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dc.contributor.authorRybacki, Lisa Jean
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 57-10, Section: B, page: 6095.;Advisors: Geoffrey Childs.
dc.description.abstractThe transcriptional regulation of the Strongylocentrotus purpuratus late H1{dollar}\beta{dollar} gene was examined to further elucidate the mechanism of stage-specific activation.;Within the regulatory regions of both the early and the late H1 genes are several common cis-sequence elements. The Upstream Sequences Elements (USE) 0, I, II and the TATA box, although divergent between families, are present in the same relative positions. However, the late H1 genes, {dollar}\beta{dollar} and {dollar}\gamma{dollar}, which are co-regulated during embryogenesis possess two additional conserved common sequence motifs, USE III and USE IV, which are located upstream of USE II. Previous studies of the SpH1{dollar}\beta{dollar} promoter indicated that USE IV was indispensable for activated mid-blastula stage transcription, and the gene encoding its binding protein, Stage-Specific Activator Protein (SSAP), was cloned.;The detailed analysis of the entire late gene specific region revealed that there were actually multiple binding sites for SSAP. DNAse I footprinting of the region extending from {dollar}-{dollar}372 to {dollar}-{dollar}152, which included all of the conserved late gene specific sequences, yielded three separate protected areas. These regions were protected by both nuclear extract and purified SSAP and corresponded to USE IV, USE III and another site between the two, termed Site 2. SSAP bound each of these sites with a discernible difference in affinity, which was approximately 100-fold greater for USE IV than for USE III or Site 2. The ability of the conserved sequence elements USE III and USE IV to function independently in vivo was assessed by microinjection of a series of H1{dollar}\beta{dollar} DNA constructs into sea urchin zygotes. Multimers, but not single copies of either USE III or USE IV were able to activate transcription to similar levels at the mid-blastula stage. The stage-specific activity of each of the multimers was dependent on the presence of the elements of the H1{dollar}\beta{dollar} basal promoter, USE I, USE 0 and the TATA box. These same multimers were inactive in the context of the H1{dollar}\beta{dollar} gene TATA box alone. Therefore, activation of the late H1 genes is regulated by a dual mechanism, where multiple SSAP binding sites are necessary for maximal activation, and these sites, which constitute the temporal enhancer, require the presence of factors that interact with the basal promoter.;A separate but convergent approach to examining H1{dollar}\beta{dollar} gene activation concentrates on the ability of SSAP to activate transcription in vivo. SSAP, although present at all developmental stages, undergoes a change in state from monomer to dimer at about the same time in development that the H1{dollar}\beta{dollar} gene is activated. Overexpression of SSAP by microinjection of synthetic mRNA into zygotes resulted in 3 to 4-fold transactivation of target CAT reporter genes linked to USE IV enhancer sequences. This transactivation began only at and subsequent to the mid-blastula stage. These results provided evidence that SSAP was directly involved in activating transcription of the H1{dollar}\beta{dollar} gene, and suggest that SSAP must be modified in a stage-specific manner to do so. (Abstract shortened by UMI.).
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.titleStudies on the mechanism of stage-specific activation of the sea urchin H1-beta gene

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