We have used nascent strand determination analysis to map start sites of DNA replication in the mouse ribosomal gene cluster in which individual copies of the ribosomal genes are separated by intergenic spacer regions. One origin of bidirectional replication (OBR) was localized within a 3 kb region centered about 1.6 kb upstream of the rDNA transcription start site. At least one additional initiation site is situated near the 3' end of the transcription unit. Adjacent to the OBR at the transcription start site are located two amplification-promoting sequences, i.e., APS1 and APS2. Nuclease-hypersensitive sites were identified in both of the two APSs as well as in the OBR region, thus indicating that these sequences have an altered chromatin structure. In the OBR an intrinsically bent region, a purine-rich element and other prospective initiation zone components are found.
Die als Synthesebausteine wertvollen 2-siloxysubstituierten Cyclopropancarbonsaure-methylester C werden in groBer Vielfalt aus Silylenolethern E und Diazoessigsaure-methylester (D) unter Kupfersalzkatalyse in guten Ausbeuten erhalten. Wahrend die regio-und stereochemischen Eigenschaften von E voll auf die Cyclopropane C ubertragen werden, ist die Stereoselektivitat beziiglich der Stellung der Methoxycarbonylgruppe nur gering. Die zur Konfigurationsbestimmung der Verbindungen C herangezogenen 'H-und 'k-Kriterien werden diskutiert.Synthesis of 2-Siloxy-substituted Methyl Cyclopropanecarboxylates 2-Siloxy-substituted methyl cyclopropanecarboxylates C -useful building blocks in synthesisare obtained in great variety and with good yields from the silyl enol ethers E and methyl diazoacetate (D) under copper salt catalysis. Whereas the regiochemical and stereochemical properties of E are completely transferred to the cyclopropanes C, the stereoselectivity concerning the position of the methoxycarbonyl group is low. The 'H and I3C data used to determine the configurations of compounds C are discussed.Zum Aufbau von funktionalisierten Kohlenstoffgerusten zog man in den letzten Jahren immer haufiger Cyclopropanderivate heran'-'). Dabei werden im wesentlichen zwei Typen verwendet: Acceptor-substituierte Cyclopropane A werden von Nucleophilen gedffnet (Homo-Michael-System) 2), wahrend Donorcyclopropane B mit Elektrophilen reagieren (Homo-Enolat-System) '). Seebach hat diese Verfahrensweise treffend als Umpolung mittels ,,Cyclopropantrick" bezeichnet Ib'. Obwohl der vermutlich erste Vertreter der vicinal Donor-Acceptor-substituierten Cyclopropane AB schon 1935 beschrieben wurde'), fand diese Verbindungsklasse wenig Beachtung. Erst Wenkerf 5, erkannte, daR derartige Cyclopropane als Vorstufen fur die synthetisch wertvollen 1,4-DicarbonyIverbindungen eingesetzt werden kdnnen; auf diese Weise baute er elegant und effizient Terpen-und Alkaloidgeruste auf 3c). Allerdings verwendete er in der Regel Alkylenolether als Ausgangsmaterial (Donorkomponente), deren Nachteile gegenuber den von uns eingesetzten Silylenolethern unten geschildert werden.
A replication fork barrier at the 3'-end of mouse ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. This barrier is an inherent property of a defined DNA-protein complex including transcription termination factor I, and specific protein-protein interactions occur between this factor and protein(s) of the replication machinery. Here we report that a second DNA-binding protein is essential for barrier activity. We have purified and functionally characterised the protein from HeLa cells. The final preparation contained two polypeptides with molecular masses of 70 and 86 kDa, respectively. Both polypeptides interact with a GC-stretch adjacent to the binding site of transcription termination factor I. The specificity of binding to the barrier DNA was demonstrated in an electrophoretic mobility shift assay. The biochemical properties of this protein resemble that of Ku antigen, a human nuclear DNA-binding heterodimer that is the target of autoimmune-antibodies in several autoimmune diseases. Recombinant Ku protein, purified as heterodimer from co-infected insect cells, is able to partially rescue the barrier activity in Ku-depleted HeLa cell extracts. These data demonstrate that transcription termination factor I and Ku act synergistically to prevent head-on collision between the replication and the transcription machinery.
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