by Lee Tsz On. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 148-155). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgments --- p.v / Contents --- p.vi / List of figures --- p.xii / List of table --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Growth hormone (GH) --- p.1 / Chapter 1.1.1. --- Introduction to GH --- p.1 / Chapter 1.1.2. --- Actions of GH --- p.2 / Chapter 1.1.3. --- Structure of GH --- p.3 / Chapter 1.1.4. --- The sequence of GH --- p.5 / Chapter 1.2. --- Growth hormone receptor (GHR) --- p.6 / Chapter 1.2.1 --- Introduction to GHR --- p.6 / Chapter 1.2.2. --- Structure of the extracellular domain of GHR --- p.9 / Chapter 1.2.3. --- The regulation of GHR --- p.12 / Chapter 1.2.4. --- GHR biosynthesis --- p.13 / Chapter 1.2.5. --- Tissue distribution of GHR --- p.14 / Chapter 1.3. --- Signal transduction mechanisms of GHR --- p.15 / Chapter 1.3.1. --- Dimerization of GH and GHR complex --- p.15 / Chapter 1.3.2. --- The Jak and Stat pathway --- p.18 / Chapter 1.3.3. --- The ras and other signaling pathways --- p.20 / Chapter 1.4. --- Project aim --- p.22 / Chapter Chapter 2 --- Material and Methods / Chapter 2.1. --- Preparation of ribonuclease free reagents and apparatus --- p.23 / Chapter 2.2. --- Isolation of total RNA --- p.23 / Chapter 2.3. --- Isolation of mRNA --- p.24 / Chapter 2.4. --- Spectrophotometric quantification and qualification of DNA and RNA --- p.24 / Chapter 2.5. --- First strand cDNA synthesis --- p.25 / Chapter 2.6. --- Agarose gel electrophoresis of DNA --- p.25 / Chapter 2.7. --- Formaldehyde agarose gel electrophoresis of RNA --- p.26 / Chapter 2.8. --- Vacuum transfer of DNA to a nylon membrane --- p.26 / Chapter 2.9. --- Nucleic acids purification by MicroSpin (S-200HR) columns --- p.27 / Chapter 2.10. --- DNA radioactive labeling by nick translation --- p.27 / Chapter 2.11. --- Southern blot analysis --- p.28 / Chapter 2.12. --- Autoradiography and molecular imager --- p.28 / Chapter 2.13 . --- Linearization and dephosphorylation of plasmid DNA --- p.29 / Chapter 2.14. --- Purification of DNA from agarose using QIAEX II kit --- p.29 / Chapter 2.15. --- 3'End modification of PCR amplified DNA --- p.30 / Chapter 2.16. --- Ligation of DNA fragments to linearized vector --- p.30 / Chapter 2.17. --- Preparation of Escherichia coli competent cells --- p.31 / Chapter 2.18. --- Transformation --- p.31 / Chapter 2.19. --- Mini preparation of plasmid DNA --- p.32 / Chapter 2.20. --- Maxi preparation of plasmid DNA --- p.34 / Chapter 2.21 . --- PCR sequencing --- p.35 / Chapter 2.22. --- cDNA library screening --- p.36 / Chapter 2.23. --- Preparation and sterilization of culture medium --- p.38 / Chapter 2.24. --- Preparation of frozen stock of culture cells --- p.39 / Chapter 2.25. --- Cell passage of CHO-Kl --- p.39 / Chapter 2.26. --- Counting of cells --- p.40 / Chapter 2.27. --- Proliferation assay performed on CHO-K1 cells (MTT method) --- p.40 / Chapter 2.28. --- Luciferase assay --- p.41 / Chapter 2.29. --- SDS-PAGE preparation --- p.42 / Chapter 2.30. --- SDS-PAGE analysis of proteins --- p.42 / Chapter 2.31 . --- Recombinant protein expression --- p.43 / Chapter 2.32. --- Small scale purification of recombinant proteins --- p.44 / Chapter 2.33. --- Restriction digestion of DNA --- p.45 / Chapter 2.34. --- Purification of PCR product using QIAquick PCR purification kit --- p.45 / Chapter 2.35. --- TA cloning of PCR fragment --- p.45 / Chapter 2.36. --- Transfection of plasmid into CHO-K1 cells --- p.46 / Chapter 2.37. --- Sources of hormones --- p.46 / Chapter 2.38. --- Buffer and reagents --- p.47 / Chapter Chapter 3 --- "Cloning, expression and tissue distribution of Xenopus laevis GHR" / Chapter 3.1. --- Introduction --- p.50 / Chapter 3.2. --- Materials and methods --- p.51 / Chapter 3.2.1. --- Molecular cloning of xGHR cDNA / Chapter 