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

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 6 uppsatser.

Recombinant DNA techniques were used to clone and isolate Cellulo- monas fimi cellulase genes. A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant plasmids carrying cellulase genes. The screening procedure is based on binding cellulases and other proteins released from lysed clones to CNBr-acti- vated paper. The paper is treated with anti-cellulase antibody and the antigen-antibody complex is detected by autoradiography using ¹²⁵I-labeled protein A from Staphylococcus aureus. This- immunoassay, was used to identify recombinant plasmids containing strains, carrying at least two different cellulase genes. The enzymes present in extracts of E. coli cellulase clones were active in catalysing the hydrolysis of carboxymethy1 eel 1ulose as indicated by the production of reducing sugars. Osmotic shock treatment of one E. coli cellulase clone revealed that the majority of the cellulase enzyme synthesized by this clone was transported to the periplasmic space. Cellulase encoding plasmids were characterized by the presence of either a 6.6 or a 5.0 kilobase C. fimi DNA gene fragment. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 117-130). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2004. / Typescript. Vita. Includes bibliographical references (leaves 117-130). Also available on the Internet.

A group of Escherichia coli clones harbouring recombinant pBR322 plasmid, containing Cellulomonas fimi DNA inserts, that reacted with antiserum to C.fimi culture supernatant, was screened for their ability to hydrolyze carboxymethyl cellulose (CMC) and 4-methylumbeliferyll-β-D-cellobioside (MUC). A clone, pEC62, hydrolyzed MUC but did not hydrolyze CMC. The recombinant enzyme encoded by pEC62 was shown to be a β-glucosidase (cellobiase). C.fimii itself was shown to encode an extracellular β-glucosidase in C.fimi. This is the first report of an extracellular β-glucosidase from a bacterium. Deletion analysis localized the cloned gene (cbg)to the tet promoter proximal region of the 7.0 kilobase insert of pEC62. Further analysis and sequence data showed a highly active derivative of pEC62 contained a translational gene fusion between lacZ of pUC13 and cbg. From this data, a location for the cbg start site was proposed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

No description available.

by Lee Kai Fai, Calvin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 159-167). / ACKNOWLEDGMENTS --- p.i / ABSTRACT --- p.ii / CONTENTS --- p.iv / ABBREVIATIONS --- p.xi / Chapter CHAPTER ONE --- General Introduction --- p.1 / Chapter 1.1 --- The Phenomenon of Host-controlled Restriction --- p.1 / Chapter 1.2 --- Classification of Restriction and Modification Systems --- p.2 / Chapter 1.2.1 --- Type I Restriction-Modification Systems --- p.2 / Chapter 1.2.2 --- Type II Restriction-Modification Systems --- p.3 / Chapter 1.2.3 --- Type III Restriction-Modification Systems --- p.4 / Chapter 1.2.4 --- Type IV Restriction-Modification Systems --- p.5 / Chapter 1.3 --- Occurrence of Restriction-Modification Systems --- p.6 / Chapter 1.4 --- Effect of Methylation --- p.7 / Chapter 1.5 --- Alternation of