http://hdl.handle.net/10033/6824 AG Epigenetische Regulationsmechanismen (EPI) Wed, 22 May 2013 11:35:15 GMT 2013-05-22T11:35:15Z http://hdl.handle.net/10033/246152 Title: YY1-Binding Sites Provide Central Switch Functions in the PARP-1 Gene Expression Network. Authors: Doetsch, Martina; Gluch, Angela; Poznanović, Goran; Bode, Juergen; Vidaković, Melita Abstract: Evidence is presented for the involvement of the interplay between transcription factor Yin Yang 1 (YY1) and poly(ADP-ribose) polymerase-1 (PARP-1) in the regulation of mouse PARP-1 gene (muPARP-1) promoter activity. We identified potential YY1 binding motifs (BM) at seven positions in the muPARP-1 core-promoter (-574/+200). Binding of YY1 was observed by the electrophoretic supershift assay using anti-YY1 antibody and linearized or supercoiled forms of plasmids bearing the core promoter, as well as with 30 bp oligonucleotide probes containing the individual YY1 binding motifs and four muPARP-1 promoter fragments. We detected YY1 binding to BM1 (-587/-558), BM4 (-348/-319) and a very prominent association with BM7 (+86/+115). Inspection of BM7 reveals overlap of the muPARP-1 translation start site with the Kozak sequence and YY1 and PARP-1 recognition sites. Site-directed mutagenesis of the YY1 and PARP-1 core motifs eliminated protein binding and showed that YY1 mediates PARP-1 binding next to the Kozak sequence. Transfection experiments with a reporter gene under the control of the muPARP-1 promoter revealed that YY1 binding to BM1 and BM4 independently repressed the promoter. Mutations at these sites prevented YY1 binding, allowing for increased reporter gene activity. In PARP-1 knockout cells subjected to PARP-1 overexpression, effects similar to YY1 became apparent; over expression of YY1 and PARP-1 revealed their synergistic action. Together with our previous findings these results expand the PARP-1 autoregulatory loop principle by YY1 actions, implying rigid limitation of muPARP-1 expression. The joint actions of PARP-1 and YY1 emerge as important contributions to cell homeostasis. Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/10033/246152 2012-01-01T00:00:00Z http://hdl.handle.net/10033/76653 Title: Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties. Authors: Qiao, Junhua; Oumard, André; Wegloehner, Wolfgang; Bode, Juergen Abstract: Site-specific recombinases have revolutionized the systematic generation of transgenic cell lines and embryonic stem cells/animals and will ultimately also reveal their potential in the genetic modification of induced pluripotent stem cells. Introduced in 1994, our Flp recombinase-mediated cassette exchange strategy permits the exchange of a target cassette for a cassette with the gene of interest, introduced as a part of an exchange vector. The process is "clean" in the sense that it does not co-introduce prokaryotic vector parts; neither does it leave behind a selection marker. Stringent selection principles provide master cell lines permitting subsequent recombinase-mediated cassette exchange cycles in the absence of a drug selection and with a considerable efficiency (approximately 10%). Exemplified by Chinese hamster ovary cells, the strategy proves to be successful even for cell lines with an unstable genotype. Fri, 24 Jul 2009 00:00:00 GMT http://hdl.handle.net/10033/76653 2009-07-24T00:00:00Z http://hdl.handle.net/10033/36092 Title: T(3;7)(q27;q32) fuses BCL6 to a non-coding region at FRA7H near miR-29. Authors: Schneider, B; Nagel, S; Kaufmann, M; Winkelmann, S; Bode, J; Drexler, H G; MacLeod, R A F Sun, 01 Jun 2008 00:00:00 GMT http://hdl.handle.net/10033/36092 2008-06-01T00:00:00Z http://hdl.handle.net/10033/19353 Title: The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon. Authors: Moes, Lorin; Wirth, Manfred Abstract: BACKGROUND: Bovine viral diarrhea virus (BVDV) is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA 1. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein 2. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV) and the more distantly related Hepatitis C virus (HCV) pseudoknot function in translation has been demonstrated. RESULTS: To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical swine fever virus, a pestivirus, and the hepatitis C viruses, another genus of the Flaviviridae. CONCLUSION: The IRES of the non-cytopathic BVDV SD-1 strain displays features known from other pestivirus IRESes. The predicted pseudoknot in the 5'UTR of BVDV SD-1 virus represents an important structural element in BVDV translation. Mon, 01 Jan 2007 00:00:00 GMT http://hdl.handle.net/10033/19353 2007-01-01T00:00:00Z