Thesis (Master of Applied Sciences in Chemistry)--Cape Peninsula University of Technology, 2018. / Antimicrobial Peptides (AMPs) are short peptides of about 45 - 54 amino acids that exhibit antibacterial and antifungal activities. Plant defensin is a type of AMP in plants which belong to a family of cationic peptides with a characteristic 3D folding pattern held in place by four disulfide bridges. AMPs especially defensins have been identified to have a huge biotechnological potential and are being patented for many applications. The aim of this work was to clone an antimicrobial peptide from Vigna subterranea and characterise it with bioinformatics analysis. 4 sets of primers were synthesized according to the sequences of conserved regions in AMPs i.e. defensins from legumes like Vigna unguiculata, Vigna radiata, Cicer arietinum and Cajanus cajan, amongst others, which have defensins with only a few sequence differences. The primers were designated VsDef P1 to P4. Using Vigna subterranea total genomic DNA as a template, fragments of expected sizes were successfully amplified and cloned into the pDRIVE vector and used to transform Escherichia coli JM109 cells in each case. Representative clones were sequenced and analysed using BLAST from National Center for Biotechnology Information. However, only the VIG clone was shown to be a bona fide defensin (over 90% identity, E-value of 1ex102, 99% query coverage of the nucleotide sequence, compared to Vigna unguiculata defensin). Based on this high sequence identity, a new pair of primers VsDef P5 was designed based on the Vigna unguiculata defensin sequence to specifically amplify the complete Vigna subterranea defensin gene, hereafter called VsDef1. Attempts to clone VsDef1 were however unsuccessful, and evidence of clone deletion and insert re-arrangement of insert DNA was observed. Direct sequencing of the PCR product demonstrated that it was indeed the complete VsDef1 pre-protein, composed of 433 nucleotides. In silico translation and analysis showed that VsDef1 has an intron at position 105 − 259 of the nucleotide sequences and encodes for a 78 amino acid peptide. Phylogenetic analysis revealed to be similar to the sequence of the defensins for Vigna unguiculata (96%), Vigna radiata (95%), Vigna angularis (95%) and Phaseolus vulgaris (93%) on the NCBI database. The three - dimensional structure of the peptide was modelled with SWISS-MODEL expasy and the structure was found to include one α- and three β domains, similar to those of other defensins. The failure to identify VsDef1 clone in a V. subterranea library and the failure to recover its cDNA clone are consistent with the hypothesised toxicity of VsDef1 to Escherichia coli. It is suggested that a different host, such as yeast, should be used in the future. The VsDef1 mRNA levels in germinating V. subterranea seeds was however successfully investigated using real-time reverse transcription quantitative PCR. VsDef1 mRNA is present in both the testa and embryo of dry seed and will persist through the early stages of seedling growth. This demonstrates the importance of VsDef1 in fighting off infection during germination in order to ensure successful germination. It is therefore essential to characterise more antimicrobial peptides from V. subterranea. The diversity of AMPs and their patterns of expressed genes will enable understanding of complex regulatory networks, which will likely enable identifying of genes involved in diseases and new biological processes.

A antitrombina de B. jararaca foi isolada por meio de cromatografia de afinidade em coluna HiTrap Heparin HP (GE Healthcare). A análise da interação da antitrombina humana ou de B. jararaca com a heparina em sistema BIAcoreT200 demonstrou que a antitrombina de B. jararaca apresenta maior afinidade pela heparina que a antitrombina humana. Com relação à sua atividade anti-inflamatória, os resultados obtidos evidenciaram o efeito anti-inflamatório do pré e do pós-tratamento com a antitrombina de B. jararaca na resposta inflamatória aguda. A análise proteômica do exsudato inflamatório de camundongos identificou algumas proteínas possivelmente relacionadas ao mecanismo de inibição da antitrombina, como a enzima C3 do sistema complemento, a sorotransferrina, a a1-antitripsina, a apolipoproteína AI, o fibrinogênio, o cininogênio e a albumina. O processo de clonagem permitiu a obtenção da sequência completa da antitrombina de B. jararaca e apesar da longa distância evolutiva entre serpentes e humanos, diversas características da antitrombina encontram-se conservadas. / B. jararaca antithrombin was isolated by affinity chromatography using HiTrap Heparin HP column (GE Healthcare). The interaction analysis of human or B. jararaca antithrombin with heparin using a BIAcoreT200 system (GE Healthcare) demonstrated that the affinity of B. jararaca antithrombin for heparin is higher than human antithrombin. Regarding the anti-inflammatory activity of B. jararaca antithrombin, the results showed the anti-inflammatory effect of pre- and post-treatment with this molecule in acute inflammation. The proteomic analysis of inflammatory exudate of mice identified some proteins possibly related to the mechanism of inhibition of antithrombin, such as C3 complement, serum transferrin, a1-antitrypsin, apolipoprotein AI, fibrinogen, albumin and kininogen. The molecular cloning process allowed the determination of the complete sequence of B. jararaca antithrombin and despite the evolutionary distance between snakes and human, a number of characteristics are preserved in antithrombin molecule.

