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
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_320659 |
| Date | January 1995 |
| Contributors | Lo, Agnes Shuk Yee., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Publisher | Chinese University of Hong Kong |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, 172, [33] leaves : ill. (some mounted 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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