Cloning of pollutant inducible genes from common carp, cyprinus carpio.

• Other Authors: Chan, Pat Chun.
• Format: Others
• Language: English
• Published: Chinese University of Hong Kong 1996
• Subjects: Metallothionein; Cytochrome P-450; Cloning; Carp > Physiology
• Online Access: http://library.cuhk.edu.hk/record=b5888789; http://repository.lib.cuhk.edu.hk/en/item/cuhk-321577

Chan Pat Chun. === Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. === Includes bibliographical references (leaves 153-177). === Acknowledgments --- p.i === Presentations Derived from the Present thesis Work --- p.ii === Abstract --- p.iii === Abbreviations --- p.v === Abbreviation Table for Amino Acids --- p.viii === List of Figures --- p.ix === List of Tables --- p.xi === Contents --- p.xii === Chapter 1 --- Introduction --- p.1 === Chapter 1.1 --- Environmental Pollutants --- p.1 === Chapter 1.2 --- Pollutant Inducible Genes (PIGs) --- p.1 === Chapter 1.2.1 --- Classification of PIGS --- p.2 === Chapter 1.2.1.1 --- Drug Metabolizing Enzymes/Proteins --- p.2 === Chapter 1.2.1.2 --- Stress Proteins --- p.5 === Chapter 1.2.1.3 --- Antioxidant Enzymes --- p.6 === Chapter 1.2.1.4 --- "Hormones, Growth Factors and Their Receptors" --- p.6 === Chapter 1.2.1.5 --- Enzymes/Proteins Involved in Bioenergetics --- p.6 === Chapter 1.2.2 --- PIGs as a Field of Study --- p.8 === Chapter 1.2.2.1 --- Study of the Mechanism of Detoxification and Toxication --- p.8 === Chapter 1.2.2.2 --- Biomarker Study --- p.9 === Chapter 1.2.2.3 --- Study of Regulation of Gene Expression --- p.11 === Chapter 1.2.2.4 --- Study of Evolution --- p.12 === Chapter 1.3 --- Aims and Rational of the Present Study --- p.12 === Chapter 2 --- General Methodology --- p.15 === Chapter 2.1 --- Materials --- p.15 === Chapter 2.2.1 --- Reagents --- p.15 === Chapter 2.1.1.1 --- Preparation of Plasmid DNA --- p.15 === Chapter 2.1.1.2 --- Preparation of Genomic DNA --- p.15 === Chapter 2.1.1.3 --- Purification of Total RNA --- p.16 === Chapter 2.1.1.4 --- Restriction Enzyme Digestion --- p.16 === Chapter 2.1.1.5 --- Capillary Blotting of DNA (Southern Blotting) --- p.16 === Chapter 2.1.1.6 --- Capillary Blotting of Total RNA (Northern Blotting) --- p.17 === Chapter 2.1.1.7 --- Hybridization --- p.17 === Chapter 2.1.1.8 --- Library Screening --- p.18 === Chapter 2.1.1.9 --- Polymerase Chain Reaction --- p.18 === Chapter 2.1.1.10 --- Transformation of E. coli Competent Cells --- p.19 === Chapter 2.1.1.11 --- Nucleotide Sequence Determination --- p.19 === Chapter 2.1.2 --- List of Primers --- p.20 === Chapter 2.1.2.1 --- Primers used for Nucleotide Sequence Determination --- p.20 === Chapter 2.1.2.2 --- Primer Used for First Strand cDNA Synthesis --- p.20 === Chapter 2.1.2.3 --- Primers for Amplifying Actin cDNA Fragment --- p.20 === Chapter 2.1.2.4 --- Common Carp MT Specific Primers --- p.20 === Chapter 2.1.2.5 --- Teleost CYP1A Specific Primers --- p.21 === Chapter 2.1.2.6 --- Common Carp CYP1A Specific Primers --- p.21 === Chapter 2.1.2.7 --- Primers and Cassettes for the Cloning of5' Upstream Regions of MT Genes --- p.21 === Chapter 2.1.3 --- Accession Numbers of Selected P450 and MT Nucleotide and Amino Acid Sequences in the Genebank --- p.21 === Chapter 2.1.3.1 --- MTs of Different Teleost Species --- p.21 === Chapter 