by Patrick, Tik-wan Law. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 144-155). / Acknowledgements --- p.i / Contents --- p.ii / Abstract --- p.vi / Abbreviations --- p.viii / List of figures --- p.ix / List of tables --- p.xiv / Chapter Chapter One: --- Introduction --- p.1-17 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- The Human genome project --- p.2 / Chapter 1.3 --- The EST approach --- p.3 / Chapter 1.4 --- Significance of the EST approach --- p.3 / Chapter 1.5 --- Human heart cDNA sequencing --- p.5 / Chapter 1.6 --- Significance of the human adult heart EST project --- p.7 / Chapter 1.7 --- Ribosomal proteins --- p.8 / Chapter 1.7.1 --- The ribosomal constituents --- p.8 / Chapter 1.7.2 --- Eukaiyotic ribosomal proteins --- p.10 / Chapter 1.8 --- Mammalian ribosomal proteins --- p.11 / Chapter 1.8.1 --- Evolution of mammalian ribosomal proteins --- p.11 / Chapter 1.8.2 --- Significance of mammalian ribosomal proteins --- p.12 / Chapter 1.9 --- Possible functional roles of ribosomal protein --- p.14 / Chapter 1.10 --- Nomenclature of ribosomal proteins --- p.16 / Chapter 1.11 --- The theme of the thesis --- p.17 / Chapter Chapter Two: --- Materials and Methods --- p.18-49 / Chapter 2.1 --- Cycle sequencing --- p.18 / Chapter 2.1.1 --- Plating out the cDNA library --- p.18 / Chapter 2.1.2 --- Amplification of the cDNA clones by PCR --- p.19 / Chapter 2.1.3 --- Purification and quantitation of the PCR product --- p.20 / Chapter 2.1.4 --- Cycle DNA sequencing --- p.20 / Chapter 2.2 --- Cloning of hrpL29 in pUC 18 cloning vector --- p.21 / Chapter 2.2.1 --- Amplification of the phage by plate lysate --- p.21 / Chapter 2.2.2 --- Amplification of the insert by PCR --- p.22 / Chapter 2.3 --- Screening for hrpL29 transformant --- p.22 / Chapter 2.3.1 --- Mini-preparation of plasmid DNA (Sambrook et al，1989) --- p.22 / Chapter 2.3.2 --- Large scale preparation of plasmid DNA --- p.24 / Chapter 2.4 --- Primer design for cloning of an intron of hrpL29 --- p.26 / Chapter 2.5 --- Isolation of the intron of hrpL29 by PCR --- p.26 / Chapter 2.6 --- Restricted endonuclease digestion --- p.27 / Chapter 2.7 --- Purification of DNA from the agarose gel --- p.27 / Chapter 2.8 --- Dephosphorylation of linearized plasmid DNA --- p.29 / Chapter 2.9 --- DNA ligation --- p.29 / Chapter 2.10 --- "Preparation of competent bacterial cells for transformation (Hanahan,1985)" --- p.30 / Chapter 2.11 --- Plasmid DNA Transformation --- p.31 / Chapter 2.12 --- Unicycle DNA sequencing by T7 polymerase (Pharmacia) --- p.32 / Chapter 2.13 --- Synthesis of radiolabelled DNA probe --- p.33 / Chapter 2.14 --- "Oligonucleotide synthesis, deprotection and purification" --- p.34 / Chapter 2.14.1 --- Oligonucleotide synthesis --- p.34 / Chapter 2.14.2 --- Deprotection and purification of oligonucleotides --- p.35 / Chapter 2.15 --- Southern analysis --- p.36 / Chapter 2.15.1 --- "Isolation of genomic DNA from leukocytes (Ciulla et al,1988)" --- p.36 / Chapter 2.15.2 --- Restricted digestion and fractionation of genomic DNA --- p.37 / Chapter 2.15.3 --- Southern transfer of DNA onto a membrane support --- p.37 / Chapter 2.15.4 --- Prehybridization of the Southern blot --- p.40 / Chapter 2.15.5 --- Hybridization of the Southern blot --- p.40 / Chapter 2.16 --- Northern analysis --- p.41 / Chapter 2.16.1 --- "Isolation of total RNA by using the AGPC-RNA method (Chomczynski and