published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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Nitric oxide (NO) has been extensively studied due to its importance as a signaling agent. More recently, the pharmacological benefits of nitroxyl (HNO) in the treatment of cardiovascular disease, cancer, and alcoholism have been discovered.That HNO readily dimerizes complicates analysis and necessitates the use of donors. Diazeniumdiolates (NONOates), which can release either NO or HNO, are particularly attractive in this regard. NONOates from primary amines release HNO at physiological pH, and since the few existing examples have relatively short half-lives, a major research goal was to extend the lifetime range. The effect of amine structure on the lifetimes of ionic primary amine NONOates having the general structure Na(RN(H)[N(O)NO]) was unexpectedly small. This prompted the use of O2-protecting group methodology as an alternate method to stabilize donors toward decomposition. A detailed analysis of the decomposition mechanisms of a representative ionic primary amine NONOate and its O2-protected derivative is presented.NONOates were used as analytical tools to compare several commonly-used methods for detection of HNO. While these methods are used routinely for qualitative analysis of HNO, optimization for quantitative measurements was difficult. To improve method sensitivity, an HPLC assay using the fluorogenic reagent o-phthalaldehyde was developed, which may ultimately allow detection of endogenously-produced HNO.HNO donors such as cyanamide have been utilized in the treatment of alcoholism through the inhibition of aldehyde dehydrogenase (AlDH), which is critical for ethanol metabolism. Cyanamide also releases cyanide, and alternate HNO donors are thus desired for this clinical use. The efficacy of NONOates in the inhibition of AlDH was assayed in purified yeast AlDH and in mouse liver homogenate. However, efficacy was limited in a mouse model, perhaps due to a lack of selective delivery. This drug discovery project provided useful information for the future development of potentially liver-selective HNO-releasing NONOates.Together, these studies demonstrate the utility of NONOates as biomedical research tools, with synthetic modifications allowing for the modulation of decomposition profiles. As analytical tools for the development of HNO detection methods and potential pharmaceuticals in the treatment of alcoholism, NONOates provide convenience and control as donors of NO and HNO.

Aberrant metabolism has become an increasingly interesting area of cancer biology. In many cancers including lower grade glioma, glioblastomas and some leukemias, a mutation in the metabolic enzyme Isocitrate Dehydrogenase (IDH), has been found in more than 70% of cases and has been shown to lead to a distinct hypermethylator phenotype. IDH commonly converts isocitrate to alpha-ketoglutarate in normal cell metabolism. Three isoforms of this enzyme are found in humans: IDH1, IDH2 and IDH3. Studies on IDH1, the cytosolic isoform, have revealed that mutations in the enzyme’s binding site lead to a novel gain of function: the synthesis of an oncogenic metabolite, 2-hydroxyglutarate (2HG). 2HG competitively inhibits alpha-ketoglutarate dependent enzymes such as the TET 5-methylcytosine (5mC) oxygenases and histone demethylases. These oxygenases are responsible for the hydroxymethylation (5hmC) of cytosine residues, ultimately leading to demethylation and gene re-expression. Thus, mutant IDH may lead to an elevation in 5mC levels producing the hypermethylator phenotype described. A similar gain-of-function mutation in IDH2, the mitochondrial isoform of IDH1, has been associated with leukemias and gliomas lacking IDH1 mutations. Mutations in IDH1, IDH2 and TET2 are mutually exclusive, and each yields a similar hypermethylator phenotype. IDH2, along with IDH3, is primarily involved in the TCA cycle and energy production for the cell. Recently, the Taylor lab has uncovered evidence of 5mC and 5hmC residues in mitochondrial DNA, established and maintained by mtDNMT1 and TET2. Changing levels of mtDNMT1 appears to alter the patterns and levels of mtDNA transcription from the mitochondrial genome. We hypothesized that mutant IDH would produce a similar effect on the mitochondrial genome as that found in the nuclear genome and result in a decrease in the level of 5-hydroxymethylcytosine, as well as a subsequent increase in the level of 5-methylcytosine caused by the competitive inhibition of the TET enzymes by 2-hydroxyglutarate accumulation. Using molecular biology techniques such as Western blots and MeDIP (methylated DNA immunoprecipitation) I aim to uncover the role of IDH mutation on mitochondrial DNA methylation and its effect on energy production in mammalian cells.