Recognition Specificities --- p.7 / Chapter 1.5.1 --- Cross Protection by DNA Methyltransferase --- p.8 / Chapter 1.5.2 --- A-Assisted Restriction Endonuclease (RARE) Cleavage --- p.9 / Chapter 1.5.3 --- Site-specific Cleavage mediated by Triple-helix formation --- p.9 / Chapter 1.5.4 --- Site-specific Cleavage of Duplex DNA with a λ repressor- Staphylococcal Nuclease Hybrid --- p.10 / Chapter 1.5.5 --- Achilles' heel Cleavage --- p.10 / Chapter 1.5.6 --- Chimeric Restriction Endonuclease --- p.11 / Chapter 1.6 --- Cloning of Restriction and Modification Systems --- p.11 / Chapter 1.6.1 --- Selection based on Modification --- p.11 / Chapter 1.6.2 --- Other Cloning Strategies --- p.12 / Chapter 1.6.2.1 --- Sub-Cloning of Plasmids --- p.12 / Chapter 1.6.2.2 --- Selection based on Restriction --- p.13 / Chapter 1.6.2.3 --- Multi-step Cloning --- p.13 / Chapter 1.6.2.4 --- Cloning in AP1-200 and AP1-200-9 strain --- p.13 / Chapter 1.6.2.5 --- Direct Cloning of Restriction gene by 'endo-blue' method --- p.14 / Chapter 1.7 --- Genetic Location of Restriction-Modification Systems --- p.14 / Chapter 1.8 --- Sequences of Restriction-Modification Systems --- p.15 / Chapter 1.9 --- Catalytic Properties of Type II Restriction-Modification Systems --- p.17 / Chapter 1.10 --- Crystallography of Type II Restriction and Modification Enzymes --- p.19 / Chapter 1.11 --- Evolution of Type II Restriction and Modification Enzymes --- p.22 / Chapter 1.12 --- Aim of Study --- p.23 / Chapter CHAPTER TWO --- Materials and Methods --- p.24 / Chapter 2.1 --- Bacterial Strains --- p.24 / Chapter 2.2 --- General Techniques --- p.25 / Chapter 2.2.1 --- Phenol/Chloroform Extraction --- p.25 / Chapter 2.2.2 --- Ethanol Precipitation --- p.25 / Chapter 2.2.3 --- Spectrophotometry --- p.25 / Chapter 2.2.4 --- Restriction digestion of DNA --- p.26 / Chapter 2.2.5 --- Agarose Gel Electrophoresis of DNA --- p.26 / Chapter 2.2.6 --- Recovery of DNA fragment from Agarose gel --- p.26 / Chapter 2.2.7 --- Minipreparation of Plasmid --- p.27 / Chapter 2.2.8 --- Large-Scale Preparation of Plasmid DNA --- p.28 / Chapter 2.2.8A --- By Equilibrium Centrifugation in Cesium Chloride- Ethidium Bromide Gradient --- p.28 / Chapter 2.2.8B --- By Using Qiagen-tip 100 Cartridge --- p.29 / Chapter 2.2.9 --- Preparation of Competent Cells --- p.30 / Chapter 2.2.10 --- Transformation of Competent Cells --- p.31 / Chapter 2.2.11 --- Screening of Recombinant Plasmids --- p.32 / Chapter 2.2.11A --- Using Selective media --- p.32 / Chapter 2.2.11B --- Rapid Alkaline Lysis Method --- p.32 / Chapter 2.2.12 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.33 / Chapter 2.2.13 --- Size Exclusion Chromatography --- p.34 / Chapter 2.2.14 --- Electroblotting of Protein on Polyvinylidene Difluoride (PVDF) membrane --- p.35 / Chapter 2.2.15 --- Isoelectric Focusing (EEF) --- p.36 / Chapter 2.2.16 --- Protein Assay --- p.37 / Chapter 2.3 --- DNA Sequencing --- p.37 / Chapter 2.3.1 --- Isolation of a template DNA --- p.38 / Chapter 2.3.2 --- DNA Denaturation and Annealing Reaction --- p.38 / Chapter 2.3.3 --- Labeling and Termination Reaction --- p.38 / Chapter 2.3.4 --- DNA Sequencing Electrophoresis --- p.39 / Chapter 2.3.5 --- Autoradiography --- p.40 / Chapter CHAPTER THREE --- Purification and Characterization of Restriction Endonuclease from Escherichia coli HK31 --- p.41 / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Materials and Methods --- p.42 / Chapter 3.2.1 --- Preparation of Crude enzyme Extract --- p.42 / Chapter 3.2.2 --- Purification of R.EcoHK31I --- p.42 / Chapter 3.2.3 --- Characterization of Restriction endonuclease --- p.43 / Chapter 3.2.3.1 --- Enzyme Activity assay --- p.43 / Chapter 3.2.3.2 --- "Optimal pH, Temperature, Metal Ion and Salt concentration of R.EcoHK31I" --- p.43 / Chapter 3.2.3.3 --- Assay for the Purity of R.EcoHK31I --- p.43 / Chapter 3.2.3.4 --- Determination of Recognition Specificity --- p.44 / Chapter 3.2.3.5 --- Determination of the Cleavage Specificity --- p.44 / Chapter 3.3 --- Results and Discussion --- p.45 / Chapter 3.3.1 --- Purification ofR.EcoHK31I from Escherichia coli HK31 --- p.45 / Chapter 3.3.2 --- "Optimal pH,Temperature, Metal ions and Salt concentration of R.EcoHK31I" --- p.46 / Chapter 3.3.3 --- Unit Definition --- p.51 / Chapter 3.3.4 --- Purity of the R.EcoHK31I --- p.51 / Chapter 3.3.5 --- Recognition Site of the R.EcoHK31I --- p.51 / Chapter 3.3.6 --- Sensitivity of the R.EcoHK31I to dcm Methylation --- p.52 / Chapter 3.3.7 --- Cleavage Specificity of R.EcoHK31I --- p.52 / Chapter CHAPTER FOUR --- Cloning of EcoEK31I Restriction and Modification (R-M) System from Escherichia coli HK31 --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.2 --- Materials and Methods --- p.58 / Chapter 4.2.1 --- Extraction of genomic DNA from E. coli HK31 --- p.58 / Chapter 4.2.2 --- Extraction of Extra-Chromosomal DNA from E. coli HK31 --- p.59 / Chapter 4.2.3 --- Restriction Digestion of the Total DNA --- p.59 / Chapter 4.2.4 --- Preparation of Linearized and Dephosphorylated Vector --- p.60 / Chapter 4.2.5 --- Fill-in Reaction --- p.60 / Chapter 4.2.6 --- Ligation between Vector and Digested Chromosomal DNA --- p.61 / Chapter 4.2.7 --- Selection of Clones Harboring Methyltransferase gene --- p.61 / Chapter 4.2.8 --- Screening of the Survival Clones --- p.62 / Chapter 4.3 --- Results --- p.62 / Chapter 4.3.1 --- Construction of Genomic Libraries --- p.62 / Chapter 4.3.2 --- Selection of the Methyltransferase Gene --- p.66 / Chapter 4.3.3 --- In vitro Detection of R.EcoHK31I activity --- p.67 / Chapter 4.3.4 --- Functional Localization of EcoHK31I --- p.67 / Chapter 4.3.5 --- Subcloning of the Complete EcoHK31I R-M System --- p.72 / Chapter 4.4 --- Discussion --- p.72 / Chapter 4.4.1 --- Construction of Genomic Libraries --- p.72 / Chapter 4.4.2 --- Cloning of EcoHK31I Restriction and Modification System --- p.75 / Chapter 4.4.2.1 --- Selecting Endonuclease --- p.75 / Chapter 4.4.2.2 --- Detection of Restriction Endonuclease Activity --- p.76 / Chapter 4.4.3 --- Functional Localization of the R-M System --- p.76 / Chapter CHAPTER FIVE --- The Nucleotide Sequences of the EcoHK31I R-M System --- p.78 / Chapter 5.1 --- Introduction --- p.78 / Chapter 5.2 --- Materials and Methods --- p.79 / Chapter 5.2.1 --- Sequencing Strategies --- p.79 / Chapter 5.2.2 --- DNA Sequencing --- p.80 / Chapter 5.2.3 --- Sequence Analysis --- p.80 / Chapter 5.3 --- Results and Discussion --- p.80 / Chapter 5.3.1 --- Nucleotide Sequences and Deduced Amino Acid