by Lee Sau Yat. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 89-94). / Acknowledgements --- p.I / Abbreviation --- p.II / Abstract --- p.III / Table of content --- p.IV / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Historical background in the studies of LDL and LDLR --- p.1 / Chapter 1.2 --- Homeostasis of Cholesterol in Man --- p.4 / Chapter 1.2.1 --- Origin and catabolism of low density lipoprotein --- p.4 / Chapter 1.2.2 --- The LDL receptor --- p.6 / Chapter 1.2.3 --- LDL pathway --- p.7 / Chapter 1.2.4 --- Feedback regulatory action of LDL receptor --- p.10 / Chapter 1.3 --- Gene structure of LDL receptor promoter --- p.11 / Chapter 1.3.1 --- The LDL receptor promoter --- p.11 / Chapter 1.3.2 --- The responsive element in LDL receptor promoter --- p.13 / Chapter 1.4 --- Eukaryotic transcription factor --- p.15 / Chapter 1.5 --- Role of Gel-shifted assay in studying DNA binding protein --- p.17 / Chapter 1.6 --- Objective of the present thesis --- p.20 / Chapter Chapter 2 --- Materials and Methods --- p.21 / Chapter 2.1 --- Oligonucleotide synthesis and purification --- p.21 / Chapter 2.1.1 --- Primer construction --- p.21 / Chapter 2.1.2 --- Purification of oligonucleotides --- p.22 / Chapter 2.2 --- Recombinant plasmid construction --- p.24 / Chapter 2.2.1 --- Preparation of competent cell --- p.24 / Chapter 2.2.2 --- Preparation of phage DNA --- p.24 / Chapter 2.2.3 --- Amplification and purification of LDLR-promoter by PCR techniques --- p.26 / Chapter 2.2.3.1 --- Amplification and restriction site construction of LDLR- promoter --- p.26 / Chapter 2.2.3.2 --- Purification of the PCR product --- p.27 / Chapter 2.2.4 --- Preparation of plasmid pGCAT-A --- p.27 / Chapter 2.2.5 --- Recombinant plasmid pLDLRP-GCAT- A construction --- p.29 / Chapter 2.2.6 --- Transformation of DNA to competent cell --- p.29 / Chapter 2.2.7 --- Screening of positive clone pLDLRP- GCAT-A --- p.30 / Chapter 2.3 --- DNA sequencing --- p.31 / Chapter 2.3.1 --- Denaturing the double strand template --- p.31 / Chapter 2.3.2 --- Annealing reaction --- p.31 / Chapter 2.3.3 --- Labeling reaction --- p.32 / Chapter 2.3.4 --- Termination reaction --- p.32 / Chapter 2.3.5 --- Running and fixing the gel --- p.32 / Chapter 2.4 --- Cell culture and passage of different cell lines --- p.34 / Chapter 2.4.1 --- "Routine subculture of HepG 2, CHO, FSF, Cos-7 and FSF" --- p.34 / Chapter 2.4.2 --- "BMN, RTGH-1" --- p.34 / Chapter 2.5 --- Preparation of human LDL and LPDS --- p.36 / Chapter 2.5.1 --- Purification of LDL --- p.36 / Chapter 2.5.2 --- Purification of LPDS --- p.37 / Chapter 2.6 --- DNA transfection and CAT assay --- p.38 / Chapter 2.6.1 --- Transfection of recombinant plasmid to eukaryotic cells --- p.38 / Chapter 2.6.2 --- CAT assay --- p.39 / Chapter 2.7 --- 125I-LDL binding assay --- p.41 / Chapter 2.7.1 --- Radioactive iodination of LDL --- p.41 / Chapter 2.7.2 --- Purification of iodinated LDL --- p.41 / Chapter 2.7.3 --- Down regulation of LDL receptorin different cell lines --- p.41 / Chapter 2.7.4 --- Different Drugs treatment in HepG2 Cell line --- p.42 / Chapter 2.7.5 --- 125I-LDL binding assay --- p.43 / Chapter 2.8 --- Gel-shift mobility assay --- p.44 / Chapter 