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by Ng Yuen Keng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 141-148). / Table of contents --- p.1 / List of figures --- p.5 / List of tables --- p.12 / List of abbreviations --- p.13 / Abbreviation table for amino acids --- p.16 / Chapter Chapter 1 --- Literature Review --- p.17 / Chapter 1.1 --- Introduction --- p.17 / Chapter 1.2 --- The Hematopoietin/cytokine receptor superfamily --- p.20 / Chapter 1.3 --- The PRLR protein --- p.22 / Chapter 1.3.1 --- The receptor size --- p.22 / Chapter 1.3.2 --- Primary structure --- p.22 / Chapter 1.3.3 --- Structure of the extracellular domain --- p.26 / Chapter 1.3.4 --- Structure of the cytoplasmic domain --- p.30 / Chapter 1.3.5 --- Characteristics of specific PRL binding to PRLR --- p.32 / Chapter 1.5 --- The PRLR gene --- p.33 / Chapter 1.6 --- Heterogeneity of PRLR --- p.33 / Chapter 1.7 --- Signal transduction of PRLR --- p.35 / Chapter 1.7.1 --- JAK: a novel family of cytoplasmic protein tyrosine kinases --- p.35 / Chapter 1.7.2. --- Interaction between JAK2 and PRLR --- p.37 / Chapter 1.7.3 --- STAT proteins: mediators of PRL-dependent gene transcription --- p.37 / Chapter 1.7.4 --- Other signaling pathways of PRLR --- p.38 / Chapter 1.7.5 --- Future prospects on PRLR signaling --- p.38 / Chapter 1.8 --- Regulation of PRLR gene expression --- p.39 / Chapter 1.9 --- Objective of cloning the PRLR cDNA in male Syrian golden hamster --- p.42 / Chapter Chapter 2 --- PCR cloning of hamster PRLR cDNA fragment from adult male hamster liver --- p.44 / Chapter 2.1. --- Introduction --- p.44 / Chapter 2.2. --- Materials and Methods --- p.45 / Chapter 2.2.1 --- Primer design and PCR strategy --- p.45 / Chapter 2.2.2 --- Collection of liver --- p.46 / Chapter 2.2.3 --- Reverse transcription of polyadenylated RNA --- p.46 / Chapter 2.2.4 --- Nested PCR --- p.47 / Chapter 2.2.5 --- Southern analysis of the PCR products --- p.48 / Chapter 2.2.6 --- Subcloning of PCR product --- p.49 / Chapter 2.2.7 --- Sequence determination of the positive recombinant clone --- p.49 / Chapter 2.2.8 --- Sequence alignment and homology comparison --- p.50 / Chapter 2.3 --- Results --- p.55 / Chapter 2.3.1 --- Nucleotide sequence alignment and primer design --- p.55 / Chapter 2.3.2 --- Nested PCR --- p.55 / Chapter 2.3.3 --- Subcloning of the PCR product --- p.56 / Chapter 2.3.4 --- Analysis of nucleotide and predicted amino acid sequences --- p.56 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3 --- Nucleotide sequence determination of the 5' and the 3' ends of hamster PRLR cDNA --- p.69 / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- Materials and Methods --- p.71 / Chapter 3.2.1 --- Collection of liver --- p.71 / Chapter 3.2.2 --- Total RNA preparation and poly (A) + RNA isolation --- p.72 / Chapter 3.2.3 --- Double stranded cDNA synthesis --- p.73 / Chapter 3.2.4 --- Adaptor ligation --- p.74 / Chapter 3.2.5 --- 5´ة and 3' RACE PCR --- p.74 / Chapter 3.2.6 --- Cloning of the RACE PCR products --- p.76 / Chapter 3.2.7. --- Sequence determination of the RA CE PCR products --- p.77 / Chapter 3.2.8. --- Sequence analysis of the RACE PCR products --- p.78 / Chapter 3 .2.9 --- Northern blot analysis of hamster PRLR mRNA in male hamster tissues --- p.79 / Chapter 3.3 --- Results --- p.79 / Chapter 3.1.1 --- RNA preparation and double stranded cDNA synthesis --- p.79 / Chapter 3.3.2 --- RACE PCRfor the 5' and the 3' ends of hamster PRLR cDNA --- p.84 / Chapter 3.3.3 --- Cloning of the 5' and 3'RACE PCR products --- p.92 / Chapter 3.3.4 --- Sequence determination of the RACE PCR products --- p.92 / Chapter 3.3.5 --- Nucleotide sequence analysis of hamster PRLR full length cDNA --- p.101 / Chapter 3.3.6 --- Northern blot analysis of hamster PRLR --- p.101 / Chapter 3.4 --- Discussion --- p.106 / Chapter Chapter 4 --- Attempts to study the PRLR gene expression in male hamster tissues --- p.113 / Chapter 4.1 --- Introduction --- p.113 / Chapter 4.2 --- Materials and Methods --- p.115 / Chapter 4.2.1 --- Collection of tissues --- p.115 / Chapter 4.2.2 --- Total RNA preparation and poly (A)+ RNA isolation --- p.116 / Chapter 4.2.3 --- Reverse Transcription --- p.116 / Chapter 4.2.4 --- Polymerase chain reaction for detecting the presence of hamster PRLR cDNA in various tissues --- p.117 / Chapter 4.2.5 --- Nested PCR for detecting heterogeneity in PRLR cDNA sizes in various tissues --- p.117 / Chapter 4.2.6 --- Analysis and quantitation of PCR products --- p.118 / Chapter 4.3 --- Results --- p.119 / Chapter 4.4 --- Discussion --- p.134 / Chapter Chapter 5 --- General Discussion --- p.137 / References --- p.141 / Appendices --- p.149 / Chapter I. --- "Stock solution preparation (Sambrook et al., 1989)" --- p.149 / Chapter II. --- List of primers --- p.152 / Primers for sequence determination --- p.152 / "Primer for first strand cDNA synthesis and 3' RACE PCR (Frohman et al., 1988 and Loh et al.,1989)" --- p.152 / "Primers for amplifying the actin cDNA fragment (Chan et al.,1995)" --- p.152 / Primers used for PCR-cloning and semi-quantitative analysis of hamster PRLR cDNA --- p.153 / Chapter III. --- "First strand cDNA synthesis primer, cDNA adaptor and adaptor primers used in the 5' and3' end sequence determinations of hamster PRLR cDNA" --- p.154 / Chapter IV. --- "Multiple cloning sites of the pCRII (Invitorgen), pUC 18 (Pharmacia) and pBluescript SK+ vectors (Clontech)" --- p.155 / Chapter VI. --- Nucleic acid molecular weight size markers --- p.158