Isolation and characterization of polyoma virus mutants able to develop in embryonal carcinoma cells.

Vasseur, Marc; Kress, Chantal; Montreau, Nicole; Blangy, Daniel

doi:10.1073/pnas.77.2.1068

Cited by 56 publications

(35 citation statements)

References 24 publications

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“…Rearrangement of the enhancer B domain of polyomavirus may be required to identify the EC host range mutant (12,21,22,25,32,37). The unique property of fPyF9, persistence in an episomal state, seems to be due to box DNA, since the effects of insertion of box DNA in the control region differ from those thus far reported for EC mutants.…”

Section: Discussionmentioning

confidence: 98%

Negative transcriptional regulatory element that functions in embryonal carcinoma cells.

et al. 1989

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We have cloned the polyomavirus mutant fPyF9, which persists in an episomal state in F9 embryonal carcinoma cells (K. Ariizumi and H. Ariga, Mol. Cell. Biol. 6:3920-3927, 1986). fPyF9 carries three copies of exogenous sequences, the prototype of which is a 21-base-pair repeat (box DNA), in the region of the enhancer B domain of wild-type polyomavirus DNA. The consensus sequence, GCATTCCATTGTT, is 13 base pairs long. The box DNA inserted into fPyF9 appeared to come from a cellular sequence and was present in many kinds of DNAs, including F9 chromosomal DNA. The biological function of box DNA was analyzed by chloramphenicol acetyltransferase expression assays, using chimeric plasmids containing box DNA conjugated with simian virus 40 promoter elements. The results showed that box DNA repressed the activities both of the simian virus 40 promoter and enhancer only in transfected undifferentiated F9 cells and not in differentiated LTK-cells. Box DNA functioned independently of orientation and position with respect to the promoter in an enhancerlike manner, although the effect of box DNA was opposite that of the enhancer. The XhoI linker insertion into the consensus sequences of box DNA abolished the repression activity, and the protein(s) recognizing the consensus sequences was identified only in F9 cells, not in L cells. These analyses suggest that box DNA may be a negative regulatory element that functions in undifferentiated cells.Eucaryotic enhancer sequences play important roles in the regulation of a variety of viral and cellular genes (23). An enhancer is defined as an element that stimulates the level of transcription of a linked gene independently of position and orientation with respect to the promoter (23). It has recently been shown that many enhancers function in specific cells or tissues or in response to an inducing signal; the immunoglobulin heavy-chain gene enhancer functions only in B lymphocytes (4,14), and the expression of the cytochrome P1-450 gene is induced by dioxin (20).On the other hand, a negative enhancer has recently been discovered. This regulatory element functions in cis, and its mode of action is similar to that of a positive enhancer. This element, now called a silencer or dehancer, was initially discovered in the MAT locus of Saccharomyces cerevisiae, the locus that determines mating type (8). Furthermore, elements with similar properties have been shown to be located in upstream regions of the c-myc (26), p53 (7), and human 1-interferon (15) genes and in the long terminal repeat of human T-cell lymphotropic virus type III (27). Enhancers in the long terminal repeat (32, 33) and 1-interferon gene (15, 40), however, are also present close to the negative regulatory sequence. Both sequences appear to play a key role in transcriptional control of the gene linked to the 3' end. Cellular genes are generally thought to be cooperatively regulated by negative and positive enhancers, with the exception of a housekeeping gene such as the actin gene (34). To understand the mechanism of gene r...

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Section: Discussionmentioning

confidence: 98%

Negative transcriptional regulatory element that functions in embryonal carcinoma cells.

et al. 1989

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“…For example, Segal et al (1979) noted that the restrictive interaction of simian virus 40 (SV40) with non-differentiated teratocarcinoma cells was related to failure in viral mRNA splicing. For polyoma virus (Vasseur et al, 1980) and cytomegalovirus (Dutko & Oldstone, 1981), a block in replication at the transcriptional level was reported in undifferentiated embryonal carcinoma cells (ECC). D 'Auriol et al (1981) suggested that the Rauscher murine leukaemia virus proviral DNA failed to integrate into the genome of ECC to account for the restriction in virus replication.…”