Geok-yen Yeo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 225-233). / Title page --- p.i / Members of Thesis Advisory Committee --- p.ii / Abstract --- p.iii -iv / Acknowledgments --- p.v / Dedication --- p.vi / Table of Contents --- p.vii -xi / Chapter CHAPTER 1 --- INTRODUCTION --- p.1-31 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Fermentation --- p.1 / Chapter 1.3 --- Growth in Escherichia coli --- p.3 / Chapter 1.3.1 --- Aerobic growth in Escherichia coli --- p.3 / Chapter 1.3.2 --- The regulation of enzyme synthesis during cell metabolism --- p.7 / Chapter 1.3.3 --- Anaerobic growth in E. coli --- p.8 / Chapter 1.3.4 --- Anaerobic regulation by the transcriptional regulator Fnr --- p.12 / Chapter 1.3.5 --- "The case for ""Pasteur Control Proteins"" (PCP)" --- p.13 / Chapter 1.4 --- The family of alcohol dehydrogenases : An overview --- p.15 / Chapter 1.4.1 --- Molecular characteristics of alcohol dehydrogenases --- p.17 / Chapter 1.4.2 --- Residue conservation in alcohol dehydrogenases --- p.24 / Chapter 1.4.3 --- The effect of amino acid substitution on substrate specificity --- p.25 / Chapter 1.5 --- Alcohol dehydrogenases in bacteria --- p.28 / Chapter 1.5.1 --- Alcohol dehydrogenase in E. coli --- p.28 / Chapter 1.6 --- Aims of this study --- p.30 / Chapter CHAPTER 2 --- MATERIALS & METHODS --- p.32 -90 / Chapter 2.1 --- Bacterial strains --- p.32 / Chapter 2.2 --- Plasmids --- p.32 / Chapter 2.2.1 --- "Low copy number plasmid, pTJS75Km" --- p.32 / Chapter 2.2.2 --- "High copy number plasmid, pUC18" --- p.33 / Chapter 2.3 --- Bacterial culture media and solutions --- p.39 / Chapter 2.3.1 --- Luria Bertani (LB) medium --- p.39 / Chapter 2.3.2 --- L-Broth + MOPS --- p.39 / Chapter 2.3.3 --- "R medium, containing Triphenyltetrazolium chloride-ethanol (TTC-EtOH)" --- p.40 / Chapter 2.3.4 --- SOB and SOC media --- p.41 / Chapter 2.3.5 --- M9 Glucose medium --- p.42 / Chapter 2.3.6 --- Terrific Broth (TB) --- p.42 / Chapter 2.3.7 --- Rich Broth (RB) --- p.43 / Chapter 2.3.8 --- Antibiotic solutions --- p.43 / Chapter 2.4 --- Restriction endonucleases and other enzymes --- p.44 / Chapter 2.5 --- Isolation of chromosomal DNA --- p.45 / Chapter 2.5.1 --- Preparation of chromosomal DNA by spooling --- p.45 / Chapter 2.5.2 --- Preparation of chromosomal DNA by cesium chloride density gradient --- p.48 / Chapter 2.6 --- Isolation of plasmid DNA --- p.50 / Chapter 2.6.1 --- Large-scale preparation of plasmid by CsCl density gradient --- p.50 / Chapter 2.6.2 --- Small-scale preparation of plasmid DNA --- p.54 / Chapter 2.6.2. --- A Boiling method --- p.54 / Chapter 2.6.2. --- B Alkaline Lysis method --- p.55 / Chapter 2.6.3 --- Preparation of plasmid DNA by Qiagen columns --- p.56 / Chapter 2.7 --- Purification of DNA --- p.59 / Chapter 2.7.1 --- Ethanol precipitation --- p.59 / Chapter 2.7.2 --- Concentration and desalting using Centricon columns --- p.59 / Chapter 2.7.3 --- Purification of DNA by Geneclean procedure --- p.61 / Chapter 2.8 --- DNA cloning techniques --- p.63 / Chapter 2.8.1 --- Restriction endonuclease digestion --- p.63 / Chapter 2.8.2 --- Agarose-ethidium bromide gel electrophoresis --- p.65 / Chapter 2.8.2. --- A Gel loading buffer --- p.66 / Chapter 2.8.2. --- B Electro-elution of DNA --- p.67 / Chapter 2.8.3 --- Size fractionation --- p.68 / Chapter 2.8.3. --- A Salt gradient fractionation --- p.68 / Chapter 2.8.3. --- B Sucrose gradient --- p.70 / Chapter 2.8.4 --- Dephosphorylation of restriction-enzyme digested vector plasmid using calf intestinal phosphatase (CIP) --- p.71 / Chapter 2.8.5 --- Ligation of vector and insert --- p.72 / Chapter 2.8.6 --- Preparation of competent cells --- p.73 / Chapter 2.8.7 --- DNA transformation --- p.75 / Chapter 2.8.7.A --- By heat shock --- p.75 / Chapter 2.8.7.B --- By electroporation --- p.75 / Chapter 2.9 --- Screening for adhC transformants --- p.78 / Chapter 2.9.1 --- Screening for adhC clones --- p.78 / Chapter 2.9.2 --- Screening for pUC18 transformants --- p.79 / Chapter 2.10 --- Confirmation of adhC clones --- p.80 / Chapter 2.10.1 --- Reproduction of red colonies on R plates and antibiotic resistance --- p.80 / Chapter 2.10.2 --- T7 phage test for E. coli strains --- p.80 / Chapter 2.10.3 --- Plasmid size determination --- p.82 / Chapter 2.10.4 --- Re-transformation into E. coli host strains --- p.82 / Chapter 2.10.5 --- Physiological study of adhC clones --- p.83 / Chapter 2.10.6 --- Alcohol dehydrogenase assay --- p.84 / Chapter 2.11 --- The dye-binding method of protein determination --- p.87 / Chapter 2.12 --- Special procedures --- p.88 / Chapter 2.12.1 --- Generation of adh clones with deletions --- p.88 / Chapter 2.12.2 --- Sequencing reactions --- p.89 / Chapter CHAPTER 3 --- RESULTS: PART I Cloning and Restriction Mapping of the adhC mutation in a low copy number plasmid vector --- p.91 -122 / Chapter 3.1 --- Introduction: Cloning strategy --- p.91 / Chapter 3.2 --- Cloning of the adh mutation from strain CC2807B (an ADH overproducing mutant strain) in pTJS75Km --- p.93 / Chapter 3.2.1 --- Construction of the 'HK' clones --- p.93 / Chapter 3.3 --- Restriction mapping of the adh clones --- p.101 / Chapter 3.4 --- Subcloning the adhC insert --- p.110 / Chapter 3.4.1 --- Construction of plasmid pHK14 --- p.110 / Chapter 3.4.2 --- Construction of plasmid pHK15 --- p.115 / Chapter 3.4.3 --- Construction of plasmid pSS22 --- p.121 / Chapter 3.5 --- Remarks concerning the clones --- p.121 / Chapter CHAPTER 4 --- RESULTS:PART II Cloning and Sequencing of the adhC mutation in a high copy number plasmid vector --- p.123 -148 / Chapter 4.1 --- Introduction --- p.123 / Chapter 4.1.1 --- Choice of sequencing strategy --- p.123 / Chapter 4.1.2 --- An attempt to eliminate clone instability --- p.124 / Chapter 4.2 --- Subcloning of adh insert in pUC18 --- p.125 / Chapter 4.2.1 --- Study of adh clone EPR --- p.125 / Chapter 4.2.2 --- Re-construction of plasmid pEPR ( = pEE5) --- p.126 / Chapter 4.2.3 --- Construction of plasmids pEH2 and pEH3 --- p.127 / Chapter 4.2.4 --- Construction of a nested deletion library --- p.138 / Chapter CHAPTER 5 --- RESULTS : PART III Sequencing of the Mutation --- p.149 -177 / Chapter 5.1 --- Nucleotide sequencing --- p.149 / Chapter 5.2 --- Sequencing of the cloned adhC gene insert --- p.150 / Chapter 5.3 --- Analysis of the sequenced DNA by DNASIS computer software --- p.151 / Chapter 5.3.1 --- Search for codons associated with initiation and termination of transcription using the open reading frame (ORF) search --- p.151 / Chapter 5.3.2 --- Translation of the nucleotide sequence at the open reading frame (start 223 - end 2896) --- p.152 / Chapter 5.4 --- Search for DNA sequence homology with known DNA sequences --- p.152 / Chapter 5.4.1 --- Sequence homology of the structural gene (nucleotide # 223- #28%) : Two nucleotide changes revealed in DNA sequence of the structural gene adhE of Escherichia coli --- p.153 / Chapter 5.4.2 --- adhC mutation is due to changes in two amino acids --- p.153 / Chapter 5.4.3 --- The DNA sequence 5' of the mutated structural gene (upstream sequence) --- p.155 / Chapter 5.4.4 --- The DNA sequence 3' of the mutated structural gene (downstream sequence) --- p.156 / Chapter 5.5 --- Comparisons between the computer-predicted properties of the mutant and wild-type protein --- p.156 / Chapter 5.5.1 --- Prediction of the alcohol dehydrogenase protein secondary structure by the Robson Method --- p.156 / Chapter 5.5.2 --- Isoelectric point prediction --- p.156 / Chapter CHAPTER 6 --- RESULTS : PART IV Comparative Studies of Alcohol Dehydrogenase Expressionin adhC Strains and Clones --- p.178 -203 / Chapter 6.1 --- Introduction --- p.178 / Chapter 6.1.1 --- Basis for the alcohol dehydrogenase assay --- p.178 / Chapter 6.1.2 --- Choice of assay method --- p.179 / Chapter 6.1.3 --- Points to consider for ADH assay --- p.179 / Chapter 6.2 --- General growth characteristics of bacterial strains --- p.181 / Chapter 6.2.1 --- Plate cultures --- p.181 / Chapter 6.2.2 --- Overnight liquid cultures --- p.183 / Chapter 6.2.3 --- Batch liquid cultures --- p.183 / Chapter 6.2.4 --- ADH activity of strain CC2807B --- p.190 / Chapter 6.2.5 --- Comparison of ADH activity --- p.192 / Chapter 6.3 --- Investigating the mutated ADH enzyme --- p.197 / Chapter 6.3.1 --- Oxygen inactivation of the mutated enzyme --- p.197 / Chapter 6.3.2 --- Thermostability of the mutated enzyme --- p.201 / Chapter CHAPTER 7 --- DISCUSSION --- p.204 -220 / Chapter 7.1 --- Cloning of the adhC mutation --- p.204 / Chapter 7.1.1 --- Instability of clones in plasmid vector pUC18 --- p.204 / Chapter 7.1.2 --- Eliminating 'toxic' genes adjacent to adh locus --- p.207 / Chapter 7.1.3 --- Cloning in pTJS75Km low copy number vector --- p.208 / Chapter 7.2 --- DNA sequence of the adhC clones --- p.211 / Chapter 7.2.1 --- The basis for sequencing pUC 18-derived clones --- p.211 / Chapter 7.2.2 --- Homology to known alcohol dehydrogenases (ADH) sequences --- p.213 / Chapter 7.3 --- Findings concerning the adhC mutation --- p.217 / Chapter 7.3.1 --- How amino acid substitutions may affect an enzyme --- p.217 / Chapter 7.3.2 --- Physiological aspects of the bacterial cell due to the mutated enzyme --- p.218 / Chapter 7.4 --- Conclusions --- p.220 / APPENDICES --- p.221 -224 / REFERENCES --- p.225 -233

Hui, Lai Shan. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 83-87.

by Ho Ka-Pong, Bosco. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 81-100). / Chapter CHAPTER 1: --- INTRODUCTION --- p.1 / Chapter CHAPTER 2: --- PURIFICATION OF HUMAN CLASS I LIVER ADH --- p.19 / Chapter CHAPTER 3: --- DISSOCIATION AND REASSOCIATION OF HUMAN CLASS I ADH BY FREEZE/THAW TECHNIQUE --- p.36 / Chapter CHAPTER 4: --- "DISSOCIATION AND REASSOCIATION OF HUMAN CLASS I ADH BY USING UREA, GdmCl,HIGH SALT AND LOW pH" --- p.51 / Chapter CHAPTER 5: --- GENERAL DISCUSSION --- p.77 / REFERENCES --- p.81

by Agnes, Lo Shuk Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves [27-33] (2nd gp.)). / Chapter PART 1: --- Human Heart cDNA Library Sequencing / Chapter A) --- Introduction of human heart cDNA library sequencing / Chapter A.1 --- Human genome project / Chapter A.2 --- The aim of human genome project / Chapter A.3 --- Automatic sequencing / Chapter A.4 --- Cycle sequencing reaction / Chapter A.5 --- Human heart cDNA library sequencing project / Chapter B) --- Methods and materials / Chapter (I) --- Preparation of plating bacterial-Y1090 / Chapter (II) --- Plating the bacteriophage with blue-white visual selection / Chapter (III) --- Amplification of bacteriophage cDNA clones by PCR / Chapter (IV) --- Purification and quantitation of PCR products / Chapter (V) --- Cycle DNA sequencing of PCR products / Chapter (VI) --- Casting the sequencing gel / Chapter (VII) --- Sequencing by Pharmacia LKB A.L.F. DNA Sequencer / Chapter (VIII) --- Editing and saving the DNA sequence / Chapter (IX) --- Sending the DNA sequence to Genbank by E-mail / Chapter (X) --- Usage of the Genbank database / Chapter C) --- Results / Chapter D) --- Discussions / Chapter D.1 --- Application of human genomic project / Chapter D.2 --- Interpretation of the sequencing