Molecular Cloning and Functional Studies of Cyprinid Calmodulin

This dissertation, "Molecular Cloning and Functional Studies of Cyprinid Calmodulin" by Longfei, Huo, 霍龍飛, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled MOLECULAR CLONING AND FUNCTIONAL STUDIES OF CYPRINID CALMODULIN Submitted by Huo Longfei for the degree of Doctor of Philosophy at The University of Hong Kong in May 2004 2+ Calmodulin (CaM) is a Ca -binding protein essential for eukaryotic cells. In mammals, modulation of CaM gene expression in regulating the functionality of the brain-pituitary-axis has not been characterized. In lower vertebrates, functional studies on CaM gene expression in the pituitary still await investigation. By using the members of the Cyprinid family as animal models, the role of CaM gene expression in the pituitary in mediating the signal input from the hypothalamus and the expression of growth hormone (GH) gene was examined. Firstly, CaM cDNAs were isolated from the pituitaries of goldfish and grass carp. The ORF of these cDNAs encodes the same CaM protein as that of mammals. These cDNAs can be grouped with the β-subtype of fish CaM cDNAs and are structurally related to the mammalian CaM-I gene subfamily. In both goldfish and grass carp, CaM mRNA is ubiquitously expressed. In goldfish pituitary cells, activation of cAMP- or PKC-dependent pathways increased CaM mRNA levels, whereas the opposite was true 2+ for the induction of Ca entry. Basal levels of CaM mRNA were accentuated by GnRH and PACAP but were suppressed by dopaminergic stimulation through pituitary D2 receptors. These results demonstrate for the first time that pituitary CaM gene expression can be regulated by functional interactions among hypothalamic factors. To study the role of CaM in pituitary hormone gene expression, the possible involvement of CaM expression in IGF feedback on GH gene transcription was tested in carp pituitary cells. In this case, IGF-I/-II increased the amount of CaM mRNA with a concurrent drop in the GH mRNA levels. The stimulatory effect was not mimicked by insulin. CaM antagonism also increased GH mRNA expression in grass carp pituitary cells whereas CaM over-expression could reduce GH promoter activity. These results, as a whole, imply that the attenuation of GH gene expression by IGF is mediated by CaM gene expression in grass carp pituitary cells. To further examine the mechanisms of regulating CaM gene expression in fish, the full-length CaM gene was cloned in grass carp by intron trapping and genome- walking. This CaM gene is 12-Kb in size and has the same structural organization as that of human CaM genes. The 5'-promoter of this carp CaM gene is AT rich rather than GC rich and contains a TATA box, indicating that the gene is not a typical "house- keeping" gene. 5'-Deletion analysis has revealed that a strong silencer element is located within the region from -1509bp to -1369bp in the CaM promoter. In addition, the 5'-UTR has strong influence on the carp CaM promoter activity. By the use of a reporter gene approach, the grass carp CaM promoter could be activated in α-T3-1 cells by IGFs, insulin, and PKC activators, but was suppressed by PACAP, forskolin, cAMP analog, CaM antagonist, and CaMK-II inhibitor. Transcription factors Sp1 and Sp3, but not Pit-1, were also effective in suppressing the CaM promoter activity. These results indicate that CaM gene transcription in the fish model is regulated by hormones, probably through various signaling pathways and transcription factors. DOI: