RESUMO
The cell-specific regulation of DNA replication has important implications for the molecular strategy of cellular gene control. Mouse polyomavirus (Py) DNA replication is examined as a model of cell-specific replication control. Using an FM3A-derived mouse cell line which expresses early viral proteins (FOP cells), we determined the minimal sequence requirements for viral DNA replication. FOP cells were observed to have much simpler enhancer requirements than 3T6 and many other cells and did not need a B enhancer for high levels of DNA replication. Using these cells, we show that the individual or tandem binding sites for several unrelated trans-acting factors which are generally subfunctional as transcriptional enhancers (simian virus 40 A core, TGTGGAATG; EBP20, TGTGGTTTT; PEA1 [an AP-1 analog], GTGACTAA; PEA2, GACCGCAG; and PEA3, AGGAAG) stimulated low levels of Py DNA replication. The ordered dimeric combination of PEA3 and PEA1 factor-binding sites, however, acted synergistically to stimulate viral DNA replication to high wild-type levels. This is in contrast to prior results in which much larger enhancer sequences were necessary for high-level viral DNA replication. PEA3/PEA1-stimulated DNA replication showed a distance and orientation independence relative to the origin, which disagrees with some but not other prior analyses of enhancer-dependent DNA replication. It therefore appears that trans-acting factor-binding sites (enhansons) can generally activate DNA replication and that the AP-1 family of sites may act synergistically with other associated trans-acting factors to strongly affect Py DNA replication in specific cells.
Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Polyomavirus/genética , Vírus 40 dos Símios/genética , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core , Vetores Genéticos , Camundongos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-jun , Fator de Transcrição AP-2RESUMO
The relationship of viral DNA replication to the activation of viral gene expression is usually considered with respect to late genes. In this report we examine the replication activation of the polyomavirus early promoter. Using origin active and inactive mutants to drive luciferase gene expression from the polyomavirus early promoter, we show that the early promoter is also subjected to a replication dependent activation. The degree of activation can be up to a hundred fold greater than that seen without replication and is about 13 fold on a per template basis. This replication based activation is, however, cell type dependent and was seen in FOP cells but not in 3T6 cells. Analysis of the requirements of cis acting DNA show that these enhancer elements affect early transcription predominantly through the activation of replication, although some replication independent stimulation can also be seen. The implications of this result for the regulation of polyomavirus early gene regulation are considered.
Assuntos
Replicação do DNA/genética , Regulação Viral da Expressão Gênica/genética , Polyomavirus/genética , Regiões Promotoras Genéticas/genética , Sequência de Bases , Southern Blotting , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Transfecção/genética , Células Tumorais Cultivadas , Replicação ViralRESUMO
Recent molecular cloning of cDNA for the alpha subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called Tr and Tc, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, we have identified a G-protein alpha subunit, which we refer to as Gz alpha, by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine Tr alpha-subunit cDNA. The deduced amino acid sequence of Gz alpha is 41-67% identical with those of other known G-protein alpha subunits. However, the 355-residue Gz alpha lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein alpha subunits. We suggest that Gz alpha, which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.