3.2.1.1. --- Animals and tissues --- p.51 / Chapter 3.2.1.2. --- Reverse transcribed´ؤpolymerase chain reaction (RT-PCR) --- p.51 / Chapter 3.2.1.3. --- Subcloning of PCR amplified DNA fragment --- p.53 / Chapter 3.2.1.4. --- Library screening of xGHR --- p.53 / Chapter 3.2.1.5. --- 5 'Rapid amplification of cDNA end (5' RACE) --- p.55 / Chapter 3.2.2. --- Tissue distribution of xGHR / Chapter 3.2.2.1. --- Animals and tissues --- p.56 / Chapter 3.2.2.2. --- RT-PCR and Southern blot --- p.56 / Chapter 3.2.3. --- Eukarytoic expression of xGHR and functional assay of xGHR / Chapter 3.2.3.1. --- Subcloning ofxGHR into pRc/CMV --- p.57 / Chapter 3.2.3.2. --- Expression of xGHR in CHO-K1 cell --- p.58 / Chapter 3.2.3.3. --- Proliferation assay --- p.58 / Chapter 3.3. --- Results --- p.60 / Chapter 3.3.1. --- RT-PCR of the partial fragment --- p.60 / Chapter 3.3.2. --- Library screening of xGHR cDNA library --- p.61 / Chapter 3.3.3. --- 5' RACE --- p.64 / Chapter 3.3.4. --- The full-length cDNA sequence of xGHR --- p.65 / Chapter 3.3.5. --- Tissue distribution of xGHR mRNA --- p.69 / Chapter 3.3.6. --- Functional assay of xGHR in CHO-K1 cells --- p.71 / Chapter 3.4. --- Discussion --- p.74 / Chapter Chapter 4 --- Cloning and expression of Xenopus laevis GH-A and GH-B / Chapter 4.1. --- Introduction --- p.78 / Chapter 4.2. --- Materials and Methods --- p.79 / Chapter 4.2.1. --- PCR amplification of xGH-A and xGH-B partial fragments --- p.79 / Chapter 4.2.2. --- cDNA library screening of xGH-A and xGH-B --- p.80 / Chapter 4.2.3. --- Rapid amplification of cDNA ends of xGH-B / Chapter 4.2.3.1. --- 3'RACE --- p.80 / Chapter 4.2.3.2. --- 5'RACE --- p.81 / Chapter 4.2.4. --- Expression of xGH-A and xGH-B / Chapter 4.2.4.1 --- Construction of the expression vector --- p.84 / Chapter 4.2.4.2. --- Protein expression of xGH-A and xGH-B --- p.85 / Chapter 4.2.5. --- Purification of recombinant xGH-A and xGH-B --- p.85 / Chapter 4.3. --- Results --- p.87 / Chapter 4.3.1. --- PCRof xGH-A and xGH-B partial fragment --- p.87 / Chapter 4.3.2. --- Library screening of xGH-A --- p.87 / Chapter 4.3.3. --- 5' RACE and 3' RACE of xGH-B --- p.91 / Chapter 4.3.4. --- Sequence analysis of xGH-A and xGH-B --- p.93 / Chapter 4.3.5. --- Protein expression and purification of recombinant xGH-A and xGH-B --- p.100 / Chapter 4.4. --- Discussion --- p.102 / Chapter Chapter 5 --- Molecular cloning and function expression of goldfish GHR / Chapter 5.1. --- Introduction --- p.105 / Chapter 5.2. --- Materials and methods --- p.106 / Chapter 5.2.1. --- Molecular cloning of the partial fragment of gfGHR / Chapter 5.2.1.1. --- Primer design --- p.106 / Chapter 5.2.1.2. --- Library PCR of gfGHR partial fragment --- p.108 / Chapter 5.2.2. --- Library PCR of gfGHR cDNA sequence --- p.110 / Chapter 5.2.3. --- Determination of 3' End and 5' End sequences of gfGHR cDNA --- p.112 / Chapter 5.2.4. --- Tissue distribution of gfGHR / Chapter 5.2.4.1. --- Animals and tissues --- p.115 / Chapter 5.2.4.2. --- Semi-quantitative R T-PCR --- p.115 / Chapter 5.2.5. --- Functional expression of gfGHR in CHO-K1 cell / Chapter 5.2.5.1. --- Construction of an expression vector containing gfGHR --- p.116 / Chapter 5.2.5.2. --- Functional assay of gfGHR expression on CHO-K1 cells --- p.117 / Chapter 5.2.5.3. --- Proliferation assay --- p.118 / Chapter 5.2.5.4. --- Spi luciferase assay --- p.118 / Chapter 5.3. --- Results --- p.120 / Chapter 5.3.1. --- PCR amplification of the partial sequence of gfGHR --- p.120 / Chapter 5.3.2. --- The library PCR of gfGHR cDNA sequence --- p.122 / Chapter 5.3.3. --- The sequence of gfGHR --- p.124 / Chapter 5.3.4. --- Tissue distribution of gfGHR --- p.131 / Chapter 5.3.5. --- Proliferation assay --- p.133 / Chapter 5.3.6. --- Spi luciferase assay --- p.135 / Chapter 5.4. --- Discussion --- p.137 / Chapter Chapter 6 --- General discussion and future works --- p.145 / References --- p.148 / Appendix --- p.156

