No description available.

The aim of this project was to clone the gene encoding the catalytic subunit of the rat DNA replication enzyme, DNA polymerase alpha. A strategy was adopted in which cDNA clones expressing the catalytic subunit sequences would be identified using anti-DNA polymerase antibodies. DNA polymerase alpha was partially purified from regenerating rat liver and exponentially growing rat Y3 myeloma cells. The catalytic subunit was identified as a 170-180kD polypeptide by activity gel analysis of partially purified Y3 cell fractions. The catalytic subunit was found to be susceptible to degradation but without loss of polymerase activity. Glycerol gradient analysis indicated a two stage degradation of DNA polymerase in vivo. Sera were collected from mice immunised with partially purified DNA polymerase alpha from regenerated rat liver. These sera cross-reacted with Western-blotted Y3 cell fractions; removed polymerase activity from solution in plate binding assays and bound alpha polymerase activity (140-180kD) on an immuno-adsorption column cDNA was synthesised using size selected mRNA from exponentially growing Y3 cells and cloned into the expression vectors pUC8 and ?gtll, both of which utilise the lac Z gene to express cloned DNA sequences. Immunoscreening of the ?gtll library was frustrated by non-specific binding of the serum. This non-specific binding was overcome by pre-adsorbing the serum against a lysate of E. coli JM 83. Screening of the pUC8 library revealled 27 out of 2.25x104 colonies which bound pre-adsorbed anti-DNA polymerase alpha serum.

No description available.

Chack-yung Yu. / Bibliography: leaves 160-175 / Thesis (M.Phil.) -- Chinese University of Hong Kong, 1983

by Ka-ming Pang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 95-100. / Abstract --- p.i / Introduction --- p.1 / Literature review --- p.2 / Chapter 1. --- Mode of Respiration in Enteric Bacteria --- p.2 / Chapter 2. --- Nitrate Respiration --- p.4 / Chapter 3 . --- Biochemistry of Nitrate Reductase --- p.7 / Chapter 4 . --- Regulation of Nitrate Reductase --- p.8 / Chapter 5. --- Genetics and Regulation of chlC (nitrate Reductase) --- p.10 / Chapter 6. --- chlC in Salmonella typhimurium --- p.14 / Chapter 7. --- Purpose of study --- p.16 / Materials and Methods / Chapter 1 . --- Strains --- p.18 / Chapter 2. --- Media --- p.18 / Chapter 3 . --- Solution --- p.20 / Chapter 4. --- β-galactosidase assay --- p.23 / Chapter 5. --- Preparation of chromosome DNA --- p.24 / Chapter 6. --- Small scale plasmid preparation --- p.25 / Chapter 7. --- Large scale plasmid preparation --- p.26 / Chapter 8. --- Preparation of lambda DNA --- p.27 / Chapter 9. --- Preparation of M13 ssDNA for sequencing --- p.30 / Chapter 10. --- Digestion of DNA with restriction enzymes --- p.31 / Chapter 11 . --- Agarose gel electrophoresis --- p.31 / Chapter 12. --- Ligation --- p.31 / Chapter 13. --- Transformation --- p.32 / Chapter 14. --- Direct gel test and C-test of ssDNA of M13 clones --- p.34 / Chapter 15. --- DNA sequencing --- p.34 / Results / Chapter 1 . --- β-galactosidase assay of HSK1001 --- p.37 / Chapter 2. --- Preparation of HSK1001 DNA --- p.37 / Chapter 3. --- Sau3A partial digestion of HSK1001 chromosomal DNA --- p.37 / Chapter 4 . --- Ligation of HSK1001 DNA to