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Cloning and characterization of goldfish activin bA subunit and regulation of goldfish gonadotropin gene expression by activin.

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323220
Date January 2000
ContributorsYam, Kwan Mei., Chinese University of Hong Kong Graduate School. Division of Biology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xx, 129 leaves : ill. (some col.) ; 30 cm.
RightsUse of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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