sequences --- p.80 / Chapter 5.3.2 --- Comparison of Amino Acid Sequences --- p.85 / Chapter CHAPTER SIX --- Purification and Characterization of EcoHK31I Methyltransferase from E. coli K802 [pEcoHK31E] --- p.91 / Chapter 6.1 --- Introduction --- p.91 / Chapter 6.2 --- Materials and Methods --- p.92 / Chapter 6.2.1 --- Preparation of Crude enzyme Extract --- p.92 / Chapter 6.2.2 --- Purification of M.EcoHK31I --- p.92 / Chapter 6.2.3 --- Characterization of EcoHK31I Methyltransferase --- p.93 / Chapter 6.2.3.1 --- Enzyme Activity assay --- p.93 / Chapter 6.2.3.2 --- Determination of Methylation specificity --- p.93 / Chapter 6.2.3.3 --- Determination of Molecular weight of M.EcoHK31I --- p.94 / Chapter 6.2.3.4 --- Determination ofM.EcoHK31I Kinetics --- p.94 / Chapter 6.3 --- Results and Discussion --- p.96 / Chapter 6.3.1 --- Purification of EcoHK31I Methyltransferase --- p.96 / Chapter 6.3.2 --- M.EcoHK31I Modification Specificity --- p.99 / Chapter 6.3.3 --- "Determination of Molecular Weight ofM,EcoHK31I" --- p.99 / Chapter 6.3.4 --- Catalytic Properties of EcoHK31I Methyltransferase --- p.103 / Chapter 6.3.5 --- A Novel m5C-MTase M.EcoHK31I --- p.103 / Chapter CHAPTER SEVEN --- Over-expression and Characterization of EcoHK31I Restriction and Modification Enzymes --- p.106 / Chapter 7.1 --- Introduction --- p.106 / Chapter 7.1.1 --- Expression Vector pTrc series --- p.107 / Chapter 7.1.2 --- Expression Vector pET series --- p.107 / Chapter 7.2 --- Materials and Methods --- p.109 / Chapter 7.2.1 --- General technique --- p.109 / Chapter 7.2.2 --- Polymerase Chain Reaction --- p.110 / Chapter 7.2.3 --- Construction of plysSM13 --- p.110 / Chapter 7.2.4 --- Construction of pTrc99A-R36 --- p.110 / Chapter 7.2.5 --- Construction of pET3a-M38 --- p.111 / Chapter 7.2.6 --- Construction of pET3a-C23 --- p.111 / Chapter 7.2.7 --- Expression of Recombinant Proteins in E. coli hosts --- p.115 / Chapter 7.2.8 --- Purification of Recombinant R.EcoHK31I --- p.115 / Chapter 7.2.9 --- Determination of Molecular Weight of Recombinant R. EcoHK31I --- p.115 / Chapter 7.2.10 --- Polyclonal Antibodies against R.EcoHK31I --- p.116 / Chapter 7.2.11 --- Western Blotting --- p.116 / Chapter 7.2.12 --- Purification of Recombinant M.EcoHK31I polypeptide α --- p.117 / Chapter 7.2.13 --- Purification of Recombinant M.EcoHK31I polypeptide β --- p.118 / Chapter 7.2.14 --- In vitro Complementation Methylation Activity --- p.118 / Chapter 7.2.15 --- Incorporation of [3H]-AdoMet to non-methylated Lambda DNA --- p.119 / Chapter 7.3 --- Results and Discussion --- p.119 / Chapter 7.3.1 --- Expression of Recombinant R. EcoHK31I --- p.119 / Chapter 7.3.2 --- Purification and Characterization of Recombinant R.EcoHK31I --- p.120 / Chapter 7.3.2.1 --- Purification of Recombinant R.EcoHK31I --- p.120 / Chapter 7.3.2.2 --- Characterization of Recombinant R.EcoHK31I --- p.122 / Chapter 7.3.2.2.1 --- Molecular Weight and Isoelectric point of the Recombinant R.EcoHK31I --- p.122 / Chapter 7.3.2.2.2 --- Antibodies to Recombinant R.EcoHK31I --- p.125 / Chapter 7.3.3 --- Expression and Purification of M.EcoHK31Ipolypeptide α --- p.127 / Chapter 7.3.4 --- Expression and Purification of M.EcoHK31I polypeptide β --- p.127 / Chapter 7.3.5 --- Characterization of