Anthony C.T. Liang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 73-76). / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Introduction to antibiotics --- p.2 / Chapter 1.2 --- Use of antibiotics in antimicrobial chemotherapy --- p.3 / Chapter 1.3 --- Drug resistance in bacteria --- p.4 / Chapter 1.4 --- Genetic of infections drug resistance --- p.6 / Chapter 1.5 --- Clinical importance of drug resistance --- p.8 / Chapter 1.6 --- Resistance studies on a clinically- isolated Shigella Species --- p.9 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Culture media for bacteria growth --- p.10 / Chapter 2.2 --- Large scale plasmid preparation by CsCl density gradient centrifugation --- p.12 / Chapter 2.3 --- Minipreps of plasmid DNA by the alkaline lysis method --- p.14 / Chapter 2.4 --- Elution of DNA using the Geneclean Kit --- p.16 / Chapter 2.5 --- Transformation of plasmid DNA into E.coli DH5α --- p.17 / Chapter 2.6 --- Antibiotic sensitivity test and screening of resistance colonies --- p.19 / Chapter 2.7 --- Agarose electrophoresis of DNA --- p.20 / Chapter 2.8 --- Restriction and ligation --- p.21 / Chapter 2.9 --- Protocol for studying substrate profiles of aminoglycoside-modifying enzyme (AME) --- p.23 / Chapter 2.10 --- DNA sequencing using the T7 sequencing Kit from Pharmacia --- p.26 / Chapter Chapter 3 --- Antibiotic resistance studies on multiple resistant Shigella spp / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.2 --- Conjugation and transformation experiment --- p.35 / Chapter 3.3 --- Extraction of plasmid DNA from Shigella 2731and transconjugant 14R525(2731) --- p.37 / Chapter 3.4 --- Discussion --- p.39 / Chapter Chapter 4 --- Cloning and characterization of beta-lactamase gene of Shigella2731 / Chapter 4.1 --- Introduction --- p.40 / Chapter 4.2 --- Cloning of the beta-lactam gene in Shigella2731 --- p.42 / Chapter 4.3 --- "Resistance pattern of El, E2, and S1" --- p.43 / Chapter 4.4 --- "Plasmid DNA extraction of El, E2, and S1" --- p.44 / Chapter 4.5 --- Restriction mapping of the plasmid pSFlOO --- p.47 / Chapter 4.6 --- Discussion --- p.51 / Chapter Chapter 5 --- Cloning and characterization of the aminoglycoside resistance gene / Chapter 5.1 --- Introduction --- p.53 / Chapter 5.2 --- Cloning of the aminoglycoside resistance genes --- p.56 / Chapter 5.3 --- Substrate profile studies on aminoglycoside- modifying enzyme (AME) activity on transformant G and S --- p.57 / Chapter 5.4 --- Subcloning of plamsid DNA from transformant S --- p.60 / Chapter 5.5 --- "DNA sequencing of fragments A, B, and C" --- p.65 / Chapter 5.6 --- Discussion --- p.68 / Chapter Chapter 6 --- Conclusion --- p.79 / Chapter Chapter 7 --- Reference --- p.73

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

Mok Yu-Keung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (Leaves 195-201). / Abstract --- p.i / Acknowledgements --- p.iii / List of Abbreviations --- p.iv / Table of contents --- p.v / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- The need to increase the specificity and variety of restriction endonucleases --- p.1 / Chapter 1.2 --- Classification of methods used for increasing the specificity and variety of restriction endonculeases --- p.2 / Chapter 1.3 --- Isolation and characterization of restriction endonucleases from natural sources --- p.3 / Chapter 1.4 --- Modification of DNA substrate to produce new cleavage specificities --- p.6 / Chapter 1.4.1 --- Methylation of the DNA substrate --- p.6 / Chapter 1.4.1.1 --- Achilles' hell cleavage-The use of canonical methylation to produce novel specificities --- p.10 / Chapter 1.4.1.2 --- Cross protection-The use of non-canonical methylation to generate new cleavage specificity --- p.14 / Chapter 1.4.1.2.1 --- Recognition sequence of a restriction endonuclease and a methylase partially overlap --- p.14 / Chapter 1.4.1.2.2 --- Methylase recognizing a subset of the degenerate sequence of the restriction endonuclease --- p.16 / Chapter 1.4.1.2.3 --- Methylase-limited partial digestion --- p.16 / Chapter 1.4.1.3 --- The use of methylation dependent