2.1.3.2 --- MTs of Other Vertebrate Species' --- p.22 === Chapter 2.1.3.3 --- P450s of Different Teleost Species --- p.22 === Chapter 2.1.3.4 --- CYP1s of Different Mammalian Species --- p.22 === Chapter 2.2 --- Methods --- p.23 === Chapter 2.2.1 --- Preparation of Plasmid --- p.23 === Chapter 2.2.2 --- Preparation of Genomic DNA --- p.23 === Chapter 2.2.3 --- Purification of Total RNA --- p.24 === Chapter 2.2.4 --- Restriction Enzyme Digestion --- p.25 === Chapter 2.2.5 --- Capillary Blotting of DNA (Southern Blotting) --- p.25 === Chapter 2.2.5.1 --- Semi-dry Capillary Blotting --- p.25 === Chapter 2.2.5.2 --- Alkaline Transfer --- p.25 === Chapter 2.2.5.3 --- Transfer of Digested Genomic DNA on to Nylon Membrane --- p.26 === Chapter 2.2.6 --- Capillary Blotting of Total RNA (Northern Blotting) --- p.26 === Chapter 2.2.7 --- Radioactive Labeling of Nucleic Acid Probes --- p.26 === Chapter 2.2.8 --- Hybridization --- p.27 === Chapter 2.2.9 --- Library Screening --- p.27 === Chapter 2.2.9.1 --- Construction of Liver cDNA Library of Adult Common Carp --- p.27 === Chapter 2.2.9.2 --- Preparation of Plating Cells --- p.27 === Chapter 2.2.9.3 --- Phage Tittering --- p.27 === Chapter 2.2.9.4 --- Primary Screening --- p.28 === Chapter 2.2.9.5 --- Secondary Screening === Chapter 2.2.9.6 --- Conversion of Phage DNA to Phagemid by invivo Excision --- p.28 === Chapter 2.2.10 --- First Strand cDNA Synthesis --- p.29 === Chapter 2.2.11 --- Polymerase Chain Reaction --- p.29 === Chapter 2.2.12 --- Ligation of DNA with Linearized Plasmid --- p.30 === Chapter 2.2.13 --- Transformation of E. coli Competent Cell --- p.30 === Chapter 2.2.14 --- Nucleotide Sequence Determination --- p.31 === Chapter 2.2.15 --- Densitometric Analysis --- p.31 === Chapter 3 --- "Cloning of Common Carp MT cDNA and Gene, and Induction of MT mRNA Expression" --- p.32 === Chapter 3.1 --- Introduction --- p.32 === Chapter 3.1.1 --- Metals in Biological System --- p.32 === Chapter 3.1.2 --- Metallothionein --- p.33 === Chapter 3.1.2.1 --- Functions of MT --- p.26 === Chapter 3.1.2.2 --- Regulation of MT Expression --- p.39 === Chapter 3.1.3 --- Fish MTs --- p.44 === Chapter 3.1.3.1 --- Detection of MT in Teleost --- p.46 === Chapter 3.1.3.2 --- MT Studies in Common Carp --- p.47 === Chapter 3.1.4 --- Specific Aims of This Chapter --- p.49 === Chapter 3.2 --- Strategies --- p.50 === Chapter 3.3 --- Specific Methods --- p.50 === Chapter 3.3.1 --- Cloning of MT cDNAs of Common Carp --- p.50 === Chapter 3.3.2 --- Analysis of MT cDNA Sequences --- p.51 === Chapter 3.3.3 --- Southern Blot Analysis of Common Carp Genomic DNA --- p.52 === Chapter 3.3.4 --- Amplification of MT Gene Fragments Using PCR --- p.52 === Chapter 3.3.5 --- Amplification of the 5' Upstream Regions of MT Genes Using PCR --- p.52 === Chapter 3.3.6 --- Endogenous MT mRNA Expression of Juvenile and Adult Common Carp --- p.54 === Chapter 3.3.7 --- Induction of MT mRNA of Juvenile Common Carp Injected with Cadmium --- p.55 === Chapter 3.4 --- Results --- p.56 === Chapter 3.4.1 --- Cloning of Common Carp MT cDNAs --- p.56 === Chapter 3.4.2 --- Analysis of the MT cDNA Sequences --- p.57 === Chapter 3.4.3 --- Southern Blot Analysis of the Common Carp Genomic DNA --- p.59 === Chapter 3.4.4 --- Amplification of the MT Gene Fragments of Common