Integration of SV40 DNA into the cell genome and viral mutagenesis

Integration of DNA of a temperature-sensitive SV40 mutant (tsA239) into the cell genome was studied. The viral A gene (the oncogene) encodes the tumour T antigen which is ts in the mutant and is devoid of mutagenic and transforming activity under non-permissive conditions (40 degrees C). Clones of Chinese hamster cells infected by tsA239 mutant were analysed. Those infected by wild-type SV40 served as controls. As shown by dot-hybridization, SV40 DNA was detected in cells of 14 out of 18 clones infected by tsA mutant and incubated at 40.5 degrees C, and in all 20 clones infected by tsA mutant and incubated under permissive conditions (33 degrees C), the difference between the two groups being insignificant (p greater than 0.05). By means of blot-hybridization it was established that viral DNA was integrated into the cell genome of all 12 clones analysed, belonging to the three experimental series: infection by tsA mutant, incubation at 40.5 and 33 degrees C, infection by wt SV40, incubation at 40.5 degrees C. The number of integration sites ranged from one to four in different clones. Integration of SV40 DNA in tandems was observed. The data presented allow to conclude that integration per se does not play a crucial role in determining the mutagenic and transforming effect of the virus. Obviously, what matters is the activity of viral oncogene product - the T antigen.     

Expression of SV40 tumor antigen in SV40 transformed teratocarcinoma-derived cell lines

The relative importance of viral tumor antigen expression and the cellular background in the maintenance of a transformation phenotype was examined in five SV40-transformed teratocarcinoma-derived cell lines. These cell lines show qualitative differences in growth characteristics associated with transformation, and vary in their state of differentiation. Viral T antigen expression was evaluated by two criteria: 1) the amount of immunoprecipitated antigen in growing cells, and 2) the amount and rate of antigen synthesis in density-inhibited cells. There was no direct correlation found between retention, or rate of synthesis, of the viral T antigen and the degree of transformation. These findings imply that the cellular environment has a more important influence on the growth properties of a stably transformed cell than the quantitative levels of viral T antigen expression.     

Integration sites and sequence arrangement of SV40 DNA in a homogeneous series of transformed rat fibroblast lines

The state and organization of viral DNA sequences present in independently isolated rat cell lines transformed with SV40 were investigated using restriction-enzyme cleavage of the cellular DNA and blot hybridization with a viral probe. The transformed lines were established under conditions as identical as possible, except for a limited number of variables (multiplicity of infection, physiological state of the cells after infection and procedures used for selecting the transformed derivatives). They were characterized after a limited number of generations in culture. Two distinct types of organization were found: covalently integrated viral genomes were present either as single inserts or as head-to-tail oligomeric structures. The latter was observed among transformants derived from cells maintained after infection under growth-inhibiting conditions (suspension in agarose medium, confluency on a solid substrate). Single inserts were observed only among cell lines isolated after an initial period of active growth. Recurrent patterns of hybridizaton were observed in independently isolated lines, indicating that the sites of the integrative recombinations were close enough, both in the viral and the cellular sequences, not to be distinguished at the level of sensitivity of the technique (more than +/- 100 bp). Among cell lines with multiple integration sites, only part of the inserts were found in several instances to be identical to inserts observed in other transformed lines.     

Association of SV40 DNA sequences with nuclear matrix in SV40 transformed hamster fibroblasts

Nuclear matrices were isolated by the high-salt, non-ionic detergent method from SV40-transformed hamster fibroblasts (TSV5 cell line), and from hamster tumours derived from these cells. DNA isolated from matrices and total nuclei was hybridized with nick-translated SV40 DNA. The enrichment of matrix DNA with SV40 DNA sequences was observed in all five experiments with matrix DNA of TSV5 cells but only in five out of nine matrix DNA isolated from tumour cells.     

Studies on mannose-containing glycopeptides from a normal and an SV40 transformed human cell

The oligosaccharide moiety of cell-surface mannose-labelled glycopeptides from a normal (WI38) and an SV40 transformed cell (WI18 Va) have been investigated using specific glycosidases. Partially purified mannose-containing glycopeptides were separated into acidic and neutral species by high voltage paper electrophoresis. Endo-β-N-acetylglucosaminidase D, in the presence of three exoglycosidases, released from the acidic glycopeptides of non-growing cells a product completely absent in growing cells. However, the acidic species from growing WI18 Va and WI38 were found to be similar in the products released by enzyme digestion. The neutral species from growing normal cells contained a proportion of the glycopeptides resistant to endoglycosidase D while those from the non-growing cells were almost free of these resistant species. The SV40 transformed cells were further enriched, when compared to normal cells (WI38), in these neutral resistant species. We suggest that the oligomannosyl core of the majority of the susceptible species contains three mannose residues while that of the resistant species contains between six and eught.     