restriction endonucleases and methylases to generate new specificity --- p.17 / Chapter 1.4.1.4 --- Sequential double-methylation-A two step methylation procedure to generate new specificities --- p.20 / Chapter 1.4.2 --- The generation of a universal restriction endonuclease by combining a Type IIS restriction enzyme moiety and an oligonucleotide adaptor --- p.22 / Chapter 1.4.2.1 --- General principle for generating a universal restriction endonuclease --- p.22 / Chapter 1.4.2.2 --- Factors that affect the cleavage efficiency of universal restriction endonuclease --- p.25 / Chapter 1.4.2.3 --- Modifications and potential applications of the universal restriction endonuclease --- p.29 / Chapter 1.4.3 --- DNA triple helix formation-enhance restriction enzyme specificity by site-specific inhibition of restriction/modification enzymes --- p.32 / Chapter 1.5 --- Modification of the cleaving agent to produce new specificities --- p.36 / Chapter 1.5.1 --- Sequence-specific artificial endonucleases --- p.36 / Chapter 1.5.1.1 --- Oligonucleotides as sequence-specific ligand --- p.37 / Chapter 1.5.1.2 --- Protein or peptide as sequence-specific ligand --- p.40 / Chapter 1.5.1.3 --- General limitations and applications of artificial endonucleases --- p.42 / Chapter 1.5.2 --- Molecular cloning and protein engineering of the restriction-modification system of bacteria --- p.43 / Chapter 1.5.2.1 --- Molecular cloning of the bacterial restriction-modification systems --- p.43 / Chapter 1.5.2.1.1 --- The strategies used to clone and screen restriction-modification systems --- p.45 / Chapter 1.5.2.2 --- Protein engineering of the restriction-modification systems of bacteria --- p.50 / Chapter 1.5.2.2.1 --- Pre-requisites for protein engineering on the restriction-modification systems --- p.51 / Chapter 1.5.2.2.2 --- Effects of protein engineering on the activity and specificity of restriction endonuclease and methylase --- p.53 / Chapter 1.6 --- Variation of restriction endonuclease specificity by altering the reaction condition --- p.56 / Chapter 1.6.1 --- Effects of organic solvents --- p.57 / Chapter 1.6.2 --- Effects of pH and ionic environment on restriction endonuclease specificity --- p.58 / Chapter 1.6.3 --- Remarks on the use of star activity to introduce new specificity --- p.59 / Chapter 1.7 --- Aim of study --- p.59 / Chapter Chapter 2 --- Purification and characterization of thermophilic restriction endonucleases from soil Bacillus spp / Chapter 2.1 --- Materials and methods --- p.61 / Chapter 2.1.1 --- Purification of thermophilic restriction endonucleases from soil Bacillus spp --- p.61 / Chapter 2.1.1.1 --- Preparation of crude enzyme extract --- p.61 / Chapter 2.1.1.2 --- Purification of BsiB I and BsiE 1 --- p.63 / Chapter 2.1.1.3 --- Purification of BsiY I --- p.63 / Chapter 2.1.1.4 --- Preparation of BsiG I and BsiU I --- p.64 / Chapter 2.1.1.5 --- Concentration and storage of the purified restriction endonucleases --- p.64 / Chapter 2.1.1.6 --- Regeneration of the columns --- p.64 / Chapter 2.1.2 --- Characterization of restriction endonucleases --- p.65 / Chapter 2.1.2.1 --- Assay for the working temperature and ionic requirement for the restriction enzymes --- p.65 / Chapter 2.1.2.2 --- Unit determination of the restriction endonucleases --- p.66 / Chapter 2.1.2.3 --- Assay for the purities of restriction endonucleases --- p.66 / Chapter 2.1.2.4 --- Determination of recognition specificity --- p.67 / Chapter 2.1.2.5 --- Determination of the restriction endonuclease's sensitivity to dam and dcm methylation --- p.68 / Chapter 2.1.2.6 --- Determination of the cleavage specificities of restriction endonucleases --- p.70 / Chapter 2.1.2.7 --- Sequencing using Deaza dGTP --- p.73 / Chapter 2.2 --- Results --- p.73 / Chapter 2.2.1 --- Purification of thermophilic restriction endonucleases from soil Bacillus spp --- p.73 / Chapter 2.2.1.1 --- Strain identification --- p.74 / Chapter 2.2.1.2 --- Elution properties of the restriction endonucleases from columns --- p.74 / Chapter 2.2.1.2.1 --- BsiB I --- p.74 / Chapter 2.2.1.2.2 --- BsiE I --- p.77 / Chapter 2.2.1.2.3 --- BsiY 1 --- p.78 / Chapter 2.2.1.3 --- The working digestion temperature and ionic strength requirement --- p.81 / Chapter 2.2.1.4 --- Unit determination --- p.82 / Chapter 2.2.1.5 --- Purities of the purified restriction endonucleases --- p.83 / Chapter 2.2.1.6 --- Recognition sites of the purified restriction endonucleases --- p.83 / Chapter 2.2.1.6.1 --- BsiB I --- p.83 / Chapter 2.2.1.6.2 --- BsiE I --- p.85 / Chapter 2.2.1.6.3 --- BsiY 1 --- p.87 / Chapter 2.2.1.6.4 --- BsiU I and BsiG I --- p.88 / Chapter 2.2.1.7 --- Sensitivity of restriction endonucleases to dam and dcm methylation --- p.90 / Chapter 2.2.1.8 --- Cleavage specificities of the purified restriction endonucleases --- p.91 / Chapter 2.2.1.8.1 --- BsiB I --- p.91 / Chapter 2.2.1.8.2 --- BsiE I --- p.92 / Chapter 2.2.1.8.3 --- BsiY I --- p.93 / Chapter 2.2.1.9 --- Sequencing of a wrongly sequenced site in pACYC177 using Deaza-dGTP --- p.94 / Chapter Chapter 3 --- The use of Xcm I and BsiY I as an universal restriction endonuclease / Chapter 3.1 --- Materials and methods --- p.98 / Chapter 3.1.1 --- Assay of universal restriction endonuclease using ss DNAs --- p.98 / Chapter 3.1.1.1 --- Annealing reaction between adaptors and ss DNAs --- p.99 / Chapter 3.1.1.2 --- Digestion of the annealed DNA complex --- p.100 / Chapter 3.1.1.3 --- Assay of the digested ss DNA on alkaline denaturing agarose gel --- p.100 / Chapter 3.1.2 --- Assay system involving 5' end-labelled oligonucleotide --- p.101 / Chapter 3.1.2.1 --- Purification of oligonucleotides using preparative polyacrylamide gel electrophoresis --- p.102 / Chapter 3.1.2.2 --- 5'end-labelling of the oligonucleotide DNA substrate --- p.104 / Chapter 3.1.2.3 --- The annealing between adaptors and oligonucleotide DNA substrate and the digestion condition --- p.104 / Chapter 3.1.2.4 --- Assay of the labelled oligonucleotides in polyacrylamide gel after digestion --- p.105 / Chapter 3.2 --- Results --- p.106 / Chapter 3.2.1 --- Xcm I adaptors #2 and #4 --- p.106 / Chapter 3.2.1.1 --- Assay conditions used for the universal restriction endonucleases --- p.107 / Chapter 3.2.1.1.1 --- Conditions used for hybridization --- p.107 / Chapter 3.2.1.1.2 --- Conditions used for digestion --- p.108 / Chapter 3.2.1.2 --- Methods used to maximize the cleavage of M13mp7 with Xcm I adaptor #4 --- p.110 / Chapter 3.2.1.2.1 --- Methods used to optimize the hybridization process --- p.110 / Chapter 3.2.1.2.2 --- Methods used to relax the secondary DNA structures --- p.112 / Chapter 3.2.1.2.2.1 --- Linearization of M13mp7 with BamH I befor annealing the adaptor --- p.113 / Chapter 3.2.1.2.2.2 --- Relaxation of secondary structure using boiling and NaOH denaturation --- p.114 / Chapter 3.2.1.2.3 --- Methods used to optimize the digestion process --- p.115 / Chapter 3.2.1.2.3.1 --- Addition of BSA --- p.115 / Chapter 3.2.1.2.3.2 --- Addition of the restriction endonuclease in separate batches --- p.115 / Chapter 3.2.1.3 --- Digestion of ss M13mpl8 and ssM13mpl9 DNA using Xcm I adaptor #2 and adaptor #4 --- p.116 / Chapter 3.2.2 --- Xcm I adaptor #1 and #3 --- p.118 / Chapter 3.2.2.1 --- Methods used to maximize the cleavage of M13mp7 with Xcm I adaptor #1 and adaptor #3 --- p.119 / Chapter 3.2.2.1.1 --- Methods used to relax the secondary structure --- p.119 / Chapter 3.2.2.1.1.1 --- Linearization of M13mp7 with BamH I before the annealing reaction --- p.120 / Chapter 3.2.2.1.1.2 --- Relaxation of secondary structure by NaOH denaturation --- p.121 / Chapter 3.2.2.1.1.3 --- Relaxation of secondary structure by adding DMSO and urea --- p.122 / Chapter 3.2.2.1.2 --- Methods used to optimize the digestion and hybridization processes --- p.123 / Chapter 3.2.2.1.2.1 --- Annealing of M13mp7 with a different amount of adaptor #3 and digesting the DNA complex with Xcm I at different temperatures --- p.123 / Chapter 3.2.2.1.2.2 --- Optimization of digestion by adding Xcm I in separate batches --- p.124 / Chapter 3.2.3 --- BsiY I adaptor --- p.124 / Chapter 3.2.3.1 --- Methods used to optimize the cleavage of M13mp7-BsiY I adaptor