Dimitris Zouzias

Human fibroblasts transformed by the early region of SV40 DNA: Analysis of “free” viral DNA sequences

Abstract Human fibroblastic cells (HF) were transformed with the early region of the simian virus 40 genome (0.15 – 0.73 map units) by using the DNA-calcium phosphate coprecipitation technique of F. L. Graham and A. J. Van der Eb (1973, Virology 52 , 456–467). Transformation resulted in altered morphology and ability to grow in agarose. The SV40-transformed human fibroblasts (SVHF-A) have a limited life span and reach “senescence” after 10–11 passages. Analysis of the low molecular weight DNA extracted from SVHF-A cells shows a relatively high amount of free viral DNA sequences in circular supercoiled form. These circular molecules are very heterogeneous in size and contain sequences corresponding to the early region of the SV40 genome. Part of them may contain cellular DNA sequences as well. In situ hybridization experiments indicate that a minority of the SVHF-A cells (2–3%) are spontaneously induced to synthesize free viral DNA molecules and their frequency is increased by mitomycin C treatment. Immunofluorescence staining for SV40 T antigens also indicates that the cells producing free viral DNA contain higher T-antigen levels than the rest of the population. Our data suggest that the “free” viral DNA molecules derive from integrated viral sequences following replication in a minority of the cells rather than originating from a persistent extrachromosomal replication in every cell.

Isolation and preliminary characterization of the small circular DNA present in African green monkey kidney (BSC-1) cells.

Abstract The small polydisperse circular DNA (spc-DNA) previously identified in SV40-infected African green monkey kidney (BSC-1) cells ( M. G. Rush, R. Eason, and J. Vinograd, 1971 , Biochim. Biophys. Acta 228, 585–594.) has been isolated in pure form from uninfected cells. This double-stranded, covalently closed circular DNA contains species ranging in molecular weight from about 0.1 to 4 × 106, although most of the molecules are distributed in an apparently polydisperse population with molecular weights of less than 1 × 106. There are approximately 1000 to 2000 covalently closed small DNA molecules per cell, and their average buoyant density does not appear to differ significantly from that of chromosomal and mitochondrial DNAs. This spc-DNA was resolved by polyacrylamide gel electrophoresis into three distinct bands containing comparatively homogeneous circular DNAs with molecular weights of 200,000, 520,000, and 780,000. However, the reassociation rate of in vitro labeled, denatured spc-DNA suggested a molecular complexity in the range of 1 × 108, and the ability of BSC-1 chromosomal DNA to accelerate greatly the reassociation of about one third of this material indicated the presence of some repetitive chromosomal DNA sequences in spc-DNA.

Extrachromosomal DNA of Mycoplasma hominis

Abstract An extrachromosomal circular duplex DNA has been isolated from Mycoplasma hominis. This plasmid DNA has a molecular weight of about 17.8 · 106, a buoyant density of 1.680 g/ml and appears to be unrelated to the similar sized Staphylococcus aureus penicillinase plasmids. Since Mycoplasma are far simpler than other living systems, and appear to fall morphologically between viruses and bacteria, the presence of extrachromosomal DNA does not appear to be limited to the more structurally complex biological systems.

Differential post-translational modification of human type I keratins synthesized in a rabbit reticulocyte cell-free system.

The translation products synthesized in a rabbit reticulocyte lysate system, from total cellular mRNA from the human epithelial cell-line ME-180, have been examined. Keratin proteins are prominent among these translation products, and they precisely coelectrophorese in sodium dodecyl sulfate-polyacrylamide gels with keratins purified from the cells. Type-I, acidic, keratins which are acetylated in vivo, are also acetylated by the reticulocyte lysate. Examination by two-dimensional electrophoresis, of two acidic keratins known to be phosphorylated in vivo reveals that only one of these proteins is phosphorylated in the lysate system. Phosphorylation of this protein occurs after release of the completed polypeptide chain from the ribosome. The protein phosphorylated by the lysate is known to be the only ME-180 phosphokeratin modulated by cyclic AMP, reflecting in vitro the differential modification of ME-180 keratins in vivo.

