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Chum Wing Yan, Winnie. / Thesis submitted in 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 157-178). / Abstracts in English and Chinese. / English Abstract --- p.i / Chinese Abstract --- p.iii / Acknowledgements --- p.v / Abbreviations --- p.vi / Table of Contents --- p.vii / List of Figures --- p.x / List of Tables --- p.xiii / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Life cycle --- p.2 / Chapter 1.3 --- Nutritional value --- p.4 / Chapter 1.4 --- Medicinal value --- p.4 / Chapter 1.4.1 --- Antitumor ability --- p.5 / Chapter 1.4.2 --- Antimicrobial ability --- p.5 / Chapter 1.4.3 --- Hypocholesterolaemic effect --- p.6 / Chapter 1.4.4 --- Anti-viral effect --- p.6 / Chapter 1.4.5 --- Anticaries effects --- p.7 / Chapter 1.6 --- Commercial value --- p.7 / Chapter 1.6.1 --- Biodecolorization --- p.7 / Chapter 1.6.2 --- Bioconversion --- p.7 / Chapter 1.6.3 --- Biodegradation --- p.8 / Chapter 1.6.4 --- Indicator --- p.9 / Chapter 1.7 --- Cultivation --- p.9 / Chapter 1.8 --- Content --- p.10 / Chapter 1.9 --- Transformation --- p.11 / Chapter 1.10 --- Gene regulation for growth and fruiting body development --- p.13 / Chapter 1.11 --- Serial Analysis of Gene Expression --- p.18 / Chapter 1.11.1 --- Introduction --- p.18 / Chapter 1.11.2 --- Principles --- p.20 / Chapter 1.11.3 --- SAGE Application in Cancer and Immunology Studies --- p.22 / Chapter 1.11.4 --- Improvement of SAGE --- p.23 / Chapter 1.11.5 --- Bioinformatics --- p.24 / Chapter 1.12 --- DNA Microarray --- p.27 / Chapter 1.12.1 --- Introduction --- p.27 / Chapter 1.12.2 --- Application --- p.28 / Chapter 1.12.3 --- Method of cDNA Microarray --- p.28 / Chapter Chapter Two --- Serial Analysis of Gene Expression / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Material and Methods --- p.32 / Chapter 2.2.1 --- Mushroom cultivation and RNA extraction --- p.32 / Chapter 2.2.2 --- RNA Quality Estimation --- p.33 / Chapter 2.2.3 --- mRNA Isolation --- p.34 / Chapter 2.2.4 --- Serial Analysis of Gene Analysis (SAGE) --- p.34 / Chapter 2.2.4.1 --- Binding mRNA to magnetic beads for cDNA synthesis --- p.34 / Chapter 2.2.4.2 --- DNA synthesis verification --- p.35 / Chapter 2.2.4.3 --- NlaIII digestion --- p.36 / Chapter 2.2.4.4 --- NlaIII digestion verification --- p.36 / Chapter 2.2.4.5 --- Adapters ligation --- p.37 / Chapter 2.2.4.6 --- Cleaving with tagging enzyme --- p.37 / Chapter 2.2.4.7 --- Ditags creation --- p.38 / Chapter 2.2.4.8 --- PCR optimization and scale-up --- p.38 / Chapter 2.2.4.9 --- Polyacrylamide gel electrophoresis --- p.39 / Chapter 2.2.4.10 --- Eluting DNA from the gel --- p.40 / Chapter 2.2.4.11 --- NlaIII Cleavage and polyacrylamide gel electrophoresis --- p.40 / Chapter 2.2.4.12 --- Concatemers Ligation --- p.41 / Chapter 2.2.4.13 --- Cloning Concatemers into pZErO®-1 --- p.42 / Chapter 2.2.5 --- One Shot® TOP 10 Electrocomp´ёØ E. Coli transformation --- p.42 / Chapter 2.2.6 --- PCR Screening and Sequencing --- p.44 / Chapter 2.2.7 --- Sequence Analysis --- p.44 / Chapter 2.3 --- Results --- p.45 / Chapter 2.3.1 --- RNA Extraction --- p.45 / Chapter 2.3.2 --- cDNA Synthesis --- p.45 / Chapter 2.3.3 --- NlaIII digestion --- p.45 / Chapter 2.3.4 --- PCR amplification --- p.46 / Chapter 2.3.5 --- Gel-purification of the 100bp Ditags --- p.46 / Chapter 2.3.6 --- Isolation of the 26bp Ditags --- p.46 / Chapter 2.3.7 --- Concatemers Generation --- p.47 / Chapter 2.3.8 --- PCR Screening --- p.47 / Chapter 2.3.9 --- The Abundance and Identity of SAGE Tags --- p.48 / Chapter 2.4 --- Discussion --- p.86 / Chapter 2.4.1 --- RNA Extraction --- p.86 / Chapter 2.4.2 --- cDNA Synthesis and NlaIII digestion --- p.86 / Chapter 2.4.3 --- PCP amplification --- p.87 / Chapter 2.4.4 --- SAGE Tags Analysis --- p.88 / Chapter Chapter Three --- Microarray / Chapter 3.1 --- Introduction --- p.96 / Chapter 3.2 --- Materials and Methods --- p.97 / Chapter 3.2.1 --- Microarray chip preparation --- p.97 / Chapter 3.3.2 --- Sample preparation --- p.97 / Chapter 3.2.3 --- cDNA Synthesis and Sample labeling --- p.98 / Chapter 3.2.4 --- cDNA Purification --- p.99 / Chapter 3.2.5 --- cDNA analysis --- p.99 / Chapter 3.2.6 --- Array Hybridization --- p.102 / Chapter 3.2.6.1 --- Sample Preparation --- p.102 / Chapter 3.2.6.2 --- Hybridization Procedure --- p.102 / Chapter 3.2.7 --- Stringency Washes --- p.103 / Chapter 3.2.8 --- Detection with TSA --- p.103 / Chapter 3.2.9 --- Scanning and Analysis --- p.105 / Chapter 3.3 --- Results --- p.109 / Chapter 3.4 --- Discussion --- p.120 / Chapter Chapter Four --- Full Length Sequencing / Chapter 4.1 --- Introduction --- p.124 / Chapter 4.2 --- Material and Methods --- p.124 / Chapter 4.3 --- Results and Discussion --- p.125 / Chapter Chapter Five --- General Discussion --- p.149 / Appendix --- p.155 / References --- p.157

