published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

by Cheung Yeuk Siu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 141-153). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 摘要 --- p.iv / List of abbreviations --- p.v / Table of contents --- p.viii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Prolactin (PRL) --- p.1 / Chapter 1.1.1 --- General introduction --- p.1 / Chapter 1.1.2 --- Genomic organization of teleost PRL gene --- p.2 / Chapter 1.1.3 --- Conserved domains of fish PRL --- p.3 / Chapter 1.1.4 --- Structure of teleost PRL --- p.6 / Chapter 1.1.5 --- Tissue sources of PRL --- p.8 / Chapter 1.2 --- Prolactin receptor (PRLR) --- p.9 / Chapter 1.2.1 --- Tissue distribution in teleosts --- p.9 / Chapter 1.2.2 --- Receptor structure and multiple forms of PRLR --- p.11 / Chapter 1.2.3 --- Possible action mechanisms of PRL --- p.14 / Chapter 1.3 --- Control of PRL release --- p.17 / Chapter 1.4 --- Biological functions of PRL in vertebrates --- p.19 / Chapter 1.4.1 --- Biological effects on teleosts --- p.19 / Chapter 1.4.1.1 --- Osmoregulatory roles --- p.20 / Chapter 1.4.1.2 --- Non-osmoregulatory roles --- p.29 / Chapter 1.5 --- Aim of the present study --- p.32 / Chapter Chapter 2 --- "Recombinant goldfish (Carassius auratus) prolactin: subcloning, expression, purification and refolding of the recombinant protein" / Chapter 2.1 --- Introduction --- p.35 / Chapter 2.2 --- Materials --- p.38 / Chapter 2.3 --- Methods --- p.48 / Chapter 2.3.1 --- Subcloning of the gfPRL cDNA --- p.48 / Chapter 2.3.1.1 --- PCR cloning of gfPRL cDNA --- p.48 / Chapter 2.3.1.2 --- DNA sequencing of the subcloned fragment --- p.51 / Chapter 2.3.1.3 --- Subcloning of the gfPRL cDNA fragment into the expression vector --- p.52 / Chapter 2.3.2 --- Expression and purification of rgfPRL --- p.53 / Chapter 2.3.2.1 --- Transformation of pRSETA/gfPRL into BL21(DE3)pLysS cells --- p.53 / Chapter 2.3.2.2 --- Prokaryotic expression of rgfPRL --- p.53 / Chapter 2.3.2.3 --- Affinity purification of rgfPRL --- p.55 / Chapter 2.3.2.4 --- Western blot analysis of the purified rgfPRL --- p.57 / Chapter 2.3.2.5 --- Protein concentration determination of the rgfPRL --- p.59 / Chapter 2.3.3 --- Protein refolding --- p.60 / Chapter 2.4 --- Results --- p.61 / Chapter 2.4.1 --- Subcloning and DNA sequencing of the gfPRL --- p.61 / Chapter 2.4.2 --- Expression and purification of rgfPRL --- p.63 / Chapter 2.4.2.1 --- Prokaryotic expression of rgfPRL --- p.63 / Chapter 2.4.2.2 --- Affinity purification of rgfPRL --- p.66 / Chapter 2.4.2.3 --- Western blot analysis of the purified rgfPRL --- p.68 / Chapter 2.4.2.4 --- Protein concentration determination of the rgfPRL --- p.68 / Chapter 2.4.3 --- Protein refolding --- p.70 / Chapter 2.5 --- Discussion --- p.72 / Chapter Chapter 3 --- Production of polyclonal antibodies against rgfRPL / Chapter 3.1 --- Introduction --- p.81 / Chapter 3.2 --- Materials --- p.82 / Chapter 3.3 --- Methods --- p.84 / Chapter 3.3.1 --- Immunization of rabbits --- p.84 / Chapter 3.3.2 --- Collection of the polyclonal antisera --- p.85 / Chapter 3.3.3 --- Purification of IgG from the polyclonal antisera --- p.86 / Chapter 3.3.4 --- Enzyme linked immunosorbent assay (ELISA) --- p.87 / Chapter 3.3.5 --- Western blot analysis for cross-reactivity --- p.88 / Chapter 3.4 --- Results --- p.90 / Chapter 3.4.1 --- Isolation and purification of IgG from the polyclonal antisera --- p.90 / Chapter 3.4.2 --- ELISA --- p.93 / Chapter 3.4.3 --- Western blot analysis for cross-reactivity --- p.96 / Chapter 3.5 --- Discussion --- p.98 / Chapter Chapter 