Rifampicin is one of the major antibiotics used in the treatment of Mycobacterium tuberculosis. This organism causes tuberculosis. Other related nocardioform bacteria which include the Rhodococci are opportunistic pathogens in AIDS patients. These organisms cause tuberculosis-like disease and are currently treated with rifampicin and other drugs. The presence of a low level rifampicin resistance mechanism was identified in seven rhodococcal strains and five other related and unrelated bacteria. Abbreviated Abstract. Open document to view full version] / GR2017

by Pong Tsang Wai Hai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 98-105). / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations --- p.v / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Biological functions and structure of GH --- p.1 / Chapter 1.2 --- Application of recombinant GH --- p.2 / Chapter 1.3 --- Expression of eukaryotic gene in E.coli --- p.4 / Chapter 1.4 --- Methods for increasing expression of a cloned gene --- p.6 / Chapter 1.4.1 --- Changing the 5' end codons of the cDNA to E.coli preferred codons --- p.6 / Chapter 1.4.2 --- Optimization of distance between SD sequence and the initiation codons --- p.6 / Chapter 1.4.3 --- "Construction of a short ""dummy"" cistron at the 5' end of the cloned gene to improve attachment of ribosome" --- p.7 / Chapter 1.4.4 --- Increasing the copy number of recombinant expression plasmid --- p.8 / Chapter 1.4.5 --- Optimizing high density cell expression --- p.9 / Chapter 1.5 --- Quantitating the expression of cloned gene --- p.10 / Chapter 1.6 --- Inclusion bodies formation --- p.11 / Chapter 1.7 --- The purification of eukaryotic polypeptides synthesized as inclusion bodies --- p.12 / Chapter 1.7.1 --- Solubilization of the inclusion bodies --- p.13 / Chapter 1.7.2 --- Refolding the polypetides and disulfide bond formation --- p.13 / Chapter 1.8 --- Expression of secreted recombinant protein --- p.14 / Chapter 1.9 --- Purpose of present study --- p.15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- General techniques --- p.16 / Chapter 2.1.1 --- Chemical Synthesis of DNA linkers and primers --- p.16 / Chapter 2.1.2 --- Manipulation of DNA --- p.16 / Chapter 2.1.3 --- Electro-elution of DNA from Agarose Gel --- p.17 / Chapter 2.1.4 --- Preparation of Competent Cells and Transformation --- p.18 / Chapter 2.1.5 --- Screening of the Expressed Clones --- p.19 / Chapter 2.1.6 --- Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.21 / Chapter 2.1.7 --- Western blot analysis --- p.21 / Chapter 2.2 --- Purification procedures --- p.23 / Chapter 2.2.1 --- Growing up the cells in large scale --- p.23 / Chapter 2.2.2 --- Harvesting of cells from large scale culture --- p.23 / Chapter 2.2.3 --- Sonication of the cells --- p.24 / Chapter 2.2.4 --- Washing of the inclusion body --- p.24 / Chapter 2.2.5 --- Solubilization of the inclusion bodies --- p.25 / Chapter 2.2.6 --- Renaturation of r-gcGH --- p.26 / Chapter 2.2.6.1 --- Step down dilution mehtod --- p.26 / Chapter 2.2.6.2 --- Rapid dilution method --- p.26 / Chapter 2.2.7 --- Separation by reverse phase chromatography --- p.27 / Chapter 2.2.7.1 --- Octadodecylsilica (ODS) column separation --- p.27 / Chapter 2.2.7.2 --- Fast performance Liquid Chromatography(FPLC) --- p.28 / Chapter 2.3 --- Characterization methods --- p.29 / Chapter 2.3.1 --- Radioimmunoassay --- p.29 / Chapter 2.3.1.1 --- Iodination of r-gcGH --- p.29 / Chapter 2.3.1.2 --- Binding assay --- p.31 / Chapter 2.3.2 --- Preparation of anti-r-gcGH serum --- p.32 / Chapter 2.3.3 --- Determination of amino acid composition and N-terminal sequence of r-gcGH --- p.32 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Recombinant plasmids construction --- p.34 / Chapter 3.1.1 --- Basic construction of plasmid producing gcGH in E.coli --- p.34 / Chapter 3.1.2 --- N-terminal