Suzanne Mousset

Isolation and partial characterization of a nuclear RNA polymerase - SV40 DNA complex

Several laboratories have reported the isolation of nucleoprotein complexes from polyoma (py) or simian virus 40 (SV40) infected cells [ 151. A role for these complexes in viral transcription has not yet been demonstrated. Girard et al. [6] have recently described late SV40 transcriptional intermediates constituted by nascent viral mRNA molecules hydrogen-bound to the DNA templates; the method of isolation of these molecules should prevent the RNA polymerase activity in their preparation. The anionic detergent sarkosyl has been shown to cause a several-fold enhancement of endogenous RNA polymerase activity in mouse nuclei. This stimulatory effect was ascribed to at least two factors: an increased initial activity of RNA polymerase II and a prolongation of the total polymerase reaction [7]. Sarkosyl causes the release of nearly all the proteins from cellular DNA [7], but apparently does not affect RNA polymerase that has already initiated RNA synthesis PI. The effect of sarkosyl on cellular transcriptional activity [7-91 and the efficient extraction of viral DNA with this detergent [lo] allowed us to isolate a SV40 transcription complex from late infected monkey cells. The SV40 DNA-RNA polymerase complex we describe here has an endogenous transcriptional activity, which is highly sensitive to cy-amanitin, a specific inhibitor of nucleoplasmic polymerase B or II [ 111; this should prove of great interest in the study of the regulation of viral gene expression.

Transformation of established murine fibroblasts with an activated cellular Harvey-ras oncogene or the polyoma virus middle T gene increases cell permissiveness to parvovirus minute-virus-of-mice.

Transformation of permanent rodent fibroblast cells by the polyoma virus middle T gene or the activated human Harvey-ras oncogene results in increased cellular permissiveness to the autonomous parvovirus minute-virus-of-mice. Parvoviral DNA amplification is restricted in the untransformed parental cell lines. Analysis of various parameters of the parvoviral life cycle shows that this block is partially overcome in the transformed lines.

Cooperation of oncogenes in cell transformation and sensitization to killing by the parvovirus minute virus of mice

The established line of normal Fisher rat fibroblasts (FR3T3) is naturally resistant to the parvovirus minute virus of mice (MVM), and was used as a model system to study the influence of stepwise transformation on the susceptibility of cells to this virus. When transformed with genes encoding the class I nuclear oncoproteins large T antigen of polyomavirus (PyLT) or v-myc, cells retained a normal appearance, but acquired some ability to form colonies in soft agar. On the other hand, the class II transforming oncogenes encoding the middle T antigen of polyomavirus (PyMT) and c-Ha-ras-1 induced both morphological alterations and a high capacity for anchorage-independent growth in transfected cells. The concomitant expression of oncogenes from both classes (PyLT-(+)PyMT; v-myc+c-Ha-ras-1) induced a supertransformed phenotype characterized by the piling-up of cells into poorly adherent foci, even in low density cultures. The progressive transformation of this cellular system was found to coincide with a gradual increase in its susceptibility to MVMp (MVM prototype strain) infection. Compared to parental cells, class I, class II and double transformants proved to be sensitized to killing by MVMp to a low, moderate and large extent, respectively. Thus, oncogenes from different functional classes appeared to cooperate in the responsiveness of cells to parvovirus attack. Interestingly, this cooperation exacerbated both the killing of infected cells and their capacity to produce viral non-structural (NS) proteins, in agreement with the reported cytotoxic activity of NS polypeptides. Therefore, in this system, parameters of the parvovirus life cycle may serve as indications of the overall progression of the transformation process.

Passive replication of heteroimmune bacteriophage in a λ-driven replicon

Abstract Thermal induction of λ c 857 -434 hy and λ c 857 -80 hy tandem dilysogens results in replication not only of the λ, which is induced, but also of the other phage. Nevertheless, the latter remains under immunity control, since: (a) a superinfecting phage of that immunity fails to replicate; (b) the genes under direct immunity control are not expressed. Replication of the non-induced phage takes place only if it is adjacent to the λ, and only if the λ itself is able to replicate: however, this replication does not require that the replicative enzymes are interchangeable between λ and the other phage. Clearly, one deals here with passive replication of a phage, as a part of a λ-driven replicon. Function Int frequently excises the whole tandem of prophages, presumably as a double length circle. Replication from this structure, followed by the action of function Ter, accounts for our results as well as for those of Whitfleld & Appleyard (1958). The situation described here is in many respects similar to that analyzed by Gottesman & Yarmolinsky (1968). In their case, however, passive replication takes place in situ because int − mutations in the prophages prevent early excision, and particles of only one immunity are found in the yield.

Susceptibility to parvovirus minute virus of mice as a function of the degree of host cell transformation: little effect of simian virus 40 infection and phorbol ester treatment

Transformation of mouse fibroblasts by simian virus 40 (SV40) or certain oncogenes enhances their susceptibility to the lytic replication of Minute virus of mice (MVM), a non-defective parvovirus. It was investigated whether this cytotoxic action of MVM can also be potentiated by two types of incomplete and reversible cell transformation induced either by SV40 infection or by exposure to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Although the latter two treatments trigger the expression of several traits of the transformed phenotype, they do not significantly enhance cell permissiveness to MVM.

FIXATION AND EXPRESSION OF RECESSIVE MUTATIONS IN MAMMALIAN CELLS AS A MODEL FOR THE STUDY OF CARCINOGENESIS

ABSTRACT TPA, a powerful tumour promoter is shown to induce sister chromatid exchanges (SCEs), whilst the non-promoting 4–0–Me TPA derivative does not. Two classes of inhibitors of tumour promotion, inhibit such TPA induced SCEs. Thus, TPA is the first agent reported to induce SCEs in the absence of DNA damage, chromosome aberrations and mutagenesis. Since TPA is known to induce several gene functions we would like to speculate that it might also induce enzymes involved in genetic recombination. TPA is also shown to stimulate segregation of a recessive genetic trait, 6-thioguanine resistance, from a heterozygous hybrid cell. Such data support the hypothesis that tumour promotion might be the result of an aberrant mitotic chromosomal segregation event leading to the expression of carcinogen/mutagen induced recessive genetic or epigenetic chromosomal changes.