Studies on the promoters of two prolactin receptors in black seabream (Acanthopagrus schlegeli).

Sham, Wai Yan.Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.Includes bibliographical references (leaves 173-205).Abstracts in English and Chinese.Declaration of Originality --- p.iThesis / Assessment Committee --- p.iiAbstract --- p.iii摘要 --- p.viAcknowledgements --- p.viiiTable of Contents --- p.xList of Figures --- p.xivList of Tables --- p.xviiList of Abbreviations --- p.xixChapter Chapter I --- Introduction --- p.1Chapter 1.1 --- Habitats and diversity of fish --- p.1Chapter 1.2 --- Stress accompanied with osmotic changes --- p.2Chapter 1.3 --- Physiological consequences associated with osmoregulation --- p.4Chapter 1.3.1 --- Energy cost (energy metabolism) --- p.4Chapter 1.3.2 --- Food intake --- p.5Chapter 1.3.3 --- Reproduction and development --- p.5Chapter 1.3.4 --- Growth --- p.6Chapter 1.3.5 --- Immunity --- p.6Chapter 1.4 --- Osmoregulatory systems in teleosts --- p.7Chapter 1.4.1 --- Ion transport --- p.7Chapter 1.4.1.1 --- Mechanism in freshwater --- p.8Chapter 1.4.1.2 --- Mechanism in seawater --- p.8Chapter 1.4.2 --- Water channel --- p.11Chapter 1.4.3 --- Accumulation of compatible osmolytes --- p.11Chapter 1.5 --- Kinetics on ion transporters --- p.12Chapter 1.5.1 --- Na+K+ATPase --- p.12Chapter 1.5.2 --- Other ion transporters --- p.14Chapter 1.5.3 --- Other factors related to controlling ion transport --- p.15Chapter 1.6 --- Pleiotropic hormones involved in osmoregulation --- p.17Chapter 1.6.1 --- Growth hormone/ insulin-like factor-1 axis in seawater adaptation --- p.17Chapter 1.6.2 --- Prolactin in freshwater adaptation --- p.19Chapter 1.6.3 --- "Dual osmoregulatory role of cortisol, the major corticosteroid in fish" --- p.20Chapter 1.7 --- "Growth hormone receptor (GHR), prolactin receptor (PRLR) and signaling pathways" --- p.22Chapter 1.8 --- Involvement of sex hormones on osmoregulation --- p.25Chapter 1.8.1 --- Estrogens --- p.25Chapter 1.8.2 --- Testosterone --- p.26Chapter 1.9 --- Biological actions of GH through GHR and PRL through PRLR --- p.27Chapter 1.9.1 --- Regulation of GHR --- p.27Chapter 1.9.2 --- Regulation of PRLR --- p.28Chapter 1.9.3 --- Promoter characterization --- p.30Chapter 1.10 --- Adaptation to tonicity-ORE/OREBP pathway --- p.32Chapter 1.11 --- Animal model --- p.35Chapter 1.12 --- Rationale and objectives of the project --- p.35Chapter Chapter II --- Materials and methods --- p.38Chapter 2.1 --- Animals --- p.38Chapter 2.2 --- In vivo experiments --- p.38Chapter 2.3 --- In vitro experiments --- p.38Chapter 2.4 --- RNA preparation --- p.39Chapter 2.5 --- RNA integrity - formaldehyde agarose gel electrophoresis of mRNA --- p.40Chapter 2.6 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.40Chapter 2.7 --- Polymerase chain reaction PCR --- p.41Chapter 2.8 --- Quantitative Real-time PCR --- p.41Chapter 2.9 --- Cell culture --- p.43Chapter 2.10 --- Bioinformatics search --- p.43Chapter 2.11 --- Plasmid constructions --- p.43Chapter 2.12 --- Transient transfection --- p.47Chapter 2.13 --- Luciferase assay --- p.47Chapter 2.14 --- Site-directed mutagenesis of sbPRLRl and sbPRLR2 gene promoters --- p.48Chapter 2.14.1 --- One-round PCR amplification --- p.48Chapter 2.14.2 --- Sequential PCR amplification --- p.48Chapter 2.15 --- Nuclear extract preparation for electrophoretic mobility shift assays (EMSA) --- p.52Chapter 2.16 --- Electrophoretic