modification of gcGH cDNA --- p.38 / Chapter 3.1.3 --- Constuction of a short 'dummy' cistron at the 5'end of gcGH cDNA --- p.40 / Chapter 3.1.4 --- Optimization of distance between ribosomal binding site and initiation codon --- p.42 / Chapter 3.1.5 --- Increasing expression level by increasing plasmid copy number --- p.44 / Chapter 3.1.6 --- Optimizing the high density expression by changing the promoter --- p.48 / Chapter 3.1.7 --- Construction of excretion plasmid for gcGH production from E. coli --- p.48 / Chapter 3.2 --- Quantitation and qualitation of the expressed protein --- p.51 / Chapter 3.3 --- Effect of IPTG on induction of r-gcGH in pp5 --- p.57 / Chapter 3.4 --- Stability of overproducing strain pp5 during continuous culture --- p.59 / Chapter 3.5 --- Stability of overproducing strain ppADH4 during continuous culture --- p.61 / Chapter 3.6 --- "Optimization of culture condition for high level expression strains,pp5 and ppADH4" --- p.64 / Chapter 3.7 --- Purification of r-gcGH --- p.67 / Chapter 3.7.1 --- Distribution of r-gcGH as Soluble and insoluble protein in E. coli --- p.67 / Chapter 3.7.2 --- Isolation and cleaning of the inclusion bodies --- p.69 / Chapter 3.7.3 --- Solubilization and renaturation of r-gcGH --- p.71 / Chapter 3.7.4 --- Purification of r-gcGH by chromatography --- p.73 / Chapter 3.8 --- Characterization of r-gcGH --- p.78 / Chapter 3.8.1 --- Amino acid composition and N-terminal sequence determination --- p.78 / Chapter 3.8.2 --- Immunological property of r-gcGH --- p.81 / Chapter 3.8.3 --- Physical Property of r-gcGH --- p.84 / Chapter 3.8.4 --- Stability of r-gcGH --- p.84 / Chapter 3.9 --- Expression and purification of r-gcGH in excretion vector ppSP14 --- p.86 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Evaluation of expression strains --- p.88 / Chapter 4.1.1 --- Strain pKgcGH2 --- p.88 / Chapter 4.1.2 --- Strain pKgcGH2-17 --- p.88 / Chapter 4.1.3 --- Strain pSD78 --- p.89 / Chapter 4.1.4 --- "Strains pLl,pL2 and pL4" --- p.90 / Chapter 4.1.5 --- "Strains pp5,pplA,pp2I and pp4Q" --- p.90 / Chapter 4.1.6 --- Strain ppADH4 --- p.91 / Chapter 4.1.7 --- Strain ppSP14 --- p.91 / Chapter 4.2 --- Disulfide bond formation during refolding process --- p.92 / Chapter 4.2.1 --- Renaturaion in the presence of L-arginine and thiol reagent in oxidized form --- p.93 / Chapter 4.2.2 --- Renaturation in the presence of thiol reagent and 3M guanidine hydrochloride --- p.94 / Chapter 4.3 --- Stability of the r-gcGH --- p.94 / Chapter 4.4 --- Further studies --- p.96 / References --- p.98

by Kwong-Kwok Wong. / Thesis (Ph.D)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 171-183. / TITLE PAGE --- p.I / ABSTRACT --- p.II / STATEMENT --- p.V / ACKNOWLEGEMENTS --- p.VI / ABBREVIATIONS --- p.VII / TABLE OF CONTENTS --- p.VIII / LIST OF TABLES --- p.XIII / LIST OF FIGURES --- p.XVI / Chapter Chapter 1. --- Introduction and Literature Review --- p.1 / Chapter I . --- Introduction --- p.1 / Chapter A. --- General introduction --- p.1 / Chapter B. --- Purpose of study --- p.5 / Chapter II. --- Literature review --- p.6 / Chapter A. --- Global control of aerobic-anaerobic shift --- p.6 / Chapter B. --- Identified anaerobiosis-inducible genes --- p.8 / Chapter C. --- Genetics of anaerobic regulation --- p.15 / Chapter i. --- Redox control --- p.15 / Chapter ii. --- DNA conformation --- p.15 / Chapter iii. --- fnr (oxrA) regulatory gene --- p.16 / Chapter iv. --- narL gene --- p.18 / Chapter v. --- Other regulatory genes --- p.19 / Chapter vi. --- Proposed FNR and NarL recognition sequences --- p.20 / Chapter D. --- future prospect --- p.23 / Chapter Chapter 2. --- Isolation of Anaerobiosis-inducible Promoters --- p.25 / Chapter I . --- Introduction --- p.25 / Chapter A. --- Properties of promoter-probe plasmid pKK232.8 --- p.26 / Chapter B. --- Properties of promoter-probe