Studies on mannose-containing glycopeptides from a normal and an SV40 transformed human cell.

The oligosaccharide moiety of cell-surface mannose-labelled glycopeptides from a normal (WI38) and an SV40 transformed cell (W118Va) have been investigated using specific glycosidases. Partially purified mannose-containing glycopeptides were separated into acidic and neutral species by high voltage paper electrophoresis. Endo-beta-N-acetylgucosaminidase D, in the presence of three exoglycosidases, released from the acidic glycopeptides of non-growing cells a product completely absent in growing cells. However, the acidic species from growing WI18 Va and WI38 were found to be similar in the products released by enzyme digestion. The neutral species from growing normal cells contained a proportion of the glycopeptides resistant to endoglycosidase D while those from the non-growing cells were almost free of these resistant species. The SV40 transformed cells were further enriched, when compared to normal cells (WI38), in these neutral resistant species. We suggest that the oligomannosyl core of the majority of the susceptible species contains three mannose residues while that of the resistant species contains between six and eight.     

Reconstitution of damage DNA excision reaction from SV40 minichromosomes with purified nucleotide excision repair proteins

We previously constructed the cell-free nucleotide excision repair (NER) assay system with UV-irradiated SV40 minichromosomes to analyze the mechanism of NER reaction on chromatin DNA. Here we investigate the factor that acts especially on nucleosomal DNA during the damage excision reaction, and reconstitute the damage excision reaction on SV40 minichromosomes. NER-proficient HeLa whole cell extracts were fractionated, and the amounts of known NER factors involved in the column fractions were determined by immunoblot analyses. The column fractions were quantitatively and systematically replaced by highly purified NER factors. Finally, damage DNA excision reaction on SV40 minichromosomes was reconstituted with six highly purified NER factors, XPA, XPC-HR23B, XPF-ERCC1, XPG, RPA and TFIIH, as those essential for the reaction with naked DNA. Further analysis showed that the damages on chromosomal DNA were excised as the same efficiency as those on naked DNA for short incubation. At longer incubation time, however, the damage excision efficiency on nucleosomal DNA was decreased whereas naked DNA was still vigorously repaired. These observations suggest that although the six purified NER factors have a potential to eliminate the damage DNA from SV40 minichromosomes, the chromatin structure may still have some repressive effects on NER.     

Ultrastructural and cytochemical features of SV 40 transformed hypothalamic cell lines

Summary A continuous cell line was previously obtained by Simian Virus (SV) 40 transformation of primary cultures of dissociated mouse fetal hypothalami. One clone from this cell line has been previously shown to possess some of the ultrastructural features, immunological properties and synthesizing capacities of magnocellular hypothalamic neurons which secrete vasopressin and neurophysins. The present paper reports on the morphological characterization of 14 other clones or subclones of the original cell line, using the following criteria: phase contrast microscopy, electron microscopy, Gomori's aldehyde fuchsin staining, cytochemical detection of -glucuronidase, immunochemical staining with antisera against bovine neurophysin I, bovine neurophysin II, lys-vasopressin, oxytocin, LH-RH and TRH.The results allowed the conclusion that the clones as well as the subclones can be distributed into two groups: 1) neurosecretory neurons which all possess several of the ultrastructural and cytochemical features of the neurophysin-vasopressin synthesizing clone, and 2) primitive nerve cells which are devoid of such features but display numerous bundles of filaments. In addition some clones were found to display intermediate features between groups 1 and 2. A similar diversity was observed within clones of the original strain and subclones of a neurosecretory clone. It is suggested that the primitive clones could represent precursors of the neurosecretory clones.This work is dedicated to Professor W. Bargmann in honour of his 70th birthday     

Cyclic AMP levels in temperature sensitive SV40 transformed cell lines

The cAMP levels of the 3T3 cell line and its transformed derivatives were determined. The level was found to be lower in transformed cell lines than in parental 3T3 and to decrease at confluence in all cell lines tested. SV40 transformed 3T3 cell lines which are temperature sensitive in growth control were found to have lower cAMP levels than 3T3, but these levels were not temperature dependent. Retransformation of these cell lines by murine sarcoma virus did not markedly affect their cAMP levels.     

