by Wong Kwan Po, Gary. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 132-153). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Abbreviations --- p.vi / Abbrevation Table for Amino Acids --- p.ix / List of Figures --- p.x / List of Tables --- p.xiii / Table of Contents --- p.xiv / Chapter Chapte r One --- General Introduction --- p.1 / Chapter 1.1 --- Structures of PRL --- p.1 / Chapter 1.2 --- PRL receptor and its mechanism of action --- p.7 / Chapter 1.3 --- Biosynthesis of PRL --- p.11 / Chapter 1.4 --- Biological functions of PRL --- p.13 / Chapter 1.5 --- Organization and regulation of PRL gene --- p.16 / Chapter 1.6 --- Aims of this study --- p.25 / Chapter Chapter Two --- PCR Cloning of gfPRL Gene --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Buffers and Reagents --- p.27 / Chapter 2.2.2 --- Methods --- p.30 / Chapter 2.2.2.1 --- PCR of the 5'-flanking region of gfPRL gene --- p.30 / Chapter 2.2.2.2 --- Genomic PCR of gfPRL gene --- p.31 / Chapter 2.2.2.3 --- Spectrophotometric quantification and qualification of DNA and RNA --- p.31 / Chapter 2.2.2.4 --- Agarose gel electrophoresis of DNA --- p.31 / Chapter 2.2.2.5 --- DNA radioactive labeling by random priming --- p.32 / Chapter 2.2.2.6 --- Vacuum transfer of DNA fragments to a nylon membrane --- p.32 / Chapter 2.2.2.7 --- Southern blot analysis --- p.33 / Chapter 2.2.2.8 --- Molecular Imager Analysis --- p.33 / Chapter 2.2.2.8 --- Phosphorylation of PCR amplified DNA --- p.34 / Chapter 2.2.2.9 --- Ligation of DNA fragment to linearized vector --- p.34 / Chapter 2.2.2.10 --- Preparation of Escherichia coli competent cells --- p.34 / Chapter 2.2.2.11 --- Bacterial transformation by heat stock --- p.35 / Chapter 2.2.2.12 --- Automated PCR sequencing with Sequencing Ready Reaction Kit --- p.35 / Chapter 2.2.2.13 --- Primer extension using reverse transcription --- p.36 / Chapter 2.3 --- Results --- p.38 / Chapter 2.3.1 --- Cloning of the 5'-flanking region of gfPRL gene --- p.38 / Chapter 2.3.2 --- PCR cloning of gfPRL gene --- p.43 / Chapter 2.3.3 --- Identification of the transcription initiation site --- p.47 / Chapter 2.4 --- Discussion --- p.51 / Chapter 2.4.1 --- Sequence analysis of the gfPRL gene --- p.51 / Chapter 2.4.2 --- Analysis of the exon-intron boundaries --- p.53 / Chapter 2.4.3 --- Analysis of the 5'flanking region of gfPRL gene --- p.53 / Chapter 2.4.4 --- Identification of the transcription initiation site --- p.54 / Chapter 2.5 --- Conclusion --- p.54 / Chapter Chapter Three --- Promoter Analysis of the gfPRL Gene --- p.55 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Preparation of Luciferase reporter constructs --- p.56 / Chapter 3.2.2 --- Preparation of frozen stock of culture cells --- p.56 / Chapter 3.2.3. --- Cell culture --- p.56 / Chapter 3.2.4 --- Transfection of mammalian cells for transient gene expression study --- p.57 / Chapter 3.2.5 --- Luciferase assay --- p.57 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- Tissue-specific transcription of gfPRL promoter --- p.58 / Chapter 3.3.2 --- Identification of regulatory regions of gfPRL gene promoter --- p.61 / Chapter 3.3.3 --- Inhibitory effect of DA on gfPRL promoter transcription activity --- p.63 / Chapter 3.3.4 --- GfPRL promoter sequences that specifically confer negative regulation by DA --- p.65 / Chapter 3.3.5 --- The action of TRH on gfPRL promoter --- p.67 / Chapter 3.3.6 --- Investigation of gfPRL promoter sequence responsiveness towards TRH --- p.69 / Chapter 3.4 --- Discussion --- p.71 / Chapter 3.4.1 --- Tissue-specific transcription of gfPRL promoter --- p.71 / Chapter 3.4.2 --- Identification of regulatory regions of goldfish prolactin gene promoter --- p.72 / Chapter 3.4.3 --- Dopamine inhibits gfPRL promoter activity --- p.73 / Chapter 3.4.4 --- TRH action on gfPRL promoter --- p.76 / Chapter 3.5 --- Conclusion --- p.78 / Chapter Chapter Four --- Seasonal Study on gfPRL and gfGH expression --- p.80 / Chapter 4.1 --- Introduction --- p.80 / Chapter 4.2 --- Materials and Methods --- p.81 / Chapter 4.2.1 --- Blood samples and radioimmunoassay --- p.81 / Chapter 4.2.2 --- Preparation of ribonuclease free reagents and apparatus --- p.81 / Chapter 4.2.3 --- Isolation of total RNA --- p.81 / Chapter 4.2.4 --- Formaldehyde agarose gel electrophoresis of RNA --- p.82 / Chapter 4.2.5 --- First strand cDNA synthesis --- p.82 / Chapter 4.2.6 --- RT-PCR --- p.83 / Chapter 4.2.7 --- Analysis of RT-PCR --- p.86 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Tissue-specific expression of gfPRL transcript --- p.88 / Chapter 4.3.2 --- Sexual maturity of goldfish throughout the reproductive cycle --- p.90 / Chapter 4.3.3 --- Serum gfGH levels throughout the year of 2000 --- p.91 / Chapter 4.3.4 --- Serum gfPRL levels throughout the year of 2000 --- p.92 / Chapter 4.3.5 --- The variation of gfGHR and gfPRLR mRNA in the brain throughout the reproductive cycle --- p.93 / Chapter 4.3.6 --- The variation of gfGHR mRNA in the liver throughout the reproductive cycle --- p.94 / Chapter 4.3.7 --- The variation of gfGHR and gfPRLR mRNA in the kidney throughout the reproductive cycle --- p.95 / Chapter 4.3.8 --- The variation of gfGHR and gfPRLR mRNA in the gonads throughout the reproductive cycle --- p.96 / Chapter 4.4 --- Discussion --- p.98 / Chapter 4.4.1 --- Tissue-specific expression of gfPRL transcript --- p.98 / Chapter 4.4.2 --- Sexual maturity of goldfish throughout the reproductive cycle --- p.98 / Chapter 4.4.3 --- Serum gfGH and gfPRL level throughout the reproductive cycle --- p.99 / Chapter 4.4.4 --- The variation of gfGHR and gfPRLR mRNA in the brain throughout the reproductive cycle --- p.100 / Chapter 4.4.5 --- The variation of gfGHR mRNA in the liver throughout ´Øthe reproductive cycle --- p.101 / Chapter 4.4.6 --- The variation of gfGHR and gfPRLR mRNA in the kidney throughout the reproductive cycle --- p.102 / Chapter 4.4.7 --- The variation of gfGHR and gfPRLR mRNA in the gonads throughout the reproductive cycle --- p.102 / Chapter 4.5 --- Conclusion --- p.105 / Chapter Chapter Five --- Recombinant gfPRL Production --- p.106 / Chapter 5.1 --- Introduction --- p.106 / Chapter 5.2 --- Materials and Methods --- p.108 / Chapter 5.2.1 --- Buffers and Reagents --- p.108 / Chapter 5.2.2 --- Methods --- p.112 / Chapter 5.2.2.1 --- Recombinant protein expression --- p.112 / Chapter 5.2.2.2. --- Purification of the recombinant protein by XpressTM System Protein Purification (Invitrogen) --- p.112 / Chapter 5.2.2.3 --- SDS-PAGE preparation --- p.112 / Chapter 5.2.2.4 --- SDS-PAGE analysis of proteins --- p.113 / Chapter 5.2.2.5 --- Western blot analysis --- p.114 / Chapter 5.2.2.6 --- Protein refolding --- p.114 / Chapter 5.2.2.7 --- Alkaline Extraction --- p.115 / Chapter 5.2.2.8 --- Size Exclusion Chromatography --- p.115 / Chapter 5.2.2.9 --- ELISA analysis of the fractions --- p.115 / Chapter 5.2.2.10 --- Anion Exchange Chromatography --- p.116 / Chapter 5.3 --- Results --- p.117 / Chapter 5.3.1 --- Prokaryotic expression of recombinant gfPRL --- p.117 / Chapter 5.3.2 --- "Purification of reombinant gfPRL: SDS-PAGE, western blot and BCA analysis of purified recombinant gfPRL" --- p.119 / Chapter 5.3.3 --- Purification of native gfPRL and gfGH: Native hormone purification by size exclusion chromatography --- p.119 / Chapter 5.3.4 --- Native gfPRL purification by anion exchange chromatography --- p.122 / Chapter 5.3.5 --- Study the biological activity of refolded recombinant gfPRL --- p.126 / Chapter 5.4 --- Discussion --- p.127 / Chapter 5.4.1 --- Prokaryotic expression of recombinant gfPRL --- p.127 / Chapter 5.4.2 --- Purification of recombinant gfPRL --- p.128 / Chapter 5.4.3 --- Refolding of recombinant gfPRL --- p.129 / Chapter 5.4.4 --- Purification of native gfPRL --- p.130 / Chapter 5.4.5 --- Study the biological activity of recombinant gfPRL --- p.130 / Chapter 5.5 --- Conclusion --- p.131 / References --- p.132