plasmid pFZYl --- p.28 / Chapter II. --- Materials and methods --- p.30 / Chapter A. --- Bacterial strains and plasmids --- p.30 / Chapter B. --- Media --- p.30 / Chapter C. --- Solutions --- p.31 / Chapter D. --- Small scale prepartaion of plasmid DNA --- p.32 / Chapter E. --- Large scale preparation of plasmid DNA --- p.33 / Chapter F. --- Digestion of DNA with restriction endonucleases --- p.35 / Chapter G. --- Analysis of DNA samples with agarose gel electrophoresis --- p.36 / Chapter H. --- Dephosphorylation of DNA fragments --- p.37 / Chapter I. --- Partial digestion of Chromosomal DNA with restriction enzyme Sau3A. --- p.37 / Chapter J. --- Ligation of DNA --- p.38 / Chapter K. --- Preparation of competent cells --- p.38 / Chapter L. --- Transformation --- p.40 / Chapter M. --- Chloramphenicol resistance levels test for promoter clones with plasmid pKK232.8 --- p.41 / Chapter N. --- Preparation of crude cell extract for chloramphenicol acetyltransferase (CAT) assays --- p.41 / Chapter O. --- CAT assay --- p.42 / Chapter P. --- Protein assay --- p.42 / Chapter Q. --- β-galactosidase assay --- p.43 / Chapter III . --- Results --- p.45 / Chapter A. --- Molecular cloning of anaerobiosis-inducible promoters with promoter-probe plasmid pKK232.8 --- p.45 / Chapter B. --- Molecular cloning of anaerobiosis-inducible promoters with promoter-probe plasmid pFZYl --- p.54 / Chapter IV. --- Summary and Discussion --- p.70 / Chapter A. --- Cloning with promoter-probe plasmid pKK232.8 --- p.70 / Chapter B. --- Cloning with promoter-probe plasmid pFZYl --- p.71 / Chapter C. --- Number of anaerobiosis inducible promoters --- p.73 / Chapter Chapter 3. --- Subcloning and Sequencing --- p.74 / Chapter I . --- Introduction --- p.74 / Chapter II. --- Materials and methods --- p.74 / Chapter A. --- Bacterial strains and bacteriophages --- p.74 / Chapter B. --- Preparation of M13mp RF plasmid --- p.75 / Chapter C. --- DNA sequencing by the chain termination method --- p.75 / Chapter D. --- Polymerase chain reaction (PCR) for the amplification of DNA fragments cloned in plasmid pFZYl --- p.79 / Chapter E. --- Using Exonuclease III to construct unidirectional deletions to generate nested clones --- p.80 / Chapter F. --- Direct gel electrophoresis --- p.81 / Chapter G. --- C-testiscreening for the orientation of insert in M13 phage --- p.81 / Chapter III . --- Results --- p.82 / Chapter A. --- Subcloning and sequencing of pFE29 and pFE117 --- p.82 / Chapter B. --- Subcloning and sequencing of pHSKl --- p.90 / Chapter C. --- Subcloning and sequencing of pHSK8 --- p.100 / Chapter D. --- "Subclonig and sequencing of pFSl, pFS22 and pFS3 4" --- p.109 / Chapter IV. --- Summary and Discussion --- p.113 / Chapter A. --- Trimming down size of DNA fragments to smaller fragments which still contained anaerobiosis-inducible promoters --- p.113 / Chapter B. --- Nucleotide sequencing --- p.113 / Chapter C. --- Sucloning and sequencing strategy --- p.115 / Chapter Chapter 4. --- Expression of Anaerobiosis-inducible Promoters --- p.120 / Chapter I . --- Introduction --- p.120 / Chapter II. --- Materials and methods --- p.122 / Chapter A. --- Bacterial strains and phages --- p.122 / Chapter B. --- Media --- p.125 / Chapter C. --- Transformation in Salmonella typhimurium --- p.125 / Chapter D. --- Genetic techniques --- p.126 / Chapter III. --- Results --- p.129 / Chapter A. --- Expression of Escherichia coli qlpT promoter --- p.129 / Chapter B. --- "Expression of Salmonella typhimurium anaerobiosis-inducible promoters cloned in pHSK8, pFS22 and pFS34" --- p.134 / Chapter IV. --- Summary and Discussion --- p.137 / Chapter A. --- A pair of divergent promoters were both regulated by anaerobiosis and glucose. --- p.137 / Chapter B. --- fnr(oxrA) dependent and independent promoters --- p.137 / Chapter C. --- Effect of nitrate on anaerobiosis