Law Tak Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 178-213). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Contents --- p.v / List of Tables --- p.xiii / List of Figures --- p.xiv / Abbreviations --- p.xvii / Preface --- p.1 / Chapter 1 --- All about OTC / Chapter 1.1 --- The OTC Gene --- p.2 / Chapter 1.1.1 --- Mapping --- p.2 / Chapter 1.1.2 --- Isolation --- p.2 / Chapter 1.1.3 --- Structure --- p.2 / Chapter 1.1.4 --- OTC in other species --- p.4 / Chapter 1.2 --- From Gene to Protein --- p.5 / Chapter 1.2.1 --- Synthesis of pOTC --- p.5 / Chapter 1.2.2 --- Transport of pOTC into the mitochondria matrix --- p.6 / Chapter 1.2.3 --- Processing of pOTC into mature OTC --- p.7 / Chapter 1.2.4 --- Assembly of OTC monomer into trimer --- p.8 / Chapter 1.3 --- The OTC Protein --- p.9 / Chapter 1.3.1 --- Structure --- p.9 / Chapter 1.3.2 --- Distribution and location --- p.10 / Chapter 1.3.3 --- OTC in different species and its role --- p.10 / Chapter 1.4 --- Role of OTC in Urea Cycle --- p.14 / Chapter 1.4.1 --- The urea cycle --- p.14 / Chapter 1.4.2 --- Catalytic mechanism of OTC --- p.15 / Chapter 1.5 --- OTCD --- p.17 / Chapter 1.5.1 --- Urea cycle disorders --- p.17 / Chapter 1.5.2 --- OTCD --- p.17 / Chapter 1.5.3 --- Prevalence of OTCD --- p.17 / Chapter 1.6 --- Symptoms of OTCD --- p.19 / Chapter 1.6.1 --- Symptoms --- p.19 / Chapter 1.6.2 --- OTCD in males --- p.20 / Chapter 1.6.3 --- OTCD in females --- p.21 / Chapter 1.7 --- Diagnosis of OTCD --- p.23 / Chapter 1.7.1 --- Diagnosis --- p.23 / Chapter 1.7.2 --- Differential diagnosis --- p.24 / Chapter 1.7.3 --- Carrier testing by protein loading test or allopurinol test --- p.25 / Chapter 1.7.4 --- Prenatal diagnosis --- p.26 / Chapter 1.7.5 --- Preimplantation genetic diagnosis --- p.26 / Chapter 1.8 --- Treatments of OTCD --- p.27 / Chapter 1.8.1 --- Diet and supplements --- p.27 / Chapter 1.8.2 --- Alternative pathway therapy --- p.28 / Chapter 1.8.2.1 --- Sodium phenylbutyrate / phenylacetate --- p.28 / Chapter 1.8.2.2 --- Sodium benzoate --- p.28 / Chapter 1.8.3 --- Dialysis --- p.29 / Chapter 1.8.4 --- Liver transplantation --- p.30 / Chapter 1.8.5 --- Other medications --- p.31 / Chapter 1.8.6 --- Counseling --- p.32 / Chapter 1.8.7 --- Gene therapy --- p.32 / Chapter Part I --- DNA Mutation Analysis of OTCD Patients / Chapter 2 --- Introduction / Chapter 2.1 --- Mutations - Cause of OTCD --- p.35 / Chapter 2.1.1 --- Type of mutations --- p.35 / Chapter 2.1.2 --- How mutations cause OTCD? --- p.35 / Chapter 2.2 --- Properties of OTC Mutations --- p.36 / Chapter 2.2.1 --- Heterogeneity --- p.36 / Chapter 2.2.2 --- Neonatal vs late onset --- p.36 / Chapter 2.2.3 --- Recurrent mutations --- p.36 / Chapter 2.2.4 --- Gonadal mosaicism --- p.36 / Chapter 2.2.5 --- Sporadic mutations --- p.37 / Chapter 2.2.6 --- Polymorphic presentations --- p.37 / Chapter 2.3 --- Polymorphisms in OTC --- p.38 / Chapter 2.4 --- Diagnosis of OTCD by Molecular Genetic Methods --- p.39 / Chapter 2.4.1 --- Restriction fragment length polymorphisms --- p.39 / Chapter 2.4.2 --- Single-strand conformation polymorphism --- p.40 / Chapter 2.4.3 --- Denaturing gradient gel electrophoresis --- p.40 / Chapter 2.4.4 --- Chemical mismatch cleavage --- p.41 / Chapter 2.4.5 --- Linkage analysis --- p.41 / Chapter 2.4.6 --- DNA sequencing --- p.41 / Chapter 2.5 --- Advantages of Molecular Genetic Diagnosis --- p.43 / Chapter 2.5.1 --- Diagnosis --- p.43 / Chapter 2.5.2 --- Carrier testing --- p.43 / Chapter 2.5.3 --- Prenatal diagnosis --- p.43 / Chapter 3 --- Materials & Methods / Chapter 3.1 --- Genomic DNA Extraction from OTCD Patients by QIAamp® DNA Blood Mini Kit --- p.57 / Chapter 3.1.1 --- Materials --- p.58 / Chapter 3.1.1.1 --- Patients --- p.58 / Chapter 3.1.1.2 --- QIAamp® DNA blood mini kit --- p.58 / Chapter 3.1.2 --- Methods --- p.59 / Chapter 3.1.2.1 --- Genomic DNA extraction --- p.59 / Chapter 3.2 --- OTC Exons Amplification by PCR --- p.60 / Chapter 3.2.1 --- Materials --- p.61 / Chapter 3.2.1.1 --- Chemicals and reagents for agarose gel electrophoresis --- p.61 / Chapter 3.2.1.2 --- Chemicals and reagents for PCR --- p.61 / Chapter 3.2.1.3 --- MicroSpińёØ S-400 HR columns --- p.62 / Chapter 3.2.2 --- Methods --- p.63 / Chapter 3.2.2.1 --- Primer design and synthesis --- p.63 / Chapter 3.2.2.2 --- Polymerase chain reaction --- p.63 / Chapter 3.2.2.3 --- PCR product purification --- p.64 / Chapter 3.3 --- DNA sequencing --- p.65 / Chapter 3.3.1 --- Materials --- p.66 / Chapter 3.3.1.1 --- Chemicals and reagents for sequencing --- p.66 / Chapter 3.3.2 --- Methods --- p.67 / Chapter 3.3.2.1 --- Sequencing reaction --- p.67 / Chapter 3.3.2.2 --- Sequencing product purification --- p.67 / Chapter 3.3.2.3 --- Sequencing --- p.67 / Chapter 4 --- Results / Chapter 4.1 --- 7Mutations and 1 Questionable Polymorphism were Identified in OTCD Patients --- p.68 / Chapter 4.11 --- Patient 1 carried an Arg26Gln mutation --- p.70 / Chapter 4.12 --- Patient 2 carried a Leu 111 Pro substitution --- p.72 / Chapter 4.13 --- Patient 3 carried a Glul22Gly mutation --- p.73 / Chapter 4.14 --- Patient 4 carried an Argl 29His mutation --- p.75 / Chapter 4.15 --- Patient 5 carried a Lys 144Term mutation --- p.77 / Chapter 4.16 --- Patient 6 carried a Thrl78Met mutation --- p.78 / Chapter 4.17 --- Patient 7 carried an Asnl98Ile mutation --- p.79 / Chapter 4.18 --- Patient 8 carried an IVS 5 + 1 G→T mutation --- p.80 / Chapter 5 --- Discussion / Chapter 5.1 --- Heterogeneity of OTC mutations --- p.82 / Chapter 5.2 --- Two Novel Mutations were Identified: Asnl98Ile and IVS 5 + 1 G →T --- p.83 / Chapter 5.2.1 --- Asnl98Ile --- p.83 / Chapter 5.2.2 --- IVS5+1G→T --- p.83 / Chapter 5.3 --- "Five Known Mutations were Identified, Four of which were Identified in Chinese for the First Time" --- p.84 / Chapter 5.3.1 --- Arg26Gln --- p.84 / Chapter 5.3.2 --- Glul22Gly --- p.84 / Chapter 5.3.3 --- Argl29His --- p.84 / Chapter 5.3.4 --- Lysl44Term --- p.87 / Chapter 5.3.5 --- Thrl78Met --- p.87 / Chapter 5.4 --- A Questionable Polymorphism was Identified: Leu111 Pro --- p.87 / Chapter 5.5 --- Role of DNA Sequencing as a Direct Diagnosis of OTCD --- p.89 / Chapter 5.6 --- A Questionable Polymorphism: Leu101Phe with Allele Frequency --- p.90 / Chapter Part II --- Protein Expression Study of OTC Mutants / Chapter 6 --- Introduction / Chapter 6.1 --- Site-Directed Mutagenesis --- p.92 / Chapter 6.2 --- Protein Expression Systems --- p.95 / Chapter 6.2.1 --- Bacteria --- p.95 / Chapter 6.2.2 --- Yeast --- p.95 / Chapter 6.2.3 --- Baculovirus --- p.96 / Chapter 6.2.4 --- Mammalian cells --- p.96 / Chapter 6.2.5 --- Cell free expression --- p.97 / Chapter 6.3 --- OTC Enzyme Assay --- p.99 / Chapter 7 --- Materials & Methods / Chapter 7.1 --- Obtaining the OTC Clone --- p.101 / Chapter 7.1.1 --- Materials --- p.102 / Chapter 7.1.1.1 --- Chemicals for preparing low salt LB medium / agar with ZeocinTM --- p.102 / Chapter 7.1.1.2 --- AutoSe´qёØ G-50 --- p.103 / Chapter 7.1.1.3 --- GeneStorm® expression-ready clone --- p.103 / Chapter 7.1.1.4 --- QIAprep® miniprep kit --- p.103 / Chapter 7.1.1.5 --- TempliPhíёØ 100 amplification kit --- p.104 / Chapter 7.1.2 --- Methods / Chapter 7.1.2.1 --- Small-scale preparation of pcDNA3.1/OTC by QIAprep® miniprep kit --- p.105 / Chapter 7.1.2.2 --- Amplification of pcDNA3.1-OTC by TempliPhíёØ --- p.107 / Chapter 7.1.2.3 --- Primer design and synthesis for sequencing --- p.107 / Chapter 7.1.2.4 --- DNA sequencing --- p.108 / Chapter 7.2 --- Entering into the Gateway System --- p.109 / Chapter 7.2.1 --- Materials --- p.110 / Chapter 7.2.1.1 --- Chemicals and reagents for PCR --- p.110 / Chapter 7.2.1.2 --- Chemicals and reagents for preparing LB medium/agar with kanamycin --- p.110 / Chapter 7.2.1.3 --- pENTR Directional TOPO® cloning kit --- p.110 / Chapter 7.2.2 --- Methods --- p.112 / Chapter 7.2.2.1 --- Primer design and synthesis --- p.112 / Chapter 7.2.2.2 --- Polymerase chain reaction --- p.113 / Chapter 7.2.2.3 --- TOPO® cloning reaction --- p.114 / Chapter 7.2.2.4 --- Transformation --- p.114 / Chapter 7.2.2.5 --- Spreading plates --- p.115 / Chapter 7.3 --- Investigation of Subcellular Localization of OTC --- p.116 / Chapter 7.3.1 --- Materials --- p.117 / Chapter 7.3.1.1 --- Chemicals and reagents for cell culture --- p.117 / Chapter 7.3.1.2 --- 4% paraformaldehyde in PBS --- p.118 / Chapter 7.3.1.3 --- Ampicillin --- p.118 / Chapter 7.3.1.4 --- Cells --- p.119 / Chapter 7.3.1.5 --- pcDNA-DEST47 --- p.119 / Chapter 7.3.1.6 --- QuikChange® II XL site-directed mutagenesis kit --- p.119 / Chapter 7.3.1.7 --- LipofectaminéёØ2000 --- p.120 / Chapter 7.3.1.8 --- LR Clonase reaction mix --- p.120 / Chapter 7.3.1.9 --- MitoTracker® Red CMXRos --- p.120 / Chapter 7.3.1.10 --- Nikon Optiphot-2 component microscope --- p.120 / Chapter 7.3.2 --- Methods --- p.121 / Chapter 7.3.2.1 --- Swapping OTC gene from pENTR/D-TOPO to pcDNA-DEST4´7ёØ by LR ClonaséёØ reaction --- p.121 / Chapter 7.3.2.2 --- Site-directed mutagenesis of OTC by QuikChange® II XL site-directed mutagenesis kit --- p.123 / Chapter 7.3.2.2.1 --- Primer design and synthesis --- p.123 / Chapter 7.3.2.2.2 --- Mutant strand synthesis --- p.123 / Chapter 7.3.2.2.3 --- DpnI digestion of parental strand --- p.124 / Chapter 7.3.2.2.4 --- Cloning --- p.124 / Chapter 7.3.2.3 --- Cell culture --- p.125 / Chapter 7.3.2.4 --- Transfection of OTC into Hep3B and HepG2 by LipofectaminéёØ2000 --- p.126 / Chapter 7.3.2.5 --- Staining of mitochondria by MitoTracker® --- p.127 / Chapter 7.3.2.6 --- Fixation of cells --- p.127 / Chapter 7.3.2.7 --- Fluorescence microscopy --- p.128 / Chapter 7.4 --- Investigation of Enzyme Activity of OTC Mutants Expressed in a Cell-free System --- p.129 / Chapter 7.4.1 --- Materials --- p.130 / Chapter 7.4.1.1 --- Chemical and reagents for His-tag protein stain --- p.130 / Chapter 7.4.1.2 --- Chemicals and reagents for OTC enzyme assay --- p.130 / Chapter 7.4.1.3 --- Chemicals and reagents for NuPAGE® Novex pre-cast gel system --- p.131 / Chapter 7.4.1.4 --- Chemicals and reagents for total protein stain --- p.132 / Chapter 7.4.1.5 --- Chemicals and reagents for Tris-Glycine SDS-PAGE --- p.132 / Chapter 7.4.1.6 --- ExpressWayT M plus expression system --- p.134 / Chapter 7.4.1.7 --- pEXPl-DEST vector --- p.134 / Chapter 7.4.1.8 --- β-Gal assay kit --- p.135 / Chapter 7.4.1.9 --- Rapid translation system RTS GroE supplement --- p.135 / Chapter 7.4.2 --- Methods --- p.136 / Chapter 7.4.2.1 --- Swapping OTC gene from pENTR/D-TOPO to pEXPl-DEST by LR Clonase reaction --- p.136 / Chapter 7.4.2.2 --- Site-directed mutagenesis of pEXP1-DEST/OTC by QuikChange® II XL site-directed mutagenesis kit --- p.136 / Chapter 7.4.2.3 --- Cell-free expression by ExpressWayT M plus expression system --- p.137 / Chapter 7.4.2.4 --- Preparation of proteins for SDS PAGE --- p.138 / Chapter 7.4.2.5 --- SDS-PAGE --- p.138 / Chapter 7.4.2.6 --- Staining of His-tagged fusion protein by In VisiońёØ His-tag In-gel stain --- p.139 / Chapter 7.4.2.7 --- OTC enzyme assay --- p.140 / Chapter 7.4.2.7.1 --- Validation of OTC enzyme assay by normal subjects' sera --- p.140 / Chapter 7.4.2.7.2 --- Determination of linear range by OTC and citrulline --- p.140 / Chapter 7.4.2.7.3 --- Enzyme assay of cell-free expressed WT OTC --- p.140 / Chapter 7.4.2.8 --- β -Gal assay --- p.141 / Chapter 8 --- Results / Chapter 8.1 --- The OTC Gene in GeneStorm® Expression-Ready Clone Showed 3 Mismatches with the Published OTC cDNA Sequence (GenBank Accession Number NM_00531) --- p.143 / Chapter 8.2 --- OTC and its Mutants Showed a Mitochondrial Distribution in Hep3B and HepG2 --- p.145 / Chapter 8.2.1 --- pcDNA-DEST47/OTC with desired mutations generated --- p.147 / Chapter 8.2.2 --- OTC and its mutants showed a mitochondrial distribution in Hep3B --- p.149 / Chapter 8.2.3 --- OTC and its mutants showed a mitochondrial distribution in HepG2 --- p.151 / Chapter 8.3 --- Cell-free Expression is not a Feasible Method for Expressing Active OTC --- p.153 / Chapter 8.3.1 --- pEXPl/OTC2 with desired mutations generated --- p.156 / Chapter 8.3.2 --- OTC and its mutants were expressed by the cell-free system as shown in SDS-PAGE analysis --- p.158 / Chapter 8.3.3 --- OTC and its mutants expression were confirmed by His-Tag In-gel stain --- p.160 / Chapter 8.3.4 --- Setup of OTC assay was validated --- p.161 / Chapter 8.3.4.1 --- Validation of OTC enzyme assay with normal subjects' sera --- p.161 / Chapter 8.3.4.2 --- Establishment of the linear relationship of enzymatic reaction in Streptococcus faecalis OTC enzyme assay --- p.163 / Chapter 8.3.4.3 --- Establishment of the linear relationship of colorimetric reaction in OTC enzyme assay --- p.165 / Chapter 8.3.4.4 --- OTC synthesized by cell-free expression system was not active --- p.167 / Chapter 9 --- Discussion / Chapter 9.1 --- "Mutations in OTC, Including Arg26Gln, have no Effect on the Subcellular Localization of OTC in Hep3B and HepG2" --- p.169 / Chapter 9.1.1 --- Arg26Gln --- p.170 / Chapter 9.1.2 --- "Leu 101 Phe, Leu111 Pro, Thrl78Met, and Asnl98Ile" --- p.172 / Chapter 9.2 --- Arg129His Mutant may be unstable --- p.173 / Chapter 9.3 --- Cell-free Expression may not be a Feasible Method for Expression of Active OTC --- p.174 / Bibliography --- p.178 / Appendices / Chapter A.1 --- DNA Sequence of OTC --- p.214 / Chapter A.2 --- Amino Acid Sequence of OTC --- p.215 / Chapter A.3 --- Splicing Sites in OTC --- p.216 / Chapter A.4 --- Multiple Alignments of OTC Protein from 45 Species --- p.217 / Chapter A.5 --- Summary of Patients' Information --- p.218 / Chapter A.6 --- Primers Used in PCR and Sequencing of Patients' Genomic DNA and the Sequence Amplified --- p.223 / Chapter A.7 --- Primers Used in GeneStorm© Expression-Ready Clone and the Sequence Amplified --- p.227 / Chapter A.8 --- Primers Used in pENTR Directional TOPO® Cloning Kit --- p.228 / Chapter A.9 --- Primers Used in Mutagenesis and the Codon Changed --- p.229 / Chapter A.10 --- Vector Information of pcDNA-DEST47 --- p.231 / Chapter A.11 --- Vector Information of pcDNA/GW-47/CAT --- p.232 / Chapter A.12 --- Vector Information of pcDNA3.1/GS --- p.233 / Chapter A.13 --- Vector Information of pEXPl-DEST --- p.234 / Chapter A.14 --- Vector Information of pEXPl-GW/lacZ --- p.235 / Chapter A.15 --- Vector Information of pENTR/D-TOPO --- p.236 / Chapter A.16 --- Vector Information of pIVEX Control Vector GFP --- p.237 / Chapter A.17 --- Genotype of Bacteria Cells --- p.238 / Chapter A.18 --- Details of Markers --- p.239
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324635 |
| Date | January 2004 |
| Contributors | Law, Tak Yin., Chinese University of Hong Kong Graduate School. Division of Chemical Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xix, 239 leaves : ill. (some col.) ; 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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