complex with BsiY I --- p.126 / Chapter 3.2.3.1.1 --- Optimization of hybridization using various concentrations of NaCl during the annealing reaction --- p.126 / Chapter 3.2.3.1.2 --- Optimization of digestion by binding BsiY I to the BsiY I adaptor before annealing --- p.127 / Chapter 3.2.4 --- The use of 5' end-labelled oligonucleotide DNA substrates for digestion with universal restriction endonuclease --- p.128 / Chapter Chapter 4 --- Molecular cloning of the BsiY I restriction-modification system / Chapter 4.1 --- Materials and methods --- p.132 / Chapter 4.1.1 --- Preparation of chromosomal DNA from BsiY I producing Bacillus stearothermophilus --- p.132 / Chapter 4.1.1.1 --- Restriction digestion of the chromosomal DNA --- p.134 / Chapter 4.1.1.2 --- Southern hybridization to locate the position of the DNA fragment coding for the restriction-modification system --- p.135 / Chapter 4.1.1.2.1 --- Southern transfer of DNA fragments onto nitro-cellulose paper --- p.135 / Chapter 4.1.1.2.2 --- Labelling of the probes by Nick-translation --- p.136 / Chapter 4.1.1.2.3 --- Hybridization of the nick-translated probes onto the DNA fragments fixed on NC paper --- p.137 / Chapter 4.1.2 --- Large-scale preparation of the cloning vector --- p.137 / Chapter 4.1.2.1 --- Restriction endonuclease digestion and dephosphorylation of the vector ´Ø.… --- p.139 / Chapter 4.1.3 --- Ligation between vector and DNA inserts --- p.139 / Chapter 4.1.4 --- Transformation of the ligated DNA into competent cells --- p.140 / Chapter 4.1.4.1 --- Preparation of competent cells --- p.140 / Chapter 4.1.4.2 --- Transformation of the ligated vector and insert DNA into competent cells --- p.142 / Chapter 4.1.5 --- Rapid alkaline lysis method for screening transformants that contains an insert --- p.143 / Chapter 4.1.6 --- Preparation of the genomic library and its plasmid DNA --- p.144 / Chapter 4.1.7 --- Screening procedures used to clone the BsiY I restriction-modification system --- p.144 / Chapter 4.1.7.1 --- In vitro selection using Hungarian Trick --- p.145 / Chapter 4.1.7.2 --- In vivo selection using the host strain AP1-200 and AP1-200-9 --- p.145 / Chapter 4.1.7.2.1 --- Preparation of competent AP1-200 and AP1-200-9 cells --- p.146 / Chapter 4.1.7.2.2 --- Transformation of the genomic library plasmid into competent AP 1-200 and AP1-200-9 cells --- p.146 / Chapter 4.1.8 --- Assay of BsiY I restriction endonuclease and methylase activities in the suspecting clones --- p.147 / Chapter 4.1.8.1 --- Assay to BsiY I methylase activity - resistance of the plasmid to BsiY I digestion --- p.147 / Chapter 4.1.8.2 --- Assay of BsiY I methylase activity - ability to incorporate H3-methyl group from H3-SAM into DNA substrate molecules --- p.148 / Chapter 4.1.8.3 --- Assay of BsiY I restriction endonuclease activity - ability of crude enzyme extract to cleave DNA --- p.149 / Chapter 4.2 --- Results --- p.150 / Chapter 4.2.1 --- Construction of the BamH I genomic library --- p.150 / Chapter 4.2.1.1 --- Vector and insert used --- p.150 / Chapter 4.2.1.2 --- Optimization of the ligation and transformation process --- p.151 / Chapter 4.2.1.3 --- Preparation of the BamH I library --- p.153 / Chapter 4.2.1.4 --- Methods used to screen the restriction-modification system from the plasmid library --- p.155 / Chapter 4.2.1.4.1 --- The Hungarian Trick --- p.155 / Chapter 4.2.1.4.2 --- Screening of the restriction-modification system using the strains API-200 and AP1-200-9 --- p.159 / Chapter 4.2.2 --- Construction of the Hind III library --- p.161 / Chapter 4.2.2.1 --- Vector and insert used --- p.161 / Chapter 4.2.2.2 --- Optimization of the ligation and transformation process --- p.162 / Chapter 4.2.2.3 --- Preparation of the Hind III library --- p.164 / Chapter 4.2.2.4 --- Methods used to screen the restriction-modification system from the plasmid library --- p.165 / Chapter 4.2.2.4.1 --- The Hungarian Trick --- p.165 / Chapter 4.2.2.4.2 --- Screening of the restriction-modification system using the strain AP1-200 and AP1-200-9 --- p.168 / Chapter 4.2.2.5 --- Assay of methylase