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Chan chi-wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 89-95). / Abstracts in English and Chinese. / Dedication --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Abbreviations --- p.vi / List of Figures --- p.ix / List of Tables --- p.xiii / Contents --- p.xiv / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1. --- Thesis synopsis --- p.1 / Chapter 1.2. --- hNP220 protein --- p.1 / Chapter 1.2.1. --- Domain organization --- p.1 / Chapter 1.2.2. --- Known splice variants --- p.5 / Chapter 1.2.3. --- Subcellular localization --- p.7 / Chapter 1.2.4. --- Proposed roles in transcriptional activation and RNA processing --- p.7 / Chapter 1.2.5. --- Interaction between C-terminal of hNP220 and FHL2 --- p.9 / Chapter 1.3. --- Hypothesis --- p.12 / Chapter 1.4. --- Principles of key methods --- p.14 / Chapter 1.4.1. --- RLM-RACE --- p.14 / Chapter 1.4.2. --- CytoTrap® two-hybrid system --- p.15 / Chapter CHAPTER 2 --- Materials and Methods --- p.18 / Chapter 2.1. --- Cloning protocol --- p.18 / Chapter 2.1.1. --- Amplification of DNA fragment --- p.18 / Chapter 2.1.2. --- Purification of PCR product --- p.19 / Chapter 2.1.3. --- Restriction endonuclease digestion --- p.20 / Chapter 2.1.4. --- Dephosphorylation of cloning vector 5'-termini --- p.20 / Chapter 2.1.5. --- Insert/vector ligation --- p.20 / Chapter 2.1.6. --- Preparation of chemically competent bacterial cells (E. coli strain DH5a) --- p.21 / Chapter 2.1.7. --- Transformation of ligation product into chemically competent bacterial cells --- p.22 / Chapter 2.1.8. --- Small-scale preparation of bacterial plasmid DNA --- p.22 / Chapter 2.1.9. --- Screening for recombinant clone --- p.24 / Chapter 2.1.10. --- Dideoxy DNA sequencing --- p.24 / Chapter 2.1.11. --- Midi-scale preparation of recombinant plasmid DNA --- p.25 / Chapter 2.2. --- Determination of the transcription start site (TSS) of hNP220 gene --- p.27 / Chapter 2.2.1. --- RNA ligase-mediated rapid amplification of cDNA 5'-end (5-RLM-RACE) --- p.27 / Chapter 2.3. --- Isolation and identification of the third splice variant of HNP220 (hNP220ε) --- p.29 / Chapter 2.3.1. --- PCR from human heart/testis cDNAs pool --- p.29 / Chapter 2.3.2. --- RT-PCR --- p.29 / Chapter 2.3.3. --- Northern hybridization --- p.30 / Chapter 2.4. --- Human tissue distribution of hNP220 --- p.31 / Chapter 2.4.1. --- RT-PCR --- p.31 / Chapter 2.4.2. --- Northern hybridization --- p.31 / Chapter 2.5. --- Visualization of the subcellular localization patterns of GFP-tagged hNP220ε in HepG2 cell line --- p.32 / Chapter 2.5.1. --- Cloning of hNP220a and hNP220s into vector pEGFP-Cl --- p.32 / Chapter 2.5.2. --- Transfection of GFP fusion constructs into HepG2 cell line --- p.32 / Chapter 2.5.3. --- Epi-fluorescence microscopy --- p.33 / Chapter 2.6. --- Identification of the protein-protein interaction between hNP220ε and MAPRE1 --- p.34 / Chapter 2.6.1. --- CytoTrap® XR HeLa Cell cDNA Library screening --- p.34 / Chapter 2.6.1.1. --- Cloning of hNP220ε into yeast two-hybrid bait vector pSos --- p.34 / Chapter 2.6.1.2. --- Preparation of cdc25Ha & cdc25Hα yeast competent cells --- p.34 / Chapter 2.6.1.3. --- Autonomous activation study of bait fusion construct pSos-hNP220ε --- p.36 / Chapter 2.6.1.4. --- Cotransformation of pSos-hNP220ε and CytoTrap® XR HeLa Cell cDNA Library --- p.36 / Chapter 2.6.1.5. --- Verification of interaction by yeast mating --- p.38 / Chapter 2.6.1.5.1. --- Generation of yeast plasmid segregant for mating --- p.38 / Chapter 2.6.1.5.2. --- Yeast mating in 96-well plate --- p.39 / Chapter 2.6.1.6. --- Identification of putative interaction partner --- p.39 / Chapter CHAPTER 3 --- Results --- p.42 / Chapter 3.1. --- Transcription start site of the HNP220 gene is located 312 nucleotides upstream the initiation codon --- p.42 / Chapter 3.2. --- Third splice variant of hNP220 gene hNP220s) is identified --- p.44 / Chapter 3.3. --- In silico analysis of hNP220ε --- p.54 / Chapter 3.4. --- hNP220a and hNP220s are ubiquitously expressed in human fetal and adult tissues --- p.65 / Chapter 3.5. --- hNP220ε shows a punctate subnuclear localization pattern in HepG2 cell line --- p.67 / Chapter 3.6. --- hNP220ε interacts with MAPRE1 --- p.69 / Chapter CHAPTER 4 --- Discussion --- p.71 / Chapter 4.1. --- "Identification of hNP220s, the third splice variant of hNP220 gene" --- p.71 / Chapter 4.2. --- Biological resemblance between hNP220α (hNP220) and hNP220ε --- p.73 / Chapter 4.3. --- Protein-protein interaction between hNP220ε and MAPRE1 --- p.74 / Chapter 4.3.1. --- MAPRE1 protein --- p.77 / Chapter 4.3.2. --- Wnt signaling pathway --- p.78 / Chapter 4.4. --- Potential roles of hNP220 in the regulation of chromosome stability and oncogenesis --- p.82 / Chapter 4.5. --- Summary --- p.85 / Chapter 4.6. --- Concluding questions --- p.86 / Chapter 4.7. --- Future work --- p.87 / References --- p.89 / Appendix --- p.96