results / Chapter D.3 --- Quality of cDNA libraries and representation of mRNA population / Chapter D.4 --- "Gene expression profile in three different organs-heart, brain and liver" / Chapter D.5 --- Population study of the cDNA library / Chapter D.6 --- Isolation of a large number of novel genes by substraction cDNA library / Chapter D.7 --- Screening method to find out the complete coding sequence of interesting genes / Chapter D.8 --- Technical problems encountered and managed / Chapter PART 2: --- Identification of human cytosolic malate dehydrogenase by large scale human heart cDNA library sequencing / Chapter CHAPTER 1: --- Introduction of malate dehydrogenase / Chapter 1.1 --- Malate dehydrogenase--Kreb's cycle enzyme / Chapter 1.2 --- Two stereospecific forms of dehydrogenase / Chapter 1.3 --- NAD-binding domain / Chapter 1.4 --- The active site / Chapter 1.5 --- Comparison of surface properties between cMDH and mMDH / Chapter 1.6 --- N-terminal region and mitochondrial import / Chapter 1.7 --- Subunit-subunit interactions / Chapter 1.8 --- Physiological importance of malate dehydrogenase / Chapter 1.9 --- Secondary structure-total 11 β-strands and 9 α-helixes / Chapter 1.10 --- Objectives of the thesis / Chapter CHAPTER 2: --- Cloning and sequence analysis of human cytosolic malate dehydrogenase (hcMDH) / Chapter 2.1 --- Cloning of human cytosolic malate dehydrogenase (hcMDH) / Chapter 2.1.1 --- Methods and materials / Chapter 2.1.1.1 --- Cloning full length of hcMDH into expression vector pAED4 / Chapter 2.1.1.2 --- Preparation of competent cell-JM109 for transformation / Chapter 2.1.1.3 --- Minipreparation of plasmid DNA / Chapter 2.1.1.4 --- Midi-preparation of bacteriophage λDNA by QIAGEN´ёØ / Chapter 2.1.1.5 --- Titration of bacteriophage λ of human adult heart cDNA library / Chapter 2.1.1.6 --- Preparation of soft-agarose lysates / Chapter 2.1.1.7 --- Elution of DNA from agarose gel by GENECLEAN´ёØ / Chapter 2.1.2 --- Results / Chapter 2.1.3 --- Discussions / Chapter 2.2 --- Sequence analysis of human cytosolic malate dehydrogenase (hcMDH) / Chapter 2.2.1 --- Methods and materials: Autoread sequencing / Chapter (I) --- Annealing of primer to double-stranded template / Chapter (II) --- Sequencing / Chapter 2.2.2 --- Results and discussions / Chapter 2.3 --- Amino acids and protein structure analysis of cMDH / Chapter CHAPTER 3 : --- "Protein expression, partial purification and folding experiments of human cytosolic malate dehydrogenase (hcMDH)" / Chapter 3.1 --- Protein expression of hcMDH in E. coli / Chapter 3.1.1 --- Methods and materials / Chapter 3.1.1.1 --- Protein expression induced by IPTG / Chapter 3.1.1.2 --- Isoelectric focusing (IEF)-two dimensional gel electrophoresis / Chapter (I) --- First dimensional electrofocusing / Chapter (II) --- The second dimension SDS-PAGE electrophoresis / Chapter (III) --- Sample preparation / Chapter 3.1.2 --- Results / Chapter 3.1.3 --- Discussions / Chapter 3.1.3.1 --- The properties of expressed protein of hcMDH / Chapter 3.1.3.2 --- T7 expression system / Chapter 3.1.3.3 --- Strong φ 10 promoter / Chapter 3.1.3.4 --- E.coli BL21 host cell / Chapter 3.2 --- Partial purification and folding experiments of hcMDH / Chapter 3.2.1 --- Methods and materials / Chapter 3.2.1.1 --- Partial purification of hcMDH expressed protein / Chapter (I) --- Preparation of supernatant from E.coli crude extract / Chapter (II) --- Ion-exchange column chromatography / Chapter (III) --- Affinity chromatography / Chapter (IV) --- Gel filtration on a Sepharose CL-6B column / Chapter 3.2.1.2 --- Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) / Chapter 3.2.1.3 --- Staining the protein gel by the Coomassie Blue R-250 method / Chapter 