Chan, Yuk-Hang. / "June 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 162-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

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.i / List of Contents --- p.ii / List of Figures --- p.viii / List of Tables --- p.xii / List of Primers --- p.xiii / Abbreviations --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Growth Hormone (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 --- Biosynthesis and Degradation of GHR --- p.6 / Chapter 1.2.3 --- Regulation of GHR Level --- p.7 / Chapter 1.2.4 --- The Structure of GHR --- p.9 / Chapter 1.2.5 --- The Structure of GHR Gene --- p.13 / Chapter 1.2.6 --- Growth Hormone Binding Protein (GHBP) --- p.14 / Chapter 1.2.7 --- The GH/Prolactin/Cytokine/Erythropoietin Receptor Superfamily --- p.15 / Chapter 1.2.8 --- Proposed Signal Transduction Pathway --- p.17 / Chapter 1.2.9 --- GHR Related Dwarfism --- p.22 / Chapter i). --- Substitution of certain amino acid residues in the extracellular domain --- p.22 / Chapter ii). --- Deletion of the extracellular domain --- p.23 / Chapter a). --- deletion of a small portion of the binding protein / Chapter b). --- deletion of a large portion of the binding protein / Chapter c). --- deletion of a large portion of the binding domain and the whole transmembrane domain / Chapter iii). --- Associated with normal GHBP --- p.24 / Chapter 1.3 --- Objectives of Cloning Vertebrate GHR cDNAs --- p.24 / Chapter Chapter 2 --- General Experimental Methods / Chapter 2.1 --- Preparation of Ribonuclease Free Reagents and Apparatus --- p.26 / Chapter 2.2 --- Isolation of Total RNA --- p.26 / Chapter 2.3 --- Isolation of mRNA --- p.26 / Chapter a). --- directly from tissue / Chapter b). --- from isolated total RNA / Chapter 2.4 --- Spectrophotometric Quantification and Qualification of DNA and RNA --- p.29 / Chapter 2.5 --- First Strand cDNA Synthesis --- p.29 / Chapter 2.6 --- Polymerase Chain Reaction (PCR) --- p.30 / Chapter 2.7 --- Agarose Gel Electrophoresis --- p.31 / Chapter 2.8 --- Formaldehyde Agarose Gel Electrophoresis of RNA --- p.31 / Chapter 2.9 --- Capillary Transfer of DNA/RNA to a Nylon Membrane (Southern/Northern Blotting) --- p.32 / Chapter a). --- DNA denaturing / Chapter b). --- Capillary transfer / Chapter 2.10 --- DNA Radiolabelling --- p.33 / Chapter a). --- By random primer translation / Chapter b). --- By nick translation / Chapter 2.11 --- Spuncolumn Chromatography --- p.34 / Chapter 2.12 --- Hybridization of Southern/Northern Blot --- p.35 / Chapter 2.13 --- Autoradiography --- p.35 / Chapter 2.14 --- Linearization and Dephosphorylation of Plasmid DNA --- p.36 / Chapter 2.15 --- Restriction Digestion of DNA --- p.36 / Chapter 2.16 --- Purification of DNA from Agarose Gel using GENECLEAN® Kit --- p.36 / Chapter 2.17 --- 3' End Modification of PCR Amplified DNA --- p.37 / Chapter 2.18 --- Ligation of DNA Fragments to Linearized Vector --- p.37 / Chapter 2.19 --- Preparation of Escherichia coli Competent Cells --- p.38 / Chapter 2.20 --- Transformation of the Escherichia coli Strain DH5a --- p.38 / Chapter 2.21 --- Minipreparation of Plasmid DNA --- p.39 / Chapter 2.22 --- DNA Purification by Phenol/Chloroform Extraction --- p.39 / Chapter 2.23 --- Ethanol Precipitation of DNA and RNA --- p.40 / Chapter 2.24 --- Preparation of Plasmid DNA using Wizard´ёØ Minipreps DNA Purification Kit from Promega --- p.40 / Chapter 2.25 --- Preparation of Plasmid DNA using QIAGEN-tip100 --- p.41 / Chapter 2.26 --- DNA Sequencing --- p.42 / Chapter 2.26.1 --- DNA Sequencing Reaction / Chapter a). --- T7 sequencing / Chapter b). --- PCR sequencing / Chapter 2.26.2 --- DNA Sequencing Electrophoresis --- p.44 / Chapter i). --- Preparation of 8% polyacrylamide gel solution / Chapter