Section: Introductionmentioning

confidence: 99%

Eclipse of Coxsackievirus Infectivity: the Restrictive Event for a Non-fusing Myogenic Cell Line

Schultz

Crowell

1983

Journal of General Virology

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SUMMARYCoxsackieviruses A2, A5 and B3 did not replicate in LsCL3-U cells (a non-fusing variant of the rat L s myogenic cell line) although these cells possessed a common receptor for coxsackieviruses A2 and A5, and a different receptor for coxsackievirus B3. The restriction in replication was identified as a block in viral eclipse, since 6 MLiC1 treatment permitted recovery of the coxsackievirus A2 inoculum from L8 CL3-U cells after 2 h at 37 °C, and the cells could be transfected by viral RN A. Cellular fusion which was induced in LsCL3-U cultures by herpes simplex virus type 1 (HF strain) facilitated coxsackievirus A2 and A5 replication. Differentiating myogenic L8 cells acquired full susceptibility to infection concurrently with the appearance of acetylcholine receptors, the muscle-specific isoenzyme of creatine phosphokinase, prominent myotube formation and the acquired capacity of the cells to eclipse virus.

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“…Immunofluorescence detection of T-antigen-positive cells was done 60 to 72 h after transfection with plasmid DNA as described above, according to techniques described elsewhere (27). purified by centrifugation in cesium chlorideethidium bromide density gradients.…”

Section: Methodsmentioning

confidence: 99%

Deletions of N-terminal sequences of polyoma virus T-antigens reduce but do not abolish transformation of rat fibroblasts.

Katinka¹,

Yaniv²

1982

Mol. Cell. Biol.

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Polyoma virus transforms, upon infection or DNA transfection, nonpermissive Fisher rat fibroblasts. Cloned viral DNA was deleted of sequences around the BglI site at nucleotide 86 by Bal3l nuclease treatment and then recloned in Escherichia coli. The extent of deletion for each mutant was then determined by DNA sequencing. Deletions included the early transcription control signals; others stretched into the N-terminal coding sequences of the viral tumor antigens. The transformation efficiency of 16 mutants was tested by transfecting rat fibroblasts. Expression of the T antigens was analyzed by immunofluorescence detection after transfection of rat fibroblasts, mouse secondary embryo cells, and HeLa cells. We found that the absence of the early transcription control sequences (TATA and CAAT boxes) did not significantly alter the transformation capacity of the virus. On the other hand, deletion of the initiator methionine ATG codon or further into the coding sequences did abolish the transformation capacity in some mutants, whereas others maintained a reduced transforming activity, possibly by initiation of translation in a penultimate methionine. Calif., 1978) between nt 120 and 128, the CAAT box (2) between nt 61 and 69, and the binding site for large T antigen (4) between nt 49 and 64, in the efficiency of transformation? (ii) Will viral sequences transform FR3T3 in spite of deletions in the N-terminal coding sequences of the T antigens starting at nt 173?To answer these questions, we deleted sequences of cloned Py DNA around the BglI site at nt 86 by Bal31 nuclease treatment and tested the transformation efficiencies of the altered viruses on FR3T3 cells (rat fibroblasts). The exact extent of the deletion for each cloned mutant was determined by DNA sequencing. The expression of the T antigens was also explored by immunofluorescence detection after transfection of replication-nonpermissive FR3T3 and HeLa cells as well as permissive secondary mouse embryo cells.Our results demonstrate that viral transformation capacity could be conserved when different control elements were absent. Furthermore, deletions of N-terminal coding sequences greatly reduced but did not always abolish the transformation of FR3T3 by Py. The efficiency of such transformations is not directly linked to the extent of coding sequences deleted and may instead be related to a possible initiation of translation at an internal methionine.

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Isolation and characterization of polyoma virus mutants able to develop in embryonal carcinoma cells.

Cited by 56 publications

References 24 publications

Negative transcriptional regulatory element that functions in embryonal carcinoma cells.

Negative transcriptional regulatory element that functions in embryonal carcinoma cells.

Eclipse of Coxsackievirus Infectivity: the Restrictive Event for a Non-fusing Myogenic Cell Line

Deletions of N-terminal sequences of polyoma virus T-antigens reduce but do not abolish transformation of rat fibroblasts.

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