mobility shift assays (EMSA) --- p.53Chapter 2.16.1 --- Preparation of annealed complementary oligonucleotides --- p.53Chapter 2.16.2 --- Preparation of radiolabeled oligonucleotides --- p.54Chapter 2.16.3 --- Preparation of non-denaturing polyacrylamide gel --- p.54Chapter 2.16.4 --- Electrophoretic mobility shift assay --- p.54Chapter 2.17 --- Data analysis --- p.56Chapter Chapter III --- Result: In vivo gene expression studies --- p.57Chapter 3.1 --- Aim of the chapter --- p.57Chapter 3.2 --- Results --- p.57Chapter 3.2.1 --- Validation of real-time PCR --- p.57Chapter 3.2.2 --- "Transcript levels of sbPRLRl, sbPRLR2, sbNKA α- and sbNKA β-subunit after transfer of fish from seawater to hypersaline water" --- p.61Chapter 3.2.2.1 --- Gill --- p.61Chapter 3.2.2.2 --- Kidney --- p.64Chapter 3.3 --- Discussion --- p.67Chapter 3.3.1 --- sbPRLR gene expression after seawater-to-hypersaline water transfer --- p.67Chapter 3.3.2 --- sbNKA α- and sbNKA β-subunit expression after seawater-to-hypersaline water transfer --- p.71Chapter 3.3.3 --- Overall summary --- p.76Chapter Chapter IV --- Result: In vitro gene expression studies --- p.77Chapter 4.1 --- Aim of the chapter --- p.77Chapter 4.2 --- Results --- p.77Chapter 4.2.1 --- Gill organ culture --- p.77Chapter 4.2.2 --- Kidney explants --- p.81Chapter 4.3 --- Discussion --- p.84Chapter Chapter V --- Result: Studies on the gene promoter of sbPRLRs under osmotic change --- p.88Chapter 5.1 --- Aim of the chapterChapter 5.2 --- Results --- p.88Chapter 5.2.1 --- The 5´ة-UTR region of the two sbPRLR genes contain several osmotic response elements --- p.88Chapter 5.2.2 --- Promoter activities of the 5´ة-flanking regions of the two sbPRLR genes under osmotic stress --- p.91Chapter 5.2.3 --- Identification of a DNA region required for the responses to NaCl --- p.97Chapter 5.2.4 --- Interaction of the osmotic response region and transcription factors --- p.104Chapter 5.2.4.1 --- Identification of DNA-binding activity in nuclear extract --- p.104Chapter 5.2.4.2 --- OSTF1 transactivates the sbPRLR gene promoters --- p.107Chapter 5.3 --- Discussion --- p.109Chapter Chapter VI --- Result: Studies on the gene promoters of sbPRLR under hormonal challenge --- p.125Chapter 6.1 --- Aim of the chapter --- p.125Chapter 6.2 --- Results --- p.125Chapter 6.2.1 --- The 5´ة-UTR region of the two sbPRLR genes contains several hormone response elements --- p.125Chapter 6.2.2 --- Identification of a DNA region required for the responses to steroid hormones --- p.126Chapter 6.2.2.1 --- E2-dependent regulation of sbPRLR 1 and sbPRLR2 genes use imperfect EREs --- p.126Chapter 6.2.2.2 --- An ARE motif in sbPRLR2 gene promoter --- p.132Chapter 6.2.2.3 --- An imperfect and a novel GRE present on the sbPRLRl and sbPRLR2 gene promoters respectively --- p.138Chapter 6.2.3 --- Interaction of the hormone response region and transcription factors --- p.144Chapter 6.2.3.1 --- The ER interacts with the putative ERE motifs in vitro --- p.144Chapter 6.2.3.2 --- The AR interacts with the putative ARE motif in vitro --- p.145Chapter 6.2.3.3 --- The GR interacts with the putative GRE motifs in vitro --- p.145Chapter 6.3 --- Discussion --- p.152Chapter 6.3.1. --- Sex steroids --- p.152Chapter 6.3.1.1 --- Estrogens --- p.152Chapter 6.3.1.2 --- Androgens --- p.157Chapter 6.3.2. --- Interrenal hormone ´ؤ cortisol --- p.163Chapter Chapter VII --- General conclusion and perspectives --- p.170References --- p.17