lambda EMBL3 --- p.40 / Chapter 5. --- In vitro packaging and screening of lambda clones --- p.40 / Chapter 6. --- Restriction mapping of lambda clones --- p.43 / Chapter 6.1 --- Restriction mapping of HSK4001 to HSK4006 --- p.43 / Chapter 6.2 --- Orientation of Mu dA fusion clones --- p.43 / Chapter 7 . --- "Sub-cloning of SalI-HindIII fragments of HSK4002 HSK4003, HSK4005 and HSK4006 into pFZYl" --- p.66 / Chapter 8. --- Sub-cloning of 2.4 kb HindIII fragment of HSK4005 and HSK4006 into pFZY1 --- p.67 / Chapter 9. --- Sub-cloning of 2.4 kb HindIII fragment of HSK4006 into M13mpl9 --- p.67 / Chapter 10. --- Sub-cloning of 2.4 kb HindIII fragment of HSK4005 into M13mp19 --- p.69 / Chapter 11. --- Sub-cloning of 2.5 kb SalI-HindIII fragment of HSK4006 into M13mpl8 --- p.72 / Chapter 12. --- Sub-cloning of 2.2 kb SalI-HindIII fragment of HSK4005 into M13mp19 --- p.72 / Chapter 13. --- Complementation test of M13 clones --- p.74 / Chapter 14. --- Sub-cloning of 4.3 kb HindIII-EcoRI fragment and 3.8 kb EcoRI-BamHI fragments of HSK4006 into M13 mp18 and mp19 --- p.74 / Chapter 15. --- DNA sequences of M13 clones --- p.77 / Dicussion / Chapter 1 . --- Cloning of Mu dA operon fusion to EMBL3 vector --- p.88 / Chapter 2 . --- Sequences of SalI-HindIII fragments of HSK4005 to HSK4006 --- p.92 / Reference --- p.95

by Yung Mei Hing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 130-135. / ACKNOWLEDEMENTS --- p.i / ABSTRACT --- p.ii / CONTENTS --- p.iii / ABBREVIATIONS --- p.viii / Chapter CHAPTER ONE --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Chemistry and Structure of Trichosanthin --- p.1 / Chapter 1.1.1 --- Chemistry --- p.1 / Chapter 1.1.2 --- Primary Structure --- p.2 / Chapter 1.1.3 --- Three-dimensional Structure --- p.4 / Chapter 1.2 --- Biological Activities of Trichosanthin --- p.6 / Chapter 1.2.1 --- Abortifacient Properties --- p.6 / Chapter 1.2.1A --- Termination of Mid-term Gestation --- p.6 / Chapter 1.2.1B --- Inhibition of Early Pregnancy --- p.12 / Chapter 1.2.2 --- Immunological Properties --- p.13 / Chapter 1.2.2A --- Antigenicity and Allergenicity --- p.13 / Chapter 1.2.2B --- Immunosuppressive Effects --- p.14 / Chapter 1.2.3 --- Anti-tumour Activity --- p.15 / Chapter 1.2.4 --- Ribosome-inactivating Activity --- p.16 / Chapter 1.2.5 --- Human Immunodeficiency Virus (HIV) Inhibitory Activity --- p.20 / Chapter 1.3 --- Objectives and Strategy of Cloning the cDNA of Trichosanthin --- p.21 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.24 / Chapter 2.1 --- General Techniques --- p.24 / Chapter 2.1.1 --- Extraction with Phenol --- p.24 / Chapter 2.1.2 --- Ethanol Precipitation --- p.24 / Chapter 2.1.3 --- Spectrophotometry --- p.25 / Chapter 2.1.4 --- Restriction Digestion of DNA --- p.25 / Chapter 2.1.5 --- End-labelling DNA with Recessed 3'-ends --- p.25 / Chapter 2.1.6 --- Agarose Gel Electrophoresis of DNA --- p.26 / Chapter 2.1.7 --- Elution of DNA from Agarose Gel --- p.26 / Chapter 2.1.8 --- Minipreparation of Bacteriophage A DNA from Plate Lysates --- p.27 / Chapter 2.1.9 --- Minipreparation of Plasmid DNA --- p.28 / Chapter 2.1.10 --- Large-scale Preparation of Plasmid DNA --- p.29 / Chapter 2.1.10A --- By Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradient --- p.29 / Chapter 2.1.10B --- By Using QIAGEN-pack 100 