ii). --- Casting the gel / Chapter iii). --- Electrophoresis / Chapter Chapter 3 --- Molecular Cloning of Golden Hamster (Mesocricetus auratus) GHR cDNA / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Experimental Methods / Chapter 3.2.1 --- Animals and Tissues --- p.47 / Chapter 3.2.2 --- PCR Cloning of GHR cDNA Fragments in the Cytoplasmic Domain --- p.47 / Chapter 3.2.2.1 --- Primer design and PCR strategy --- p.47 / Chapter 3.2.2.2 --- PCR studies on the hamster liver and kidney first strand cDNA --- p.49 / Chapter 3.2.2.3 --- Southern analysis of the PCR products --- p.50 / Chapter 3.2.2.4 --- Subcloning and sequencing of PCR amplified cDNA fragments --- p.50 / Chapter 3.2.3 --- Screening of a Hamster Liver cDNA Library --- p.51 / Chapter 3.2.3.1 --- Preparation of the plating bacteria --- p.51 / Chapter 3.2.3.2 --- Phage titering of the λ ZAP library --- p.51 / Chapter 3.2.3.3 --- Primary screening of the amplified hamster liver cDNA library --- p.52 / Chapter 3.2.3.4 --- Plaque uplifting and hybridization with hamster GHR cDNA fragment --- p.52 / Chapter 3.2.3.5 --- Purification of putative clones from primary screening --- p.53 / Chapter 3.2.3.6 --- Checking the size of the DNA insert --- p.53 / Chapter 3.2.3.7 --- In vitro excision to release phagemid from the phage vector --- p.54 / Chapter 3.2.3.8 --- Plasmid minipreparation of the putative clones --- p.56 / Chapter 3.2.3.9 --- Nucleotide sequencing of the DNA inserts of different clones --- p.56 / Chapter 3.2.4 --- Tissue Distribution of GHR in Hamster Tissues and the Relative Expression Level of GHR mRNAin these tissues --- p.58 / Chapter 3.2.5 --- Cloning of the Full-length GHR cDNA into a Mammalian Vector --- p.59 / Chapter 3.2.5.1 --- PCR amplification of the full-length hamster GHR cDNA --- p.59 / Chapter 3.2.5.2 --- Preparation of the hamster GHR cDNA insert for ligation --- p.60 / Chapter 3.2.5.3 --- Linearization of pRc/CMV expression vector --- p.60 / Chapter 3.2.5.4 --- Ligation of the linearized expression vector with the full-length hamster GHR cDNA --- p.61 / Chapter 3.3 --- Results / Chapter 3.3.1 --- PCR Amplification of Hamster GHR cDNA Fragments --- p.61 / Chapter 3.3.1.1 --- RT-PCR --- p.61 / Chapter 3.3.1.2 --- Southern blot analysis --- p.62 / Chapter 3.3.1.3 --- Subcloning and nucleotide sequencing of PCR amplified hamster GHR cDNA fragments --- p.64 / Chapter 3.3.2 --- Screening of an Amplified λZAP Hamster Liver cDNA Library --- p.70 / Chapter 3.3.2.1 --- Preparation of the cDNA probe and phage titering --- p.70 / Chapter 3.3.2.2 --- Screening of the cDNA library --- p.70 / Chapter 3.3.2.3 --- PCR study of the 5' and 3' regions of the DNA insert of the clones selected for secondary screening --- p.72 / Chapter 3.2.3.4 --- Nucleotide sequencing of the full-length hamster GHR cDNA --- p.73 / Chapter 3.2.3.5 --- Tissue distribution of GHR in hamster and the relative expression level of the GHR mRNA in these tissues --- p.73 / Chapter 3.2.3.6 --- Cloning of the full-length hamster GHR cDNA into a mammalian expression vector --- p.79 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Cloning of the Full-length hamster GHR cDNA --- p.81 / Chapter 3.4.2 --- Comparison of the Nucleotide and the Predicted Amino Acid Sequences of the Hamster GHR with other Cloned GHRs --- p.82 / Chapter 3.4.3 --- Tissue Distribution of GHR in Hamster and the Relative Expression Level of the GHR mRNA in these Tissues --- p.89 / Chapter 3.4.4 --- Further Studies on Hamster GHR --- p.90 / Chapter Chapter 4 --- Molecular Cloning of Chinese Bullfrog (Rana tigria rigulosa) GHR cDNA from Adult Frog Liver / Chapter 4.1 --- Introduction --- p.92 / Chapter 4.2 --- Experimental Methods / Chapter 4.2.1 --- Animal and Tissues --- p.93 / Chapter 4.2.2 --- Cloning of the Cytoplasmic Domain of Frog GHR cDNA by PCR --- p.93 / Chapter 4.2.2.1 --- RT-PCR --- p.93 / Chapter 4.2.2.2 --- Southern blot analysis of PCR amplified products --- p.95 / Chapter 4.2.2.3 --- Subcloning and sequencing of PCR amplified DNA fragments --- p.95 / Chapter 4.2.2.4 --- Restriction analysis of GHR cDNA fragment between GHR p1 and GHR p2 --- p.95 / Chapter 4.2.2.5 --- PCR cloning of other portions of frog GHR cDNA --- p.96 / Chapter 4.2.2.6 --- Subcloning and sequencing of PCR amplified GHR cDNA fragment using primers other than GHR p1 and GHR p2 --- p.97 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Cloning of the Intracellular Domain of Frog GHR cDNA by RT-PCR --- p.97 / Chapter 4.3.1.1 --- RT-PCR --- p.97 / Chapter 4.3.1.2 --- Southern blot analysis --- p.98 / Chapter 4.3.1.3 --- Subcloning and sequencing of PCR amplified DNA fragments --- p.98 / Chapter 4.3.1.4 --- Restriction enzyme analysis of GHR cDNA fragments --- p.102 / Chapter 4.3.1.5 --- PCR cloning of other portions of frog GHR cDNA --- p.103 / Chapter 4.3.1.6 --- Subcloning and sequencing of PCR products from other portions of frog GHR cDNA --- p.103 / Chapter 4.4 --- Discussion / Chapter 4.4.1 --- Cloning of the Full-length frog GHR cDNA --- p.109 / Chapter 4.4.2 --- Further Studies on Frog GHR --- p.117 / Chapter Chapter 5 --- Attempts on the Molecular Cloning of Teleost GHR cDNA / Chapter 5.1 --- Introduction --- p.119 / Chapter 5.2 --- Experimental Methods / Chapter 5.2.1 --- Animals and Tissues --- p.120 / Chapter 5.2.2 --- PCR Cloning of Teleost GHR cDNA fragments --- p.120 / Chapter 5.2.2.1 --- Design of PCR primers --- p.120 / Chapter 5.2.2.2 --- Preparation of mRNA and synthesis of first strand cDNA --- p.122 / Chapter 5.2.2.3 --- PCR studies on dace and snakehead fish liver first strand cDNA --- p.122 / Chapter 5.2.2.3.1 --- PCR studies on dace liver first strand cDNA --- p.122 / Chapter 5.2.2.3.2 --- PCR studies on snakehead fish liver first strand cDNA --- p.122 / Chapter 5.2.3 --- "Northern Analysis on Dace, Snakehead fish and Eel mRNA" --- p.123 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Molecular Studies on Dace GHR cDNA --- p.123 / Chapter 5.3.1.1 --- PCR studies on dace first strand cDNA --- p.123 / Chapter 5.3.2 --- PCR Studies on Teleost First Strand cDNA --- p.128 / Chapter 5.3.3 --- Northern Analysis on Teleost mRNA --- p.128 / Chapter 5.4 --- Discussion --- p.130 / Chapter 5.4.1 --- PCR Studies on Teleost GHR cDNA --- p.130 / Chapter 5.4.2 --- Northern Analysis on Teleost mRNA --- p.131 / Chapter Chapter 6 --- General Discussion / Chapter 6.1 --- Achievement of this Project --- p.134 / Chapter 6.1.1 --- Hamster GHR --- p.134 / Chapter 6.1.2 --- Frog GHR --- p.135 / Chapter 6.1.3 --- Teleost GHR --- p.136 / Chapter 6.2 --- Postulation on Cloned GHRs at the Molecular Level --- p.136 / Bibliography --- p.141 / Appendices --- p.150

by Simon Chan Siu Hoi. / Spine title varies. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 130-148). / Acknowledgments --- p.i / Table of Contents --- p.ii / List of Abbreviations --- p.ix / List of Figures --- p.xiii / List of Tables --- p.xvi / Page / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Growth Hormone --- p.1 / Chapter 1.2 --- Growth Hormone Receptor --- p.3 / Chapter 1.2.1 --- Cytokine/Hematopoietin Receptor Superfamily --- p.3 / Chapter 1.2.2 --- Tissue Distribution of GHR --- p.6 / Chapter 1.2.3 --- Biosynthesis and Degradation of GHR --- p.7 / Chapter 1.2.4 --- Regulation of GHR Level --- p.8 / Chapter 1.2.5 --- The GHR Protein --- p.10 / Chapter 1.2.6 --- The GHR Gene --- p.15 / Chapter 1.2.7 --- GHR Dimerization --- p.16 / Chapter 1.2.8 --- Mechanism of Signaling by GHR --- p.19 / Chapter 1.2.9 --- GH Binding Protein --- p.21 / Chapter 1.2.10 --- GHR Related Dwarfism --- p.23 / Chapter 1.3 --- Objectives of the Present Investigation --- p.25 / Chapter Chapter 2 --- Materials and Methods --- p.27 / Chapter 2.1 --- Fish Growth Hormone Radioactive Labeling --- p.27 / Chapter 2.1.1 --- Preparation of Iodogen-Coated Tubes --- p.27 / Chapter 2.1.2 --- Packing of the Sephadex G-75 Column --- p.28 / Chapter 2.1.3 --- Iodination of brGH and Purification of the Iodinated brGH --- p.28 / Chapter 2.1.4 --- Determination of the Specific Radioactivity and Percentage of 125I Incorporation --- p.29 / Chapter 2.1.5 --- Reagents and Buffers Used --- p.30 / Chapter 2.2 --- Integrity of 125I-brGH --- p.30 / Chapter 2.2.1 --- HPLC of brGH --- p.31 / Chapter 2.2.2 --- HPLC of 125I-brGH after Iodination --- p.31 / Chapter 2.2.3 --- HPLC of 125I-brGH after Receptor Binding --- p.31 / Chapter 2.3 --- Preparation of Membranes from Fish Tissues --- p.32 / Chapter 2.3.1 --- Preparation of Snakehead Fish Liver Membranes --- p.32 / Chapter 2.3.2 --- Reagents and Buffers Used --- p.33 / Chapter 2.4 --- Protein Determination of Membrane Preparations --- p.34 / Chapter 2.4.1 --- The BCA Protein Reaction Scheme --- p.34 / Chapter 2.4.2 --- BCA Protein Determination Protocol --- p.34 / Chapter 2.5 --- Receptor Binding Studies --- p.35 / Chapter 2.5.1 --- Association and Dissociation Studies --- p.36 / Chapter 2.5.2 --- pH Dependence Study --- p.36 / Chapter 2.5.3 --- Membrane Protein Dependence Study --- p.37 / Chapter 2.5.4 --- Ca2+ Dependence Study --- p.37 / Chapter 2.5.5 --- Tissue Distribution Study --- p.37 / Chapter 2.5.6 --- Displacement and Specificity Studies --- p.38 / Chapter 2.5.7 --- Dithiothreitol (DTT) Dependence Study --- p.39 / Chapter 2.5.8 --- p-Chloromercuribenzene Sulfonate (PCMBS) Pretreatment: Dose Dependence Study --- p.39 / Chapter 2.5.9 --- Scatchard Analysis of the PCMBS Pretreated and Control Snakehead Fish Liver Membranes --- p.40 / Chapter 2.5.10 --- Reversibility of the PCMBS Effect --- p.40 / Chapter 2.5.11 --- Reagents and Buffers Used --- p.41 / Chapter 2.6 --- Crosslinking Studies --- p.41 / Chapter 2.6.1 --- Crosslinking Performed on Snakehead Fish Liver Membranes --- p.41 / Chapter 2.6.2 --- Crosslinking Performed on Solubilized Snakehead Fish Liver Membranes --- p.42 / Chapter 2.6.3 --- Gel Filtration Chromatography of the Crosslinked Comp)lexes --- p.43 / Chapter 2.6.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of the Crosslinked Complexes --- p.43 / Chapter 2.6.5 --- Reagents and Buffers Used --- p.45 / Chapter 2.7 --- Western Blot Analysis of Snakehead Fish Liver GHR --- p.46 / Chapter 2.7.1 --- SDS-PAGE of Snakehead Fish Liver Membranes --- p.46 / Chapter 2.7.2 --- Transfer of Proteins onto Polyvinylidene Fluoride (PVDF) Membrane --- p.46 / Chapter 2.7.3 --- Antibody Development of PVDF Membrane --- p.47 / Chapter 2.7.4 --- Reagents and Buffers Used --- p.48 / Chapter 2.8 --- Solubilization of Snakehead Fish Liver Membranes and Solubilized Receptor Binding Studies --- p.48 / Chapter 2.8.1 --- Solubilization of Snakehead Fish Liver Membranes --- p.49 / Chapter 2.8.2 --- Solubilized Receptor Binding Assay --- p.49 / Chapter 2.8.3 --- "Solubilization of Snakehead Fish Liver Membranes: Detergent Concentration, pH, Temperature and Time Dependence" --- p.50 / Chapter 2.8.4 --- Solubilized Receptor Binding Study: Interference of Detergent --- p.50 / Chapter 2.8.5 --- Reagents and Buffers Used --- p.51 / Chapter 2.9 --- Purification of Snakehead Fish Liver GHR by Affinity Chromatography --- p.51 / Chapter 2.9.1 --- Affinity Column Preparation --- p.52 / Chapter 2.9.2 --- Snakehead Fish Liver GHR Purification --- p.52 / Chapter 2.9.3 --- Reagents and Buffers Used --- p.53 / Chapter Chapter 3 --- Results: fGH Labeling and Integrity Determination --- p.54 / Chapter 3.1 --- Introduction --- p.54 / Chapter 3.2 --- Experimental Results --- p.55 / Chapter 3.2.1 --- Iodination of fGH --- p.55 / Chapter 3.2.2 --- Integrity of 125I-fGH --- p.55 / Chapter 3.3 --- Discussion --- p.61 / Chapter