Sacchi,1987, modified)" --- p.41 / Chapter 2.16.2 --- Separation of total RNA by electrophoresis and transfer onto a membrane support --- p.43 / Chapter 2.16.3 --- Prehybridization of the Northern blot --- p.46 / Chapter 2.16.4 --- Hybridization of the Northern blot --- p.47 / Chapter 2.17 --- First strand cDNA synthesis (Pharmacia) --- p.48 / Chapter 2.18 --- PCR of the first strand cDNA --- p.48 / Chapter Chapter Three: --- Results --- p.50-113 / Chapter 3.1 --- Partial sequencing of adult human heart cDNA clones --- p.50 / Chapter 3.2 --- DNA homology searching by using the program BLASTN --- p.52 / Chapter 3.2.1 --- Catalogue of the 502 ESTs of the cardiovascular system --- p.54 / Chapter 3.2.2 --- Classification and frequency of the human adult heart cDNA clones --- p.63 / Chapter 3.3 --- Submission of the cDNA sequences to NCBI --- p.64 / Chapter 3.4 --- Pattern of gene expression in the human adult cardiovascular system --- p.66 / Chapter 3.5 --- "Sequence determination of hrpL29 (Law et. al., 1996)" --- p.72 / Chapter 3.5.1 --- Cycle Taq sequencing of hrpL29 --- p.72 / Chapter 3.5.2 --- Subcloning of the hrpL29 cDNA insert into the pUC18 DNA cloning vector --- p.75 / Chapter 3.5.3 --- Unicycle T7 sequencing of hrpL29 --- p.77 / Chapter 3.6 --- Sequence alignment and comparison of hrpL29 with other known sequences in the databases --- p.79 / Chapter 3.7 --- The primary structure of hrpL29 --- p.83 / Chapter 3.8 --- Results of RT-PCR and PCR --- p.88 / Chapter 3.9 --- Genomic analysis of hrpL29 --- p.92 / Chapter 3.9.1 --- Isolation of the first intron of hrpL29 --- p.92 / Chapter 3.9.2 --- Southern analysis of hrpL29 --- p.97 / Chapter 3.10 --- Northern analysis of hrpL29 --- p.103 / Chapter 3.10.1 --- Tissue distribution of hrpL29 mRNA in rat tissues --- p.103 / Chapter 3.10.2 --- Time course of hRPL29 expression in mouse heart --- p.106 / Chapter 3.10.3 --- Time course of hRPL29 expression in mouse brain --- p.110 / Chapter Chapter Four: --- Discussion --- p.114-139 / Chapter 4.1 --- Characterization of the ESTs --- p.114 / Chapter 4.2 --- Significance of the heart EST project --- p.116 / Chapter 4.3 --- Redundancy of the EST sequencing --- p.118 / Chapter 4.4 --- The importance of frequent database searching --- p.119 / Chapter 4.5 --- The importance of an efficient comparison algorithm --- p.120 / Chapter 4.6 --- Human ribosomal protein L29 (hRPL29) --- p.122 / Chapter 4.7 --- Internal duplication in hRPL29 --- p.124 / Chapter 4.8 --- Primary structure analysis of hRPL29 --- p.126 / Chapter 4.9 --- RT-PCR and PCR of the first strand cDNA with primers using the C095-ATG and dT primer --- p.128 / Chapter 4.10 --- Southern analysis of hrpL29 --- p.128 / Chapter 4.11 --- Northern analysis of hrpL29 --- p.133 / Chapter 4.11.1 --- Tissue distribution of the mRNA species of hrpL29 --- p.133 / Chapter 4.11.2 --- Time course of hRPL29 expression in mouse heart and brain --- p.134 / Chapter 4.12 --- Possible functional role of hRPL29 --- p.135 / Chapter 4.13 --- Further aspects --- p.137 / Appendix --- p.140-143 / References --- p.144-155
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_321603 |
| Date | January 1996 |
| Contributors | Law, Patrick Tik-wan., 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, xiv, 155 leaves : ill. (some col., 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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