Cartridge --- p.31 / Chapter 2.1.11 --- Preparation and Transformation of Competent Escherichia coli --- p.32 / Chapter 2.1.12 --- Preparation of Nucleic Acid Probes --- p.34 / Chapter 2.1.12A --- By Nick Translation --- p.34 / Chapter 2.1.12B --- By Random-primed Labelling --- p.34 / Chapter 2.1.13 --- Sephadex G-50 Spun-column Chromatography --- p.35 / Chapter 2.1.14 --- Monitoring the Progress of Probe Labelling Reactions --- p.37 / Chapter 2.1.15 --- Liquid Scintillation Counting --- p.37 / Chapter 2.1.16 --- Southern and Northern Hybridizations --- p.38 / Chapter 2.1.17 --- Autoradiography --- p.41 / Chapter 2.2 --- Techniques for Constructing a Complementary DNA (cDNA) Library --- p.42 / Chapter 2.2.1 --- Extraction and Purification of Plant RNA --- p.42 / Chapter 2.2.2 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.44 / Chapter 2.2.3 --- Ohgo(dT)-cellulose Column Chromatography --- p.46 / Chapter 2.2.4 --- In Vitro Translation in Rabbit Reticulocyte Lysate --- p.47 / Chapter 2.2.5 --- SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) of In Vitro Translation Products --- p.48 / Chapter 2.2.6 --- cDNA Synthesis --- p.50 / Chapter 2.2.6A --- First Strand Synthesis --- p.50 / Chapter 2.2.6B --- Second Strand Synthesis --- p.51 / Chapter 2.2.6C --- Purification of the Double-stranded cDNA (ds cDNA) --- p.51 / Chapter 2.2.7 --- Methylation of the cDNA with EcoRI Methylase --- p.52 / Chapter 2.2.8 --- Addition of EcoRl Cohesive Termini onto the cDNA --- p.52 / Chapter 2.2.9 --- Removal of Excess EcoRI Linkers and Size Fractionation of the cDNA --- p.53 / Chapter 2.2.10 --- Ligation of the cDNA with EcoRI-digested λgt10 --- p.55 / Chapter 2.2.11 --- In Vitro Packaging --- p.56 / Chapter 2.2.12 --- Titration of the λgt10 Library V --- p.56 / Chapter 2.3 --- Screening the cDNA Library in λgt10 with a Nucleic Acid Probe --- p.57 / Chapter 2.4 --- Subcloning DNA Fragments in pUC18 --- p.59 / Chapter 2.4.1 --- Dephosphorylation of Linearized pUC18 with Protruding 5' Termini --- p.59 / Chapter 2.4.2 --- Ligation of Foreign DNA with Linearized pUC18 with Cohesive Termini --- p.60 / Chapter 2.5 --- DNA Sequencing on Double-stranded Templates --- p.60 / Chapter 2.5.1 --- DNA Sequencing Reaction --- p.61 / Chapter 2.5.1A --- Alkaline Denaturation of Double-stranded Plasmid Template --- p.61 / Chapter 2.5.1B --- Labelling Reaction and Termination Reactions --- p.61 / Chapter 2.5.2 --- DNA Sequencing Electrophoresis --- p.62 / Chapter CHAPTER THREE --- CONSTRUCTION AND CLONAL SCREENING OF cDNA LIBRARY FROM ROOT TUBERS OF TRICHOSANTHES KIRILOWII MAXIMOWICZ --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Isolation of Total RNA from Root Tubers --- p.69 / Chapter 3.3 --- Purification of Poly(A)+ RNA from Total RNA --- p.70 / Chapter 3.4 --- Northern Blot Analysis of the Poly(A)+ RNA --- p.72 / Chapter 3.5 --- In Vitro Translation of the Poly(A)+ RNA --- p.75 / Chapter 3.6 --- cDNA Synthesis --- p.78 / Chapter 3.7 --- Southern Blot Analysis of the cDNA --- p.81 / Chapter 3.8 --- Addition of EcoRI Cohesive Termini to the cDNA --- p.81 / Chapter 3.9 --- Removal of Excess EcoRI Linkers and Size Fractionation of the cDNA vi --- p.84 / Chapter 3.10 --- Ligation of the Size Fractionated cDNA with EcoRI-digested λgt10 and In Vito Packaging of the A Hybrids --- p.85 / Chapter 3.11 --- Analysis of cDNA Inserts --- p.85 / Chapter 3.12 --- Clonal Screening of the cDNA Library --- p.88 / Chapter 3.13 --- Characterization of Positive Clones --- p.88 / Chapter 3.14 --- Subcloning Positive Inserts into pUC18 --- p.92 / Chapter 3.15 --- Discussion --- p.93 / Chapter CHAPTER FOUR --- DNA SEQUENCING OF POSITIVE CLONES --- p.99 / Chapter 4.1 --- Introduction --- p.99 / Chapter 4.2 --- Sequencing Strategies --- p.104 / Chapter 4.2.1 --- pTCS48210 Sequencing --- p.m / Chapter 4.2.2 --- pTCS5021 Sequencing --- p.107 / Chapter 4.3 --- Results and Discussion --- p.108 / Chapter 4.3.1 --- Nucleotide Sequence and Deduced Amino Acid Sequence --- p.108 / Chapter 4.3.2 --- Secondary Structure Predicted from the Amino Acid Sequence Encoded by the Trichosanthin cDNA --- p.116 / APPENDIX A --- p.124 / APPENDIX B --- p.126 / APPENDIX C --- p.129 / REFERENCES --- p.130

by Chan Yuk Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 120-137). / Acknowledgments --- p.i / Table of Content --- p.ii / List of Tables --- p.vi / List of Figures --- p.viii / List of Abbreviations --- p.xi / Chapter CHAPTER 1 --- Literature Review Page / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- "Overview of the Structures of GH, PRL and SL" --- p.2 / Chapter 1.3 --- Physiological Significance of the Hormones in Teleost --- p.12 / Chapter 1.3.1 --- Growth Hormone --- p.12 / Chapter 1.3.2 --- Prolactin --- p.12 / Chapter 1.3.3 --- Somatolactin --- p.13 / Chapter 1.4 --- Structure - Function Relationship and Receptor Binding Activities --- p.15 / Chapter 1.5 --- Aims of Thesis --- p.23 / Chapter CHAPTER 2 --- General Methodology / Chapter 2.1 --- Materials --- p.25 / Chapter 2.2 --- DNA manipulation methods --- p.28 / Chapter 2.2.1 --- Polymerase Chain Reaction (PCR) --- p.28 / Chapter 2.2.2 --- Ethanol Precipitation of DNA --- p.29 / Chapter 2.2.3 --- Agarose Gel Electrophoresis of DNA --- p.29 / Chapter 2.2.4 --- Sephadex G-50 Spun Column --- p.30 / Chapter 2.2.5 --- Nick Translation --- p.30 / Chapter 2.2.6 --- Small Scale Plasmid Preparation by Alkaline Lysis Method --- p.31 / Chapter 2.2.7 --- Large Scale Plasmid Preparation Using Magic´ёØ Maxipreps DNA Purification System (Promega) --- p.32 / Chapter 2.3 --- DNA Cloning Methods --- p.33 / Chapter 2.3.1 --- Blunt-end Ligation of PCR Product to pUC18 Vector --- p.33 / Chapter 2.3.1.1 --- Preparation and Transformation of E. coli (JM109) Competent Cell --- p.33 / Chapter 2.3.1.2 --- Recovery of DNA from Agarose Gel Using the Sephaglas Bandprep Kit (Pharmacia Biotech) --- p.34 / Chapter 2.3.1.3 --- Kinasing Reaction --- p.34 / Chapter 2.3.1.4 --- Klenow Fill-in Reaction and Ligation of DNA Fragments --- p.35 / Chapter 2.3.2 --- Screening of Lambda Phage cDNA Library --- p.35 / Chapter 2.3.2.1 --- Preparation of Host Cell for Screening of Lambda Phage cDNA Library --- p.35 / Chapter 2.3.2.2 --- Phage Stock Tittering --- p.36 / Chapter 2.3.2.3 --- Plaque Lifting and Fixation on Nylon Membranes --- p.36 / Chapter 2.3.2.4 --- Library Screening by Hybridization --- p.37 / Chapter 2.3.2.5 --- In vivo Excision --- p.37 / Chapter 2.4 --- Nucleotide Sequence Determination using Dideoxy Nucleotide Chain Termination Method --- p.38 / Chapter 2.5 --- Protein methods --- p.39 / Chapter 2.5.1 --- Bicinchoninic Acid (BCA) Assay --- p.39 / Chapter 2.5.2 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.40 / Chapter CHAPTER 3 --- Isolation and Characterization of cDNA Clones for Goldfish Prolactin / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Methods --- p.43 / Chapter 3.2.1 --- Preparation of Hormone Specific DNA Probes by PCR Cloning --- p.43 / Chapter 3.2.2 --- Construction of Goldfish Pituitary cDNA Library --- p.44 / Chapter 3.2.3 --- cDNA Library Screening for Hormone Specific cDNAs --- p.44 / Chapter 3.2.4 --- Restriction Enzyme Digestion of the cDNA Clones and Subcloning of the Restriction Fragments --- p.45 / Chapter 3.2.5 --- Nucleotide Sequences Determination using Dideoxy Nucleotide Chain Termination Method --- p.46 / Chapter 3.2.6 --- Southern Analysis of the Goldfish Genomic DNA --- p.47 / Chapter 3.2.7 --- Northern Blot Analyses of Goldfish Pituitary Total RNA --- p.47 / Chapter 3.3 --- Results --- p.49 / Chapter 3.3.1 --- Screening of the Library and Characterization of the Clones --- p.49 / Chapter 3.3.1.1 --- Analyses of the cDNA Clones --- p.49 / Chapter 3.3.1.2 --- Cross Hybridization Reactivity --- p.57 / Chapter 3.3.2 --- Genomic Southern Blot Analyses --- p.58 / Chapter 3.3.3 --- Gene Expression of the Pituitary Hormones - Northern Blot Analyses --- p.61 / Chapter 3.4 --- Discussion --- p.64 / Chapter 3.4.1 --- Cross Hybridization Reavtivities of the Probes to the Hormone cDNA Clones --- p.64 / Chapter 3.4.2 --- Structural Analyses of the Hormone cDNA Clones --- p.65 / Chapter 3.4.2.1 --- Growth Hormone --- p.65 / Chapter 3.4.2.2 --- Prolactin --- p.68 / Chapter 3.4.2.3 --- Somatolactin --- p.70 / Chapter 3.4.3 --- Secondary Structure Prediction --- p.72 / Chapter 3.4.4 --- Genomic Southern Analyses --- p.78 / Chapter 3.4.5 --- Pituitary Expression of Goldfish Hormone mRNAs --- p.79 / Chapter 3.5 --- Conclusion --- p.81 / Chapter CHAPTER 4 --- "Expression of Recombinant Goldfish Growth Hormone, Prolactin and Somatolactin in Escherichia coli (E. coli)" / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and Methods --- p.83 / Chapter 4.2.1 --- Materials --- p.83 / Chapter 4.2.2 --- Construction of Expression Vectors Carrying the Hormone Coding Regions --- p.84 / Chapter 4.2.3 --- Pilot Expression Experiment to Determine Kinetics of Expression --- p.86 / Chapter 4.2.4 --- "Large Scale Expression and Purification of Recombinant Hormones by Ni2+-NTA Affinity Column (ProBond´ёØ column, Invitrogen)" --- p.86 / Chapter 4.2.5 --- Western Blotting of the Recombinant Hormones on Polyvinylidene Fluoride (PVDF) Membrane --- p.87 / Chapter 4.2.6 --- Dot Blot Immobilisation of the Recombinant Hormones on Nitrocellulose Membrane --- p.88 / Chapter 4.2.7 --- Detection of the Blotted Protein by Enzyme-linked Immunodetaction Method --- p.88 / Chapter 4.3 --- Results --- p.90 / Chapter 4.3.1 --- Construction of the Expression Vectors --- p.90 / Chapter 4.3.2 --- Expression of the Recombinant Hormones --- p.97 / Chapter 4.3.3 --- Partial Purification and Analyses of the recombinant Hormones --- p.97 / Chapter 4.4 --- Discussion --- p.105 / Chapter 4.4.1 --- Construction of the Expression Vectors --- p.105 / Chapter 4.4.2 --- Expression of the Recombinant Hormones --- p.108 / Chapter 4.4.3 --- Partial Purification and Characterization of the Recombinant Hormones --- p.109 / Chapter 4.5 --- Conclusion --- p.114 / Chapter CHAPTER 5 --- General Discussion --- p.115 / References --- p.120

