by Nathan, Yiu-hung Yam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 139-157). / Acknowledgements --- p.i / Table of Contents --- p.ii / Abstract --- p.viii / List of Abbreviations --- p.x / List of Tables --- p.xii / List of Figures --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General introduction --- p.1 / Chapter 1.2 --- Aims of the present study --- p.2 / Chapter 1.3 --- Sequencing of an adult human heart cDNA library --- p.3 / Chapter 1.4 --- Rat/mouse CRIP --- p.5 / Chapter 1.5 --- LIM proteins --- p.13 / Chapter 1.6 --- Zinc-binding proteins --- p.17 / Chapter 1.7 --- Bacterial expression system using the pAED4 vector --- p.24 / Chapter Chapter 2 --- Identification and sequence analysis ofhCRHP --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and methods --- p.29 / Chapter 2.2.1 --- Bacterial strains and vectors --- p.29 / Chapter 2.2.2 --- "Mediums, buffers and solutions" --- p.31 / Chapter 2.2.3 --- Bacteriophage clones preparation --- p.34 / Chapter 2.2.4 --- Amplification of clones by PCR --- p.35 / Chapter 2.2.5 --- Cycle sequencing of PCR products --- p.36 / Chapter 2.2.6 --- DNA sequences analysis --- p.38 / Chapter 2.3 --- Results --- p.39 / Chapter 2.3.1 --- Sequence analysis of hCRHP --- p.39 / Chapter 2.3.2 --- Comparison of hCRHP with CRIP --- p.52 / Chapter 2.3.3 --- Comparison of hCRHP with some LIM proteins --- p.56 / Chapter 2.4 --- Discussions --- p.61 / Chapter Chapter 3 --- Study of hCRHP at the nucleic acid level --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Materials and methods --- p.66 / Chapter 3.2.1 --- Animals --- p.66 / Chapter 3.2.2 --- "Mediums, buffers, enzymes and solutions" --- p.66 / Chapter 3.2.3 --- Preparation of total RNA --- p.70 / Chapter 3.2.3.1 --- Preparation of RNA by the CsCl method --- p.70 / Chapter 3.2.3.2 --- Preparation of RNA by the AGPC method --- p.71 / Chapter 3.2.4 --- Northern hybridization of hCRHP --- p.72 / Chapter 3.2.4.1 --- Formaldehyde agarose gel electrophoresis --- p.72 / Chapter 3.2.4.2 --- Preparation of radioactive probe --- p.73 / Chapter 3.2.4.3 --- RNA transfer and Northern hybridization --- p.74 / Chapter 3.2.5 --- Preparation of human genomic DNA --- p.77 / Chapter 3.2.6 --- Southern hybridization of hCRHP --- p.78 / Chapter 3.2.6.1 --- Restriction cutting and agarose gel electrophoresis of genomic DNA --- p.78 / Chapter 3.2.6.2 --- DNA transfer and Southern hybridization --- p.79 / Chapter 3.3 --- Results --- p.80 / Chapter 3.3.1 --- Southern hybridization of hCRHP --- p.80 / Chapter 3.3.2 --- Identification of hCRHP in neonatal human heart --- p.83 / Chapter 3.3.3 --- Tissue distribution of CRIP mRNA in rat tissues --- p.85 / Chapter 3.3.4 --- Time course of CRIP expression in rat heart --- p.85 / Chapter 3.4 --- Discussions --- p.87 / Chapter Chapter 4 --- Subcloning and expression of hCRHP --- p.89 / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Materials and methods --- p.90 / Chapter 4.2.1 --- Bacterial strains and vectors --- p.90 / Chapter 4.2.2 --- "Mediums, buffers, enzymes and solutions" --- p.92 / Chapter 4.2.3 --- Subcloning of hCRHP into pAED4 --- p.98 / Chapter 4.2.3.1 --- Primers design and PCR --- p.98 / Chapter 4.2.3.2 --- Purification of PCR products by Geneclean II´ёØ (BIO 101 Inc) --- p.99 / Chapter 4.2.3.3 --- Restriction digestion of purified PCR product and pAED4 --- p.100 / Chapter 4.2.3.4 --- Ligation and transformation of hCRHP --- p.101 / Chapter 4.2.3.5 --- Amplification and purification of pAED4-hCRHP --- p.103 / Chapter 4.2.4 --- Expression of hCRHP --- p.105 / Chapter 4.2.4.1 --- Induction of hCRHP expression --- p.105 / Chapter 4.2.4.2 --- SDS-PAGE and protein detection --- p.106 / Chapter 4.3 --- Results --- p.108 / Chapter 4.3.1 --- Subcloning of hCRHP into pAED4 --- p.108 / Chapter 4.3.2 --- Induction and optimization of hCRHP expression --- p.110 / Chapter 4.4 --- Discussions --- p.117 / Chapter Chapter 5 --- Partial purification and isoelectric focusing of hCRHP --- p.120 / Chapter 5.1 --- Introduction --- p.120 / Chapter 5.2 --- Materials and methods --- p.121 / Chapter 5.2.1 --- "Mediums, buffers and mediums" --- p.121 / Chapter 5.2.2 --- Purification of hCRHP by ammonium sulphate precipitation --- p.121 / Chapter 5.2.3 --- Purification of hCRHP by hydrochloric acid extraction --- p.122 / Chapter 5.2.4 --- Purification of hCRHP by ultrafiltration --- p.123 / Chapter 5.2.5 --- Isoelectric focusing of hCRHP --- p.127 / Chapter 5.3 --- Results --- p.128 / Chapter 5.3.1 --- Partial purification of hCRHP by ammonium sulphate precipitation --- p.128 / Chapter 5.3.2 --- Partial purification of hCRHP by hydrochloric acid extraction --- p.128 / Chapter 5.3.3 --- Partial purification of hCRHP by ultrafiltration --- p.131 / Chapter 5.3.4 --- Isoelectric focusing of hCRHP --- p.133 / Chapter 5.4 --- Discussions --- p.133 / Chapter Chapter 6 --- Discussions --- p.136 / Chapter 6.1 --- The possible role(s) of hCRHP/CRIP --- p.136 / Chapter 6.2 --- Future prospects --- p.137 / References --- p.139 / Appendix 1 --- p.158
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_320526 |
| Date | January 1995 |
| Contributors | Yam, Nathan Yiu-hung., 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, xv, 167 leaves : ill. (some 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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