M.EcoHK31I polypeptides a and β --- p.129 / Chapter 7.3.5.1 --- Molecular Weight Determination --- p.129 / Chapter 7.3.5.2 --- Isoelectric Point Determination --- p.132 / Chapter 7.3.5.3 --- In vivo and in vitro Methylation Activity --- p.132 / Chapter CHAPTER EIGHT --- Generation and Activity Assay of Q193G Mutein of M.EcoHK31I Polypeptide a --- p.138 / Chapter 8.1 --- Introduction --- p.138 / Chapter 8.2 --- Materials and Methods --- p.139 / Chapter 8.2.1 --- Construction of pET3a-M38 (Q193G) --- p.139 / Chapter 8.2.2 --- Expression and Purification of Q193G protein --- p.140 / Chapter 8.2.3 --- In vivo and in vitro methylation activity of Q193G Mutein --- p.140 / Chapter 8.3 --- Results and Discussion --- p.145 / Chapter 8.3.1 --- "Construction, Expression and Purification of Q193G Mutein" --- p.145 / Chapter 8.3.2 --- Determination of Molecular Weight and Isoelectric point of Q193G --- p.145 / Chapter 8.3.3 --- In vivo and in vitro methylation activity of Q193G Mutein --- p.145 / Chapter 8.3.4 --- Recognition Specificity of Q193G Mutein --- p.147 / Chapter CHAPTER NINE --- General Discussion --- p.151 / REFERENCES --- p.159 / APPENDIX A --- p.168

Siu Shu Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 100-106). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Contents --- p.ii / Abstract / 摘要 --- p.iv / Abbreviations --- p.vi / List of figures --- p.vii / List of tables --- p.x / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- "Human EST sequencing project, the role and goal" --- p.1 / Chapter 1.2 --- Human liver cDNA sequencing --- p.2 / Chapter 1.3 --- The role of membrane-associated proteins in hepatocellular functions --- p.3 / Chapter 1.3.1 --- Outline of the liver function --- p.3 / Chapter 1.3.2 --- Basic structure of hepatocyte --- p.4 / Chapter 1.3.3 --- Category of membrane associated proteins --- p.5 / Chapter 1.4 --- Identification of human OAT2 gene --- p.7 / Chapter 1.5 --- The multispecific transporter family --- p.8 / Chapter 1.5.1 --- Classification --- p.8 / Chapter 1.5.2 --- The human OAT family --- p.9 / Chapter 1.6 --- The characteristics of rat multispecific OAT2 --- p.11 / Chapter 1.7 --- Clinical significance of organic anion transport proteins --- p.14 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1 --- Human liver EST sequencing project --- p.16 / Chapter 2.1.1 --- Plating out the adult human liver phage library --- p.16 / Chapter 2.1.2 --- PCR detection and amplification of the cDNA clone --- p.17 / Chapter 2.1.3 --- Automatic cDNA sequencing --- p.18 / Chapter 2.2 --- Cloning of hOAT2 gene into TA cloning vector pT-Adv --- p.19 / Chapter 2.2.1 --- Amplification of hOAT2 by PCR --- p.19 / Chapter 2.2.2 --- Ligation reaction --- p.19 / Chapter 2.2.3 --- Transformation of recombinant plasmid into competent cells --- p.20 / Chapter 2.3 --- Sequence analysis and structural prediction --- p.20 / Chapter 2.4 --- Cloning of the hOAT2 gene into the pQE30 expression vector --- p.21 / Chapter 2.4.1 --- PCR amplification and restriction endonuclease cutting --- p.21 / Chapter 2.4.2 --- Gene clean --- p.22 / Chapter 2.4.3 --- Preparation of bacterial competent cells --- p.23 / Chapter 2.5 --- Small scale synthesis of plasmid DNA --- p.24 / Chapter 2.6 --- Large scale synthesis