The evolution of polyoma-transformed rat cell lines during propagation in vitro

Abstract We have previously shown that the presence of a functional viral large T antigen in polyoma-transformed rat cells results in instability of the integrated viral DNA, as manifest by free viral DNA production, excision, and amplification of the integrated viral molecules. It has also been observed that polyoma tumors or transformed cell lines often do not produce functional large T antigen. To determine if an evolution toward the loss of large T-Ag function took place in vitro in polyoma-transformed cell lines, we studied the changes occurring in the integrated viral DNA sequences of a ts-a Py-transformed rat cell line, ts-a H3, upon propagation under conditions permissive (33°) or nonpermissive (39°) for large T function. H3 contains a single insertion of a partial tandem of ∼1.2 viral genomes, and at 33° produces free viral DNA and also undergoes a high frequency of excision and amplification, involving recombination between homologous portions of viral DNA. While the integrated arrangement of viral DNA sequences remains unchanged at 39°, in two independent experiments long-term propagation of H3 at 33° resulted in the emergence of a fully transformed cell population which no longer produced free viral DNA and whose integrated viral DNA pattern was modified. In one of these populations, H3 ∗ , interruption of the integrated viral coding sequences unique to large T antigen by a segment of host DNA resulted in the loss of large T antigen function. In another population, H3A ∗ -3, the integrated viral DNA had undergone some rearrangement which, however, did not apparently affect large T Ag coding sequences. Since the latter cell population did not produce free viral DNA and was unable to “rescue” integrated viral DNA sequences from a T-Ag-negative Py-transformed cell line after cell fusion, the large T antigen produced by H3A ∗ -3 is probably nonfunctional. These results are consistent with the hypothesis that the presence of large T antigen leads to instability of the viral DNA integration and that this instability is responsible for the selection of large T antigen-negative cells from Py-transformed populations. Possible mechanisms underlying this type of evolution are presented.

Preparation of radioiodinated simian virus 40 DNA for use in DNA - DNA reassociation kinetics experiments.

A method is described for the preparation of 125I-labelled SV40 DNA. Using this method, SV40 DNA can be routinely labelled to 15 - 10(6) dpm per mug; much higher specific activities are easily obtained by minor modifications of the method. Once incorporated, the radioactive label dissociates from DNA exceedingly slowly at 4 degrees C or at 68 degrees C. Iodinated SV40 DNA is shown to be useful in the quantitation of viral nucleic acid sequences in SV40-transformed 3T3 cells by DNA - DNA reassociation kinetics.

Symmetric transcription of simian virus 40 DNA in the nuclei of transformed mouse cells.

Abstract The transcription pattern of SV40 DNA in the transformed mouse 3T3 cell line SV101 has been determined by DNA-RNA hybridization studies using the separated strands of restriction endonuclease derived viral DNA fragments as molecular probes. RNA isolated from the cytoplasm of these cells contained asymmetrically transcribed base sequences complementary to only the early region of the viral genome, while RNA isolated from purified nuclei contained symmetrically transcribed sequences complementary to all regions. The presence of late, anti-early, and anti-late RNA copies in the nuclei of these transformed cells, and their absence from the cytoplasm, suggests that RNA processing and/or transport are significant control mechanisms in the expression of this eukaryotic DNA.

Nonintegrated viral DNA in rat cells doubly transformed by SV40 and polyoma virus.

Abstract Rat F2408 cells transformed by polyoma virus contain, in addition to integrated viral genomes, a small number (an average of 20–50 copies per cell) of nonintegrated viral DNA molecules. On the other hand, SV40-transformed rat cells contain only integrated viral genomes. SV40-transformed rat cells also differ from the polyoma transformants, in that they grow in soft agar medium at a much slower rate. Cells doubly transformed by polyoma and SV40 can be easily isolated following polyoma superinfection of SV40-transformed rat cells, as their rate of growth in agar is enhanced. These doubly transformed cells yield polyoma or SV40, respectively, after fusion with cells permissive for each virus. However, only polyoma-specific DNA sequences can be detected in these cells in a “free” state.

Studies on plasmid replication: II. In vivo transcription and its control in penicillinase plasmids from Staphylococcus aureus

Abstract DNA-RNA hybridization and hybridization-competition have been used to demonstrate in vivo transcription of staphylococcal penicillinase plasmids and to study its control. These studies have shown that the amount of plasmid-specific messenger RNA present in plasmid-carrying organisms is roughly proportional to the amount of plasmid DNA but can be significantly increased by simultaneous phenotypic induction of several plasmid genes. The results include the finding that there is a small but significant degree of homology between the plasmid and the staphylococcal chromosome. A mutant plasmid that is thermosensitive for replication and for the expression of several of its genes has also been examined. This expression defect has been found to be correlated with a substantial reduction in the synthesis of plasmid-specific mRNA at the restrictive temperature.