The efficiency of somatic cell nuclear transfer has been improved slightly with the use of Demecolcine as a chemical enucleant. While the reasons for this improved efficiency remain unclear, it has been hypothesized that the Demecolcine assisted enucleation procedure is less exigent to vital cell processes within the oocyte including the Anaphase-Promoting Complex (APC) dependent ubiquitination of proteins. In order to test the effect of Demecolcine on the APC, the spatial localizations of Apc11, the catalytic core of the complex, and Cdc20, a main activator of the complex, were studied in developing mouse oocytes. In control oocytes, a high concentration of Apc11 protein was observed surrounding the meiotic spindle, but this perispindular localization was not observed in oocytes treated with Demecolcine. Similarly, oocytes stained for Cdc20 also demonstrated cytoplasmic localization in control oocytes with a variation consistent with previous studies in total protein at different stages of development. However, in oocytes treated with Demecolcine, this developmental variation was not observed. These data suggest that since both Apc11 and Cdc20 localization are affected by an incubation in Demecolcine, the activity of the APC would also be affected. In order to test this theory, Rec8, a meiotic specific member of the cohesion complex, was localized in developing mouse embryos. Since the destruction of Rec8 is a downstream consequence of the ubiquitination pathway, Rec8 localization serves as an indirect indicator of APC activity. The data indicate Rec8 localization was only subtly influenced by Demecolcine, thus the magnitude of the drug's effect APC activity remains unclear.

by Yu Wai Fu, Jason. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 112-120). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iii / Chapter CHAPTER 1 --- INTORDUCTION --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.2 --- Historical Overview --- p.3 / Chapter 1.2.1 --- Insulin-Like Growth Factors I and II --- p.3 / Chapter 1.2.2 --- IGF Receptors --- p.5 / Chapter 1.2.3 --- IGF Binding Proteins --- p.7 / Chapter 1.3 --- Origin and Production of IGFs --- p.7 / Chapter 1.3.1 --- The Hypothalamo-Pituitary-GH-IGF-I Axis --- p.7 / Chapter 1.3.2 --- Factors Regulating IGF production --- p.9 / Chapter 1.3.3 --- Expression of IGFs in the Central Nervous System --- p.11 / Chapter 1.4 --- Actions of IGFs --- p.12 / Chapter 1.4.1 --- Insulin-like Metabolic Effects --- p.12 / Chapter 1.4.2 --- Mitogenic Effects --- p.13 / Chapter 1.4.3 --- Effects on Differentiation --- p.13 / Chapter 1.4.4 --- IGFs in Reproductive System --- p.14 / Chapter 1.4.5 --- IGF Actions in the Central Nervous System --- p.14 / Chapter 1.5 --- Transgenic and Knockout Animal Models for IGFs --- p.15 / Chapter 1.6 --- Molecular Biology of IGFs --- p.17 / Chapter 1.6.1 --- Structure of the IGF Genes --- p.17 / Chapter 1.6.2 --- Expression of IGF Genes --- p.21 / Chapter 1.6.3 --- Regulation of IGF Gene Expression --- p.23 / Chapter 1.7 --- IGF Receptors --- p.24 / Chapter 1.7.1 --- IGF-I Receptor --- p.24 / Chapter 1.7.2 --- IGF-II Receptor --- p.26 / Chapter 1.8 --- IGFBPs --- p.26 / Chapter 