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Cloning and expression of goldfish (Carassius auratus) pituitary polypeptide hormones.

by Chan Yuk Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 120-137). / Acknowledgments --- p.i / Table of Content --- p.ii / List of Tables --- p.vi / List of Figures --- p.viii / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- Literature Review Page / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- "Overview of the Structures of GH, PRL and SL" --- p.2 / Chapter 1.3 --- Physiological Significance of the Hormones in Teleost --- p.12 / Chapter 1.3.1 --- Growth Hormone --- p.12 / Chapter 1.3.2 --- Prolactin --- p.12 / Chapter 1.3.3 --- Somatolactin --- p.13 / Chapter 1.4 --- Structure - Function Relationship and Receptor Binding Activities --- p.15 / Chapter 1.5 --- Aims of Thesis --- p.23 / Chapter CHAPTER 2 --- General Methodology / Chapter 2.1 --- Materials --- p.25 / Chapter 2.2 --- DNA manipulation methods --- p.28 / Chapter 2.2.1 --- Polymerase Chain Reaction (PCR) --- p.28 / Chapter 2.2.2 --- Ethanol Precipitation of DNA --- p.29 / Chapter 2.2.3 --- Agarose Gel Electrophoresis of DNA --- p.29 / Chapter 2.2.4 --- Sephadex G-50 Spun Column --- p.30 / Chapter 2.2.5 --- Nick Translation --- p.30 / Chapter 2.2.6 --- Small Scale Plasmid Preparation by Alkaline Lysis Method --- p.31 / Chapter 2.2.7 --- Large Scale Plasmid Preparation Using Magic´ёØ Maxipreps DNA Purification System (Promega) --- p.32 / Chapter 2.3 --- DNA Cloning Methods --- p.33 / Chapter 2.3.1 --- Blunt-end Ligation of PCR Product to pUC18 Vector --- p.33 / Chapter 2.3.1.1 --- Preparation and Transformation of E. coli (JM109) Competent Cell --- p.33 / Chapter 2.3.1.2 --- Recovery of DNA from Agarose Gel Using the Sephaglas Bandprep Kit (Pharmacia Biotech) --- p.34 / Chapter 2.3.1.3 --- Kinasing Reaction --- p.34 / Chapter 2.3.1.4 --- Klenow Fill-in Reaction and Ligation of DNA Fragments --- p.35 / Chapter 2.3.2 --- Screening of Lambda Phage cDNA Library --- p.35 / Chapter 2.3.2.1 --- Preparation of Host Cell for Screening of Lambda Phage cDNA Library --- p.35 / Chapter 2.3.2.2 --- Phage Stock Tittering --- p.36 / Chapter 2.3.2.3 --- Plaque Lifting and Fixation on Nylon Membranes --- p.36 / Chapter 2.3.2.4 --- Library Screening by Hybridization --- p.37 / Chapter 2.3.2.5 --- In vivo Excision --- p.37 / Chapter 2.4 --- Nucleotide Sequence Determination using Dideoxy Nucleotide Chain Termination Method --- p.38 / Chapter 2.5 --- Protein methods --- p.39 / Chapter 2.5.1 --- Bicinchoninic Acid (BCA) Assay --- p.39 / Chapter 2.5.2 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.40 / Chapter CHAPTER 3 --- Isolation and Characterization of cDNA Clones for Goldfish Prolactin / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Methods --- p.43 / Chapter 3.2.1 --- Preparation of Hormone Specific DNA Probes by PCR Cloning --- p.43 / Chapter 3.2.2 --- Construction of Goldfish Pituitary cDNA Library --- p.44 / Chapter 3.2.3 --- cDNA Library Screening for Hormone Specific cDNAs --- p.44 / Chapter 3.2.4 --- Restriction Enzyme Digestion of the cDNA Clones and Subcloning of the Restriction Fragments --- p.45 / Chapter 3.2.5 --- Nucleotide Sequences Determination using Dideoxy Nucleotide Chain Termination Method --- p.46 / Chapter 3.2.6 --- Southern Analysis of the Goldfish Genomic DNA --- p.47 / Chapter 3.2.7 --- Northern Blot Analyses of Goldfish Pituitary Total RNA --- p.47 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Screening of the Library and Characterization of the Clones --- p.49 / Chapter 3.3.1.1 --- Analyses of the cDNA Clones --- p.49 / Chapter 3.3.1.2 --- Cross Hybridization Reactivity --- p.57 / Chapter 3.3.2 --- Genomic Southern Blot Analyses --- p.58 / Chapter 3.3.3 --- Gene Expression of the Pituitary Hormones - Northern Blot Analyses --- p.61 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.4.1 --- Cross Hybridization Reavtivities of the Probes to the Hormone cDNA Clones --- p.64 / Chapter 3.4.2 --- Structural Analyses of the Hormone cDNA Clones --- p.65 / Chapter 3.4.2.1 --- Growth Hormone --- p.65 / Chapter 3.4.2.2 --- Prolactin --- p.68 / Chapter 3.4.2.3 --- Somatolactin --- p.70 / Chapter 3.4.3 --- Secondary Structure Prediction --- p.72 / Chapter 3.4.4 --- Genomic Southern Analyses --- p.78 / Chapter 3.4.5 --- Pituitary Expression of Goldfish Hormone mRNAs --- p.79 / Chapter 3.5 --- Conclusion --- p.81 / Chapter CHAPTER 4 --- "Expression of Recombinant Goldfish Growth Hormone, Prolactin and Somatolactin in Escherichia coli (E. coli)" / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and Methods --- p.83 / Chapter 4.2.1 --- Materials --- p.83 / Chapter 4.2.2 --- Construction of Expression Vectors Carrying the Hormone Coding Regions --- p.84 / Chapter 4.2.3 --- Pilot Expression Experiment to Determine Kinetics of Expression --- p.86 / Chapter 4.2.4 --- "Large Scale Expression and Purification of Recombinant Hormones by Ni2+-NTA Affinity Column (ProBond´ёØ column, Invitrogen)" --- p.86 / Chapter 4.2.5 --- Western Blotting of the Recombinant Hormones on Polyvinylidene Fluoride (PVDF) Membrane --- p.87 / Chapter 4.2.6 --- Dot Blot Immobilisation of the Recombinant Hormones on Nitrocellulose Membrane --- p.88 / Chapter 4.2.7 --- Detection of the Blotted Protein by Enzyme-linked Immunodetaction Method --- p.88 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Construction of the Expression Vectors --- p.90 / Chapter 4.3.2 --- Expression of the Recombinant Hormones --- p.97 / Chapter 4.3.3 --- Partial Purification and Analyses of the recombinant Hormones --- p.97 / Chapter 4.4 --- Discussion --- p.105 / Chapter 4.4.1 --- Construction of the Expression Vectors --- p.105 / Chapter 4.4.2 --- Expression of the Recombinant Hormones --- p.108 / Chapter 4.4.3 --- Partial Purification and Characterization of the Recombinant Hormones --- p.109 / Chapter 4.5 --- Conclusion --- p.114 / Chapter CHAPTER 5 --- General Discussion --- p.115 / References --- p.120

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_321578
Date January 1996
ContributorsChan, Yuk Hang., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
PublisherChinese University of Hong Kong
Source SetsThe Chinese University of Hong Kong
LanguageEnglish
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xiii, 137 leaves : ill. (some col., some mounted) ; 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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