Duan Meijuan. / Thesis submitted in: August 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 170-194). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / Chinese abstract --- p.vi / List of Tables --- p.viii / List of Figures --- p.ix / List of Abbreviations --- p.xiv / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Literature Review --- p.3 / Chapter 2.1 --- Hybrid rice: Genetics and breeding --- p.3 / Chapter 2.1.1 --- Classification in rice --- p.5 / Chapter 2.1.2 --- Heterosis in rice --- p.6 / Chapter 2.1.2.1 --- Performance of heterosis in rice --- p.6 / Chapter 2.1.2.2. --- Genetic mechanism of heterosis in rice --- p.7 / Chapter 2.1.3 --- Utilization of heterosis in rice --- p.9 / Chapter 2.2 --- Grain quality in rice --- p.11 / Chapter 2.2.1 --- Composition of rice grain quality --- p.11 / Chapter 2.2.1.1 --- Appearance quality --- p.11 / Chapter 2.2.1.2 --- Milling quality --- p.11 / Chapter 2.2.1.3 --- Nutritional quality --- p.12 / Chapter 2.2.1.4 --- Cooking and eating quality --- p.17 / Chapter 2.2.2 --- Genetic and breeding for high nutritional quality rice --- p.17 / Chapter 2.2.3 --- "Structural, physiological and biochemical changes during rice seed development" --- p.18 / Chapter 2.3 --- Molecular biological characteristics of rice seed storage protein --- p.20 / Chapter 2.3.1 --- "Property, classification and structure" --- p.20 / Chapter 2.3.1.1 --- Property and classification --- p.20 / Chapter 2.3.1.2 --- Composition structure --- p.20 / Chapter 2.3.1.2.1 --- Glutelin --- p.21 / Chapter 2.3.1.2.2 --- Prolamin --- p.22 / Chapter 2.3.1.2.3 --- Globulin and Albumin --- p.23 / Chapter 2.3.2 --- "Structure, expression and regulation of genes encoding rice seed storage protein genes" --- p.24 / Chapter 2.3.2.1 --- Structure --- p.24 / Chapter 2.3.2.1.1 --- Glutelin gene family --- p.24 / Chapter 2.3.2.1.2 --- Prolamin gene family --- p.26 / Chapter 2.3.2.1.3 --- Albumin and globulin gene family --- p.27 / Chapter 2.3.2.2 --- Expression of storage proteins in rice seed development --- p.28 / Chapter 2.3.2.3 --- Regulation of expression of seed storage protein genes --- p.29 / Chapter 2.3.2.3.1 --- Regulation at transcriptional level --- p.29 / Chapter 2.3.2.3.2 --- Regulation at post-transcriptional level --- p.31 / Chapter 2.3.2.3.3 --- Regulation at translational level --- p.31 / Chapter 2.3.3 --- "Synthesis, processing and deposition of rice seed storage proteins" --- p.32 / Chapter 2.4 --- Molecular characteristics of starch in rice grain --- p.34 / Chapter 2.4.1 --- Property of rice starch --- p.34 / Chapter 2.4.2 --- Starch biosynthesis in rice --- p.37 / Chapter 2.4.3 --- Enzymes involved in starch biosynthesis in rice --- p.39 / Chapter 2.4.3.1 --- ADP-glucose pyrophosphorylase (AGPase) --- p.39 / Chapter 2.4.3.2 --- Soluble starch synthase (SSS) --- p.41 / Chapter 2.4.3.3 --- Granular-bound starch synthase (GBSS) --- p.42 / Chapter 2.4.3.4 --- Starch branching enzyme (SBE) --- p.44 / Chapter 2.4.3.5 --- Starch debranching enzyme (SDE) --- p.46 / Chapter 2.5 --- Aspartate family amino acid biosynthetic pathway in rice --- p.48 / Chapter 2.5.1 --- Introduction --- p.48 / Chapter 2.5.2 --- Two key regulatory enzymes involved in lysine synthesis pathway --- p.50 / Chapter 2.5.2.1 --- Aspartate kinase (AK) --- p.50 / Chapter 2.5.2.2 --- Dihydrodipicolinate synthase (DHPS) --- p.51 / Chapter 2.5.2.3. --- Regulation of lysine and the other end products of AK pathway --- p.52 / Chapter 2.6 --- Proteomics in plants --- p.53 / Chapter 2.7 --- Approaches for grain quality improvement in rice --- p.56 / Chapter 2.7.1 --- Improvement of nutrition quality --- p.56 / Chapter 2.7.2 --- Improvement of eating and cooking quality --- p.57 / Chapter 2.8 --- Objectives of my project --- p.58 / Chapter Chapter 3. --- Materials and Methods --- p.60 / Chapter 3.1 --- Materials --- p.60 / Chapter 3.1.1 --- Chemicals --- p.60 / Chapter 3.1.2 --- Apparatus and commercial kits --- p.60 / Chapter 3.1.3 --- Plant materials --- p.61 / Chapter 3.1.4 --- DNA sequencing --- p.61 / Chapter 3.1.5 --- Software --- p.61 / Chapter 3.2 --- Methods --- p.62 / Chapter 3.2.1 --- Search for protein and DNA sequences of all genes --- p.62 / Chapter 3.2.1.1 --- Genes encoding rice glutelin family --- p.62 / Chapter 3.2.1.2 --- Genes encoding rice prolamin family --- p.63 / Chapter 3.2.1.3 --- Genes encoding rice albumin family --- p.63 / Chapter 3.2.1.4 --- Genes encoding rice globulin family --- p.64 / Chapter 3.2.1.5 --- Genes encoding rice starch synthesis enzymes --- p.64 / Chapter 3.2.2 --- Alignment of homologous DNA sequence between family member genes --- p.65 / Chapter 3.2.2.1 --- Seeds storage protein gene families of rice seeds --- p.65 / Chapter 3.2.2.2 --- Rice starch synthase gene families --- p.67 / Chapter 3.2.3 --- Primer design --- p.69 / Chapter 3.2.4 --- Collection of developing hybrid rice seeds --- p.71 / Chapter 3.2.5 --- Total RNA extraction --- p.75 / Chapter 3.2.6 --- Quantification of total RNA and determination of internal control --- p.75 / Chapter 3.2.7 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.77 / Chapter 3.2.8 --- Northern blot analysis --- p.78 / Chapter 3.2.9 --- DNA sequencing --- p.79 / Chapter 3.2.10 --- Protein extraction --- p.80 / Chapter 3.2.10.1 --- Extraction of four kinds of storage proteins --- p.80 / Chapter 3.2.10.2 --- Extraction of the Wx protein --- p.81 / Chapter 3.2.11 --- Tricine SDS-PAGE --- p.82 / Chapter 3.2.12 --- "Determination of crude protein and amylose content in P64S,9311 and F1 hybrid" --- p.83 / Chapter 3.2.12.1 --- Determination of crude protein --- p.83 / Chapter 3.2.12.2 --- Determination of amylose content --- p.84 / Chapter 3.2.13 --- Two-dimension gel electrophoresis --- p.85 / Chapter 3.2.13.1 --- Clean up of protein sample for 2-D gel --- p.85 / Chapter 3.2.13.2 --- Quantification of protein samples --- p.86 / Chapter 3.2.13.3 --- First-dimension IEF (isoelectric focusing) --- p.86 / Chapter 3.2.13.4 --- IPG strips equilibration --- p.87 / Chapter 3.2.13.5 --- Second-dimension SDS PAGE --- p.87 / Chapter 3.2.13.6 --- Silver staining of 2-D gel --- p.88 / Chapter 3.2.14 --- MALDI-ToF mass spectrometry (Matrix Assisted Laser Desorption Ionization-Time of Flight) --- p.88 / Chapter 3.2.14.1 --- Sample destaining --- p.88 / Chapter 3.2.14.2 --- In-gel digestion with trypsin enzyme --- p.89 / Chapter 3.2.14.3 --- Desalination of the digested sample with Zip Tip --- p.90 / Chapter 3.2.14.4 --- Mass spectrometry --- p.90 / Chapter Chapter 4. --- Results --- p.91 / Chapter 4.1 --- Quantification of the total RNA from developing seeds at different developingstages --- p.91 / Chapter 4.2 --- Determination of internal control --- p.92 / Chapter 4.3 --- DNA sequence analysis --- p.95 / Chapter 4.4 --- Profiling the expression of genes encoding rice seed storage proteins --- p.97 / Chapter 4.4.1 --- The glutelin genes --- p.97 / Chapter 4.4.1.1 --- The Gtl (GluA-2) gene --- p.100 / Chapter 4.4.1.2 --- The Gt2 (GluA-1) gene --- p.100 / Chapter 4.4.1.3 --- The Gt3 (GluA-3) gene --- p.101 / Chapter 4.4.1.4 --- Comparison of the expression profiles of GluA subfamily genes --- p.101 / Chapter 4.4.1.5 --- The GluB-1 gene --- p.101 / Chapter 4.4.1.6 --- The GluB-2 gene --- p.102 / Chapter 4.4.1.7 --- The GluB-4 gene --- p.102 / Chapter 4.4.1.8 --- Comparing of the expression profiles of GluB subfamily genes --- p.102 / Chapter 4.4.1.9 --- Profiling the expression of glutilin family genes in developing hybrid rice seeds --- p.103 / Chapter 4.4.1.10 --- Profiling glutelin gene expression in developing seeds at protein level --- p.103 / Chapter 4.4.2 --- Profiling the expression of genes encoding prolamin familyin developing hybrid rice seeds --- p.105 / Chapter 4.4.2.1 --- The 10-kDa prolamin gene --- p.105 / Chapter 4.4.2.2 --- The RP5 gene --- p.108 / Chapter 4.4.2.3 --- The RP6 gene --- p.108 / Chapter 4.4.2.4 --- The Prol 7 gene --- p.109 / Chapter 4.4.2.5 --- The Prol 14 gene --- p.109 / Chapter 4.4.2.6 --- The Prol 17 gene --- p.109 / Chapter 4.4.2.7 --- Expression profiles of prolamin family genes --- p.110 / Chapter 4.4.2.8 --- Expression profiles of prolamin genes in developing hybrid rice seeds at protein level --- p.111 / Chapter 4.4.3 --- Profiling the expression of genes encoding globulin familyin developing hybrid rice seed --- p.113 / Chapter 4.4.3.1 --- The 26-kDa globulin (alpha-globulin) gene --- p.113 / Chapter 4.4.3.2 --- The globulin 1 gene --- p.113 / Chapter 4.4.3.3 --- The globulin 2 gene --- p.115 / Chapter 4.4.3.4 --- The Low molecular weight (LMW) globulin gene --- p.115 / Chapter 4.4.3.5 --- Profiling the expression of the globulin family genes --- p.115 / Chapter 4.4.3.6 --- Expression profiles of globulin proteins in developing hybrid rice seeds at protein level --- p.117 / Chapter 4.4.4 --- Profiling the expression of genes encoding rice albumin familyin developing hybrid rice seeds --- p.118 / Chapter 4.4.4.1 --- The RA5 gene --- p.118 / Chapter 4.4.4.2 --- The RA 14 gene --- p.119 / Chapter 4.4.4.3 --- The RA 17 gene --- p.119 / Chapter 4.4.4.4 --- Profiling the expression of the albumin family genes --- p.121 / Chapter 4.4.4.5 --- Albumin gene expression in developing hybrid rice seeds at protein level --- p.121 / Chapter 4.4.5 --- Comparison of expression pattern of all genes encoding rice seed storage proteins in developing hybrid rice seeds --- p.123 / Chapter 4.4.6 --- Profiling the total proteins in developing hybrid rice seeds --- p.126 / Chapter 4.5 --- Profiling the expression of genes encoding rice starch synthasesin developing hybrid rice seeds --- p.127 / Chapter 4.5.1 --- Rice ADP-glucose pyrophosphorylase (AGPase) genes --- p.127 / Chapter 4.5.1.1 --- The AGPase large subunit gene --- p.127 / Chapter 4.5.1.2 --- The AGPase small subunit gene --- p.127 / Chapter 4.5.2 --- "The Wx (Granule bound starch synthase, GBSS) gene" --- p.129 / Chapter 4.5.3 --- Genes encoding rice SSS (Soluble starch synthase) family --- p.132 / Chapter 4.5.3.1 --- The SSS1 gene --- p.132 / Chapter 4.5.3.2 --- The SSS II-1 gene --- p.132 / Chapter 4.5.3.3 --- The SSS II-2 gene --- p.132 / Chapter 4.5.3.4 --- The SSS II-3 gene --- p.135 / Chapter 4.5.3.5 --- The SSS III-2 gene --- p.135 / Chapter 4.5.3.6 --- The SSS IV-1 gene --- p.135 / Chapter 4.5.3.7 --- The SSS IV-2 gene --- p.135 / Chapter 4.5.3.8 --- Profiling the expression of SSS family genes --- p.136 / Chapter 4.5.4 --- Genes encoding rice starch branching enzyme (SBE) family --- p.138 / Chapter 4.5.4.1 --- The SBE-1 gene --- p.138 / Chapter 4.5.4.2 --- The SBE-3 gene --- p.138 / Chapter 4.5.4.3 --- The SBE-4 gene --- p.138 / Chapter 4.5.4.4 --- Profiling the expression of SBE family genes --- p.140 / Chapter 4.5.5 --- Genes encoding rice starch debranching enzyme (SDE) family --- p.141 / Chapter 4.5.5.1 --- The isoamylase gene --- p.141 / Chapter 4.5.5.2 --- The pullulanase gene --- p.141 / Chapter 4.5.5.3 --- Difference between isoamylose and pullulanase --- p.141 / Chapter 4.5.6 --- Comparison of the expression patterns of genes encoding the enzymes involved in starch synthesis in developing hybrid rice seeds --- p.143 / Chapter 4.6 --- Profiling the expression of genes encoding aspartate family amino acid biosynthetic pathway in rice in developing hybrid rice seeds --- p.145 / Chapter 4.6.1 --- Rice AK (aspartate kinase) gene --- p.145 / Chapter 4.6.2 --- The DHPS gene --- p.145 / Chapter 4.7 --- Two-dimension gel electrophoresis and MALDI-ToF seed proteins analysis of rice --- p.147 / Chapter Chapter 5. --- Discussion --- p.152 / Chapter 5.1 --- Super hybrid rice as experimental material and its significance --- p.152 / Chapter 5.2 --- RT-PCR and northern blotting as methods to profile gene expression --- p.153 / Chapter 5.3 --- Regulation of genes related to nutritional quality in rice --- p.155 / Chapter 5.3.1 --- Storage protein genes --- p.155 / Chapter 5.3.2 --- Lysine synthesis enzymes --- p.158 / Chapter 5.4 --- Regulation of genes related to cooking and eating quality in rice --- p.159 / Chapter 5.5 --- Heredity of genes expression in F1 hybrid --- p.161 / Chapter 5.6 --- Application of 2-D gel electrophoresis --- p.162 / Chapter 5.7 --- Future perspectives --- p.163 / Chapter Chapter 6. --- Conclusion --- p.164 / References --- p.170