of plasmid DNA --- p.25 / Chapter 2.7 --- Cloning of the hOAT2 gene into the pSecTag2B mammalian expression vector --- p.26 / Chapter 2.8 --- Cloning of the hOAT2 gene into the pEGFP-C2 fluorescent vector --- p.27 / Chapter 2.8.1 --- Tissue culture and transfection --- p.27 / Chapter 2.8.2 --- Fluorescence microscopy examination --- p.28 / Chapter 2.9 --- Chromosomal mapping of the hOAT2 gene --- p.29 / Chapter 2.9.1 --- Somatic cell hybrids mapping --- p.29 / Chapter 2.9.2 --- Radiation hybrids mapping --- p.29 / Chapter 2.10 --- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) --- p.30 / Chapter 2.11 --- Western hybridization --- p.32 / Chapter 2.11.1 --- Preparation of anti-hOAT2 antibodies --- p.32 / Chapter 2.11.1.1 --- Synthetic peptide conjugation --- p.32 / Chapter 2.11.1.2 --- Immunizing rabbit polyclonal antibodies for human OAT2 --- p.32 / Chapter 2.11.1.3 --- Purification of the rabbit polyclonal IgG antibodies --- p.33 / Chapter 2.11.2 --- Western blot analysis --- p.33 / Chapter 2.11.2.1 --- Protein isolation from rat liver --- p.33 / Chapter 2.11.2.2 --- Prote in preparation from cell lysate --- p.34 / Chapter 2.11.2.3 --- Quantitation of total proteins by Bradford protein assay --- p.35 / Chapter 2.11.2.4 --- Blotting and hybridization --- p.35 / Chapter Chapter 3: --- Results / Chapter 3.1 --- Catalogue of the 500 liver ESTs --- p.37 / Chapter 3.2 --- Nomenclature of human NLT gene --- p.47 / Chapter 3.3 --- Cloning and characterization of the hOAT2 sequence --- p.48 / Chapter 3.3.1 --- Isolation of hOAT2 cDNA from human liver cDNA library --- p.48 / Chapter 3.3.2 --- The primary and secondary structural analysis of hOAT2 --- p.53 / Chapter 3.3.3 --- Motif search and prediction --- p.61 / Chapter 3.3.4 --- Homology alignment --- p.64 / Chapter 3.4 --- Chromosomal mapping of hOAT2 gene --- p.67 / Chapter 3.4.1 --- Somatic cell hybrid mapping of hOA T2 gene --- p.67 / Chapter 3.4.2 --- Radiation hybrid mapping of hOA T2 gene --- p.69 / Chapter 3.4.3 --- Identification of partial human genomic sequence --- p.73 / Chapter 3.5 --- Detection of the hOAT2 gene expression in human tissues by RT- PCR assay --- p.76 / Chapter 3.6 --- Detection of subcellular localization of hOAT2 protein by conjugating fluorescence protein --- p.81 / Chapter 3.7 --- Immunodetection of protein extracts from cultured cells --- p.83 / Chapter Chapter 4: --- Discussion / Chapter 4.1 --- Characterization of the hepatocellular ESTs --- p.85 / Chapter 4.1.1 --- Classification and frequency distribution of the 500 ESTs --- p.85 / Chapter 4.1.2 --- The expression pattern of membrane associated proteins --- p.87 / Chapter 4.2 --- Tissue distribution and expression profiles of hOAT2 --- p.88 / Chapter 4.3 --- HOAT2 in fetal development --- p.89 / Chapter 4.4 --- Predicting the topology of membrane proteins --- p.90 / Chapter 4.5 --- Chromosomal mapping of human OAT2 --- p.91 / Chapter 4.6 --- Possible functions of hOAT2 --- p.93 / Chapter 4.6.1 --- Hepato-renal relation --- p.93 / Chapter 4.6.2 --- Substrate diversity --- p.95 / Chapter 4.7 --- Fluorescence detection for subcellular localization --- p.96 / Chapter 4.8 --- Conclusion --- p.97 / Chapter 4.9 --- Further aspects --- p.99 / References --- p.100 / Appendix --- p.107