PCR-based codon-specific random mutagenesis and site-specific mutagenesis were performed to construct a library of 18 amino acid changes at Arg276 in the conserved leucine-loop of the core catalytic domain of human uracil-DNA glycosylase (UNG). Each Arg276 mutant was then overproduced in E. coli cells and purified to apparent homogeneity by conventional chromatography. All of the R276 mutant proteins formed a stable complex with the uracil-DNA glycosylase inhibitor protein (Ugi) in vitro, suggesting that the active site structure of the mutant enzymes was not perturbed. The catalytic activity of all mutant proteins was reduced; the least active mutant, R276E, exhibited 0.6% of wild-type UNG activity, whereas the most active mutant, R276H, exhibited 43%. Equilibrium binding measurements utilizing a 2- aminopurine-deoxypseudouridine DNA substrate showed that all mutant proteins displayed greatly reduced base flipping/DNA binding. However, the efficiency of UV-catalyzed cross-linking of the R276 mutants to single-stranded DNA was much less compromised. Using a concatemeric [³²P]U·A DNA polynucleotide substrate to assess enzyme processivity, UNG was shown to use a processive search mechanism to locate successive uracil residues, and Arg276 mutations did not alter this attribute. A transient kinetics approach was used to study six different amino acid substitutions at Arg276 (R276C, R276E, R276H, R276L, R276W, and R276Y). When reacted with double-stranded uracil-DNA, these mutations resulted in a significant reduction in the rate of base flipping and enzyme conformational change, and in catalytic activity. However, these mutational effects were not observed when the mutant proteins were reacted with single-stranded uracil-DNA. Thus, mutations at Arg276 effectively transformed the enzyme into a single-strand-specific uracil-DNA glycosylase. The nuclear form of human uracil-DNA glycosylase (LTNG2) was overproduced in E. coli cells and purified to apparent homogeneity. While UNG2 retained ~9 % of UNG activity, it did form a stable complex with Ugi. Paradoxically, low concentrations of NaC1 and MgC1₂ stimulated UNG2 catalytic activity as well as the rate of rapid fluorescence quenching; however, the rate of uracil flipping was reduced. When UNG2 bound pseudouracil-containing DNA, conformational change was not detected. / Graduation date: 2005

Graduation date: 2003

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Not available / text

A hormone response element and a negative regulatory element upstream of the human insulin gene have been investigated. The hormone response element is located one kilobase upstream of the transcription start site. When isolated and placed upstream of a viral promoter, it has been found to increase transcription in response to retinoic acid and thyroid hormone. It is also able to mediate a transcriptional response to retinoic acid, and to the retinoic acid receptor in the context of the entire insulin gene promoter/enhancer region. This element is able to bind to members of the retinoid receptor family in vitro. Insulin gene transcription in isolated human islets of Langerhans was also shown to be upregulated by retinoic acid. The negative regulatory element within the human insulin gene enhancer lies between 279 and 258 base pairs upstream of the transcription start site, although it relies upon nearby insulin enhancer sequence in order to act upon a heterologous promoter. The transcriptional silencing properties of the negative element can be abolished by point mutations of critical residues. The element forms several complexes with nuclear proteins from an insulin producing cell line, one of which is related to the ubiquitous POU domain factor, Oct-1. The relevance of these findings to the control of insulin gene transcription is discussed.