activity using H3-SAM --- p.170 / Chapter 4.2.3 --- The use of Southern blotting and hybridization to find if two available probes have homology to the BsiY I restriction-modification system --- p.173 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- Purification and characterization of restriction endonucleases from Bacillus spp --- p.176 / Chapter 5.1.1 --- Methods used to purify the restriction endonuclease --- p.177 / Chapter 5.1.2 --- Characterization of the restriction endonucleases --- p.179 / Chapter 5.1.2.1 --- Determination of the purities of the purified restriction endonucleases --- p.179 / Chapter 5.1.2.2 --- Determination of the recognition site --- p.179 / Chapter 5.1.2.3 --- Determination of the cleavage site --- p.180 / Chapter 5.1.2.4 --- Sequencing using Deaza-dGTP --- p.181 / Chapter 5.2 --- The use of Xcm I and BsiY I as universal restriction endonucleases --- p.182 / Chapter 5.2.1 --- The adverse effects of hair-pin loop on the cleavage with universal restriction enzymes --- p.183 / Chapter 5.3 --- Molecular cloning of the BsiY I restriction-modification system --- p.187 / Chapter 5.3.1 --- Construction of the genomic library --- p.187 / Chapter 5.3.1.1 --- Preparation of the insert and vector --- p.188 / Chapter 5.3.1.2 --- Optimization of the ligation and transformation processes --- p.188 / Chapter 5.3.2 --- Screening strategies used to clone the BsiY I restriction-modification system --- p.189 / Chapter 5.3.2.1 --- The Hungarian Trick --- p.189 / Chapter 5.3.2.2 --- Screening using the strains AP1-200 and AP1-200-9 cells --- p.191 / Chapter 5.3.3 --- Assay of the gene products from the cloned restriction-modification system --- p.192 / Chapter 5.3.3.1 --- Methylase activity --- p.192 / Chapter 5.3.3.2 --- Restriction endonuclease activity --- p.193 / Chapter 5.4 --- Future prospects --- p.193 / References --- p.195 / Appendix --- p.201

Ng Dean Yew, Dennis. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 85-98). / Abstract --- p.i / Acknowledgments --- p.iii / Table of contents --- p.iv / List of figures --- p.vii / List of tables --- p.ix / List of abbreviation --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- History --- p.1 / Chapter 1.2. --- Classification and structure of Epstein-Barr Virus --- p.2 / Chapter 1.3. --- Genomic organization of EBV --- p.3 / Chapter 1.4. --- Replication cycle of EBV --- p.5 / Chapter 1.5. --- EBV latent and lytic cycle proteins --- p.6 / Chapter 1.6. --- Clinical diseases associated with EBV Infection --- p.11 / Chapter 1.7. --- Association of EBV and NPC --- p.13 / Chapter 1.8. --- EBV serological markers in the diagnosis of NPC --- p.13 / Chapter 1.9. --- Sources of EBV-specific DNase --- p.15 / Chapter 1.10. --- Characteristics of Epstein-Barr virus alkaline DNase --- p.15 / Chapter 1.11. --- Aim of the project --- p.18 / Chapter Chapter 2 --- Materials & Methods / Chapter 2.1. --- Molecular cloning --- p.19 / Chapter 2.1.1. --- Cell culture --- p.19 / Chapter 2.1.2. --- mRNA purification --- p.19 / Chapter 2.1.3. --- First strand cDNA synthesis --- p.21 / Chapter 2.1.4. --- Polymerase chain reaction (PCR) of cDNA --- p.21 / Chapter 2.1.5. --- Purification of PCR product after gel electrophoresis --- p.22 / Chapter 2.1.6. --- Ligation of PCR amplified DNase gene into pUC18 Sma/BAP vector --- p.23 / Chapter 2.1.7. --- Transformation by electroporation --- p.24 / Chapter 2.1.7.1. --- Cell preparation --- p.24 / Chapter 2.1.7.2. --- Electroporation procedure --- p.25 / Chapter 2.2. --- Extraction ofplasmid DNA --- p.28 / Chapter 2.2.1. --- Boiling preparation --- p.28 / Chapter 2.2.2. --- Plasmid digestion --- p.29 / Chapter 2.3. --- Large-scale purification ofplasmid --- p.29 / Chapter 2.4. --- Small-scale purification ofplasmid --- p.32 / Chapter 2.5. --- DNA sequencing --- p.33 / Chapter 2.5.1. --- Annealing of primer to template DNA --- p.33 / Chapter 2.5.2. --- Labelling reaction --- p.34 / Chapter 2.5.3. --- Sequencing termination reaction --- p.35 / Chapter 2.5.4. --- Prepartion of sequencing gel --- p.36 / Chapter 2.5.5. --- Autoradiography of sequencing gel --- p.38 / Chapter 2.6. --- Epitope mapping --- p.39 / Chapter 2.6.1. --- Processing of EBV- specific DNase peptides --- p.39 / Chapter Chapter 3 --- Results / Chapter 3.1. --- Molecular cloning --- p.41 / Chapter 3.1.1. --- Cell culture --- p.41 / Chapter 3.1.2. --- mRNA purification --- p.42 / Chapter 3.1.3. --- PCR amplification --- p.42 / Chapter 3. 