Molecular studies on growth hormone receptor complementary DNA.

• Other Authors: Lau, Kwok Fai.
• Format: Others
• Language: English
• Published: Chinese University of Hong Kong 1994
• Subjects: Somatotropin > Receptors; Antisense DNA; Molecular cloning
• Online Access: http://library.cuhk.edu.hk/record=b5895455; http://repository.lib.cuhk.edu.hk/en/item/cuhk-318261

by Lau Kwok Fai. === Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. === Includes bibliographical references (leaves 126-134). === Acknowledgments --- p.i === Abstract --- p.ii === Contents --- p.iv === Abbreviations --- p.ix === List of Figures --- p.x === List of Tables --- p.xii === List of Primers --- p.xiii === Chapter Chapter 1 --- Introduction === Chapter 1.1 --- A Brief Introduction of GH --- p.1 === Chapter 1.2 --- Growth Hormone Receptor (GHR) --- p.3 === Chapter 1.2.1 --- Tissue Distribution of GHR --- p.4 === Chapter 1.2.2 --- GHR Biosynthesis and Degradation --- p.7 === Chapter 1.2.3 --- Regulation of GHR level --- p.8 === Chapter 1.2.4 --- Structure of GHR --- p.10 === Chapter 1.2.5 --- Possible Signal Transduction Pathways of GHR --- p.13 === Chapter 1.2.6 --- GHR Related Dwarfism --- p.15 === Chapter 1.2.7 --- Significance of Cloning of GHR cDNA --- p.16 === Chapter 1.3 --- Objectives of the Present Study --- p.17 === Chapter Chapter 2 --- General Materials and Methods === Chapter 2.1 --- Ethanol Precipitation of DNA and RNA --- p.19 === Chapter 2.2 --- Spectrophotometric Determination of DNA and RNA --- p.19 === Chapter 2.3 --- Minipreparation of Plasmid DNA --- p.19 === Chapter 2.4 --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification Kit from Promega --- p.20 === Chapter 2.5 --- Preparation of Plasmid DNA using QIAGEN-tip100 --- p.21 === Chapter 2.6 --- Preparation and Transformation of Escherichia coli Competent Cell --- p.22 === Chapter 2.7 --- Rapid Screening for the Presence of Desired Plasmid --- p.23 === Chapter 2.8 --- Agarose Gel Electrophoresis --- p.23 === Chapter 2.9 --- Formaldehyde / Agarose Gel Electrophoresis --- p.24 === Chapter 2.10 --- Restriction Digestion of DNA --- p.25 === Chapter 2.11 --- Linearization and Dephosphorylation of Plasmid Vector --- p.25 === Chapter 2.12 --- Purification of DNA form Agarose Gel Using GENECLEAN II® Kit --- p.25 === Chapter 2.13 --- Purification of DNA by Phenol / Chloroform Extraction --- p.26 === Chapter 2.14 --- DNA Radiolabelling --- p.26 === Chapter 2.15 --- Spun-Column Chromatography --- p.27 === Chapter 2.16 --- Capillary Transfer of DNA/RNA to a Nylon Membrane --- p.27 === Chapter 2.16.1 --- DNA Denaturation --- p.27 === Chapter 2.16.2 --- Capillary Transfer --- p.28 === Chapter 2.17 --- Hybridization of DNA/RNA --- p.28 === Chapter 2.18 --- Autoradiography --- p.29 === Chapter 2.19 --- Preparation of Ribonuclease Free Reagents and Apparatus --- p.29 === Chapter 2.20 --- Total RNA Isolation --- p.30 === Chapter 2.21 --- mRNA Isolation --- p.31 === Chapter 2.22 --- First Strand cDNA Synthesis --- p.32 === Chapter 2.23 --- Polymerase Chain Reaction --- p.32 === Chapter 2.24 --- 3'End Modification of PCR Amplified DNA --- p.33 === Chapter 2.25 --- Ligation of DNA Fragments --- p.34 === Chapter 2.26 --- DNA Sequencing --- p.34 === Chapter 2.26.1 --- DNA Sequencing Reaction --- p.34 === Chapter 2.26.2 --- DNA Sequencing Electrophoresis --- p.35 === Chapter 2.27 --- Reagents and Buffers --- p.38 === Chapter 2.27.1 --- Media for Bacterial Culture --- p.38 === Chapter 2.27.2 --- Reagents for Preparation of Plasmid DNA --- p.38 === Chapter 2.27.3 --- Buffers for Agarose Gel Electrophoresis --- p.40 === Chapter 2.27.4 --- Buffers for Formaldehyde Gel Electrophoresis --- p.40 === Chapter 2.27.5 --- Buffers for Preparation Competent Cells --- p.41 === Chapter 2.27.6 --- Buffers for Capillary Transfer and Hybridization --- p.42 === Chapter 2.27.7 --- Buffers for Total RNA Extraction --- p.43 === Chapter 2.27.8 --- 10X CIP Buffers --- p.43 === Chapter 2.28 --- Size of DNA/RNA Molecular Weight Markers --- p.44 === Chapter Chapter 3 --- Molecular Studies on Chicken Growth Hormone Receptor === Chapter 3.1 --- Introduction --- p.45 === Chapter 3.2 --- Material and Methods --- p.46 === Chapter 3.2.1 --- Molecular Cloning of Chicken GHR cDNA by PCR --- p.46 === Chapter 3.2.1.1 --- Animals and Tissue --- p.46 === Chapter 3.2.1.2 --- Reverse Transcrbed-Polymerase Chain Reaction (RT-PCR) --- p.46 === Chapter 3.2.1.3 --- Subcloning of PCR Amplified DNA Fragments --- p.47 === Chapter 3.2.2 --- Ontogeny of GHR mRNA Expression in Chicken Liver and Brain --- p.48 === Chapter 3.2.2.1 --- Animals and Tissues --- p.48 === Chapter 3.2.2.2 --- Northern Analysis --- p.48 === Chapter 3.2.2.3 --- Quantification of GHR mRNA level --- p.49 === Chapter 3.2.3 --- Prokaryotic Expression of Chicken GHR cDNA --- p.49 === Chapter 3.2.3.1 --- Subcloning of Chicken GHR cDNA into a Prokaryotic Expression Vector --- p.49 === Chapter 3.2.3.2 --- Expression of Chicken GHR cDNAin E.coli --- p.50 === Chapter 3.2.3.3 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 === Chapter 3.2.4 --- Reagents and Buffers === Chapter 3.2.4.1 --- Medium for Bacterial Culture --- p.53 === Chapter 3.2.4.2 --- Reagents for SDS-PAGE --- p.53 === Chapter 3.2.5 --- Size of Protein Molecular Weight Markers --- p.54 === Chapter 3.3 --- Results --- p.55 === Chapter 3.3.1 --- Molecular Cloning of Chicken GHR cDNA by PCR --- p.55 === Chapter 3.3.1.1 --- RT-PCR --- p.55 === Chapter 3.3.1.2 --- Subcloning --- p.56 === Chapter 3.3.1.3 --- Nucleotide Sequence Analysis --- p.57 === Chapter 3.3.2 --- Ontogeny of GHR mRNA Expression in Chicken Liver and Brain --- p.59 === Chapter 3.3.3 --- Prokaryotic Expression of Chicken GHR cDNA --- p.64 === Chapter 3.3.3.1 --- Subcloning --- p.64 === Chapter 3.3.3.2 --- Nucleotide Sequence Analysis --- p.65 === Chapter 3.3.3.3 --- Prokaryotic Expression --- p.66 === Chapter 3.4 --- Discussion --- p.68 === Chapter 3.4.1 --- Molecular Cloning of Chicken GHR cDNA by PCR --- p.68 === Chapter 3.4.2 --- Ontogeny of GHR mRNA Expression in Chicken Liver and Brain --- p.70 === Chapter 3.4.3 --- Prokaryotic Expression of Chicken GHR cDNA --- p.71 === Chapter Chapter 4 --- Molecular Cloning of Pigeon Growth Hormone Receptor Complementary DNA by Polymerase Chain Reaction and Sequence Analysis === Chapter 4.1 --- Introduction --- p.74 === Chapter 4.2 --- Materials and Methods --- p.75 === Chapter 4.2.1 --- Animals and Tissues --- p.75 === Chapter 4.2.2 --- Cloning of Pigeon GHR cDNA Main Core by PCR --- p.75 === Chapter 4.2.2.1 --- RT-PCR --- p.75 === Chapter 4.2.2.2 --- Southern Analysis of PCR Amplified Product --- p.76 === Chapter 4.2.2.3 --- Subcloning of PCR Amplified DNA Fragment --- p.76 === Chapter 4.2.3 --- Determination of 3' End Coding Sequence of Pigeon GHR cDNA --- p.76 === Chapter 4.2.4 --- Determination of 5' End Coding Sequence of Pigeon GHR cDNA --- p.79 === Chapter 4.3 --- Results === Chapter 4.3.1 --- Cloning of Pigeon GHR cDNA Main Core by PCR --- p.82 === Chapter 4.3.1.1 --- RT-PCR --- p.82 === Chapter 4.3.1.2 --- Southern Analysis --- p.83 === Chapter 4.3.1.3 --- Subcloning of Fragment M --- p.83 === Chapter 4.3.1.4 --- Restriction Digestion of Plasmid --- p.85 === Chapter 4.3.1.5 --- Nucleotide Sequence Analysis --- p.86 === Chapter 4.3.2 --- Determination of 3' End and 5' End coding Sequences of Pigeon GHR cDNA --- p.88 === Chapter 4.3.2.1 --- Random Primer Initiated RNA-PCR --- p.88 === Chapter 4.3.2.2 --- AmpliFINDER RACE --- p.88 === Chapter 4.3.2.3 --- Subcloning of Fragment 3' and Fragment 5' --- p.90 === Chapter 4.3.2.4 --- Nucleotide Sequence Analysis --- p.92 === Chapter 4.3.3 --- Nucleotide Sequence and Predicted Amino Acid Sequence of Pigeon GHR --- p.93 === Chapter 4.4 --- Discussion --- p.100 === Chapter Chapter 5 --- Attempts on Molecular Cloning of Fish Growth Hormone Receptor Complementary DNA === Chapter 5.1 --- Introduction --- p.106 === Chapter 5.2 --- Materials and Methods --- p.107 === Chapter 5.2.1 --- Animals and Tissues --- p.107 === Chapter 5.2.2 --- Design of PCR primers --- p.107 === Chapter 5.2.3 --- RT-PCR and Subcloning of PCR Amplified DNA --- p.108 === Chapter 5.2.4 --- Northern Analysis of Dace Liver RNA --- p.110 === Chapter 5.3 --- Results === Chapter 5.3.1 --- PCR --- p.111 === Chapter 5.3.2 --- Subcloning --- p.112 === Chapter 5.3.3 --- Nucleotide Sequence Analysis --- p.114 === Chapter 5.3.4 --- Northern Analysis --- p.117 === Chapter 5.4 --- Discussion --- p.119 === Chapter Chapter 6 --- General Discussion --- p.123 === References --- p.126 === Appendix --- p.135