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Pituitary-specific transcription factor PIT-1 in Chinese grass carp : molecular cloning, functional characterization, and regulation of its transcript expression at the pituitary level

by Kwong, Ka-yee

Abstract (Summary)
(Uncorrected OCR) Abstract of thesis entitled PITUITARY-SPECIFIC TRANSCRIPTION FACTOR PIT-1 IN CHINESE GRASS CARP: MOLECULAR CLONING, FUNCTIONAL CHARACTERIZATION, AND REGULATION OF ITS TRANSCRIPT EXPRESSION AT THE PITUITARY LEVEL Submitted by Kwong Ka Yee for the degree of Master of Philosophy at The University of Hong Kong in January 2004 Pituitary-specific transcription factor-1 (Pit-l/GHF-1), a Pit-Oct-Unc (POU) family transcription factor, is known to be essential for the pituitary specific expression of prolactin and growth hormone (GH). Although the functional role of Pit-1 at the pituitary level has been well established in mammals, the neuroendocrine mechanisms regulating Pit-1 gene expression have not been fully characterized. In lower vertebrates, such as fish, it is still unclear if Pit-1 expression at the pituitary level can be regulated by hypothalamic factors. In this study, a Pit-1 cDNA was cloned in Chinese grass carp, Ctenopharyngodon idellus using 5' and 3' Rapid Amplification of cDNA Ends (RACE). Sequence analysis has revealed that this Pit-1 cDNA has a 55 bp 5' untranslated region, a 1071 bp open reading frame encoding 356 amino acids, and a 941 bp 3' untranslated region. The amino acid sequence of the encoded protein contains Serine/Threonine-rich activation (STA) domain with both B and j inserts, POU-specific domain and POU-homeodomain which are highly homologous to Pit-1 reported in other fish species. Using Northern blot and RT-PCR, a 3.5 Kb Pit-1 transcript was identified exclusively in the pituitary but not in other tissues in Chinese grass carp. Genomic Southern analysis also revealed that there were two copies of Pit-1 gene in the grass carp genome. To confirm the functional identity of grass carp Pit-1, overexpression of this newly cloned cDNA was conducted in mammalian cell lines transfected with a luciferase reporter construct carrying the 5' promoter of grass carp growth hormone (GH) gene. In these experiments, luciferase activity was significantly increased with Pit-1 overexpression, confirming that the newly cloned Pit-1 is functional in supporting the promoter activity of the grass carp GH gene. This Pit-1 cDNA was then used to set up a slot blot assay to monitor "steady-state" Pit-1 mRNA expression in primary cultures of grass carp pituitary cells. Surprisingly, the Pit-1 transcript level was transiently down-regulated by static incubation with the adenylate cyclase (AC) activator forskolin and membrane permeable cAMP analog. Both the AC inhibitor MDL 12330A and protein kinase A (PKA) inhibitor H89 were effective in blocking forskolin-induced down-regulation of Pit-1 mRNA expression, suggesting the involvement of the AC/cAMP/PKA cascade in forskolin's action. Unlike forskolin, static incubation with somatostatin (SRIF), a well documented physiological GH-release inhibitor, suppressed cAMP production but induced a rise in Pit-1 mRNA levels. These results, as a whole, have confirmed that functional Pit-1 is expressed in the pituitary of Chinese grass carp. The present study also demonstrates for the first time that cAMP regulates the Pit-1 transcript level in an opposite manner compared to GH gene expression. It is probable that SRIF can stimulate Pit-1 gene expression at the pituitary level through inhibition of the AC/cAMP/PKA pathway.
Bibliographical Information:

Advisor:

School:The University of Hong Kong

School Location:China - Hong Kong SAR

Source Type:Master's Thesis

Keywords:transcription factors genetic regulation ctenopharyngodon idella genetics

ISBN:

Date of Publication:01/01/2004

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