1.9 --- Teleost IGFs --- p.28 / Chapter 1.9.1 --- The GH-IGF-Axis in Teleost --- p.28 / Chapter 1.9.2 --- Osmoregulation and Other Biological Actions of IGFin Teleost --- p.29 / Chapter 1.9.3 --- Molecular Biology of IGFs in Teleost --- p.30 / Chapter 1.9.4 --- IGFBPs and IGF Receptors in Teleost --- p.31 / Chapter 1.10 --- Rationale and Aim of the Present Study --- p.32 / Chapter CHAPTER 2 --- SEARCH OF IGF-I PROMOTER BY GENOMIC DNA POLYMERASE CHAIN REACTION --- p.34 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.2 --- Materials --- p.35 / Chapter 2.3 --- Methods --- p.39 / Chapter 2.3.1 --- Preparation of Genomic DNA from Carp Testis --- p.39 / Chapter 2.3.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.40 / Chapter 2.3.3 --- Polymerase Chain Reaction --- p.40 / Chapter 2.3.3.1 --- Ligation of the Cassette to Digested Genomic DNA --- p.40 / Chapter 2.3.3.2 --- Amplification by PCR --- p.40 / Chapter 2.3.4 --- Agarose Gel Electrophoresis --- p.42 / Chapter 2.3.5 --- Gene Clean Using Sephaglas´ёØ BandPrep Kit (Pharamica) --- p.42 / Chapter 2.3.6 --- Cloning of PCR Products --- p.43 / Chapter 2.3.7 --- Transformation of Competent Cell (Heat Shock Method) --- p.43 / Chapter 2.3.8 --- Small Scale Alkaline Preparation of Plasmid DNA --- p.44 / Chapter 2.3.9 --- Restriction Enzyme Digestion to Release the Insert --- p.45 / Chapter 2.3.10 --- Large Scale Plasmid Preparation of the Positive Clone --- p.45 / Chapter 2.3.11 --- DNA Sequencing of the Positive Clone Using the T7 DNA Polymerase Sequencing Kit (Pharmacia) --- p.46 / Chapter 2.4 --- Results and Discussion --- p.49 / Chapter CHAPTER 3 --- ISOLATION OF GENOMIC CLONES CARRYING THE IGF-I GENE --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials --- p.56 / Chapter 3.3 --- Methods --- p.58 / Chapter 3.3.1 --- Preparation of the Plating Host Cells --- p.58 / Chapter 3.3.2 --- Phage Titering --- p.58 / Chapter 3.3.3 --- Primary Screening of Common Carp Genomic Library --- p.59 / Chapter 3.3.4 --- Preparation of Radioactive Nucleic Acid Probes --- p.60 / Chapter 3.3.5 --- Purification of the Positive Clones --- p.60 / Chapter 3.3.6 --- Purification of DNA from Lambda Phage Using Sephaglas´ёØ PhagePrep Kit (Pharmacia) --- p.61 / Chapter 3.3.7 --- Restriction Enzyme Digestion Release of Inserts --- p.62 / Chapter 3.3.8 --- Capillary Transfer of DNA to Nylon Membrane Under Alkaline Condition --- p.62 / Chapter 3.3.9 --- Southern Analysis of the 10 Positive Clones --- p.63 / Chapter 3.3.10 --- Restriction Mapping of the Clone P1 --- p.64 / Chapter 3.3.11 --- Subcloning of the Fragments of the Clone PI into Plasmid Vector --- p.64 / Chapter 3.3.12 --- IGF-I Specific PCR --- p.64 / Chapter 3.3.13 --- Amplification of Introns from the Clone P1 Using PCR --- p.67 / Chapter 3.4 --- Results and Discussion --- p.70 / Chapter CHAPTER 4 --- RNA ASSAY USING REVERSE TRANSCRIPTION- POLYMERASE CHAIN REACTION --- p.83 / Chapter 4.1 --- Introduction --- p.83 / Chapter 4.2 --- Materials --- p.85 / Chapter 4.3 --- Methods --- p.86 / Chapter 4.3.1 --- Administration of Hormones --- p.86 / Chapter 4.3.1.1 --- Injection Time Course1 --- p.86 / Chapter 4.3.1.2 --- Injection Time Course2 --- p.86 / Chapter 4.3.2 --- Total RNA Extraction --- p.87 / Chapter 4.3.2.1 --- Rapid RNA Isolation --- p.87 / Chapter 4.3.3 