3.2.1.4 --- Staining the protein gel by the Silver staining Method / Chapter 3.2.1.5 --- Quantitation of protein by the Bradford Method / Chapter 3.2.1.6 --- Native gel electrophoresis / Chapter 3.2.1.7 --- Malate dehydrogenase MDH enzyme staining method / Chapter 3.2.1.8 --- Malate dehydrogenase MDH enzyme assay / Chapter 3.2.1.9 --- Fast protein liquid chromatography (FPLC) / Chapter 3.2.1.10 --- Protein folding experiment / Chapter 3.2.1.11 --- Eukaryotic expression of hcMDH / Chapter 3.2.2 --- Results / Chapter 3.2.2.1 --- Partial purification by chromatography / Chapter 3.2.2.2 --- Native gel / Chapter 3.2.2.3 --- FPLC / Chapter 3.2.2.4 --- To aid folding of protein by adding NADH / Chapter 3.2.2.5 --- Eukaryotic expression / Chapter 3.2.3 --- Discussions / Chapter 3.2.3.1 --- Purification of malate dehydrogenase MDH / Chapter 3.2.3.2 --- "Methods for visualizing dehydrogenase enzymes, e.g. malate dehydrogenase" / Chapter 3.2.3.3 --- The presence of unfold hcMDH protein in bacteria / Chapter 3.2.3.4 --- Folding of protein by heat shock protein GroE / Chapter 3.2.3.5 --- Eukaryotic expression / Chapter CHAPTER 4: --- Master screening of single base change by PCR-SSCP (Single Strand Conformational Polymorphism) / Chapter 4.1 --- Theory of SSCP / Chapter 4.2 --- Methods and materials / Chapter 4.3 --- Results / Chapter 4.4 --- Discussions / Chapter 4.4.1 --- The procedure of SSCP / Chapter 4.4.2 --- An alternative quick detection method for polymorphism of hcMDH at position 565--by automatic sequencing / Chapter 4.4.3 --- Other detection methods-- RNA-PCR and ddF / Chapter 4.4.4 --- Parameters affecting sensitivity of SSCP / Chapter 4.4.5 --- Application of SSCP / Chapter CHAPTER 5: --- Southern hybridization and In situ hybridization / Chapter 5.1 --- Southern blot analysis of human cytosolic malate dehydrogenase (hcMDH) / Chapter 5.1.1 --- Methods and materials / Chapter (I) --- Transfer genomic DNA to Nylon membrane / Chapter (II) --- Synthesis of radiolabelling cDNA probe / Chapter (III) --- Pre-hybridization and hybridization reaction / Chapter 5.1.2 --- Results / Chapter 5.1.3 --- Discussions / Chapter 5.2 --- In situ hybridization / Chapter 5.2.1 --- Methods and materials / Chapter (I) --- Preparation of Dig labelling probe by random primed labelling / Chapter (II) --- Estimating the yield of Dig-labelled nucleic acids / Chapter (III) --- Denaturation and hybridization of the hcMDH probe with animal tissues / Chapter (IV) --- Color development of the tissue / Chapter 5.2.2 --- Results / Chapter 5.2.3 --- Discussions / Chapter 5.2.3.1 --- Cellular distribution of hcMDH / Chapter 5.2.3.2 --- The principle of in situ hybridization / Chapter 5.2.3.3 --- Specimen preparation / Chapter 5.2.3.4 --- Hybridization conditions / Chapter 5.2.3.5 --- "Ontogeny of MDH in rabbit fetal brain, heart and lung" / Appendixes: / "Appendix I: 531 random cDNA clones from clone no. J950 to K951 in human heart cDNA library sequencing project. The name of clones, accession number, the length of the partial sequence and percentage of match are listed" / Appendix II: The new accession no. of Novel clones in Genbank / "Appendix III: The enzymatic reaction, molecular weigth, specific activity and Michaelis constants of different sources of malate dehydrogenase" / Appendix IV: The full sequence of nucleic acids and amino acids of human cytosolic malate dehydrogenase hcMDH. Accession no. of hcMDH is U20352 in Genbank / Appendix V: Nucleotide sequences of the mouse cMDH gene / Appendix VI: Nucleotide sequences of the mouse mMDH gene / Appendix VII: Structural organization of the mouse cytosolic malate dehydrogenase and its comparison with that of the mouse mitochondrial malate dehydrogenase gene