Zhang Junjun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 147-160). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Statement from Author --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xiv / List of Figures --- p.xvi / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction and Literature Review --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Literature Review --- p.2 / Chapter 1.2.1 --- Rice as a Model Monocot Cereal --- p.2 / Chapter 1.2.1.1 --- Genome Size --- p.3 / Chapter 1.2.1.2 --- Synteny --- p.3 / Chapter 1.2.1.3 --- Well-mapped Genome --- p.4 / Chapter 1.2.1.4 --- Amenable to Transformation --- p.4 / Chapter 1.2.2 --- Rice Genome Sequencing --- p.4 / Chapter 1.2.3 --- Rice Functional Genomics --- p.6 / Chapter 1.2.3.1 --- International Collaboration on Rice Functional Genomics --- p.6 / Chapter 1.2.3.2 --- Recent Progress on Rice Functional Genomics in China --- p.7 / Chapter 1.2.4 --- Large-scale Expressed Sequenced Tag (EST) Analysis --- p.8 / Chapter 1.2.4.1 --- Introduction --- p.8 / Chapter 1.2.4.2 --- Large-scale EST Analysis vs. SAGE and MPSS: Advantages and Shortcomings --- p.12 / Chapter 1.2.4.3 --- Large-scale Rice EST analysis --- p.14 / Chapter 1.2.4.4 --- Seed Specific Large-scale EST Analysis --- p.16 / Chapter 1.2.4.5 --- Bioinformatics Tools Involved --- p.17 / Chapter 1.2.5 --- Profiling Gene Expression using cDNA Microarray --- p.20 / Chapter 1.2.5.1 --- Introduction --- p.20 / Chapter 1.2.5.2 --- Advantages of cDNA Microarray in Gene Expression Profiling --- p.22 / Chapter 1.2.5.3 --- Profiling Gene Expression by cDNA Microarrays --- p.24 / Chapter 1.2.5.4 --- Profiling Seed-specific Gene Expression --- p.26 / Chapter 1.2.6 --- Overview of Current Information on the Biosynthesis and Gene Regulation of Major Components during Rice Seed Formation --- p.30 / Chapter 1.2.6.1 --- Starch --- p.30 / Chapter 1.2.6.2 --- Storage Proteins --- p.38 / Chapter 1.2.6.3 --- Lipids --- p.42 / Chapter Chapter 2 --- Materials and Methods --- p.44 / Chapter 2.1 --- Materials --- p.44 / Chapter 2.1.1 --- Chemicals --- p.44 / Chapter 2.1.2 --- Facilities and Instruments --- p.45 / Chapter 2.1.3 --- Commercial Kits --- p.46 / Chapter 2.1.4 --- Oligonucleotides --- p.47 / Chapter 2.1.5 --- Plant Materials --- p.50 / Chapter 2.1.6 --- Bacterial Strains --- p.52 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- mRNA Isolation --- p.52 / Chapter 2.2.2 --- Construction of cDNA Library --- p.53 / Chapter 2.2.2.1 --- Construction of cDNA Library in Phage --- p.53 / Chapter 2.2.2.2 --- Construction of cDNA Library in E.coli --- p.56 / Chapter 2.2.3 --- Screening of cDNA Library --- p.62 / Chapter 2.2.3.1 --- In situ Hybridization --- p.62 / Chapter 2.2.3.2 --- Probe Synthesis --- p.63 / Chapter 2.2.3.3 --- Hybridization and Detection --- p.64 / Chapter 2.2.4 --- Single-pass Sequencing of cDNAs --- p.65 / Chapter 2.2.5 --- Sequence Analysis --- p.67 / Chapter 2.2.6 --- BLAST Search --- p.71 / Chapter 2.2.7 --- Contig Analysis --- p.71 / Chapter 2.2.8 --- Database Management --- p.72 / Chapter 2.2.9 --- Selection Criteria for ESTs in Different Pathways --- p.72 / Chapter 2.2.10 --- Construction of Super Hybrid Rice Seed cDNA Microarray --- p.75 / Chapter 2.2.11 --- "Probe Synthesis, Microarray Hybridization and Detection" --- p.75 / Chapter 2.2.12 --- Quality Test of External Control --- p.78 / Chapter 2.2.13 --- Scanning of Microarray --- p.79 / Chapter 2.2.14 --- Analysis of Microarray Data --- p.79 / Chapter 2.2.15 --- Normalization --- p.79 / Chapter 2.2.16 --- Northern Blot Analysis --- p.80 / Chapter Chapter 3 --- Results and Discussion --- p.82 / Chapter 3.1 --- cDNA Libraries --- p.82 / Chapter 3.1.1 --- cDNA Library in Phage --- p.82 / Chapter 3.1.2 --- cDNA Library in E.coli --- p.84 / Chapter 3.2 --- Statistic Data of Screening and Single-pass Sequencing --- p.87 / Chapter 3.3 --- Genes Expressing in Developing Rice Seeds --- p.89 / Chapter 3.4 --- Novel ESTs in the Seed EST Set --- p.94 / Chapter 3.5 --- Unique Genes in the EST Set from the Phage cDNA Library --- p.94 / Chapter 3.6 --- Mapping ESTs onto Rice Genome --- p.95 / Chapter 3.7 --- The Most Abundant Transcripts Reflected by EST Redundancy --- p.98 / Chapter 3.8 --- ESTs and Representive Genes in Different Pathways --- p.102 / Chapter 3.9 --- ESTs Involved in Carbon Flows --- p.106 / Chapter 3.10 --- Microarray Configuration and Contents --- p.109 / Chapter 3.11 --- Quantification of Total RNA from Seeds at 5 Developing Stages --- p.110 / Chapter 3.12 --- Quality Test of External Control --- p.111 / Chapter 3.13 --- Scanning of Array Hybridization --- p.113 / Chapter 3.14 --- Reproducibility of Technical Duplicates --- p.116 / Chapter 3.15 --- Summary of Gene Expression Profiles in Developing Super Hybrid Rice Seeds --- p.117 / Chapter 3.16 --- Expression Profiles of Major Storage Compounds in Rice Seeds --- p.119 / Chapter 3.16.1 --- Expression profiles of storage protein genes --- p.119 / Chapter 3.16.2 --- Expression Profiles of Starch Synthesis Related Genes --- p.122 / Chapter 3.16.3 --- Expression Profiles of Lipid Synthesis Related Genes --- p.124 / Chapter 3.16.4 --- General Expression Patterns of Major Storage Compounds --- p.126 / Chapter 3.18 --- General Discussion --- p.131 / Chapter 3.18.1 --- Cautions when Interpreting the EST Data --- p.131 / Chapter 3.18.2 --- Comparison of EST Data Sets of Developing Seeds between Arabidopsis and Rice --- p.131 / Chapter 3.18.3 --- Comparison of cDNA Library Construction by Two Methods --- p.135 / Chapter 3.18.4 --- Comparison of Different Normalization Methods --- p.138 / Chapter 3.18.5 --- Comparison between Microarray and Northern Data --- p.140 / Chapter Chapter 4 --- Conclusion --- p.142 / Chapter Chapter 5 --- Future Perspectives --- p.145 / Reference Lists --- p.147

Polyreactive immunoglobulins (Ig) exhibit a capacity to recognise multiple, structurally dissimilar antigens through a single combining site. This characteristic differentiates these Igs from monoreactive Igs which bind to a single antigen, usually with high specificity and affinity. Chronic B lymphocytic leukaemia (B CLL) is a malignancy identified by the incessant accumulation, in the peripheral circulation, of B lymphocytes of a mature and resting morphology. B CLL malignant cells generally express both surface IgM and the pan T cell antigen CD5. Moreover, the IgM on the surface of these CD5 positive B CLL cells is frequently polyreactive. This thesis examines the structural diversity found in the combining sites of B CLL derived Igs in an attempt to elucidate the structural basis of polyreactive antigen binding displayed by a significant proportion of human Igs. The genes encoding the variable (V) domains of five B CLL derived IgM antibodies (Bel, Tre, Yar, Hod and Jak) were cloned and sequenced (Chapter Two). When the light chain V domain genes were aligned with the closest germline VL and JL coding DNA sequences it was determined that there was either a complete absence of somatic mutation (Tre, Yar and Jak) or a minimal number of mutations (Bel and Hod) present in the rearranged VL domain genes. A remarkable fidelity in the splicing of VL to JL genes was noted suggesting that the diversity, normally introduced through variability of splicing VL to JL, is reduced in Igs expressed by B CLL cells. Furthermore, the markedly reduced primary structural diversity was highlighted when two of the VL domain genes (Yar and Hod) were found to be different in sequence by only four nucleotides and two amino acids. The heavy chain V domain genes of the same five Igs were sequenced in another study (Brock, 1995), however, it was interesting to analyse the sequences of the VH domain genes and compare them with the VL domain genes. The naive or gerrnline nature of the B CLL antibodies was reflected in the VH genes by either an absence or a low frequency of mutations within these sequences compared with germline immunoglobulin gene sequences. No obvious conserved motif, which could be related to polyreactivity, was observed when the primary protein sequence was analysed for distribution of identical or similar amino acids. Thus, homology modelling was used to construct three-dimensional models of the Fv (VL-VH) portions of the five B CLL IgM molecules to examine the structures of the combining sites of these Igs (Chapter Three). Framework regions were constructed using X-ray coordinates taken from highly hon~ologous human variable domain structures. Complementarity determining regions (CDR) were predicted by grafting loops, taken from known Ig structures, onto the Fv framework models. The CDR templates were selected, where possible, to be of the same length and of high residue identity or similarity. If a single template CDR was not appropriate to model a particular CDR the loop was built from loop sterns of known conformation, followed by chain closure with a p-turn. Template models were refined using standard molecular mechanics simulations. The binding