expression. --- p.138 / Chapter Chapter 5. --- Analysis of Anaerobiosis-inducible Promoter-containing DNA sequences and Final Discussion --- p.141 / Chapter I. --- Analysis of anaerobiosis-inducible promoter-containing DNA sequences --- p.141 / Chapter A. --- "Search for initiation codon, conserved ""-10"" and ""-35"" regions" --- p.141 / Chapter B. --- Search for FNR binding sites and NarL binding sites. --- p.151 / Chapter C. --- Homology search among the promoter sequences of all anaerobiosis-inducible genes. --- p.156 / Chapter II. --- Final Discussions. --- p.164 / Chapter A. --- Summary of the properties of the sequenced and characterized promoters cloned in this study --- p.164 / Chapter B. --- Further studies. --- p.167 / REFERENCES --- p.168

by Yeung Sze Mang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 123-133). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / 槪論 --- p.iii / ABBREVIATIONS --- p.iv / AMINO ACIDS SHORTHAND --- p.vi / TABLE OF CONTENTS --- p.vii-x / Chapter CHAPTER 1 --- LITERATURE REVIEW / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Structural Analysis of SL --- p.1 / Chapter 1.3 --- Location of SL-producing cells and Expression of SL --- p.5 / Chapter 1.4 --- Possible Functions of SL --- p.9 / Chapter 1.4.1 --- Adaptation to various backgrounds and Intensities of Illuminations --- p.9 / Chapter 1.4.2 --- Control of Reproduction and Maturation --- p.10 / Chapter 1.4.3 --- Responses to Stress --- p.12 / Chapter 1.4.4 --- Regulation of P034- and Ca2+ Metabolism --- p.12 / Chapter 1.4.5 --- Acid - Base Balance --- p.14 / Chapter 1.4.6 --- Regulation of Energy Metabolism --- p.15 / Chapter 1.4.7 --- Regulation of Fat Metabolism --- p.15 / Chapter 1.5 --- Regulation of SL Gene Expression --- p.19 / Chapter 1.5.1 --- Pit-1 Related Gene Regulation --- p.19 / Chapter 1.5.2 --- Regulation of Hormone Secretion --- p.21 / Chapter 1.5.2.1 --- Hypothalamic Factors --- p.21 / Chapter 1.5.2.2 --- Steroids --- p.23 / Chapter 1.6 --- Aims of Thesis --- p.23 / Chapter 1.6.1 --- Identification of SLII from Goldfish (Carassius auratus) --- p.23 / Chapter 1.6.2 --- Aims --- p.27 / Chapter CHAPTER 2 --- PCR ANALYSIS OF GFSLII GENE AND ITS EXPRESSION IN GOLDFISH TISSUE / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.2 --- Materials and Methods --- p.31 / Chapter 2.2.1 --- Materials --- p.31 / Chapter 2.2.2 --- Methods --- p.33 / Chapter 2.2.2.1 --- Subcloning and DNA Sequencing of the Goldfish SLII Amplified by PCR --- p.33 / Chapter 2.2.2.1.1 --- PCR Cloning of Goldfish SLII Gene --- p.33 / Chapter 2.2.2.1.2 --- Restriction Enzyme Digestion of the PCR Clones --- p.33 / Chapter 2.2.2.1.3 --- Subcloning of the Digested Fragments --- p.33 / Chapter 2.2.2.1.4 --- DNA Sequencing of the Subcloned Fragments --- p.34 / Chapter 2.2.2.2 --- Tissue Distribution Studies Using RNA Assay --- p.35 / Chapter 2.2.2.2.1 --- Tissue Preparation --- p.35 / Chapter 2.2.2.2.2 --- Total RNA Extraction --- p.35 / Chapter 2.2.2.2.3 --- Electrophoresis of RNA in Formadehyde Agarose Gel --- p.36 / Chapter 2.2.2.2.4 --- First Strand cDNA Synthesis --- p.37 / Chapter 2.2.2.2.5 --- Goldfish SLII Specific PCR --- p.37 / Chapter 2.2.2.2.6 --- PCR to Test DNA Contamination --- p.38 / Chapter 2.3 --- Results --- p.39 / Chapter 2.3.1 --- Subcloning and DNA Sequencing of the Goldfish SLII Amplified by PCR --- p.39 / Chapter 2.3.2 --- Tissue Distribution Studies Using RNA Assay --- p.40 / Chapter 2.4 --- Discussion --- p.45 / Chapter 2.4.1 --- Subcloning and DNA Sequencing of the Goldfish SLII Amplified by PCR --- p.45 / Chapter 2.4.2 --- Tissue Distribution Studies Using RNA Assay --- p.46 / Chapter CHAPTER 3 --- ANALYSIS OF GOLDFISH SLII GENE / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Materials and Methods --- p.49 / Chapter 3.2.1 --- Materials --- p.49 / Chapter 3.2.2 --- Methods --- p.54 / Chapter 