Carp Using PCR --- p.62 === Chapter 3.4.5 --- Amplification of the 5' Upstream Regions of MT Genes using PCR --- p.65 === Chapter 3.4.6 --- Endogenous MT mRNA Expression of Juvenile and Adult Common Carp --- p.67 === Chapter 3.4.7 --- Induction of MT mRNA of Juvenile Common Carp Injected with Cadmium --- p.68 === Chapter 3.5 --- Discussion --- p.72 === Chapter 3.5.1 --- MT cDNAs of Common Carp --- p.72 === Chapter 3.5.1.1 --- Coding Region --- p.72 === Chapter 3.5.1.2 --- The 3' Untranslated Region --- p.75 === Chapter 3.5.1.3 --- The 5' Untranslated Region --- p.76 === Chapter 3.5.2 --- MT Genes of Common Carp --- p.77 === Chapter 3.5.3 --- MT mRNA Expression of Common Carp --- p.82 === Chapter 3.5.4 --- Normalization of the Signals of Northern Blot Analysis --- p.85 === Chapter 3.5.5 --- Common Carp MT mRNA as Biomarker of Heavy Metal Exposure? --- p.87 === Chapter 3.6 --- Conclusion --- p.89 === Chapter 4 --- Cloning of Common Carp CYP1A cDNAs and Induction of CYP1A mRNA Expression --- p.90 === Chapter 4.1 --- Introduction --- p.90 === Chapter 4.1.1 --- Cytochrome P450s --- p.90 === Chapter 4.1.2 --- Cytochrome P450 1 (CYP1) --- p.93 === Chapter 4.1.3 --- AhR Mediated CYP1A1 Gene Induction --- p.94 === Chapter 4.1.3.1 --- Anthropogenic Sources of AhR Ligands --- p.95 === Chapter 4.1.3.2 --- Natural Sources of AhR Ligands --- p.97 === Chapter 4.1.3.3 --- Potency of Inducibility --- p.97 === Chapter 4.1.3.4 --- Induction of CYP1A1 Gene Transcription by AhR --- p.98 === Chapter 4.1.3.5 --- Non-AhR Mediated CYP1A1 Gene Transcription? --- p.105 === Chapter 4.1.4 --- CYP1A Studies in Teleost Species --- p.107 === Chapter 4.1.4.1 --- Regulation of CYP1A in Teleost --- p.109 === Chapter 4.1.4.2 --- Detection of CYP1A in Teleost --- p.111 === Chapter 4.1.4.3 --- CYP1A Studies of Common Carp --- p.113 === Chapter 4.1.5 --- Specific Aims of This Chapter --- p.114 === Chapter 4.2 --- Strategies --- p.115 === Chapter 4.3 --- Specific Methods --- p.119 === Chapter 4.3.1 --- RT-PCR of CYP1A cDNAs of Common Carp --- p.119 === Chapter 4.3.2 --- Determination of the Nucleotide Sequences of the CYP1A cDNAs of Common Carp --- p.119 === Chapter 4.3.3 --- Library Screening --- p.119 === Chapter 4.3.4 --- Analysis of the CYP1A Genes of Common Carp --- p.121 === Chapter 4.3.5 --- Induction of CYP1A mRNA of Common Carp Injected with 3-MC --- p.122 === Chapter 4.4 --- Results --- p.123 === Chapter 4.4.1 --- RT-PCR of CYP1A cDNAs of Common Carp --- p.123 === Chapter 4.4.2 --- Determination of the Nucleotide Sequences of the CYP1A cDNAs of Common Carp --- p.124 === Chapter 4.4.3 --- Library Screening --- p.124 === Chapter 4.4.4 --- Analysis of the CYP1A Genes of Common Carp --- p.128 === Chapter 4.4.5 --- Induction of CYP1A mRNA of Common Carp Injected with 3-MC --- p.131 === Chapter 4.5 --- Discussion --- p.134 === Chapter 4.5.1 --- On the Use of Rainbow Trout CYP1A1 cDNA Probe --- p.134 === Chapter 4.5.2 --- CYP1A cDNAs of Common Carp --- p.134 === Chapter 4.5.3 --- CYP1A Genes of Common Carp --- p.138 === Chapter 4.5.4 --- CYP1A Expression in Uninduced and Induced Tissues --- p.142 === Chapter 4.5.5 --- The Use of CYP1A cDNAs As Biomarkers --- p.146 === Chapter 4.6 --- Conclusion --- p.148 === Chapter 5 --- General Conclusion --- p.149 === Chapter 6 --- References --- p.153