Transcriptional regulation of the rat growth hormone and prolactin promoters: In vitro and in vivo studies.
Item
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Title
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Transcriptional regulation of the rat growth hormone and prolactin promoters: In vitro and in vivo studies.
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Identifier
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AAI9530909
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identifier
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9530909
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Creator
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Pan, Wayne Tze.
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Contributor
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Adviser: Carter Bancroft
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Date
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1995
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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, Molecular | Biology, Animal Physiology
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Abstract
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The rat growth hormone (GH) and prolactin (PRL) genes are evolutionarily and ontogenically related. Therefore, it is not surprising that their promoters would share common elements. Indeed, a single transcription factor, pit-1, has been identified which activates both promoters. However, under physiologic conditions, these two pituitary genes are expressed divergently. The question we attempt to address in this dissertation is how these two promoters are differentially regulated.;It had been previously demonstrated that the expression of a PRL promoter construct stably transfected into a nonpituitary cell line could be activated on cell fusion to a pituitary cell line, whereas a GH promoter construct could not. Studies described herein using other nonpituitary host cell lines, a GH promoter construct containing additional upstream promoter sequences, and a more efficient cell fusion protocol confirmed and extended those results.;Various in vitro methods were used to identify and characterize the pit-1-binding sites on both promoters. Using DNase footprinting techniques, four sites were identified on the proximal PRL promoter and two on the GH promoter. Sequence analysis of the binding sites revealed a consensus sequence, (A/T)TA(A/T)TCA. Gel-shift experiments using site-specific mutants of the consensus sequence identified nucleotide base-pairs critical for pit-1 binding.;Functional in vitro transcription experiments demonstrated the ability of a minimal PRL or GH promoter to direct cell-specific transcription. In addition, co-transfection experiments demonstrated the ability of pit-1 to activate both promoters in vivo.;Deletion analysis of the GH promoter identified two repressor elements. We characterized two DNA-binding proteins which interact specifically with the proximal repressor element (PRE). ssPREB is a ubiquitous, single-strand-specific DNA-binding protein. PREB is a double-stranded DNA-binding protein found in nonpituitary cell lines. When the PRE was cloned in front of a heterologous promoter, herpesvirus thymidine kinase (HSV-tk), each class of DNA-binding protein had a unique effect on expression of the HSV-tk promoter. In a ssPREB-rich environment, the PRE is an activator, whereas in a PREB-rich environment, the PRE acts as a repressor.
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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.