4 --- Isolation of native PRL from goldfish pituitaries / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Materials --- p.101 / Chapter 4.3 --- Methods --- p.103 / Chapter 4.3.1 --- Alkaline extraction --- p.103 / Chapter 4.3.2 --- Size exclusion chromatography --- p.104 / Chapter 4.3.3 --- Anion exchange chromatography --- p.104 / Chapter 4.4 --- Results --- p.106 / Chapter 4.4.1 --- Size exclusion chromatography --- p.106 / Chapter 4.4.2 --- Anion exchange chromatography --- p.109 / Chapter 4.4.3 --- SDS-PAGE analysis and immuno-detection of the purified protein --- p.112 / Chapter 4.5 --- Discussion --- p.114 / Chapter Chapter 5 --- Receptor binding assays / Chapter 5.1 --- Introduction --- p.115 / Chapter 5.2 --- Materials --- p.117 / Chapter 5.3 --- Methods --- p.119 / Chapter 5.3.1 --- Gill membrane preparation --- p.119 / Chapter 5.3.2 --- Radioactive labelling of the primary ligand --- p.120 / Chapter 5.3.3 --- Determination of the percentage of 125I incorporation and specific radioactivity of the radioligand --- p.121 / Chapter 5.3.4 --- Membrane protein dependence assay --- p.122 / Chapter 5.3.5 --- Receptor binding study using rgfPRL --- p.124 / Chapter 5.4 --- Results --- p.125 / Chapter 5.4.1 --- Radioactive labelling of the primary ligand --- p.125 / Chapter 5.4.2 --- Determination of the percentage of 125I incorporation and specific radioactivity of the radioligand --- p.127 / Chapter 5.4.3 --- Membrane protein dependence assay --- p.129 / Chapter 5.4.4 --- Receptor binding study using rgfPRL --- p.131 / Chapter 5.5 --- Discussion --- p.133 / Chapter Chapter 6 --- General discussion and conclusion --- p.136 / References --- p.141

by Wong Kwan Po, Gary. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 132-153). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Abbreviations --- p.vi / Abbrevation Table for Amino Acids --- p.ix / List of Figures --- p.x / List of Tables --- p.xiii / Table of Contents --- p.xiv / Chapter Chapte r One --- General Introduction --- p.1 / Chapter 1.1 --- Structures of PRL --- p.1 / Chapter 1.2 --- PRL receptor and its mechanism of action --- p.7 / Chapter 1.3 --- Biosynthesis of PRL --- p.11 / Chapter 1.4 --- Biological functions of PRL --- p.13 / Chapter 1.5 --- Organization and regulation of PRL gene --- p.16 / Chapter 1.6 --- Aims of this study --- p.25 / Chapter Chapter Two --- PCR Cloning of gfPRL Gene --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Buffers and Reagents --- p.27 / Chapter 2.2.2 --- Methods --- p.30 / Chapter 2.2.2.1 --- PCR of the 5'-flanking region of gfPRL gene --- p.30 / Chapter 2.2.2.2 --- Genomic PCR of gfPRL gene --- p.31 / Chapter 2.2.2.3 --- Spectrophotometric quantification and qualification of DNA and RNA --- p.31 / Chapter 2.2.2.4 --- Agarose gel electrophoresis of DNA --- p.31 / Chapter 2.2.2.5 --- DNA radioactive labeling by random priming --- p.32 / Chapter 2.2.2.6 --- Vacuum transfer of DNA fragments to a nylon membrane --- p.32 / Chapter 2.2.2.7 --- Southern blot analysis --- p.33 / Chapter 2.2.2.8 --- Molecular Imager Analysis --- p.33 / Chapter 2.2.2.8 --- Phosphorylation of PCR amplified DNA --- p.34 / Chapter 2.2.2.9 --- Ligation of DNA fragment to linearized vector --- p.34 / Chapter 2.2.2.10 --- Preparation of Escherichia coli competent cells --- p.34 / Chapter 2.2.2.11 --- Bacterial transformation by heat stock --- p.35 / Chapter 2.2.2.12 --- Automated PCR sequencing with Sequencing Ready Reaction Kit --- p.35 / Chapter 2.2.2.13 --- Primer extension using reverse transcription --- p.36 / Chapter 2.3 --- Results --- p.38 / Chapter 2.3.1 --- Cloning