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

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