sites were either relatively flat or contained a deep cavity at the VL-VH domain interface. Further differences in topology were the result of some CDR loops protruding into the solvent. Examination of the electrostatic molecular surface did not reveal a common structural feature within the binding sites of the five polyreactive Fv. While two of the binding cavities were positively charged the other three structures displayed either negatively charged or predominantly hydrophobic combining sites. These findings suggested that a diversity of structural mechanisms are involved in polyreactive antigen binding. Rcsidues within CDRs which have aromatic side-chains and are partially exposed to solvent were distributed across large regions of the combining sites. It is possible that these aromatic residues are responsible for the conserved binding to mouse Igs observed (Chapter Two) for the B CLL derived polyreactive IgM molecules. Two Fv molecules (Be1 and Tre) were cloned as dicistronic constructs, into the bacterial expression vector pFLAG. The expression of the Fvs was fully characterised and unfortunately the VL and VH of Be1 and Tre Igs did not associate in an appropriate manner to yield large quantities of purified Fv (Chapter Four). Expression of correctly folded and stabilised fragments of human polyreactive immunoglobulins would enable the structural basis for the polyreactive binding phenomenon to be fully explored using protein crystallography.

"October 2002" Bibliography: leaves 159-198. This thesis describes the mapping of the breakpoints of three different chromosome rearrangements, all involving 1p36, in acute myeloid leukaemia (AML) patients, and an investigation into the molecular outcomes of these rearrangements.

The PsbL, PsbM and PsbT subunits of photosystem II (PSII) are single-helix membrane-spanning proteins found at the monomer-monomer interface that may stabilize the dimeric complex. This study has characterised strains of Synechocystis sp. PCC 6803 where psbL, psbM and psbT have been interrupted by the insertion of antibiotic-resistance cassettes. The [Delta]PsbL strain exhibited slowed growth that correlated with a disruption in PSII assembly leading to an accumulation of CP43-less PSII monomers. Moreover, the [Delta]PsbL:[Delta]PsbM and [Delta]PsbL:[Delta]PsbT double mutants were not photoautotrophic. In contrast, the [Delta]PsbM and [Delta]PsbT strains grew photoautotrophically and supported oxygen evolution, albeit at reduced rates compared to wild type. S-state analyses showed that the removal of PsbM or PsbT did not affect the donor side reactions of PSII, which includes the oxidation of water, however, the removal of PsbT impaired electron flow between Q[A] and Q[B] on the acceptor side of PSII. Blue-Native PAGE revealed that removal of either PsbM or PsbT was insufficient to entirely disrupt dimer formation; however, the combined removal of PsbM and PsbT resulted in the predominance of monomeric forms of PSII in the [Delta]PsbM:[Delta]PsbT strain. Under high light (2 mE m⁻� s⁻� at 30�C), [Delta]PsbM and [Delta]PsbT cells were considerably more susceptible to photoinactivation than wild type; however, they were able to fully recover in a protein synthesis-dependent manner when returned to moderate light levels (0.03 mE m⁻� s⁻�). A requirement for Psb27 was found in the protein-synthesis-dependent recovery of photoinactivated [Delta]PsbT cells. More significantly, an absolute functional requirement was found for Psb27 in the [Delta]PsbM strain, where functional PSII complexes are not assembled in the absence of Psb27. These results suggest that Psb27 is critical for PSII assembly in the absence of PsbM, and also for the protein-synthesis-dependent recovery of PSII in the absence of PsbT. Moreover, in addition to Psb27, the PsbU subunit of the oxygen-evolving complex was also found to be an absolute functional requirement in the [Delta]PsbM strain, where functional PSII centres are not assembled when both PsbM and PsbU are absent. It appears, therefore, that PsbM has crucial functional interactions with specific extrinsic proteins located in the vicinity of the oxygen-evolving complex. Interestingly, the [Delta]PsbM strain was also found to have a high susceptibility to suppressor mutations, indicating it has important functional roles in the cyanobacterial cell.