of the 5'-flanking region of gfPRL gene --- p.38 / Chapter 2.3.2 --- PCR cloning of gfPRL gene --- p.43 / Chapter 2.3.3 --- Identification of the transcription initiation site --- p.47 / Chapter 2.4 --- Discussion --- p.51 / Chapter 2.4.1 --- Sequence analysis of the gfPRL gene --- p.51 / Chapter 2.4.2 --- Analysis of the exon-intron boundaries --- p.53 / Chapter 2.4.3 --- Analysis of the 5'flanking region of gfPRL gene --- p.53 / Chapter 2.4.4 --- Identification of the transcription initiation site --- p.54 / Chapter 2.5 --- Conclusion --- p.54 / Chapter Chapter Three --- Promoter Analysis of the gfPRL Gene --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Preparation of Luciferase reporter constructs --- p.56 / Chapter 3.2.2 --- Preparation of frozen stock of culture cells --- p.56 / Chapter 3.2.3. --- Cell culture --- p.56 / Chapter 3.2.4 --- Transfection of mammalian cells for transient gene expression study --- p.57 / Chapter 3.2.5 --- Luciferase assay --- p.57 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- Tissue-specific transcription of gfPRL promoter --- p.58 / Chapter 3.3.2 --- Identification of regulatory regions of gfPRL gene promoter --- p.61 / Chapter 3.3.3 --- Inhibitory effect of DA on gfPRL promoter transcription activity --- p.63 / Chapter 3.3.4 --- GfPRL promoter sequences that specifically confer negative regulation by DA --- p.65 / Chapter 3.3.5 --- The action of TRH on gfPRL promoter --- p.67 / Chapter 3.3.6 --- Investigation of gfPRL promoter sequence responsiveness towards TRH --- p.69 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Tissue-specific transcription of gfPRL promoter --- p.71 / Chapter 3.4.2 --- Identification of regulatory regions of goldfish prolactin gene promoter --- p.72 / Chapter 3.4.3 --- Dopamine inhibits gfPRL promoter activity --- p.73 / Chapter 3.4.4 --- TRH action on gfPRL promoter --- p.76 / Chapter 3.5 --- Conclusion --- p.78 / Chapter Chapter Four --- Seasonal Study on gfPRL and gfGH expression --- p.80 / Chapter 4.1 --- Introduction --- p.80 / Chapter 4.2 --- Materials and Methods --- p.81 / Chapter 4.2.1 --- Blood samples and radioimmunoassay --- p.81 / Chapter 4.2.2 --- Preparation of ribonuclease free reagents and apparatus --- p.81 / Chapter 4.2.3 --- Isolation of total RNA --- p.81 / Chapter 4.2.4 --- Formaldehyde agarose gel electrophoresis of RNA --- p.82 / Chapter 4.2.5 --- First strand cDNA synthesis --- p.82 / Chapter 4.2.6 --- RT-PCR --- p.83 / Chapter 4.2.7 --- Analysis of RT-PCR --- p.86 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Tissue-specific expression of gfPRL transcript --- p.88 / Chapter 4.3.2 --- Sexual maturity of goldfish throughout the reproductive cycle --- p.90 / Chapter 4.3.3 --- Serum gfGH levels throughout the year of 2000 --- p.91 / Chapter 4.3.4 --- Serum gfPRL levels throughout the year of 2000 --- p.92 / Chapter 4.3.5 --- The variation of gfGHR and gfPRLR mRNA in the brain throughout the reproductive cycle --- p.93 / Chapter 4.3.6 --- The variation of gfGHR mRNA in the liver throughout the reproductive cycle --- p.94 / Chapter 4.3.7 --- The variation of gfGHR and gfPRLR mRNA in the kidney throughout the reproductive cycle --- p.95 / Chapter 4.3.8 --- The variation of gfGHR and gfPRLR mRNA in the gonads throughout the reproductive cycle --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.4.1 --- Tissue-specific expression of gfPRL transcript --- p.98 / Chapter 4.4.2 --- Sexual maturity of goldfish throughout the reproductive cycle --- p.98 / Chapter 4.4.3 --- Serum gfGH and gfPRL level throughout the reproductive cycle --- p.99 / Chapter 