Chapter 4 --- Results: Membrane Receptor Binding Studies --- p.62 / Chapter 4.1 --- Introduction --- p.62 / Chapter 4.2 --- Experimental Results --- p.63 / Chapter 4.2.1 --- Optimal Conditions for Snakehead Fish Liver Membrane GHR Binding --- p.64 / Chapter 4.2.1.1 --- Association and Dissociation Studies --- p.64 / Chapter 4.2.1.2 --- pH Dependence Study --- p.67 / Chapter 4.2.1.3 --- Membrane Protein Dependence Study --- p.70 / Chapter 4.2.1.4 --- Ca2+ Dependence Study --- p.73 / Chapter 4.2.2 --- Localization and Specificity of Snakehead Fish GHR --- p.76 / Chapter 4.2.2.1 --- Tissue Distribution Study --- p.76 / Chapter 4.2.2.2 --- Displacement and Specificity Studies --- p.78 / Chapter 4.2.3 --- Effects of Sulfhydryl Group Reducing and Oxidizing Agents on GHR Binding --- p.81 / Chapter 4.2.3.1 --- Effect of DTT: Concentration Dependence Study --- p.81 / Chapter 4.2.3.2 --- Effect of PCMBS: Concentration Dependence Study --- p.84 / Chapter 4.2.3.3 --- Scatchard Analysis of Control and PCMBS- pretreated Membranes --- p.86 / Chapter 4.2.3.4 --- Reversibility of the PCMBS Effect --- p.88 / Chapter 4.3 --- Discussion --- p.90 / Chapter 4.3.1 --- Optimal Conditions for Snakehead Fish Liver Membrane GHR Binding --- p.90 / Chapter 4.3.2 --- Localization and Specificity of Snakehead Fish GHR --- p.93 / Chapter 4.3.3 --- Effects of Sulfhydryl Group Reducing and Oxidizing Agents on GHR Binding --- p.96 / Chapter Chapter 5 --- Results: Crosslinking and Western Blot Analysis --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.1.1 --- Crosslinking Studies --- p.101 / Chapter 5.1.2 --- Western Blot Analysis --- p.103 / Chapter 5.2 --- Experimental Results --- p.104 / Chapter 5.2.1 --- Crosslinking Studies --- p.104 / Chapter 5.2.2 --- Western Blot Analysis --- p.105 / Chapter 5.3 --- Discussion --- p.112 / Chapter Chapter 6 --- Results: Affinity Purification of Snakehead Fish Liver GHR --- p.115 / Chapter 6.1 --- Introduction --- p.115 / Chapter 6.1.1 --- Membrane Solubilization and Solubilized GHR Binding Studies --- p.115 / Chapter 6.1.2 --- Affinity Purification of Solubilized Snakehead Fish Liver GHR --- p.116 / Chapter 6.2 --- Exp erimental Results --- p.117 / Chapter 6.2.1 --- Solubilization of Snakehead Fish Liver Membranes --- p.117 / Chapter 6.2.2 --- Interference of Detergents in the Solubilized Receptor Binding Assay --- p.118 / Chapter 6.2.3 --- Affinity Purification of Solubilized Snakehead Fish Liver GHR --- p.120 / Chapter 6.3 --- Discussion --- p.122 / Chapter Chapter 7 --- General Discussion --- p.125 / References --- p.130

Chicken genomic DNA containing 5 kb of the 5$ sp prime$ end of the growth hormone (GH) receptor gene and 12 kb of the region up-stream was cloned and a restriction map was constructed. Using subcloned fragments as probes, a HindIII polymorphism was detected in both egg layer and in meat-type chickens. This polymorphic site was mapped at 7 kb up-stream of the coding region of the GH-receptor gene and a PCR assay for the polymorphism was developed to facilitate genotyping of large numbers of chickens. / Alleles of the GH-receptor gene and the GH gene were analyzed for association with traits in chicken strains of different genetic origins. In egg layers, association was significant for juvenile body weight, egg weight, feed consumption and feed efficiency for egg mass (P $<$ 0.05). In meat-type chickens, the GH-receptor allele associated with high juvenile body weight in egg layers was co-selected with leanness. A comparison of the genotype classes revealed that for several traits there was significant interaction between the GH and GH-receptor genotype. The results indicated that there are variants of the genes of the GH-axis which affect traits in White Leghorns and that the effect of a genetic variation in one gene may depend on the variation in another gene.

