Prolactin gene expression: Phosphorylation of regulatory proteins, and transcriptional kinetics.
Item
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Title
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Prolactin gene expression: Phosphorylation of regulatory proteins, and transcriptional kinetics.
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Identifier
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AAI9909401
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identifier
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9909401
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Creator
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Ashton, Robert Andrew.
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Contributor
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Adviser: Carter Bancroft
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Date
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1998
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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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Biology, Neuroscience | Biology, Molecular
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Abstract
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Prolactin (Prl) gene expression is modulated by various extracellular signals. Signals are relayed to the nucleus via diverse second messenger systems, including cAMP acting via protein kinase A (PKA). This work examines how PKA-mediated phosphorylation affects binding of several transcription factors to the Prl promoter.;One or more of the transcription factors that bind to the Prl promoter could potentially mediate PKA signal transduction. In preparation for biochemical studies, I expressed several of them in bacteria. I readily purified thyrotroph embryonic factor (TEF) and CREB, since both proteins are expressed in a soluble form in bacteria. In contrast, purification of all other transcription factors was problematic, since they formed insoluble inclusion bodies in bacteria. Optimization of several growth parameters failed to improve protein solubility. Employing a different approach, I created a thioredoxin fusion vector that reportedly increased the solubility of several other proteins. Pit-1 solubility was slightly improved in this vector, but PBLF remained completely insoluble when expressed in E. coli.;I characterized TEF phosphorylation. TEF is a good substrate for both PKA and PKC. The K{dollar}\rm\sb{lcub}M{rcub}{dollar} (18 {dollar}\mu{dollar}M) and V{dollar}\rm\sb{lcub}max{rcub}{dollar} (0.63 pmol PO{dollar}\sb4{dollar}/min) values of TEF conform with other proteins that are phosphorylated by PKA. PKA phosphorylation increased the affinity of wild type TEF for site 1P on the Prl promoter. Mutation of serine208 to alanine lowered the affinity of TEF for site 1P, and PKA phosphorylation of mutated TEF further lowered its binding affinity. Mutation of serine208 decreased the incorporation of phosphate into TEF by 50%. Peptide mapping and phosphoamino analysis were consistent with two PKA phosphorylation sites in TEF, serine208 and serine91.;Finally, I examined the kinetics of Prl gene activation by thyrotropin releasing hormone (TRH). I designed and implemented a ribonuclease protection assay to measure early transcriptional activation of the endogenous Prl gene in rat pituitary GH{dollar}\sb3{dollar} lactotropes. I optimized this assay, and then showed that TRH causes a rapid, biphasic increase in Prl gene expression. There is an initial increase in Prl transcription, peaking at about 20 minutes, followed by a decline and a second, slower increase in transcription that continues for several hours.
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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.
