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

Isogenic clonal fish lines are a powerful resource for aquaculture-related research. Fully inbred individuals, clone founders, can be produced either through mitotic gynogenesis or androgenesis and a further generation from those propagates fully inbred clonal lines. Despite rapid generation, as opposed to successive generation of sibling mating as in mice, the production of such lines may be hampered due to (i) potential residual contribution from irradiated gametes associated with poorly optimised protocols, (ii) reduced survival of clone founders and (iii) spontaneous arisal of meiotic gynogenetics with varying degree of heterozygosity, contaminating fully homozygous progenies. This research set out to address challenges and gain insights into isogenic clonal fish lines development by using double-digest RADseq (ddRADseq) to generate large numbers of genetic markers covering the genome of interest. Analysis of potential contribution from irradiated sperm indicated successful uniparental inheritance in meiotic and mitotic gynogenetics European seabass. Exclusive transmission of maternal alleles was detected in G1 progeny of Atlantic salmon (with a duplicated genome), while G2 progenies presented varying levels of sire contribution suggesting sub-optimal UV irradiation which was undetected previously with 27 microsatellite markers. Identification of telomeric markers in European seabass, with higher recombination frequencies for efficient differentiation of meiotic and mitotic gynogenetics was successful, and a genetic linkage map was generated from this data. One clear case of a spontaneous meiotic gynogenetic fish was detected among 18 putative DH fish in European seabass, despite earlier screening for isogenicity using 11 microsatellite markers. An unidentified larval DNA restriction digestion inhibition mechanism observed in Nile tilapia prevented the construction of SNP-based genetic linkage map. In summary, this study provides strong evidence on efficacy of NGS technologies for the development and verification of isogenic clonal fish lines. Reliable establishment of isogenic clonal fish lines is critical for their utility as a research tool.

Cheung, Chun Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 111-128). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iv / 摘要 --- p.vii / Table of content --- p.ix / List of Figures --- p.xvii / List of Tables --- p.xix / Abbreviations --- p.xxi / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Complementary and Alternative Medicine (CAM) and Traditional Chinese Medicine (TCM) --- p.1 / Chapter 1.2 --- The development of Traditional Chinese Medicine --- p.2 / Chapter 1.3 --- Quality control of Traditional Chinese Medicine --- p.3 / Chapter 1.4 --- Problems of adulteration --- p.5 / Chapter 1.4.1 --- Confusion by common names --- p.5 / Chapter 1.4.2 --- Erroneous and intentional adulteration --- p.6 / Chapter 1.5 --- Authentication of Traditional Chinese Medicine using DNA techniques --- p.7 / Chapter 1.6 --- Crocodile meat --- p.10 / Chapter 1.6.1 --- Crocodile meat as Traditional Chinese Medicine --- p.10 / Chapter 1.6.2 --- Crocodile meat as exotic meat --- p.10 / Chapter 1.6.3 --- Effects of crocodile meat on mice --- p.12 / Chapter 1.6.4 --- Adulteration of crocodile meat in Hong Kong --- p.13 / Chapter 1.6.5 --- Authentication of crocodile meat --- p.14 / Chapter 1.6.5.1 --- SCAR analysis --- p.14 / Chapter 1.6.5.2 --- 12S and 16S ribosomal DNA --- p.14 / Chapter 1.7 --- Fish air-bladder --- p.15 / Chapter 1.7.1 --- Fish air-bladder as Traditional Chinese Medicine --- p.15 / Chapter 1.7.2 --- A case study --- p.16 / Chapter 1.7.3 --- Authentication of fish air-bladder --- p.17 / Chapter 1.8 --- Radix Stellariae --- p.18 / Chapter 1.8.1 --- Stellaria dichotoma L. var. lanceolata Bge --- p.18 / Chapter 1.8.2 --- Radix Stellariae as Traditional Chinese Medicine --- p.19 / Chapter 1.8.3 --- Chemicals in Radix Stellariae and their uses --- p.19 / Chapter 1.8.4 --- Adulteration of Radix Stellariae --- p.20 / Chapter 1.8.5 --- Authentication of Radix Stellariae --- p.21 / Chapter 1.8.5.1 --- Internal Transcribed Spacers (ITS) --- p.21 / Chapter 1.8.5.2 --- trnH-psbA intergenic spacer --- p.23 / Chapter 1.9 --- Objectives --- p.25 / Chapter Chapter 2. --- Materials and Methods --- p.26 / Chapter 2.1 --- Samples used in the study --- p.26 / Chapter 2.1.1 --- Crocodile and monitor lizard samples --- p.26 / Chapter 2.1.2 --- Sequence from NCBI database --- p.26 / Chapter 2.1.3 --- Fish air-bladder samples --- p.30 / Chapter 2.1.4 --- Radix Stellariae samples and samples of related species --- p.33 / Chapter 2.1.5 --- Sequences from NCBI database --- p.33 / Chapter 2.2 --- Reagents and equipments --- p.36 / Chapter 2.2.1 --- Sample preparation and DNA extraction --- p.36 / Chapter 2.2.2 --- Polymerase Chain Reaction --- p.38 / Chapter 2.2.3 --- Agarose gel electrophoresis and Gene Clean --- p.39 / Chapter 2.2.4 --- Cloning --- p.40 / Chapter 2.2.5 --- Cycle sequencing --- p.41 / Chapter 2.3 --- Experimental procedures --- p.42 / Chapter 2.3.1 --- Sample preparation --- p.42 / Chapter 2.3.2 --- DNA extraction --- p.42 / Chapter 2.3.3 --- Polymerase Chain Reaction --- p.44 / Chapter 2.3.4 --- Agarose gel electrophoresis --- p.47 / Chapter 2.3.5 --- Gene Clean --- p.47 / Chapter 2.3.6 --- Cloning --- p.48 / Chapter 2.3.7 --- Cycle sequencing and sequence analyses --- p.51 / Chapter Chapter 3. --- Crocodile meat - Results and Discussion --- p.54 / Chapter 3.1 --- Results --- p.54 / Chapter 3.1.1 --- SCAR analysis --- p.54 / Chapter 3.1.2 --- Sequence analyses --- p.55 / Chapter 3.1.3 --- The dendrograms --- p.56 / Chapter 3.2 --- Discussion --- p.60 / Chapter 3.2.1 --- SCAR as a quick and inexpensive method for the authentication of crocodile meat --- p.60 / Chapter 3.2.2 --- DNA sequencing - A useful tool to identify the source species of the crocodile meat --- p.61 / Chapter 3.2.3 --- Adulteration of crocodile meat in Hong Kong --- p.63 / Chapter 3.2.4 --- Source species of the genuine crocodile meats and the adulterants --- p.63 / Chapter 3.2.5 --- Regulation of labeling of food in Hong Kong --- p.69 / Chapter 3.2.6 --- Source species of the lizard head and tail from AFCD --- p.69 / Chapter 3.3 --- Summary --- p.70 / Chapter Chapter 4. --- Fish air-bladders - Results and Discussion --- p.72 / Chapter 4.1 --- Results --- p.72 / Chapter 4.1.1 --- Identities of sample BH and F1 --- p.73 / Chapter 4.1.2 --- Identity of sample BS --- p.74 / Chapter 4.1.3 --- Identities of samples GD and ZG --- p.74 / Chapter 4.1.4 --- Identity of sample GG --- p.74 / Chapter 4.1.5 --- "Identities of samples HB, HT and SH" --- p.75 / Chapter 4.1.6 --- Identity of sample JL --- p.75 / Chapter 4.1.7 --- Identity of sample MS --- p.76 / Chapter 4.1.8 --- Identity of sample RE --- p.76 / Chapter 4.2 --- Discussion --- p.77 / Chapter 4.2.1 --- Sample RE was confirmed to have originated from rabbit ears --- p.77 / Chapter 4.2.2 --- Identities of the dry fish air-bladders sold in Hong Kong --- p.79 / Chapter 4.2.3 --- Identities of the fresh fish air-bladders sold in Hong Kong --- p.82 / Chapter 4.2.4 --- Limitations of the use of DNA sequences for source species identification --- p.83 / Chapter 4.2.5 --- Variation in prices of fish air-bladders --- p.87 / Chapter 4.3 --- Summary --- p.88 / Chapter Chapter 5. --- Radix Stellariae - Results and Discussion --- p.89 / Chapter 5.1 --- Results --- p.89 / Chapter 5.1.1 --- Sequence analyses --- p.90 / Chapter 5.1.2 --- The dendrograms --- p.90 / Chapter 5.2 --- Discussion --- p.97 / Chapter 5.2.1 --- Identities of the samples obtained from the market --- p.97 / Chapter 5.2.2 --- Identity of sample Sdl4 --- p.97 / Chapter 5.2.3 --- Identities of samples Sd02R and Sd04 --- p.100 / Chapter 5.2.4 --- Myosoton aquaticum in the Stellaria-Myosoton clade --- p.104 / Chapter 5.2.5 --- Medicinal uses of the substitutes of Radix Stellariae --- p.105 / Chapter 5.3 --- Summary --- p.106 / Chapter Chapter 6. --- Conclusion --- p.107 / Reference --- p.111 / Appendix 1. 12S rDNA sequences of crocodilian and Varanus species obtained from NCBI database for sequence analyses --- p.129 / Appendix 2. 16S rDNA sequences of crocodilian and Varanus species obtained from NCBI database for sequence analyses --- p.130 / "Appendix 3. ITS sequences of the species in the genera Arenaria, Myosoton, Silene, and Stellaria obtained from NCBI database for sequence analyses" --- p.131 / Appendix 4. 7rnH-psbA intergenic spacer sequences of Silene species obtained from NCBI database for sequence analyses --- p.132 / Appendix 5. Sequence alignment of 12S rRNA gene sequences of crocodile and monitor lizard samples --- p.133 / Appendix 6. Sequence alignment of 16S rRNA gene sequences of crocodile and lizard samples --- p.141 / Appendix 7. Sequence alignment of coxl sequences of fish air-bladder samples --- p.149 / Appendix 8. Sequence alignment of 12S rRNA gene sequences of fish air-bladder samples --- p.151 / Appendix 9. Sequence alignment of 16S rRNA gene sequences of fish air-bladder samples --- p.153 / Appendix 10. Sequence alignment of coxl region of Vibrio parahaemolyticus and the coxl primers --- p.155 / Appendix 11. Sequence alignment of ITS sequences of Radix Stellariae and related samples --- p.156 / Appendix 12. Sequence alignment of trnH-psbA of Radix Stellariae and related samples --- p.163 / Appendix 13. Search results of coxl sequences of the fish air-bladder samples in BOLD-IDS --- p.167 / Appendix 14. Search results of coxl sequences of the fish air-bladder samples in NCBI nucleotide BLAST --- p.168 / Appendix 15. Search results of 12S rDNA sequences of the fish air-bladder samples in NCBI nucleotide BLAST --- p.169 / Appendix 16. Search results of 16S rDNA sequences of the fish air-bladder samples in NCBI nucleotide BLAST --- p.170 / Appendix 17. Sequence similarities (%) of coxl sequences of the fish air-bladder samples --- p.171 / Appendix 18. Sequence similarities (%) of 12S rDNA sequences of the fish air-bladder samples --- p.172 / Appendix 19. Sequence similarities (%) of 16S rDNA sequences of the fish air-bladder samples --- p.173