4.4.4 --- The variation of gfGHR and gfPRLR mRNA in the brain throughout the reproductive cycle --- p.100 / Chapter 4.4.5 --- The variation of gfGHR mRNA in the liver throughout ´Øthe reproductive cycle --- p.101 / Chapter 4.4.6 --- The variation of gfGHR and gfPRLR mRNA in the kidney throughout the reproductive cycle --- p.102 / Chapter 4.4.7 --- The variation of gfGHR and gfPRLR mRNA in the gonads throughout the reproductive cycle --- p.102 / Chapter 4.5 --- Conclusion --- p.105 / Chapter Chapter Five --- Recombinant gfPRL Production --- p.106 / Chapter 5.1 --- Introduction --- p.106 / Chapter 5.2 --- Materials and Methods --- p.108 / Chapter 5.2.1 --- Buffers and Reagents --- p.108 / Chapter 5.2.2 --- Methods --- p.112 / Chapter 5.2.2.1 --- Recombinant protein expression --- p.112 / Chapter 5.2.2.2. --- Purification of the recombinant protein by XpressTM System Protein Purification (Invitrogen) --- p.112 / Chapter 5.2.2.3 --- SDS-PAGE preparation --- p.112 / Chapter 5.2.2.4 --- SDS-PAGE analysis of proteins --- p.113 / Chapter 5.2.2.5 --- Western blot analysis --- p.114 / Chapter 5.2.2.6 --- Protein refolding --- p.114 / Chapter 5.2.2.7 --- Alkaline Extraction --- p.115 / Chapter 5.2.2.8 --- Size Exclusion Chromatography --- p.115 / Chapter 5.2.2.9 --- ELISA analysis of the fractions --- p.115 / Chapter 5.2.2.10 --- Anion Exchange Chromatography --- p.116 / Chapter 5.3 --- Results --- p.117 / Chapter 5.3.1 --- Prokaryotic expression of recombinant gfPRL --- p.117 / Chapter 5.3.2 --- "Purification of reombinant gfPRL: SDS-PAGE, western blot and BCA analysis of purified recombinant gfPRL" --- p.119 / Chapter 5.3.3 --- Purification of native gfPRL and gfGH: Native hormone purification by size exclusion chromatography --- p.119 / Chapter 5.3.4 --- Native gfPRL purification by anion exchange chromatography --- p.122 / Chapter 5.3.5 --- Study the biological activity of refolded recombinant gfPRL --- p.126 / Chapter 5.4 --- Discussion --- p.127 / Chapter 5.4.1 --- Prokaryotic expression of recombinant gfPRL --- p.127 / Chapter 5.4.2 --- Purification of recombinant gfPRL --- p.128 / Chapter 5.4.3 --- Refolding of recombinant gfPRL --- p.129 / Chapter 5.4.4 --- Purification of native gfPRL --- p.130 / Chapter 5.4.5 --- Study the biological activity of recombinant gfPRL --- p.130 / Chapter 5.5 --- Conclusion --- p.131 / References --- p.132

Yam Kwan Mei. / Thesis submitted in: August 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 108-129). / Abstracts in English and Chinese. / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.vi / Table of Contents --- p.ix / List of Tables --- p.xiv / List of Figures --- p.xv / Symbols and Abbreviations --- p.xviii / Scientific names --- p.xx / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Gonadotropins --- p.1 / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Function --- p.3 / Chapter 1.1.3 --- Regulation --- p.9 / Chapter 1.1.3.1 --- GnRH --- p.9 / Chapter 1.1.3.2 --- Steroids --- p.11 / Chapter 1.1.3.3 --- Activin --- p.12 / Chapter 1.2 --- Activin Family of Growth Factors --- p.14 / Chapter 1.2.1 --- Structure --- p.14 / Chapter 1.2.2 --- Function --- p.17 / Chapter 1.3 --- Objectives --- p.22 / Chapter Chapter 2 --- Cloning of Goldfish Activin βA cDNA and the Expression of Its mRNA in Gonadal and Non-gonadal Tissues --- p.24 / Chapter 2.1 --- Introduction --- p.24 / Chapter 2.2 --- Materials and Methods --- p.25 / Chapter 2.2.1 --- Cloning of goldfish activin βA cDNA --- p.26 / Chapter 2.2.1.1 --- Cloning of the 5' and 3' cDNA ends --- p.26 / Chapter 2.2.1.2 --- Extension of the 5' and 3' fragments --- p.28 / Chapter 2.2.2 --- Sequencing of the cDNA --- p.28 / Chapter 2.2.2.1 --- Generation of pKS/GactβA constructs with insert in different orientations --- p.28 / Chapter 2.2.2.2 --- Generation of overlapping subclones --- p.29 / Chapter 2.2.2.3 --- Cycle sequencing --- p.30 / Chapter 2.2.2.4 --- Sequence analyses --- p.30 / Chapter 2.2.3 --- Isolation of total and messenger RNA --- p.30 / Chapter 2.2.3.1 --- Isolation of total RNA --- p.30 / Chapter 2.2.3.2 --- Isolation of messenger RNA --- p.31 / Chapter 2.2.4 --- Southern blot analysis --- p.32 / Chapter 2.2.5 --- Northern blot analysis --- p.33 / Chapter 2.2.6 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- Cloning and sequence analysis of activin β A cDNA --- p.35 / Chapter 2.3.2 --- Distribution of activin βA mRNA in different tissues --- p.49 / Chapter 2.4 --- Discussion --- p.53 / Chapter Chapter 3 --- Establishment and Characterization of Stable Cell Lines for the Recombinant Production of Goldfish Activin A --- p.59 / Chapter 3.1 --- Introduction --- p.59 / Chapter 3.2 --- Materials and Methods --- p.60 / Chapter 3.2.1 --- Construction of expression plasmid --- p.60 / Chapter 3.2.2 --- Cell culture --- p.62 / Chapter 3.2.3 --- Transfection of CHO cells --- p.62 / Chapter 3.2.4 --- G418 selection of transfected CHO cells --- p.62 / Chapter 3.2.5 --- Activin bioassay (EDF-assay) --- p.63 / Chapter 3.2.6 --- Cloning of pBK/GactβA-transfected CHO cells by limited dilution --- p.63 / Chapter 3.2.7 --- Isolation of total RNA --- p.65 / Chapter 3.2.8 --- Northern blot analysis --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Optimization of G418 concentration for selection --- p.66 / Chapter 3.3.2 --- Expression of activin activity by pBK/GactpβA- transfected CHO cells --- p.67 / Chapter 3.3.3 --- Establishment and characterization of CHO cell lines that stably produce recombinant goldfish activin A --- p.67 / Chapter 3.4 --- Discussion --- p.73 / Chapter Chapter 4 --- Differential Regulation of Goldfish Gonadotropin (GTH-Iβ and GTH-IIβ) Gene Expression by Recombinant Goldfish Activin --- p.79 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and Methods --- p.82 / Chapter 4.2.1 --- Animals --- p.82 / Chapter 4.2.2 --- Drug treatment --- p.83 / Chapter 4.2.3 --- Primary culture of dispersed pituitary cells --- p.84 / Chapter 4.2.4 --- Southern blot analysis --- p.85 / Chapter 4.2.5 --- Isolation of total RNA --- p.86 / Chapter 4.2.6 --- Northern blot analysis --- p.86 / Chapter 4.2.7 --- Dot blot analysis --- p.87 / Chapter 4.2.8 --- Data analyses --- p.87 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Probe specificity --- p.88 / Chapter 4.3.2 --- Effects of goldfish activin on pituitary GTH-Iβ and -IIβ mRNA expression --- p.88 / Chapter 4.3.3 --- Blockade of activin effects by follistatin --- p.92 / Chapter 4.4 --- Discussion --- p.96 / Chapter Chapter 5 --- General Discussion --- p.101 / Chapter 5.1 --- Overview --- p.101 / Chapter 5.2 --- Contribution of the Present Research --- p.103 / Chapter 5.2.1 --- Cloning of full-length goldfish activin βA cDNA --- p.103 / Chapter 5.2.2 --- Establishment of stable cell lines for the recombinant production of goldfish activin A --- p.104 / Chapter 5.2.3 --- Differential regulation of goldfish gonadotropin (GTH-Iβ and GTH-IIβ) gene expression by recombinant goldfish activin --- p.105 / Chapter 5.3 --- Future Research Direction --- p.107 / Chapter 5.3.1 --- Activin studies --- p.107 / Chapter 5.3.2 --- Gonadotropin studies --- p.107 / References --- p.108

published_or_final_version / Zoology / Master / Master of Philosophy