Selected strains of Streptococcus equi subsp. zooepidemicus produce a bacteriocin-like inhibitory substance called zoocin A. Zoocin A has bacteriolytic activity against closely related species of streptococci, including the etiological agents of dental caries and Streptococcus pyogenes. Zoocin A functions to hydrolyse the peptidoglycan of bacterial cell walls. Producer-cell immunity to zoocin A is encoded by zif, the zoocin A immunity factor. The zooA-zif gene locus is chromosomally encoded and flanked by two transposon-like sequences with significant similarity to the lss-lif locus encoding lysostaphin in Staphylococcus simulans. This suggested the possibility that the zooA-zif locus was acquired by S. equi subsp. zooepidemicus through a horizontal gene transfer event. Studies conducted to examine this hypothesis were unable to establish the mechanism by which zooA-zif was acquired. Examination of 24 S. equi subsp. zooepidemicus strains using random amplified polymorphic DNA analysis and pulse field gel electrophoresis showed that the species was highly heterogeneous. Examination of the 24 strains using Southern hybridisation revealed only three S. equi subsp. zooepidemicus strains contained the zooA and zif genes; however the partial open reading frames (flaR and rgg-like) flanking zooA and zif appeared to be conserved in all tested strains. Long template PCR was used to examine this chromosomal area containing the zooA and zif genes in 24 S. equi subsp. zooepidemicus strains. These results, along with comparison of sequence data from two zoocin A producing strains, and analysis of the sequenced S. equi subsp. zooepidemicus H70 and S. equi subsp. equi 4047 genomes showed the area surrounding the zooA-zif locus to be one of considerable genetic variation substantiating the hypothesis that zooA-zif were acquired via horizontal gene transfer. Studies on zoocin A production suggested that zooA expression was under carbon catabolite regulation, (CCR). In the Gram-positive bacteria CCR, is mediated by the carbon catabolite control protein (CcpA). CcpA is a DNA binding protein that can negatively regulate transcription of its target gene by binding to a catabolite-responsive element (CRE). This study reports the regulation of zif and zooA expression in response to cellular growth on different carbon sources, the presence of a ccpA-like gene in the genome of S. equi subsp. zooepidemicus, the presence of a CRE sequence in the promoter region of zooA, and the effect of a ccpA deletion on zooA expression. Zoocin A activity, zooA and zif mRNA transcript levels, and zooA and zif reporter gene activities were monitored during growth of S. equi subsp. zooepidemicus 4881 in a pH-controlled batch culture system. Zoocin A titres were maximal when the bacteria were grown on a starch-based medium and minimal when grown on a glucose-based medium. Northern hybridization showed that zif was constitutively expressed during growth regardless of the carbon source available. In contrast, zooA was transcribed at low levels in cells grown on a glucose-based medium but at much higher levels in cells grown on a starch-based medium. Use of a zooA promoter-chloramphenicol acetyl transferase (CAT) reporter demonstrated that zooA expression increased 17-fold in cells grown on a starch-based medium. Use of a zif promoter-CAT reporter showed that in cells grown on a starch-based medium zif was transcribed approximately 4 times as strongly as in cells grown on glucose. Transcription of zif was always much less than that of zooA. A ccpA deletion mutant appeared to exhibit complete de-repression of zooA under all growth conditions. Together these data showed that zoocin A expression was regulated in response to the carbon source on which the bacteria were grown.

"October 2002" / Bibliography: leaves 159-198. / xiv, 198 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis describes the mapping of the breakpoints of three different chromosome rearrangements, all involving 1p36, in acute myeloid leukaemia (AML) patients, and an investigation into the molecular outcomes of these rearrangements. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2003

Bibliography: leaves 206-263. / xviii, 313, [5] leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this project was to develop tools to assist in the construction of the ovine genome map so economic trait loci in sheep can be identified. (abstract) / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 2001