No description available.

Kong Pui Shan. / Thesis submitted in: November 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 164-185). / Abstracts in English and Chinese. / Table of Contents --- p.i / List of Figures and Tables --- p.iii / List of Abbreviations --- p.viii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Endocrine system and the pituitary gland in teleosts --- p.1 / Chapter 1.2 --- The biology of black seabream (Acanthopagrus schlegeli) --- p.4 / Chapter 1.3 --- Seasonal variation of gene expression in black seabream pituitary --- p.7 / Chapter 1.4 --- Expressed sequence tag (EST) analysis --- p.9 / Chapter 1.5 --- Basic Local Alignment Search Tool (BLAST) --- p.11 / Chapter 1.6 --- Aim of this study --- p.14 / Chapter Chapter 2: --- Materials and Methods --- p.15 / Chapter 2.1 --- Preliminary EST analysis of black seabream pituitary cDNA libraries --- p.15 / Chapter 2.2 --- Larger scale EST analysis of black seabream pituitary cDNA libraries --- p.19 / Chapter 2.3 --- "Northern blot analysis of gene expression levels of growth hormone (GH), prolactin (PRL), somatolactin (SL) and P-actin in adult female black seabream pituitaries" --- p.23 / Chapter 2.4 --- Real time-PCR analysis of seasonal expression of growth hormone family genes in black seabream pituitary --- p.25 / Chapter 2.5 --- Dot blot (miniarray) analysis of seasonal expression of different genes expressed in black seabream pituitary --- p.28 / Chapter Chapter 3: --- Preliminary EST Analysis of Black Seabream Pituitary cDNA Libraries --- p.30 / Chapter 3.1 --- Results and discussion --- p.30 / Chapter 3.1.1 --- EST analysis of black seabream pituitary cDNA libraries --- p.30 / Chapter 3.1.2 --- Full-length sequencing of different hormone genes of black seabream --- p.47 / Chapter Chapter 4: --- Larger Scale EST Analysis of Black Seabream Pituitary cDNA Libraries --- p.64 / Chapter 4.1 --- Results and discussion --- p.64 / Chapter 4.1.1 --- EST analysis of black seabream pituitary cDNA libraries --- p.64 / Chapter 4.1.2 --- Genes first time found in fish --- p.91 / Chapter Chapter 5: --- Analysis of Black Seabream Pituitary Gene Expressions in Pituitary Collected from Different Months --- p.103 / Chapter 5.1 --- Introduction --- p.103 / Chapter 5.2 --- Northern blot analysis of seasonal variation of growth hormone family genes --- p.105 / Chapter 5.3 --- Seasonal analysis of growth hormone family genes by real time-PCR --- p.110 / Chapter 5.4 --- Sexual maturity of black seabream throughout the reproductive cycle --- p.118 / Chapter 5.5 --- Seasonal analysis of black seabream pituitary genes by dot blot study --- p.119 / Chapter 5.6 --- Conclusion --- p.153 / Chapter Chapter 6: --- General Discussion --- p.154 / References --- p.164

The effect of exogenous somatotropin (eST) on bone changes were evaluated in twenty-nine juvenile horses in race training using radiographs of the third metacarpal obtained over the course of a 128 day research project. A biodensitometer was used to measure bone density, and a micrometer was used to measure cortical bone width and medullary cavity width. Fifteen horses were given daily intramuscular injections of eST and fourteen horses were given daily intramuscular injections of sterile saline and served as the control group. By day 128, the increase in total radiographic bone aluminum equivalence (RBAE) was significantly greater in the eST horses than in the control horses. The increases in RBAE in the dorsal and the medial cortices were greater in the eST horses than in the control horses, but these differences were not significant. There was a trend for changes in the ratio of RBAE in the dorsal to palmar and in the medial to lateral cortices to be greater in the eST than in the control horses. By day 128, the increases in both the dorsal and the medial cortical bone width were significantly greater in the eST than in the control group of horses. The eST horses had a significantly greater decrease in dorsal to palmar medullary cavity width, and increase in dorsal to palmar bone diameter than the control group. A computed index of dorsal cortical bone increased significantly more in the eST than in the control group. The stresses applied to bone are greater in the dorso-medial direction in racehorses. To decrease the strain, bone must either increase in bone mineral density, cortical width, and/or bone diameter. Both the eST group and the control group did make these changes in bone over time, but the eST group more effectively remodeled and modeled bone to increase the strength of the third metacarpal than did the control group of horses. In this research project, exogenous somatotropin treatment had a positive effect on the density and geometry of the third metacarpal. These changes are believed to result in a decreased risk of bone injury to the eST treated horses.

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Thesis (Ph. D.)--Ohio University, June, 1997. / Title from PDF t.p.