3.2.2.1 --- Screening of Goldfish Genomic Library --- p.54 / Chapter 3.2.2.1.1 --- Preparation of the Plating Host --- p.54 / Chapter 3.2.2.1.2 --- Preparation of the Probe --- p.54 / Chapter 3.2.2.1.3 --- Primary Screening of Goldfish Genomic Library --- p.55 / Chapter 3.2.2.1.4 --- Isolation of the Positive Clones --- p.56 / Chapter 3.2.2.1.5 --- Phage Titering --- p.56 / Chapter 3.2.2.1.6 --- Purification of the Positive Clones --- p.57 / Chapter 3.2.2.1.7 --- Phage DNA Preparation --- p.57 / Chapter 3.2.2.1.8 --- Find out the Target Gene Size of the Positive Clones --- p.58 / Chapter 3.2.2.1.9 --- Cloning of the PCR Fragments into pUC18 Vector --- p.59 / Chapter 3.2.2.1.10 --- Checking the Cloned Insert Size --- p.60 / Chapter 3.2.2.1.11 --- Restriction Enzyme Digestion to Release the Inserts --- p.61 / Chapter 3.2.2.1.12 --- Mini prep of the Positive Clones for Further Investigations --- p.61 / Chapter 3.2.2.1.13 --- DNA Sequencing of the Positive Clones --- p.61 / Chapter 3.2.2.1.14 --- Restriction Enzyme Mapping of the Positive Clones --- p.62 / Chapter 3.2.2.1.15 --- Subcloning of Clone 2A and5A / Chapter 3.2.2.1.16 --- Determination of the Promoter Region of Clone 2A Using Universal Genome Walker Kit --- p.63 / Chapter 3.2.2.2 --- Southern Blot Analysis of Goldfish and Catfish Genomic DNA --- p.66 / Chapter 3.2.2.2.1 --- Genomic DNA Preparation from Goldfish and Catfish Tissues --- p.66 / Chapter 3.2.2.2.2 --- Restriction Enzyme Digestion of the Genomic DNA --- p.67 / Chapter 3.2.2.2.3 --- Alkaline Transfer of the Digested Genomic DNA --- p.67 / Chapter 3.2.2.2.4 --- Hybridization of the Digested Genomic DNA --- p.67 / Chapter 3.3 --- Results --- p.69 / Chapter 3.3.1 --- Screening of the Goldfish Genomic Library --- p.69 / Chapter 3.3.2 --- Mapping the Target Genes --- p.69 / Chapter 3.3.3 --- DNA Sequencing of the 2 Positive Clones --- p.69 / Chapter 3.3.4 --- Southern Blot Analysis of Goldfish and Catfish Genomic DNA --- p.81 / Chapter 3.4 --- Discussion --- p.83 / Chapter CHAPTER 4 --- EXPRESSION OF RECOMBINANT GOLDFISH SOMATOLACTIN IN ESCHERICHIA COLI (E. COLI) / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Materials and Methods --- p.89 / Chapter 4.2.1 --- Materials --- p.89 / Chapter 4.2.2 --- Methods --- p.96 / Chapter 4.2.2.1 --- Transformation of the Recombinant Protein Carrying Plasmid into E. coli. (BL21) --- p.96 / Chapter 4.2.2.2 --- Small Scale Expression of Recombinant Goldfish SLII Protein --- p.96 / Chapter 4.2.2.3 --- Large Scale Expression of Recombinant Goldfish SLII Protein --- p.97 / Chapter 4.2.2.4 --- Preparation of the Recombinant Protein for Purification --- p.99 / Chapter 4.2.2.5 --- Protein Purification Using Novagen His-Bind Resin Kit --- p.99 / Chapter 4.2.2.6 --- Production of Polyclonal Antibody in Rabbits --- p.100 / Chapter 4.2.2.7 --- Enzyme Linked Immunosorbant Assay (ELISA) --- p.101 / Chapter 4.2.2.8 --- Western Blot Analysis of the Recombinant Hormones --- p.103 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- Expression of the Recombinant Goldfish SLII --- p.105 / Chapter 4.3.2 --- Purification of the Recombinant Goldfish SLII --- p.105 / Chapter 4.3.3 --- ELISA Analysis --- p.105 / Chapter 4.3.4 --- Western Blot Analysis --- p.110 / Chapter 4.4 --- Discussion --- p.113 / Chapter 4.4.1 --- Expression of the Recombinant Goldfish SLII --- p.113 / Chapter 4.4.2 --- Purification of the Recombinant Goldfish SLII --- p.114 / Chapter 4.4.3 --- Analysis of the Recombinant Goldfish SLII --- p.114 / Chapter CHAPTER 5 --- GENERAL DISCUSSION AND CONCLUSIONS --- p.116 / REFERENCES --- p.123

