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A study of tumor suppressor genes in multiple myeloma.

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

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_322502
Date January 1998
ContributorsChung, Nellie Yuk Fei., Chinese University of Hong Kong Graduate School. Division of Anatomical and Cellular Pathology.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, ix, 120 leaves : ill. (some mounted) ; 30 cm.
RightsUse 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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