2.8.1 --- Extraction of crude nuclear extracts --- p.44 / Chapter 2.8.2 --- 5'end-labeling of synthetic oligonucleotides --- p.45 / Chapter 2.8.3 --- Purification of labeled oligonucleotides --- p.45 / Chapter 2.8.4 --- Nuclear protein and DNA binding reaction --- p.46 / Chapter 2.8.5 --- Gel-shift mobility electrophoresis by PhastSystem --- p.46 / Chapter 2.9 --- Construction of λ gt 11 cDNA library of HepG 2cell --- p.48 / Chapter 2.9.1 --- Purification of mRNA from HepG 2 --- p.48 / Chapter 2.9.2 --- cDNA preparation --- p.48 / Chapter 2.9.3 --- In-vitro packaging of phage --- p.48 / Chapter 2.9.3 --- Screening the expression library --- p.49 / Chapter Chapter 3 --- Results --- p.50 / Chapter 3.1 --- Construction of recombinant plasmid --- p.50 / Chapter 3.1.1 --- hLDLR-promoter λ 34 clone --- p.50 / Chapter 3.1.2 --- Restriction site generation in LDLR- promoter by PCR --- p.52 / Chapter 3.1.3 --- Preparation of pGCAT-A reporter plasmid --- p.55 / Chapter 3.1.4 --- Screening of pGCAT-A-LDLRP recombinant --- p.55 / Chapter 3.1.5 --- Sequencing of pGCAT-A-LDLR- promoter recombinant --- p.57 / Chapter 3.2 --- CAT assay of recombinant plasmid on transfected HepG 2 cell --- p.57 / Chapter 3.3 --- 125I-LDL binding assay --- p.57 / Chapter 3.3.1 --- 125 I - LDL binding assay of different cell lines --- p.57 / Chapter 3.3.2 --- Characterization of cell surface receptor of HepG 2 cell by different drugs treatment --- p.61 / Chapter 3.4 --- Gel shift mobility assay --- p.63 / Chapter 3.4.1 --- Binding effect of Repeat 2 to different cell lines --- p.63 / Chapter 3.4.2 --- Optimizing the binding reactionin HepG 2 cell by poly(dI.dC) --- p.63 / Chapter 3.4.3 --- Specificity of Repeat 2 in binding to HepG 2 cell nuclear protein --- p.66 / Chapter 3.4.4 --- LDL dose response treatment in HepG2 cell --- p.68 / Chapter 3.4.4.1 --- Binding of Repeat 2 to specific nuclear protein --- p.68 / Chapter 3.4.4.2 --- Binding of Repeat 2 to a non- specific cell nuclear protein from cells treated with LDL --- p.68 / Chapter 3.4.5 --- Effect of different drugs on the binding of Repeat 2 to nuclear proteins in HepG 2 cell --- p.71 / Chapter 3.5 --- HepG 2 cell cDNA library screening --- p.73 / Chapter Chapter 4 --- Discussion --- p.77 / Chapter 4.1 --- Strategy on construction of reporter plasmid pLDLRP-GC AT-A --- p.77 / Chapter 4.2 --- The expression of CAT in HepG 2 cell --- p.78 / Chapter 4.3 --- Identification of a DNA binding protein for Repeat 2 in HepG 2 cell --- p.80 / Chapter 4.3.1 --- Binding effect of nuclear protein to Repeat 2 --- p.82 / Chapter 4.3.1.1 --- LDL dose response relationships --- p.82 / Chapter 4.3.1.2 --- Effect of protein inhibitors --- p.83 / Chapter 4.3.1.3 --- Effect of Shanzha --- p.86 / Chapter 4.3.2 --- Screening of the cDNA library of HepG 2 cells --- p.86 / Chapter Chapter 5 --- Further Studies --- p.88 / References --- p.89 / Appendix --- p.95
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_319102 |
| Date | January 1993 |
| Contributors | Lee, Sau Yat., 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, x, 99 leaves : ill. (chiefly 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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