Immunologic selection of simian virus 40 (SV40) T-antigen-negative tumor cells which arise by excision of early SV40 DNA.

A clonal line of highly oncogenic spontaneously transformed mouse cells (104C) was transformed in tissue culture by simian virus 40 (SV40) and subsequently recloned (106CSC). This 106CSC cell line expressed T antigen and transplantation antigen but was about 100 times less tumorigenic than the 104C parent. When 10(5) 106CSC cells were injected into immunocompetent syngeneic mice, tumors were produced. From such tumors, cell lines were established in culture, all of which were consistently negative for T antigen. We found previously by solution DNA hybridization methods that the tumor cells were depleted in the early region of SV40 DNA which codes for the T antigen. We postulated that this loss occurs through a DNA rearrangement of unknown mechanism in one or a few 106CSC cells and that the tumors are then produced from such a cell or cells, whereas all the T-antigen-positive 106CSC cells are rejected by immunologic means. In this investigation we showed by the DNA transfer method using appropriately selected SV40 DNA probes that indeed the tumor cell clone (130CSCT) we selected to investigate came from one 106CSC cell in which the T-antigen-coding SV40 DNA sequences (but not all the early SV40 DNA sequences) were lost by an excision and recombination mechanism. We also showed that the 130CSCT cells, which are highly tumorigenic, could again be transformed by SV40 and that the resulting T-antigen-positive cloned derivative cells became much less tumorigenic (approximately 10(5)-fold), apparently again because of immunologic recognition and rejection. Indeed, when 10(7) T-antigen-positive cloned cells were injected, all the T-antigen-positive cells were rejected and the tumor was produced again from one or more T-antigen-negative cells. Thus, a one-step in vivo transplantation experiment allowed a selection (for tumorigenicity and against the SV40 T antigen) of a mutant mammalian cell with a DNA deletion at a definable site.     

Temperature dependence of strand separation of the DNA molecules containing integrated SV40 DNA in transformed cells.

It has previously been demonstrated that the DNA molecules containing a genetic marker from one region of a bacterial genome undergo complete strand separation at temperatures usually different from the molecules containing a genetic marker from another region of the genome. The experiments also showed that if a group of molecules undergo complete strand separation over a narrow temperature range, of the order of one to three degrees, it is highly likely that they all come from one region of the bacterial genome. The purpose of the work reported here was to establish appropriate procedures for doing a similar analysis of the DNA molecules containing the integrated SV40 DNA in transformed mouse cells. One result of interest is that in the 11A8 cell line about 40 per cent of the integrated SV40 DNA detectable by an RNA-DNA hybridization assay can be accounted for by a group of molecules which undergo complete strand separation within a 1.0 degree C interval.     

Genomic rearrangements in a mouse cell line containing integrated SV40 DNA

In the SV40-transformed mouse embryo fibroblast cell line SVT2/S, genomic rearrangements involving the SV40 DNA and flanking host sequences were identified by Southern blot hybridization using viral DNA as probe. No rearrangements of SV40 DNA integrated into nonpermissive mouse cells have been previously described. The standard arrangement found in the majority of subclones was mapped with 20 restriction enzymes, 10 of which cleave sites within the SV40 DNA. A single copy of a defective integrated viral genome is present, in which the late region is missing from about nucleotide 200 clockwise to about nucleotide 1750. The rest of the viral genome including the origin of replication and T antigen binding region is present and colinear with SV40 DNA, except for an internal repeat of about 1750 bp located between nucleotides 2750 and 4500. Rearrangements were found in 4 out of 20 random subclones of the parental SVT2/S cell line and 3 of the 4 continued to rearrange. The thioguanine-resistant cell line 281-1-4, derived from SVT2/S, remained stable on subculture but a chloramphenicol-resistant mutant, 107-6-4, derived from 281-1-4, was highly unstable. In 107-6-4, unique rearrangements were found in 6 of 31 subclones of a population that had undergone abut 25 doublings from a single-cell isolate. The high rate of rearrangement and the sporadic expression of rearrangement potential are characteristic of the transposable controlling elements discovered by McClintock.     

Coupled endo-exodeoxyribonuclease activity from HeLa cell nuclei interacts with specific regions of the SV40 mini chromosome

A recently described endo-exodeoxyribonuclease activity (1) was determined to interact preferentially with the origin/enhancer region and the 5' end of the late promoter region of the SV40 mini chromosome. The enzymatic activity has no preferential site of interaction with naked DNA, which suggests, the chromatin organization is responsible for this site directed interaction.