by Lee Tsz On. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 148-155). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgments --- p.v / Contents --- p.vi / List of figures --- p.xii / List of table --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction / Chapter 1.1. --- Growth hormone (GH) --- p.1 / Chapter 1.1.1. --- Introduction to GH --- p.1 / Chapter 1.1.2. --- Actions of GH --- p.2 / Chapter 1.1.3. --- Structure of GH --- p.3 / Chapter 1.1.4. --- The sequence of GH --- p.5 / Chapter 1.2. --- Growth hormone receptor (GHR) --- p.6 / Chapter 1.2.1 --- Introduction to GHR --- p.6 / Chapter 1.2.2. --- Structure of the extracellular domain of GHR --- p.9 / Chapter 1.2.3. --- The regulation of GHR --- p.12 / Chapter 1.2.4. --- GHR biosynthesis --- p.13 / Chapter 1.2.5. --- Tissue distribution of GHR --- p.14 / Chapter 1.3. --- Signal transduction mechanisms of GHR --- p.15 / Chapter 1.3.1. --- Dimerization of GH and GHR complex --- p.15 / Chapter 1.3.2. --- The Jak and Stat pathway --- p.18 / Chapter 1.3.3. --- The ras and other signaling pathways --- p.20 / Chapter 1.4. --- Project aim --- p.22 / Chapter Chapter 2 --- Material and Methods / Chapter 2.1. --- Preparation of ribonuclease free reagents and apparatus --- p.23 / Chapter 2.2. --- Isolation of total RNA --- p.23 / Chapter 2.3. --- Isolation of mRNA --- p.24 / Chapter 2.4. --- Spectrophotometric quantification and qualification of DNA and RNA --- p.24 / Chapter 2.5. --- First strand cDNA synthesis --- p.25 / Chapter 2.6. --- Agarose gel electrophoresis of DNA --- p.25 / Chapter 2.7. --- Formaldehyde agarose gel electrophoresis of RNA --- p.26 / Chapter 2.8. --- Vacuum transfer of DNA to a nylon membrane --- p.26 / Chapter 2.9. --- Nucleic acids purification by MicroSpin (S-200HR) columns --- p.27 / Chapter 2.10. --- DNA radioactive labeling by nick translation --- p.27 / Chapter 2.11. --- Southern blot analysis --- p.28 / Chapter 2.12. --- Autoradiography and molecular imager --- p.28 / Chapter 2.13 . --- Linearization and dephosphorylation of plasmid DNA --- p.29 / Chapter 2.14. --- Purification of DNA from agarose using QIAEX II kit --- p.29 / Chapter 2.15. --- 3'End modification of PCR amplified DNA --- p.30 / Chapter 2.16. --- Ligation of DNA fragments to linearized vector --- p.30 / Chapter 2.17. --- Preparation of Escherichia coli competent cells --- p.31 / Chapter 2.18. --- Transformation --- p.31 / Chapter 2.19. --- Mini preparation of plasmid DNA --- p.32 / Chapter 2.20. --- Maxi preparation of plasmid DNA --- p.34 / Chapter 2.21 . --- PCR sequencing --- p.35 / Chapter 2.22. --- cDNA library screening --- p.36 / Chapter 2.23. --- Preparation and sterilization of culture medium --- p.38 / Chapter 2.24. --- Preparation of frozen stock of culture cells --- p.39 / Chapter 2.25. --- Cell passage of CHO-Kl --- p.39 / Chapter 2.26. --- Counting of cells --- p.40 / Chapter 2.27. --- Proliferation assay performed on CHO-K1 cells (MTT method) --- p.40 / Chapter 2.28. --- Luciferase assay --- p.41 / Chapter 2.29. --- SDS-PAGE preparation --- p.42 / Chapter 2.30. --- SDS-PAGE analysis of proteins --- p.42 / Chapter 2.31 . --- Recombinant protein expression --- p.43 / Chapter 2.32. --- Small scale purification of recombinant proteins --- p.44 / Chapter 2.33. --- Restriction digestion of DNA --- p.45 / Chapter 2.34. --- Purification of PCR product using QIAquick PCR purification kit --- p.45 / Chapter 2.35. --- TA cloning of PCR fragment --- p.45 / Chapter 2.36. --- Transfection of plasmid into CHO-K1 cells --- p.46 / Chapter 2.37. --- Sources of hormones --- p.46 / Chapter 2.38. --- Buffer and reagents --- p.47 / Chapter Chapter 3 --- "Cloning, expression and tissue distribution of Xenopus laevis GHR" / Chapter 3.1. --- Introduction --- p.50 / Chapter 3.2. --- Materials and methods --- p.51 / Chapter 3.2.1. --- Molecular cloning of xGHR cDNA / Chapter 3.2.1.1. --- Animals and tissues --- p.51 / Chapter 3.2.1.2. --- Reverse transcribed´ؤpolymerase chain reaction (RT-PCR) --- p.51 / Chapter 3.2.1.3. --- Subcloning of PCR amplified DNA fragment --- p.53 / Chapter 3.2.1.4. --- Library screening of xGHR --- p.53 / Chapter 3.2.1.5. --- 5 'Rapid amplification of cDNA end (5' RACE) --- p.55 / Chapter 3.2.2. --- Tissue distribution of xGHR / Chapter 3.2.2.1. --- Animals and tissues --- p.56 / Chapter 3.2.2.2. --- RT-PCR and Southern blot --- p.56 / Chapter 3.2.3. --- Eukarytoic expression of xGHR and functional assay of xGHR / Chapter 3.2.3.1. --- Subcloning ofxGHR into pRc/CMV --- p.57 / Chapter 3.2.3.2. --- Expression of xGHR in CHO-K1 cell --- p.58 / Chapter 3.2.3.3. --- Proliferation assay --- p.58 / Chapter 3.3. --- Results --- p.60 / Chapter 3.3.1. --- RT-PCR of the partial fragment --- p.60 / Chapter 3.3.2. --- Library screening of xGHR cDNA library --- p.61 / Chapter 3.3.3. --- 5' RACE --- p.64 / Chapter 3.3.4. --- The full-length cDNA sequence of xGHR --- p.65 / Chapter 3.3.5. --- Tissue distribution of xGHR mRNA --- p.69 / Chapter 3.3.6. --- Functional assay of xGHR in CHO-K1 cells --- p.71 / Chapter 3.4. --- Discussion --- p.74 / Chapter Chapter 4 --- Cloning and expression of Xenopus laevis GH-A and GH-B / Chapter 4.1. --- Introduction --- p.78 / Chapter 4.2. --- Materials and Methods --- p.79 / Chapter 4.2.1. --- PCR amplification of xGH-A and xGH-B partial fragments --- p.79 / Chapter 4.2.2. --- cDNA library screening of xGH-A and xGH-B --- p.80 / Chapter 4.2.3. --- Rapid amplification of cDNA ends of xGH-B / Chapter 4.2.3.1. --- 3'RACE --- p.80 / Chapter 4.2.3.2. --- 5'RACE --- p.81 / Chapter 4.2.4. --- Expression of xGH-A and xGH-B / Chapter 4.2.4.1 --- Construction of the expression vector --- p.84 / Chapter 4.2.4.2. --- Protein expression of xGH-A and xGH-B --- p.85 / Chapter 4.2.5. --- Purification of recombinant xGH-A and xGH-B --- p.85 / Chapter 4.3. --- Results --- p.87 / Chapter 4.3.1. --- PCRof xGH-A and xGH-B partial fragment --- p.87 / Chapter 4.3.2. --- Library screening of xGH-A --- p.87 / Chapter 4.3.3. --- 5' RACE and 3' RACE of xGH-B --- p.91 / Chapter 4.3.4. --- Sequence analysis of xGH-A and xGH-B --- p.93 / Chapter 4.3.5. --- Protein expression and purification of recombinant xGH-A and xGH-B --- p.100 / Chapter 4.4. --- Discussion --- p.102 / Chapter Chapter 5 --- Molecular cloning and function expression of goldfish GHR / Chapter 5.1. --- Introduction --- p.105 / Chapter 5.2. --- Materials and methods --- p.106 / Chapter 5.2.1. --- Molecular cloning of the partial fragment of gfGHR / Chapter 5.2.1.1. --- Primer design --- p.106 / Chapter 5.2.1.2. --- Library PCR of gfGHR partial fragment --- p.108 / Chapter 5.2.2. --- Library PCR of gfGHR cDNA sequence --- p.110 / Chapter 5.2.3. --- Determination of 3' End and 5' End sequences of gfGHR cDNA --- p.112 / Chapter 5.2.4. --- Tissue distribution of gfGHR / Chapter 5.2.4.1. --- Animals and tissues --- p.115 / Chapter 5.2.4.2. --- Semi-quantitative R T-PCR --- p.115 / Chapter 5.2.5. --- Functional expression of gfGHR in CHO-K1 cell / Chapter 5.2.5.1. --- Construction of an expression vector containing gfGHR --- p.116 / Chapter 5.2.5.2. --- Functional assay of gfGHR expression on CHO-K1 cells --- p.117 / Chapter 5.2.5.3. --- Proliferation assay --- p.118 / Chapter 5.2.5.4. --- Spi luciferase assay --- p.118 / Chapter 5.3. --- Results --- p.120 / Chapter 5.3.1. --- PCR amplification of the partial sequence of gfGHR --- p.120 / Chapter 5.3.2. --- The library PCR of gfGHR cDNA sequence --- p.122 / Chapter 5.3.3. --- The sequence of gfGHR --- p.124 / Chapter 5.3.4. --- Tissue distribution of gfGHR --- p.131 / Chapter 5.3.5. --- Proliferation assay --- p.133 / Chapter 5.3.6. --- Spi luciferase assay --- p.135 / Chapter 5.4. --- Discussion --- p.137 / Chapter Chapter 6 --- General discussion and future works --- p.145 / References --- p.148 / Appendix --- p.156

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

