Cloning and characterization of a novel DNA-binding protein that can mediate the cellular transcriptional action of protein kinase A.
Item
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Title
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Cloning and characterization of a novel DNA-binding protein that can mediate the cellular transcriptional action of protein kinase A.
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Identifier
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AAI9807930
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identifier
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9807930
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Creator
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Fliss, Makiko Suzuki.
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Contributor
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Adviser: Carter Bancroft
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Date
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1997
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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
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Abstract
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A novel transcription factor, Ickis, was cloned from a pituitary GC cell expression library, using the prolactin (PRL) gene regulatory-element site1P as a probe. Ickis binds specifically to the distal end of site1P, adjacent to the site for the pituitary-specific factor, Pit-1, implying a potential interaction between these proteins. The complete Ickis cDNA (1919 bp), obtained using 5{dollar}\sp\prime{dollar}-RACE PCR, encodes a novel protein containing three WD-motifs, but no classical DNA-binding motif. Genomic southern analyses revealed that Ickis is a single copy gene that is well-conserved from yeast to human, suggesting an important physiological role for Ickis.;Northern analysis showed that Ickis mRNA accumulates in GH3 pituitary cells with a size of circa 1.9 kb, corresponding well with the length of the cloned Ickis cDNA. Analysis of a human organ blot revealed tissue-specific forms of Ickis mRNA, which might be generated from alternative splicing and/or promoter usage. RT-PCR analysis of human tumors revealed that Ickis mRNA is present in many pituitary cell-types but in only a subset of neuronal cells. Ickis may thus play a regulatory role outside of the pituitary, and exhibit tissue-specific activity via isoforms.;Western analysis using GH3 (pituitary) cells showed nuclear accumulation of Ickis protein circa 46 KDa in size, which corresponds well with the molecular mass deduced from the Ickis coding region. Transient co-transfection analysis showed that Ickis can activate the PRL promoter in heterologous C6 cells. Ickis but not Pit-1 was further activated by PKA, suggesting that Ickis may be a downstream effector for PKA regulation of PRL. Ickis and Pit-1 exhibited additive activity on this promoter. Similar analyses were performed using a GAL4-Ickis chimera, in order to examine Ickis action in the homologous GH3 cells. GAL4-Ickis activated a GAL4 promoter construct, indicating that Ickis contains a functional transactivation domain. This fusion protein was also able to reconstitute PKA activation in the homologous cellular environment. In summary, Ickis is a novel transcription factor that can activate the PRL promoter via site1P, and can also mediate transcriptional actions of PKA.
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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.
