Promoter analysis of the Xenopus laevis oocyte beta-tubulin gene.
Item
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Title
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Promoter analysis of the Xenopus laevis oocyte beta-tubulin gene.
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Identifier
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AAI9432353
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identifier
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9432353
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Creator
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Lopingco, Maria Cristina T.
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Contributor
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Adviser: James J. Bieker
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Date
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1994
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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The formation of a particular microtubule structure is dictated in part by the ratio of tubulin variants within a cell. This ratio is established by steady-state message levels that are controlled by transcriptional and autoregulatory mechanisms. The Xenopus laevis oocyte expresses at least two {dollar}\beta{dollar}-tubulin mRNAs, one (Xlot) more abundantly than the other. Xlot (Xenopus laevis oocyte tubulin) is classified as a class IVb {dollar}\beta{dollar}-tubulin based on its tissue distribution and carboxy terminus sequence. It is expressed at low levels in all tissues examined and is upregulated in germ-cells.;A portion of the Xlot promoter was cloned and partially sequenced. In addition to the TATA and CAAT boxes, numerous potential cis-elements are present, several of which are also found in the human class IVb {dollar}\beta{dollar}-tubulin promoter.;The Xlot promoter was shown to be capable of driving a significant level of transcription of a reporter gene when microinjected into Xenopus oocytes. 336 bp of promoter sequence directly upstream of the transcription start site was characterized by deletion analysis. The minimal promoter is within the first 177 bp. In addition, there are at least 2 regions between nucleotide positions {dollar}-{dollar}177 and {dollar}-{dollar}336 that modulate minimal promoter activity.;This limited promoter region was divided into the proximal ({dollar}-{dollar}121 to +22) and distal ({dollar}-{dollar}272 to {dollar}-{dollar}116) fragments. Each fragment was shown to form 2 complexes with oocyte proteins. It appears that the protein(s) responsible for complex formation with the proximal and distal fragments are similar, as judged by competition analysis. Potential cis-acting elements identified by sequence comparison of the two fragments were analyzed.;In addition to transcriptional control, oocytes appear to utilize the co-translational autoregulatory mechanism in order to maintain the appropriate level of mRNA. This mechanism requires that the message to be degraded codes for MREI at the amino terminus, and that translation be ongoing beyond residue 41. Message levels are low in oocytes microinjected with constructs encoding tubulin mRNAs with the appropriate signal for autoregulation. When the constructs contain the sequence MVYI at the amino terminus or a stop codon at position nine, steady-state message levels are elevated.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