Chloroplast genomes of land plants show conservation of structure and gene arrangement. The spinach chloroplast genome is comprised of a covalently closed. circular DNA molecule of 150 kilobases and is typical of these plants. Approximately 20% of the proteins found in the spinach chloroplast are encoded by the chloroplast genome and translated on chloroplast ribosomes. The remainder are encoded on chromosomes in the nucleus, translated on cytoplasmic ribosomes and transported into the chloroplast. Spinach chloroplast DNA was isolated from crude 2 chloroplast preparations by a new method. Chloroplasts were lysed with alkaline sodium dodecyl sulfate, contaminating macromolecules precipitated with acidified potassium acetate and plastid DNA was purified by phenol:chloroform extraction and ethanol:ammonium acetate precipitation. The yield was approximately 50 ug chloroplast DNA per 100 grams leaf material. The DNA consisted of 10% circular molecules and 90% linear molecules. The chloroplast DNA was digested with restriction enzyme PstI and the fragments were cloned into the plasmid vector pUC9. Several recombinant plasmids were isolated and the chloroplast DNA inserts identified. The recombinant plasmid pRD105 containing the PstI #5 fragment was subjected to further investigation. The ClaI restriction sites of the PstI #5 fragment were mapped and the insert was subcloned into the plasmid vector pGEM4, which bears bacteriophage SP6 and T7 RNA polymerase promoter sequences.

Copy of author's previously published work inserted. Bibliography: leaves 139-160.

Copy of author's previously published work inserted. / Bibliography: leaves 139-160. / 160, [10] leaves, [41] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2001?

Interest in the mechanisms of homeo domain specificity led to a screen that identified Drosophila proteins able to bind a consensus homeo domain site. One clone isolated in this screen produced no homeo domain and was selected for further characterisation as a protein with an unknown DNA binding domain and the potential to interact with homeo domain proteins on the DNA. This thesis describes the characterisation of the Drosophila gene dead ringer ( dri ) corresponding to this clone. Isolation of overlapping cDNA clones and sequence analysis allowed the identification of a complete open reading frame in the dri message that gave a predicted protein of 901 amino acids. Database searches and multiple sequence alignment revealed a widely conserved motif in the Dri sequence that is found in proteins from organisms as diverse as yeast, nematodes, flies and humans. Biochemical analysis of the properties of this conserved motif revealed that it could function as a DNA binding domain when expressed in a fusion protein. The in vitro specificity of the Dri DNA binding domain was determined by selection and sequencing of target sites. The Dri consensus site obtained was strikingly similar to that of the Qfo class of homeo domains, although the sequence and predicted secondary structure of the Dri DNA binding domain do not resemble a homeo domain. Analysis of the developmental expression pattern of dri showed a ubiquitous maternal deposit gradually refined to localisation in the mesoderm at germ band extension, then further restriction to a diverse set of tissues including the salivary gland ducts, parts of the gut and a subset of the central nervous system. The phenotype of P - element insertion and deletion mutations of dri were identified as causing embryonic lethality preceded by a disruption of the hindgut and loss of Dri expression in the ring gland. The identification of the novel, conserved DNA - binding domain in Dead ringer offers an explanation for the regulatory activity of several important related proteins and presents an opportunity to use the advantages of the Drosophila model system to clarify the role of these proteins in transcriptional control. / Thesis (Ph.D.)--Departments of Biochemistry and Genetics, 1996.