement fixation, to the isolated subunits and to the intact enzyme, in the absence and presence of ligands, such as inhibitors, nucleotides, nucleosides, oligo- and poly-nucleotides, and DNA of different composition. In many cases the results show a subunit-specific dependence of complement fixation upon the presence of a ligand and suggest a functional topography of the interaction between the subunits alpha, beta, and beta' of RNA polymerase and defined nucleotide sequences and small ligands.     

Isolation and characterization of the fission yeast gene Sprpa12+ reveals that the conserved C-terminal zinc-finger region is dispensable for the function of its product

RNA polymerase I of Saccharomyces cerevisiae contains a small subunit, A12.2, encoded by RPA12, that was previously shown to be involved in the assembly and/or stabilization of the largest subunit, A190, of RNA polymerase I. To examine whether an equivalent subunit is present in another eukaryotic RNA polymerase I, we have cloned a Schizosaccahromyces pombe cDNA that is able to complement the rpa12 mutation in S. cerevisiae. The gene, named Sprpa12+, encodes a polypeptide of 119 amino acids that shows 55% identity to S. cerevisiae A12. 2 over its entire length, including two zinc-finger motifs. Disruption of the chromosomal Sprpa12+ gene shows that it is required for growth at higher temperatures but not at lower temperatures. Expression of Sprpa190+/nuc1+, which encodes the largest subunit of the S. pombe RNA polymerase I, from a multicopy plasmid can partially suppress the growth defect of the Sprpa12 disruptant at higher temperatures. These findings suggest that A12.2 subunit is functionally and structurally conserved between S. cerevisiae and S. pombe. Finally, the analysis of mutants suggests that SpRPA12 requires the zinc-finger domain in the N-terminal region but not the one in the C-terminal region for its function.     

Cell cycle-dependent expression of early viral genes in one group of simian virus 40-transformed rat cells.

SV40-transformed FR 3T3 rat cells were previously shown to exhibit different patterns of accumulation of the virus-coded T-antigen. One group of transformants accumulates T-antigen throughout the cell cycle, whereas in another group, only the cells in the G2 phase of the cell cycle are stained by immunofluorescence with anti-T antigen antibodies. We investigated the mechanism involved by determining the amounts of early SV40 RNA during the cell cycle. Cells in the various phases of the cell cycle were sorted from an asynchronously growing population using a flow cytofluorimeter. Determination of the amounts of viral RNA in the different nuclear and cytoplasmic RNA fractions showed that in transformants with a G2-restricted accumulation of T-antigen, viral RNA was present in G2, to some extent in S, but could not be detected in cells in G1. In contrast, equivalent amounts of viral RNA were detected in all the phases of the cell cycle in the other group of transformants. Cell sorting, performed after pulse-labeling the cells for 2 h with [35S]methionine, confirmed that translation of the viral mRNAs occurred only in G2 in the first group of transformants, and throughout the cell cycle in the second group.     

Interaction of Simian Virus 40 chromatin with Simian Virus 40 T-antigen.

We have studied the binding of the tumor antigen (T-antigen) of simian virus 40 to simian virus 40 chromatin (minichromosomes). The minichromosomes isolated from infected cells by a modification of standard techniques were relatively free of contaminating RNA and cellular DNA and had a ratio (by weight) of protein to DNA of approximately 1; their DNA was 50 to 60% digestible to an acid-soluble form by staphylococcal nuclease. Cleavage of this chromatin with restriction endonucleases indicated that the nuclease-resistant regions were randomly distributed in the population of minichromosomes, but were not randomly distributed within minichromosomes. Only 20 to 35% of these minichromosomes adsorbed nonspecifically to nitrocellulose filters, permitting binding studies between simian virus 40 T-antigen and chromatin to be performed. Approximately two to three times as much T-antigen was required to bind chromatin as to bind an equivalent amount of free DNA. When T-antigen was present in excess, both chromatin and free DNA were quantitatively retained on the filters. On the other hand, when DNA or chromatin was present in excess, only one-third as much chromatin as DNA was retained. We suggest that T-antigen-chromatin complexes may be formed by the cooperative binding of T-antigen to chromatin, whereas T-antigen-DNA complexes may be formed by simple bimolecular interactions.