by Nellie Yuk Fei Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 111-120). / Abstract also in Chinese. / Abstract --- p.i / List of Abbreviations --- p.iii / Acknowledgements --- p.iv / Publication of this study --- p.vi / Table of Contents --- p.vii / Chapter Chapter1: --- Introduction --- p.1 / Chapter 1.1 --- Multiple Myeloma --- p.2 / Chapter 1.2 --- The Problem --- p.2 / Chapter Chapter2: --- Literature Review --- p.5 / Chapter 2.1 --- Molecular Genetics of Multiple Myeloma --- p.6 / Chapter 2.1.1 --- Cytogenetics --- p.6 / Chapter 2.2 --- Alterations of Proto-Oncogenes --- p.9 / Chapter 2.2.1 --- c-myc --- p.9 / Chapter 2.2.2 --- Ras --- p.10 / Chapter 2.2.3 --- Bcl-2 and Related Protein --- p.10 / Chapter 2.3 --- Alteration of Tumor-Suppressor genes --- p.11 / Chapter 2.3.1 --- p53 Gene Mutations --- p.11 / Chapter 2.3.2 --- Retinoblastoma (Rb) Gene --- p.11 / Chapter 2.3.3 --- p16 and p15 Genes --- p.13 / Chapter Chapter3: --- DNA Methylation and Cancers --- p.14 / Chapter 3.1 --- Role of DNA Methylation --- p.15 / Chapter 3.2 --- CpG Islands --- p.15 / Chapter 3.3 --- Abnormalities of DNA Methylation in Neoplasia --- p.16 / Chapter 3.3.1 --- DNA Hypomethylation in Cancer --- p.16 / Chapter 3.3.2 --- DNA Methyltransferase Activity in Cancer --- p.17 / Chapter 3.4 --- Regional DNA Hypermethylation in Cancer --- p.17 / Chapter 3.4.1 --- p16 and p15 Genes in Solid Tumors --- p.18 / Chapter 3.4.2 --- The p16 and p15 Genes in Leukemia and other Hematopoietic Malignancies --- p.19 / Chapter 3.4.3 --- Retinoblastoma Gene --- p.20 / Chapter 3.5 --- Mechanism Underlying the DNA Methylation Changes --- p.21 / Chapter Chapter4: --- Background of Study --- p.23 / Chapter 4.1 --- Background of Study --- p.24 / Chapter 4.2 --- Project Objectives --- p.27 / Chapter Chapter5: --- Materials and Methods --- p.29 / Chapter 5.1 --- Patients Samples --- p.30 / Chapter 5.2 --- Normal Controls --- p.30 / Chapter 5.3 --- Storage of the Samples --- p.32 / Chapter 5.4 --- Materials --- p.32 / Chapter 5.4.1 --- Chemicals --- p.32 / Chapter 5.4.2 --- Primers --- p.33 / Chapter 5.4.3 --- Enzymes --- p.35 / Chapter 5.5 --- Methods --- p.35 / Chapter 5.5.1 --- Cloning of p16 and p15 Exon 1 Probes for Southern Analysis --- p.35 / Chapter 5.5.1.1 --- PCR Amplification of p16 and p15 exon1 Probes from Normal Blood DNA --- p.35 / Chapter 5.5.1.2 --- Recovery and Purification of p16 and p15 Exon 1 DNA Fragment --- p.36 / Chapter 5.5.1.3 --- Ligation --- p.37 / Chapter 5.5.1.4 --- Transformation --- p.37 / Chapter 5.5.1.5 --- Plating --- p.38 / Chapter 5.5.1.6 --- Screening of Recombinant Plasmid --- p.38 / Chapter 5.5.1.7 --- Confirmation of Cloned DNA by Sequencing --- p.42 / Chapter 5.5.2 --- DNA Extraction and Purification --- p.45 / Chapter 5.5.2.1 --- DNA Extraction from Bone Marrow Aspirate and Peripheral Blood --- p.45 / Chapter 5.5.2.2 --- Isolation of Plasmid DNA from Transformant Cutures --- p.46 / Chapter 5.5.2.3 --- Qualification and Quantification of DNA --- p.49 / Chapter 5.5.3 --- Detection of Hypermethylation by Southern Analysis --- p.50 / Chapter 5.5.3.1 --- Restriction Enzyme Digestion --- p.50 / Chapter 5.5.3.2 --- Agarose Gel Electrophoresis --- p.51 / Chapter 5.5.3.3 --- Southern Transfer --- p.51 / Chapter 5.5.3.4 --- Membrane Fixation --- p.51 / Chapter 5.5.3.5 --- Recovery and Purification of p16 and p15 Exon 1 Probes from Plasmid --- p.52 / Chapter 5.5.3.6 --- Probe Labeling --- p.54 / Chapter 5.5.3.7 --- Purification of Radioactive labeled DNA --- p.54 / Chapter 5.5.3.8 --- Southern Hybridization --- p.55 / Chapter 5.5.3.9 --- Post Hybridization --- p.55 / Chapter 5.5.3.10 --- Autoradiography --- p.56 / Chapter 5.5.4 --- Polymerase Chain Reaction-Single Strand Conformational Polymorphism Analysis (PCR-SSCP) --- p.56 / Chapter 5.5.4.1 --- 5'- end Radioactive Labeling of Primer --- p.56 / Chapter 5.5.4.2 --- Amplification of Target Sequence by PCR --- p.57 / Chapter 5.5.4.3 --- Non-denaturing Polyacrylamide Gel Electrophresis --- p.57 / Chapter 5.5.4.4 --- Direct DNA Sequence of PCR Products --- p.58 / Chapter 5.5.5 --- Prevention of Overall Contamination in PCR --- p.60 / Chapter 5.5.6 --- "Sensitivity, Specificity Controls" --- p.62 / Chapter Chapter6: --- Results --- p.64 / Chapter 6.1 --- Patient Characteristics --- p.65 / Chapter 6.1.1 --- General Patient Characteristics --- p.65 / Chapter 6.1.2 --- Clinical and Laboratory Features --- p.65 / Chapter 6.2 --- Southern Blot Analysis of p16/p15 and Rb --- p.79 / Chapter 6.2.1 --- Absence of Deletions or hypermethylationin Normal Controls --- p.79 / Chapter 6.2.2 --- Absence of Homozygous Deletions or Mutationsin p16/15 and Rb among all MM Patients --- p.79 / Chapter 6.2.3 --- Hypermethylation of p16 --- p.89 / Chapter 6.2.4 --- Hypermethylation of p15 --- p.92 / Chapter 6.3 --- Hypermethylation of p16/p15 and Clinico-pathologic Correlation --- p.94 / Chapter Chapter7: --- Discussion --- p.97 / Chapter 7.1 --- "Absence of Homozygous Deletions, Gene Rearrangements and Mutations in p16/p15 and Rb" --- p.98 / Chapter 7.2 --- Hypermethylation of p16/p15-An Alternative Way for Gene Inactivation --- p.100 / Chapter 7.2.1 --- Methylation of p15 Gene --- p.101 / Chapter 7.2.2 --- Methylation of 5'-CpG Island of p16/p15 and Lack of Gene Expression --- p.102 / Chapter 7.2.3 --- Comparison of Methylation Status of Primary Samples and Cell Lines in MM --- p.103 / Chapter 7.2.4 --- Progressive Gene Inactivation by Random Methylation Errors --- p.104 / Chapter 7.2.5 --- The Lack of Correlation of Tumor Contents Revealed by the Southern Analysis and Morphologic Assessment --- p.105 / Chapter 7.3 --- Knudson's Two-hit Model of Tumorigenesis --- p.106 / Chapter 7.4 --- Inverse Relationship of p16 and Rb --- p.107 / Chapter 7.5 --- Implications of Our Findings --- p.109 / Chapter 7.6 --- Future Studies --- p.109 / References --- p.111
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322502 |
| Date | January 1998 |
| Contributors | Chung, Nellie Yuk Fei., Chinese University of Hong Kong Graduate School. Division of Anatomical and Cellular Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, ix, 120 leaves : ill. (some mounted) ; 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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