by Tsui Kwok-wing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 188-216). / ACKNOWLEDGEMENTS --- p.ii / ABSTRACT --- p.iii / TABLE OF CONTENTS --- p.v / ABBREVIATIONS --- p.ix / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Human genome project --- p.5 / Chapter 1.3 --- Organization of human genome --- p.7 / Chapter 1.4 --- Adult human heart cDNA library --- p.9 / Chapter 1.5 --- Gene expression in adult human heart --- p.10 / Chapter 1.6 --- Polymerase chain reaction --- p.12 / Chapter 1.7 --- Purification of PCR products --- p.15 / Chapter 1.8 --- Automated DNA sequencing --- p.17 / Chapter 1.9 --- Sequence analysis by electronic mail server --- p.21 / Chapter 1.10 --- Effects of agar and agarose on Vent´ёØ and Taq DNA polymerases --- p.23 / Chapter 1.11 --- Transcription factors and zinc finger proteins --- p.25 / Chapter 1.12 --- LIM domain --- p.28 / Chapter 1.13 --- Cysteine-rich intestinal protein --- p.30 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Plating out the adult human heart cDNA library --- p.32 / Chapter 2.2 --- Amplification by polymerase chain reaction --- p.33 / Chapter 2.3 --- Purification of the PCR products by Millipore filters --- p.35 / Chapter 2.4 --- Elimination of the purification of the PCR products before sequencing --- p.36 / Chapter 2.5 --- Cycle sequencing --- p.37 / Chapter 2.6 --- Unicycle sequencing --- p.38 / Chapter 2.7 --- Sequencing by T7 polymerase --- p.39 / Chapter 2.8 --- Gel electrophoresis in the automated A.L.F. sequencer --- p.41 / Chapter 2.9 --- Sequence analysis by commercially available softwares --- p.42 / Chapter 2.10 --- Sequence analysis through electronic mail server --- p.44 / Chapter 2.11 --- Database for storing the result of each clone --- p.46 / Chapter 2.12 --- Effects of agar and agarose on Vent´ёØ and Taq DNA polymerase --- p.47 / Chapter 2.13 --- Mini-preparation of plasmid DNA --- p.50 / Chapter 2.14 --- Large scale preparation of plasmid DNA --- p.51 / Chapter 2.15 --- Cloning the human cysteine rich heart protein (hCRHP) into the pAED4 vector --- p.53 / Chapter 2.16 --- Expression of hCRHP in E coli --- p.56 / Chapter 2.17 --- Northern hybridization --- p.58 / Chapter 2.18 --- Partial protein sequencing of hCRHP --- p.59 / Chapter CHAPTER 3 --- RESULTS / Chapter 3.1 --- The sequencing results of adult human heart cDNA clones --- p.60 / Chapter 3.2 --- Accuracy of sequencing results --- p.63 / Chapter 3.3 --- Catalogues of genes expressed in the adult human heart --- p.65 / Chapter 3.4 --- Effects of agar and agarose on Vent´ёØ and Taq DNA polymerases --- p.94 / Chapter 3.5 --- Elimination of the purification of the PCR products before sequencing --- p.102 / Chapter 3.6 --- Sequence analysis of hCRHP --- p.104 / Chapter 3.7 --- Northern hybridization of hCRHP --- p.109 / Chapter 3.8 --- Expression of hCRHP in E. coli --- p.112 / Chapter CHAPTER 4 --- DISCUSSION / Chapter 4.1 --- Random sequencing of adult human heart cDNA clones --- p.118 / Chapter 4.2 --- Catalogues of genes expressed in the adult human heart --- p.130 / Chapter 4.3 --- Gene expression in the adult human heart --- p.137 / Chapter 4.4 --- Importance of nonhuman matches --- p.170 / Chapter 4.5 --- Effects of agar and agarose on Vent´ёØ and Taq DNA polymerases --- p.177 / Chapter 4.6 --- Elimination of the purification of the PCR products before sequencing --- p.180 / Chapter 4.7 --- The possible role of CRIP and hCRHP --- p.184 / Chapter 4.8 --- Future prospect --- p.186 / REFERENCE --- p.188

by Lam Wai Yip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 184-195). / Contents --- p.I - IV / Abstract --- p.V / Abbreviations --- p.VI / List of Tables and Figures --- p.VII - XV / Chapter Chapter One: --- Introduction / Part I / Chapter 1.1 --- Human genome project --- p.1 / Chapter 1.2 --- Progress of human genome project --- p.2 / Chapter 1.3 --- Human heart cDNA sequencing --- p.3 / Chapter 1.4 --- Significance of the human heart cDNA library project --- p.5 / Chapter 1.5 --- Homology search tools for cDNA sequences alignment --- p.5 / Part II / Chapter 1.6 --- Investigation of a human heart cDNA clone A076 --- p.7 / Chapter 1.7 --- General introduction of Heat Shock Proteins (HSPs) --- p.7 / Chapter 1.7.1 --- Definition of HSP --- p.8 / Chapter 1.7.2 --- Discovery of HSP --- p.10 / Chapter 1.7.3 --- Transcriptional regulation of heat shock genes --- p.11 / Chapter 1.7.4 --- Nomenclature of HSPs --- p.13 / Chapter 1.7.5 --- HSP110 --- p.13 / Chapter 1.7.6 --- HSP90 --- p.14 / Chapter 1.7.7 --- HSP70 --- p.15 / Chapter 1.7.8 --- HSP60 --- p.17 / Chapter 