1.4 --- DNA purification of PCR product --- p.42 / Chapter 3.1.5. --- Molecular cloning of PCR amplified DNase gene into pUC18 SmaI/BAP vector --- p.44 / Chapter 3.1.6. --- Transformation by electroporation --- p.46 / Chapter 3.1.7. --- Extraction of plasmid DNA --- p.48 / Chapter 3.1.7.1. --- Boiling preparation --- p.48 / Chapter 3.1.8. --- Plasmid digestion --- p.51 / Chapter 3.2. --- DNA sequencing --- p.51 / Chapter 3.2.1. --- Comparison of B95-8 EBV-speicific DNase gene with gene sequence of EBV in GeneBank --- p.50 / Chapter 3.2.2. --- Comparison of 5' end of Raji & B95-8 EBV derived EBV-specific DNase gene --- p.57 / Chapter 3.2.3. --- Comparison of the 3'end of the Raji and B95-8 denved EBV-specific DNase gene --- p.63 / Chapter 3.2.4. --- Amino acid sequence homology between B95-8 & Raji EBV-specific DNase --- p.64 / Chapter 3.2.5. --- Amino acid sequence comparison between the 3' end of the B95-8 EBV DNase protein with that of the Raji EBV DNase protein --- p.62 / Chapter 3.3. --- Epitope mapping --- p.67 / Chapter 3.3.1. --- Amino acid key --- p.67 / Chapter 3.3.2. --- Amino acid sequence of peptides --- p.73 / Chapter 3.3.2. --- O.D. readings at 492nm of five histologically proven NPC sera --- p.74 / Chapter Chapter 4 --- Discussions / Chapter 4.1. --- Overall strategy --- p.75 / Chapter 4 2 --- Significance of EBV-specific DNase as marker for NPC --- p.76 / Chapter 4.3. --- Characterization of EBV-specific DNase --- p.76 / Chapter 4.4. --- Molecular cloning of PCR amplified gene into PUC18 SmaI/BAP vector --- p.77 / Chapter 4.4.1. --- Cell culture --- p.77 / Chapter 4.4.2. --- PCR amplification --- p.73 / Chapter 4.4.3. --- "Blunting,kinasing and ligation of EBV-specific DNase cDNA into pUC18 vector" --- p.78 / Chapter 4.4 .4 --- .Transformation by electroporation --- p.80 / Chapter 4.4.5. --- Restriction enzyme digestion of pUC18/EBV-DNase plasmid … --- p.81 / Chapter 4.5. --- DNA sequencing --- p.81 / Chapter 4.6. --- Epitope mapping --- p.83 / Reference --- p.85

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

Tse Chui-ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 130-146). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 論文撮要 --- p.iii / List of Figures and Tables --- p.iv / Abbreviations --- p.vi / Table of contents --- p.vii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Literature review --- p.1 / Chapter 1.1.1 --- An overview of IGFs --- p.3 / Chapter 1.1.2 --- Molecular biology of IGFs --- p.5 / Chapter 1.1.2.1 --- IGF-I and IGF-II genes and mRNAs --- p.5 / Chapter 1.1.2.2 --- Amino acid sequences of IGF-II --- p.8 / Chapter 1.1.2.3 --- Imprinting of IGF-II --- p.12 / Chapter 1.1.3 --- IGF distribution in tissues and body fluids --- p.14 / Chapter 1.1.3.1 --- IGF in serum --- p.14 / Chapter 1.1.3.2 --- IGF binding proteins --- p.16 / Chapter 1.1.4 --- IGF receptors --- p.19 / Chapter 1.1.4.1 --- Structures of the IGF receptors --- p.20 / Chapter 1.1.4.2 --- Ligand binding of the IGF receptors --- p.21 / Chapter 1.1.4.3 --- Signal transduction and biological response --- p.22 / Chapter 1.1.5 --- Biological effects of IGF --- p.24 / Chapter 1.1.6 --- Expression of recombinant IGF --- p.28 / Chapter 1.2 --- Rationale and Objective --- p.29 / Chapter Chapter II --- Methodology --- p.33 / Chapter 2.1 --- Design of degenerate primers --- p.33 / Chapter 2.2 --- Cloning --- p.35 / Chapter 2.2.1 --- DNA extraction from agarose gel --- p.35 / Chapter 2.2.2 --- Linearization and dephosphorylation of plasmid DNA --- p.35 / Chapter 2.2.3 --- Blunt-end ligation of amplicon with linearized plasmid --- p.36 / Chapter 2.2.4 --- T/A ligation of amplicon with linearized plasmid --- p.37 / Chapter 2.2.5 --- Sticky end ligation of foreign DNA with linearized