Molecular cloning of vertebrate growth hormone receptor complementary DNAs.

by Yam Kwok Fai.Year shown on spine: 1997.Thesis (M.Phil.)--Chinese University of Hong Kong, 1996.Includes bibliographical references (leaves 141-149).Acknowledgments --- p.iList of Contents --- p.iiList of Figures --- p.viiiList of Tables --- p.xiiList of Primers --- p.xiiiAbbreviations --- p.xivChapter Chapter 1 --- IntroductionChapter 1.1 --- Growth Hormone (GH) --- p.1Chapter 1.2 --- Growth Hormone Receptor (GHR) --- p.3Chapter 1.2.1 --- Tissue Distribution of GHR --- p.4Chapter 1.2.2 --- Biosynthesis and Degradation of GHR --- p.6Chapter 1.2.3 --- Regulation of GHR Level --- p.7Chapter 1.2.4 --- The Structure of GHR --- p.9Chapter 1.2.5 --- The Structure of GHR Gene --- p.13Chapter 1.2.6 --- Growth Hormone Binding Protein (GHBP) --- p.14Chapter 1.2.7 --- The GH/Prolactin/Cytokine/Erythropoietin Receptor Superfamily --- p.15Chapter 1.2.8 --- Proposed Signal Transduction Pathway --- p.17Chapter 1.2.9 --- GHR Related Dwarfism --- p.22Chapter i). --- Substitution of certain amino acid residues in the extracellular domain --- p.22Chapter ii). --- Deletion of the extracellular domain --- p.23Chapter a). --- deletion of a small portion of the binding proteinChapter b). --- deletion of a large portion of the binding proteinChapter c). --- deletion of a large portion of the binding domain and the whole transmembrane domainChapter iii). --- Associated with normal GHBP --- p.24Chapter 1.3 --- Objectives of Cloning Vertebrate GHR cDNAs --- p.24Chapter Chapter 2 --- General Experimental MethodsChapter 2.1 --- Preparation of Ribonuclease Free Reagents and Apparatus --- p.26Chapter 2.2 --- Isolation of Total RNA --- p.26Chapter 2.3 --- Isolation of mRNA --- p.26Chapter a). --- directly from tissueChapter b). --- from isolated total RNAChapter 2.4 --- Spectrophotometric Quantification and Qualification of DNA and RNA --- p.29Chapter 2.5 --- First Strand cDNA Synthesis --- p.29Chapter 2.6 --- Polymerase Chain Reaction (PCR) --- p.30Chapter 2.7 --- Agarose Gel Electrophoresis --- p.31Chapter 2.8 --- Formaldehyde Agarose Gel Electrophoresis of RNA --- p.31Chapter 2.9 --- Capillary Transfer of DNA/RNA to a Nylon Membrane (Southern/Northern Blotting) --- p.32Chapter a). --- DNA denaturingChapter b). --- Capillary transferChapter 2.10 --- DNA Radiolabelling --- p.33Chapter a). --- By random primer translationChapter b). --- By nick translationChapter 2.11 --- Spuncolumn Chromatography --- p.34Chapter 2.12 --- Hybridization of Southern/Northern Blot --- p.35Chapter 2.13 --- Autoradiography --- p.35Chapter 2.14 --- Linearization and Dephosphorylation of Plasmid DNA --- p.36Chapter 2.15 --- Restriction Digestion of DNA --- p.36Chapter 2.16 --- Purification of DNA from Agarose Gel using GENECLEAN® Kit --- p.36Chapter 2.17 --- 3' End Modification of PCR Amplified DNA --- p.37Chapter 2.18 --- Ligation of DNA Fragments to Linearized Vector --- p.37Chapter 2.19 --- Preparation of Escherichia coli Competent Cells --- p.38Chapter 2.20 --- Transformation of the Escherichia coli Strain DH5a --- p.38Chapter 2.21 --- Minipreparation of Plasmid DNA --- p.39Chapter 2.22 --- DNA Purification by Phenol/Chloroform Extraction --- p.39Chapter 2.23 --- Ethanol Precipitation of DNA and RNA --- p.40Chapter 2.24 --- Preparation of Plasmid DNA using Wizard´ёØ Minipreps DNA Purification