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.88 / Chapter 4.3.4 --- Rapid Isolation of PolyA+ mRNA from Total RNA --- p.89 / Chapter 4.3.5 --- IGF-I Specific RT-PCR --- p.90 / Chapter 4.4 --- Results and Discussion --- p.92 / Chapter CHAPTER 5 --- SEARCH FOR IGF-II GENE USING GENOMIC SOUTHERN BLOT ANALYSIS --- p.101 / Chapter 5.1 --- Introduction --- p.101 / Chapter 5.2 --- Materials --- p.103 / Chapter 5.3 --- Methods --- p.104 / Chapter 5.3.1 --- Preparation of Genomic DNA from Carp Testis --- p.104 / Chapter 5.3.2 --- Restriction Enzyme Digestion of Genomic DNA --- p.104 / Chapter 5.3.3 --- Southern Blotting of the Digested Genomic DNA --- p.104 / Chapter 5.3.4 --- Preparation of the Trout IGF-II Specific Probe --- p.104 / Chapter 5.3.5 --- Genomic Southern Hybridization --- p.105 / Chapter 5.4 --- Results and Discussion --- p.106 / Chapter CHAPTER 6 --- GENERAL DISCUSSION AND CONCLUSION --- p.109 / REFERENCES --- p.112

The auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) has a pleiotropic phenotype including a lack of lateral roots and reduced gravitropic response, apical dominance, vascular development, and fruit growth. The dgt mutation reduces the auxin sensitivity of only a subset of auxin responses while levels, metabolism, and transport of auxin appear normal, suggesting that the Dgt gene encodes a component in an auxin-signaling pathway. This dissertation reports isolation and characterization of the Dgt gene. Delineation of three microsyntenic regions in the Arabidopsis genome containing genes homeologous to genetic markers near the Dgt gene allowed isolation of additional ESTs from the corresponding tomato region, significantly reducing the mapping distance to the dgt locus. Further analysis determined that the Dgt gene encodes a cyclophilin (LeCYP1), a previously unidentified component of auxin signaling. Each known dgt allele contains a unique mutation in the coding sequence of LeCyp1. In addition, the wild-type Dgt gene can complement dgt mutant plants. Cyclophilins characteristically have peptidylprolyl cis-trans isomerase (PPIase) activity, but it is unclear whether that activity is necessary for all of their biological functions. Each allelic dgt mutation reduces or nullifies PPIase activity of LeCYP1 fusion proteins in vitro. Immunoblot analysis indicates that all three dgt mutations are null mutations. Phylogenetic comparisons of tomato and Arabidopsis cytosolic-type cyclophilins could not identify any single Arabidopsis member as orthologous to LeCYP1/DGT. Five T-DNA insertion mutants were analyzed to determine if mutations in Arabidopsis cytosolic-type cyclophilins phenocopy the pleiotropic dgt phenotype. Overall seedling growth and morphology appear normal in the mutants, however, their gravitropic response is slow. The lack of exact phenocopy may be due to the redundant nature of Cyp genes in Arabidopsis, which has over twice as many Cyp genes as tomato. In tomato, the cyclophilin inhibitor cyclosporin A (CsA) inhibits auxin-induced adventitious root initiation and expression of two early auxin response genes, LeIAA10 and 11, that are also affected by the dgt mutation. Taken together, these results suggest that the cyclophilin encoded by the Dgt gene plays an important role in auxin signal transduction. / Graduation date: 2004