plasmid --- p.37 / Chapter 2.2.6 --- Preparation of competent of E. coli stain DHI5α cells --- p.38 / Chapter 2.2.7 --- Transformation of plasmid vector into competent cells (heat-shock/ electroporation) --- p.39 / Chapter 2.2.8 --- "Spread single colony, PCR check clone and inoculation" --- p.40 / Chapter 2.2.9 --- Small scale alkali preparation of plasmid DNA --- p.41 / Chapter 2.2.10 --- Large scale preparation of plasmid DNA --- p.41 / Chapter 2.2.11 --- Nucleotide sequencing --- p.41 / Chapter 2.2.11.1 --- Manual sequencing --- p.41 / Chapter 2.2.11.2 --- PCR sequencing --- p.43 / Chapter 2.3 --- Northern blot --- p.45 / Chapter 2.4 --- Preparation of radio-labeled probe and hybridization of radio-labeled probe to nylon immobilized nucleic acid --- p.46 / Chapter 2.5 --- RACE --- p.48 / Chapter 2.5.1 --- Design of gene-specific primer --- p.51 / Chapter 2.5.2 --- First strand cDNA synthesis --- p.51 / Chapter 2.5.3 --- TdT tailing of cDNA --- p.52 / Chapter 2.6 --- Poly-A tract extraction --- p.53 / Chapter 2.7 --- Tissue distribution of mRNA --- p.53 / Chapter 2.7.1 --- Tissue preparation --- p.53 / Chapter 2.7.2 --- Total RNA extraction --- p.54 / Chapter 2.7.3 --- Formaldehyde agarose gel electrophoresis of RNA --- p.54 / Chapter 2.8 --- RNAse protection assay --- p.55 / Chapter 2.8.1 --- Antisense probe generation --- p.56 / Chapter 2.8.2 --- Preparation of the sample RNA --- p.58 / Chapter 2.8.3 --- Hybridization --- p.58 / Chapter 2.8.4 --- RNase digestion of hybridized probe and sample RNA --- p.59 / Chapter 2.8.5 --- Preparation of radioactive marker --- p.60 / Chapter 2.8.6 --- Separation and detection of protected fragments --- p.60 / Chapter 2.8.7 --- Data processing and statistical analysis --- p.61 / Chapter 2.9 --- Injection of GH --- p.62 / Chapter 2.10 --- Recombinant protein expression --- p.62 / Chapter 2.10.1 --- Plasmid construction --- p.62 / Chapter 2.10.2 --- Expression --- p.63 / Chapter 2.11 --- Resolution of proteins on SDS-PAGE --- p.63 / Chapter 2.12 --- Purification --- p.64 / Chapter 2.13 --- Western transfer --- p.64 / Chapter 2.14 --- Immunodetection --- p.65 / Chapter Chapter III --- Results & Discussion --- p.67 / Chapter 3.1 --- Isolation and characterization of IGF-II cDNA and its gene organization --- p.67 / Chapter 3.1.1 --- Introduction --- p.67 / Chapter 3.1.2 --- Results --- p.68 / Chapter 3.1.2.1 --- Generation of a fragment of the common carp IGF-II cDNA by PCR --- p.68 / Chapter 3.1.2.2 --- Isolation of the full length common carp IGF-II cDNA by RACE. --- p.69 / Chapter 3.1.2.3 --- Nucleotide sequence analysis --- p.74 / Chapter 3.1.2.4 --- Relationship of common carp IGF-II to common carp IGF-I and insulin --- p.78 / Chapter 3.1.2.5 --- Confirmation of the presence of IGF-II in common carp --- p.79 / Chapter 3.1.2.6 --- Multiple mRNA forms of common carp IGF-I and IGF-II --- p.80 / Chapter 3.1.2.7 --- Gene organization of the common carp IGF-II gene --- p.83 / Chapter 3.1.3 --- Discussion --- p.86 / Chapter 3.2 --- Tissue specific distribution of IGF-I and IGF-II mRNA and their hormonal regulation --- p.90 / Chapter 3.2.1 --- Introduction --- p.90 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.2.1 --- RNase protection assay measurement of tissue mRNA levels in juvenile and adult common carp --- p.94 / Chapter 3.2.2.2 --- Expression of IGF-II mRNA during larval development --- p.99 / Chapter 3.2.2.3 --- Effect of GH on IGF-I and IGF-II mRNA levels in brain and Hver of juvenile common carp --- p.102 / Chapter 3.2.3 --- Discussion --- p.106 / Chapter 3.3 --- Recombinant common carp IGF-II expressed in E. coli --- p.110 / Chapter 3.3.1 --- Introduction --- p.110 / Chapter 3.3.2 --- Results --- p.112 / Chapter 3.3.2.1 --- Product of recombinant common carp IGF-II --- p.112 / Chapter 3.3.2.2 --- Purification of common carp IGF-II --- p.115 / Chapter 3.3.2.3 --- Immunodetection --- p.117 / Chapter 3.3.3 --- Discussion --- p.118 / Chapter Chapter IV --- General conclusion --- p.120 / Appendix: Reagents --- p.124 / Reference list --- p.130

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