Kit from Promega --- p.40Chapter 2.25 --- Preparation of Plasmid DNA using QIAGEN-tip100 --- p.41Chapter 2.26 --- DNA Sequencing --- p.42Chapter 2.26.1 --- DNA Sequencing ReactionChapter a). --- T7 sequencingChapter b). --- PCR sequencingChapter 2.26.2 --- DNA Sequencing Electrophoresis --- p.44Chapter i). --- Preparation of 8% polyacrylamide gel solutionChapter ii). --- Casting the gelChapter iii). --- ElectrophoresisChapter Chapter 3 --- Molecular Cloning of Golden Hamster (Mesocricetus auratus) GHR cDNAChapter 3.1 --- Introduction --- p.46Chapter 3.2 --- Experimental MethodsChapter 3.2.1 --- Animals and Tissues --- p.47Chapter 3.2.2 --- PCR Cloning of GHR cDNA Fragments in the Cytoplasmic Domain --- p.47Chapter 3.2.2.1 --- Primer design and PCR strategy --- p.47Chapter 3.2.2.2 --- PCR studies on the hamster liver and kidney first strand cDNA --- p.49Chapter 3.2.2.3 --- Southern analysis of the PCR products --- p.50Chapter 3.2.2.4 --- Subcloning and sequencing of PCR amplified cDNA fragments --- p.50Chapter 3.2.3 --- Screening of a Hamster Liver cDNA Library --- p.51Chapter 3.2.3.1 --- Preparation of the plating bacteria --- p.51Chapter 3.2.3.2 --- Phage titering of the λ ZAP library --- p.51Chapter 3.2.3.3 --- Primary screening of the amplified hamster liver cDNA library --- p.52Chapter 3.2.3.4 --- Plaque uplifting and hybridization with hamster GHR cDNA fragment --- p.52Chapter 3.2.3.5 --- Purification of putative clones from primary screening --- p.53Chapter 3.2.3.6 --- Checking the size of the DNA insert --- p.53Chapter 3.2.3.7 --- In vitro excision to release phagemid from the phage vector --- p.54Chapter 3.2.3.8 --- Plasmid minipreparation of the putative clones --- p.56Chapter 3.2.3.9 --- Nucleotide sequencing of the DNA inserts of different clones --- p.56Chapter 3.2.4 --- Tissue Distribution of GHR in Hamster Tissues and the Relative Expression Level of GHR mRNAin these tissues --- p.58Chapter 3.2.5 --- Cloning of the Full-length GHR cDNA into a Mammalian Vector --- p.59Chapter 3.2.5.1 --- PCR amplification of the full-length hamster GHR cDNA --- p.59Chapter 3.2.5.2 --- Preparation of the hamster GHR cDNA insert for ligation --- p.60Chapter 3.2.5.3 --- Linearization of pRc/CMV expression vector --- p.60Chapter 3.2.5.4 --- Ligation of the linearized expression vector with the full-length hamster GHR cDNA --- p.61Chapter 3.3 --- ResultsChapter 3.3.1 --- PCR Amplification of Hamster GHR cDNA Fragments --- p.61Chapter 3.3.1.1 --- RT-PCR --- p.61Chapter 3.3.1.2 --- Southern blot analysis --- p.62Chapter 3.3.1.3 --- Subcloning and nucleotide sequencing of PCR amplified hamster GHR cDNA fragments --- p.64Chapter 3.3.2 --- Screening of an Amplified λZAP Hamster Liver cDNA Library --- p.70Chapter 3.3.2.1 --- Preparation of the cDNA probe and phage titering --- p.70Chapter 3.3.2.2 --- Screening of the cDNA library --- p.70Chapter 3.3.2.3 --- PCR study of the 5' and 3' regions of the DNA insert of the clones selected for secondary screening --- p.72Chapter 3.2.3.4 --- Nucleotide sequencing of the full-length hamster GHR cDNA --- p.73Chapter 3.2.3.5 --- Tissue distribution of GHR in hamster and the relative expression level of the GHR mRNA in these tissues --- p.73Chapter 3.2.3.6 --- Cloning of the full-length hamster GHR cDNA into a mammalian expression vector --- p.79Chapter 3.4 --- DiscussionChapter 3.4.1 --- Cloning