1.7.9 --- Ubiquitin - HSP8 --- p.19 / Chapter 1.7.10 --- HSP27 --- p.20 / Chapter 1.8 --- The theme of this thesis --- p.28 / Chapter Chapter Two: --- Method and Materials / Chapter 2.1 --- The human heart cDNA library --- p.29 / Chapter 2.2 --- Plating out the cDNA library --- p.29 / Chapter 2.3 --- DNA amplification --- p.31 / Chapter 2.4 --- DNA sequencing reaction - Cycle sequencing reaction --- p.32 / Chapter 2.5 --- Operation of the A.L.F. DNA sequencer --- p.33 / Chapter 2.5.1 --- Preparation of the gel cassette --- p.33 / Chapter 2.5.2 --- Preparation of the acrylamide gel --- p.34 / Chapter 2.5.3 --- Fitting the gel cassette into the electrophoresis unit --- p.35 / Chapter 2.5.4 --- Settings of electrophoresis --- p.36 / Chapter 2.6 --- Comparison of DNA sequences to databases --- p.37 / Chapter 2.7 --- Programming for sending cDNA sequences to NCBI --- p.38 / Chapter 2.8 --- Storage of sequence data --- p.39 / Chapter 2.9 --- Synthesis and purification of primers --- p.40 / Chapter 2.10 --- Connection of cDNA clones using Polymerase Chain Reaction (PCR) --- p.41 / Chapter 2.11 --- Purification of DNA fragment from agarose gels by GENECLEAN´ёØ --- p.42 / Chapter 2.12 --- "Preparation of competent Escherichia coli for transformation (Hanahan, 1986)" --- p.43 / Chapter 2.13 --- Transformation of Plasmid into Competent Escherichia coli --- p.44 / Chapter 2.14 --- "Small scale preparation of plasmid DNA (Sambrook et al.,1989" --- p.45 / Chapter 2.15 --- Large scale plasmid preparation by QIAGEN´ёØ --- p.46 / Chapter 2.16 --- DNA sequencing reaction - Unicycle sequencing reaction --- p.48 / Chapter 2.17 --- Synthesis of Radiolabeled DNA probe --- p.49 / Chapter 2.18 --- "Isolation of genomic DNA from human blood cells (Thomas A. Ciulla, 1988)" --- p.51 / Chapter 2.19 --- Southern blotting --- p.52 / Chapter 2.20 --- Prehybridization and hybridization procedure for Southern blot analysis --- p.54 / Chapter 2.21 --- "AGPC-RNA extraction method (Chomczynski and Sacchi 1987, modifed)" --- p.56 / Chapter 2.22 --- Electrophoresis of RNA through gels containing formaldehyde --- p.58 / Chapter 2.23 --- First-Strand cDNA synthesis --- p.59 / Chapter 2.24 --- Use of T7 RNA polymerase to direct expression of the cloned hsp27b gene (A076&B490) --- p.60 / Chapter 2.25 --- "Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (Laemmli, 1970)" --- p.61 / Chapter 2.26 --- Staining of the Gel by the Commassie Blue Method --- p.63 / Chapter Chapter Three: --- Results / Part I / Chapter 3.1 --- Sample results of Sequencing a few clones --- p.64 / Chapter 3.2 --- A Catalogue of 497 cDNA clones obtained from human heart cDNA sequencing --- p.71 / Chapter 3.3 --- Submission of novel sequences to genbank --- p.81 / Chapter 3.4 --- A Catalogues of genes that are expressed in the adult human heart --- p.83 / Chapter 3.5 --- The use of the programmes to assist the sending and receiving of sequence data E-mail message --- p.90 / Chapter 3.5.1 --- The use of the SENDMAIL.EXE programme --- p.91 / Chapter 3.5.2 --- "The use of the EDITBLN.EXE, ALLFILE.EXE and DATABASE.EXE" --- p.95 / Part II / Chapter 3.6 --- DNA sequence profiles of cDNA clones A076 and B490 --- p.105 / Chapter 3.7 --- Ligation of cDNA clones using Polymerase Chain Reaction (PCR) --- p.112 / Chapter 3.8 --- Cloning of the PCR product A076&B490 into the pAED4 expression vector --- p.117 / Chapter 3.9 --- Unicycle sequencing of the subcloned insert A076&B490 --- p.121 / Chapter 3.10 --- Southern hybridization of hsp27b (A076&B490) --- p.125 / Chapter 3.11 --- Results of RT-PCR and PCR --- p.127 / Chapter 3.12 --- Expression pAED4-A076&B490 in E.coli --- p.133 / Chapter Chapter Four: --- Discussion / Part I / Chapter 4.1 --- EST characterization --- p.138 / Chapter 4.2 --- Further investigation --- p.140 / Chapter 4.3 --- Disadvantage of randomly picked cDNA sequencing --- p.141 / Chapter 4.4 --- Problem of GenBank database searching --- p.141 / Part II / Chapter 4.5 --- The DNA sequence of A076 and B490 --- p.143 / Chapter 4.6 --- Ligation of HSP27B by using PCR --- p.144 / Chapter 4.7 --- Analysis of the DNA and protein sequence ofhsp27b (A076&B490) --- p.145 / Chapter 4.8 --- Southern hybridization of human hsp27b --- p.153 / Chapter 4.9 --- "RT-PCR and PCR of first strand cDNA with primers A076-ATG, A076-mid and oligo dT" --- p.153 / Chapter 4.10 --- Expression of human hsp27b --- p.154 / Chapter 4.11 --- The possible roles of human hsp27b --- p.156 / Chapter 4.12 --- Further analysis --- p.160 / Appendix I --- p.161-182 / Appendix II --- p.183 / References --- p.184-195