by Yu Wai Fu, Jason. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 112-120). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iii / Chapter CHAPTER 1 --- INTORDUCTION --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Historical Overview --- p.3 / Chapter 1.2.1 --- Insulin-Like Growth Factors I and II --- p.3 / Chapter 1.2.2 --- IGF Receptors --- p.5 / Chapter 1.2.3 --- IGF Binding Proteins --- p.7 / Chapter 1.3 --- Origin and Production of IGFs --- p.7 / Chapter 1.3.1 --- The Hypothalamo-Pituitary-GH-IGF-I Axis --- p.7 / Chapter 1.3.2 --- Factors Regulating IGF production --- p.9 / Chapter 1.3.3 --- Expression of IGFs in the Central Nervous System --- p.11 / Chapter 1.4 --- Actions of IGFs --- p.12 / Chapter 1.4.1 --- Insulin-like Metabolic Effects --- p.12 / Chapter 1.4.2 --- Mitogenic Effects --- p.13 / Chapter 1.4.3 --- Effects on Differentiation --- p.13 / Chapter 1.4.4 --- IGFs in Reproductive System --- p.14 / Chapter 1.4.5 --- IGF Actions in the Central Nervous System --- p.14 / Chapter 1.5 --- Transgenic and Knockout Animal Models for IGFs --- p.15 / Chapter 1.6 --- Molecular Biology of IGFs --- p.17 / Chapter 1.6.1 --- Structure of the IGF Genes --- p.17 / Chapter 1.6.2 --- Expression of IGF Genes --- p.21 / Chapter 1.6.3 --- Regulation of IGF Gene Expression --- p.23 / Chapter 1.7 --- IGF Receptors --- p.24 / Chapter 1.7.1 --- IGF-I Receptor --- p.24 / Chapter 1.7.2 --- IGF-II Receptor --- p.26 / Chapter 1.8 --- IGFBPs --- p.26 / Chapter 1.9 --- Teleost IGFs --- p.28 / Chapter 1.9.1 --- The GH-IGF-Axis in Teleost --- p.28 / Chapter 1.9.2 --- Osmoregulation and Other Biological Actions of IGFin Teleost --- p.29 / Chapter 1.9.3 --- Molecular Biology of IGFs in Teleost --- p.30 / Chapter 1.9.4 --- IGFBPs and IGF Receptors in Teleost --- p.31 / Chapter 1.10 --- Rationale and Aim of the Present Study --- p.32 / Chapter CHAPTER 2 --- SEARCH OF IGF-I PROMOTER BY GENOMIC DNA POLYMERASE CHAIN REACTION --- p.34 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.2 --- Materials --- p.35 / Chapter 2.3 --- Methods --- p.39 / Chapter 2.3.1 --- Preparation of Genomic DNA from Carp Testis --- p.39 / Chapter 2.3.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.40 / Chapter 2.3.3 --- Polymerase Chain Reaction --- p.40 / Chapter 2.3.3.1 --- Ligation of the Cassette to Digested Genomic DNA --- p.40 / Chapter 2.3.3.2 --- Amplification by PCR --- p.40 / Chapter 2.3.4 --- Agarose Gel Electrophoresis --- p.42 / Chapter 2.3.5 --- Gene Clean Using Sephaglas´ёØ BandPrep Kit (Pharamica) --- p.42 / Chapter 2.3.6 --- Cloning of PCR Products --- p.43 / Chapter 2.3.7 --- Transformation of Competent Cell (Heat Shock Method) --- p.43 / Chapter 2.3.8 --- Small Scale Alkaline Preparation of Plasmid DNA --- p.44 / Chapter 2.3.9 --- Restriction Enzyme Digestion to Release the Insert --- p.45 / Chapter 2.3.10 --- Large Scale Plasmid Preparation of the Positive Clone --- p.45 / Chapter 2.3.11 --- DNA Sequencing of the Positive Clone Using the T7 DNA Polymerase Sequencing Kit (Pharmacia) --- p.46 / Chapter 2.4 --- Results and Discussion --- p.49 / Chapter CHAPTER 3 --- ISOLATION OF GENOMIC CLONES CARRYING THE IGF-I GENE --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials --- p.56 / Chapter 3.3 --- Methods --- p.58 / Chapter 3.3.1 --- Preparation of the Plating Host Cells --- p.58 / Chapter 3.3.2 --- Phage Titering --- p.58 / Chapter 3.3.3 --- Primary Screening of Common Carp Genomic Library --- p.59 / Chapter 3.3.4 --- Preparation of Radioactive Nucleic Acid Probes --- p.60 / Chapter 3.3.5 --- Purification of the Positive Clones --- p.60 / Chapter 3.3.6 --- Purification of DNA from Lambda Phage Using Sephaglas´ёØ PhagePrep Kit (Pharmacia) --- p.61 / Chapter 3.3.7 --- Restriction Enzyme Digestion Release of Inserts --- p.62 / Chapter 3.3.8 --- Capillary Transfer of DNA to Nylon Membrane Under Alkaline Condition --- p.62 / Chapter 3.3.9 --- Southern Analysis of the 10 Positive Clones --- p.63 / Chapter 3.3.10 --- Restriction Mapping of the Clone P1 --- p.64 / Chapter 3.3.11 --- Subcloning of the Fragments of the Clone PI into Plasmid Vector --- p.64 / Chapter 3.3.12 --- IGF-I Specific PCR --- p.64 / Chapter 3.3.13 --- Amplification of Introns from the Clone P1 Using PCR --- p.67 / Chapter 3.4 --- Results and Discussion --- p.70 / Chapter CHAPTER 4 --- RNA ASSAY USING REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION --- p.83 / Chapter 4.1 --- Introduction --- p.83 / Chapter 4.2 --- Materials --- p.85 / Chapter 4.3 --- Methods --- p.86 / Chapter 4.3.1 --- Administration of Hormones --- p.86 / Chapter 4.3.1.1 --- Injection Time Course1 --- p.86 / Chapter 4.3.1.2 --- Injection Time Course2 --- p.86 / Chapter 4.3.2 --- Total RNA Extraction --- p.87 / Chapter 4.3.2.1 --- Rapid RNA Isolation --- p.87 / Chapter 4.3.3 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.88 / Chapter 4.3.4 --- Rapid Isolation of PolyA+ mRNA from Total RNA --- p.89 / Chapter 4.3.5 --- IGF-I Specific RT-PCR --- p.90 / Chapter 4.4 --- Results and Discussion --- p.92 / Chapter CHAPTER 5 --- SEARCH FOR IGF-II GENE USING GENOMIC SOUTHERN BLOT ANALYSIS --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.2 --- Materials --- p.103 / Chapter 5.3 --- Methods --- p.104 / Chapter 5.3.1 --- Preparation of Genomic DNA from Carp Testis --- p.104 / Chapter 5.3.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.104 / Chapter 5.3.3 --- Southern Blotting of the Digested Genomic DNA --- p.104 / Chapter 5.3.4 --- Preparation of the Trout IGF-II Specific Probe --- p.104 / Chapter 5.3.5 --- Genomic Southern Hybridization --- p.105 / Chapter 5.4 --- Results and Discussion --- p.106 / Chapter CHAPTER 6 --- GENERAL DISCUSSION AND CONCLUSION --- p.109 / REFERENCES --- p.112
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322676 |
| Date | January 1997 |
| Contributors | Yu, Wai Fu Jason., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, vii, 120 leaves : ill. ; 30 cm. |
| Rights | Use 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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