of the Full-length hamster GHR cDNA --- p.81Chapter 3.4.2 --- Comparison of the Nucleotide and the Predicted Amino Acid Sequences of the Hamster GHR with other Cloned GHRs --- p.82Chapter 3.4.3 --- Tissue Distribution of GHR in Hamster and the Relative Expression Level of the GHR mRNA in these Tissues --- p.89Chapter 3.4.4 --- Further Studies on Hamster GHR --- p.90Chapter Chapter 4 --- Molecular Cloning of Chinese Bullfrog (Rana tigria rigulosa) GHR cDNA from Adult Frog LiverChapter 4.1 --- Introduction --- p.92Chapter 4.2 --- Experimental MethodsChapter 4.2.1 --- Animal and Tissues --- p.93Chapter 4.2.2 --- Cloning of the Cytoplasmic Domain of Frog GHR cDNA by PCR --- p.93Chapter 4.2.2.1 --- RT-PCR --- p.93Chapter 4.2.2.2 --- Southern blot analysis of PCR amplified products --- p.95Chapter 4.2.2.3 --- Subcloning and sequencing of PCR amplified DNA fragments --- p.95Chapter 4.2.2.4 --- Restriction analysis of GHR cDNA fragment between GHR p1 and GHR p2 --- p.95Chapter 4.2.2.5 --- PCR cloning of other portions of frog GHR cDNA --- p.96Chapter 4.2.2.6 --- Subcloning and sequencing of PCR amplified GHR cDNA fragment using primers other than GHR p1 and GHR p2 --- p.97Chapter 4.3 --- ResultsChapter 4.3.1 --- Cloning of the Intracellular Domain of Frog GHR cDNA by RT-PCR --- p.97Chapter 4.3.1.1 --- RT-PCR --- p.97Chapter 4.3.1.2 --- Southern blot analysis --- p.98Chapter 4.3.1.3 --- Subcloning and sequencing of PCR amplified DNA fragments --- p.98Chapter 4.3.1.4 --- Restriction enzyme analysis of GHR cDNA fragments --- p.102Chapter 4.3.1.5 --- PCR cloning of other portions of frog GHR cDNA --- p.103Chapter 4.3.1.6 --- Subcloning and sequencing of PCR products from other portions of frog GHR cDNA --- p.103Chapter 4.4 --- DiscussionChapter 4.4.1 --- Cloning of the Full-length frog GHR cDNA --- p.109Chapter 4.4.2 --- Further Studies on Frog GHR --- p.117Chapter Chapter 5 --- Attempts on the Molecular Cloning of Teleost GHR cDNAChapter 5.1 --- Introduction --- p.119Chapter 5.2 --- Experimental MethodsChapter 5.2.1 --- Animals and Tissues --- p.120Chapter 5.2.2 --- PCR Cloning of Teleost GHR cDNA fragments --- p.120Chapter 5.2.2.1 --- Design of PCR primers --- p.120Chapter 5.2.2.2 --- Preparation of mRNA and synthesis of first strand cDNA --- p.122Chapter 5.2.2.3 --- PCR studies on dace and snakehead fish liver first strand cDNA --- p.122Chapter 5.2.2.3.1 --- PCR studies on dace liver first strand cDNA --- p.122Chapter 5.2.2.3.2 --- PCR studies on snakehead fish liver first strand cDNA --- p.122Chapter 5.2.3 --- "Northern Analysis on Dace, Snakehead fish and Eel mRNA" --- p.123Chapter 5.3 --- ResultsChapter 5.3.1 --- Molecular Studies on Dace GHR cDNA --- p.123Chapter 5.3.1.1 --- PCR studies on dace first strand cDNA --- p.123Chapter 5.3.2 --- PCR Studies on Teleost First Strand cDNA --- p.128Chapter 5.3.3 --- Northern Analysis on Teleost mRNA --- p.128Chapter 5.4 --- Discussion --- p.130Chapter 5.4.1 --- PCR Studies on Teleost GHR cDNA --- p.130Chapter 5.4.2 --- Northern Analysis on Teleost mRNA --- p.131Chapter Chapter 6 --- General DiscussionChapter 6.1 --- Achievement of this Project --- p.134Chapter 6.1.1 --- Hamster GHR --- p.134Chapter 6.1.2 --- Frog GHR --- p.135Chapter 6.1.3 --- Teleost GHR --- p.136Chapter 6.2 --- Postulation on Cloned GHRs at the Molecular Level --- p.136Bibliography --- p.141Appendices --- p.15