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|Titolo:||Inhibition of polyoma gene expression in transformed mouse cells by hypermethylation|
|Autori interni:||LIBOI, Elio Maria|
|Data di pubblicazione:||1984|
|Abstract:||The evolution of mouse cells transformed by a recombinant plasmid containing the genome of the tsA mutant of polyoma virus (Py) cloned at the BamHI site into the plasmid pML, whose sequences therefore interrupt the Py late region, has been studied. Clones of transformed cells were selected at 39 degrees (nonpermissive temperature for large T antigen). Under these conditions viral DNA integration is stable and the cells display a uniformed transformed phenotype. Also studied in detail was the evolution of one of these cell lines (A4) upon shift to a temperature permissive for large T-Ag function (33 degrees); immediately after shift, 90% of the population became intensely positive for T-Ag and a considerable amount of free-viral DNA was produced, accompanied by a clear cytopathic effect. Surviving cells proliferated actively after 4 weeks at 33 degrees and showed a decreased expression of large T-Ag (only 2-3% of the population was T-Ag positive by immunofluorescence), a drastic reduction in the amount of free-viral DNA produced, but no apparent change in the pattern of integration of Py DNA in the host chromosomes. Analysis of the high-molecular-weight DNA with the restriction enzymes HpaII and MspI revealed that the cytosines in the recognition sequences of these enzymes were methylated. Accordingly, treating the cells with 5-Azacytidine, a methylation inhibitor, results in the expression of viral T-Ags in more than 80% of the cell population. Analysis of DNA transcription revealed a dramatic reduction of virus-specific poly(A)+ mRNA in the methylated cells; in addition, the phenotype of the 33 degrees A4 populations was much less transformed than that of the original cultures. The block of Py expression by methylation is not complete; approximately 2% of the cells remain T-Ag positive and viral transcription is not completely suppressed. This could be explained by an incomplete methylation which randomly leaves unmethylated sequences essential for Py gene expression, or by the fact that methylation is not sufficient to block transcription completely. Possible mechanisms underlying this type of evolution are discussed.|
|Appare nelle tipologie:||01.01 Articolo in Rivista|
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