Thesis (doctoral)--Lund University, 1996. / Added t.p. with thesis statement inserted.

Thesis (Ph.D.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 201-225.

Thesis (doctoral)--Lund University, 1996. / Added t.p. with thesis statement inserted.

Thesis (Ph.D.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 150 pages. Includes Vita. Includes bibliographical references.

Murine cytomegalovirus (MCMV) is used as a biological model for human cytomegalovirus (HCMV). Latency, persistence and reactivation are same of the important aspects of the murine model that share analogies with human CMV infections. In order to elucidate the molecular mechanisms leading to these events, in-depth analyses of the murine model are required at the transcriptional level. During the MCMV replication cycle, there is a sequential expression of different regions of the viral genome, hence the transcripts are divided into three kinetic classes; the immediate early (IE), early (E) and late (L). This study presents the analyses of MCMV (Smith strain) transcripts of the major IE and E transcriptional units, and a more detail analysis of one of the major E regions, E10. The IE and E transcripts were studied by probing them with Ctoitplementary DNAs (cDNAs). The cDNAs were prepared from mRNA isolated from the IE and E phases of the viral replication cycle and cloned into the bacteriophage Lambda gt10. Ten E cDNAs were mapped to specific locations of the virus genome, and these represented transcripts from the major E regions in Hindlll fragments A, B, E, F, and I-J. Five E cDNAs, each representing a different major E region, and two IE cDNAs representing the major IE region, were applied as probes in one of the studies to determine the relative transcript levels during the course of infection of 3T3L1 fibroblast cells with MCMV. The major E transcriptional units were investigated further in a study where Northern blots of RNAs, isolated from different phases of the viral replication cycle, were probed with the five E cDNAs. This study revealed transcripts that were temporally regulated since they were present only during the E and usually L phases of the viral replication cycle. In addition, the quantities of these transcripts varied depending on the phase. However, all five cDNAs detected more than one transcript which indicates complex splicing events, overlapping genes, multiple initiation sites and/or the presence of gene(s) in the complementary DNA strand. One of the E cDNAs, E10, corresponding to a transcript from a major E region of Hindlll fragment I-J, was selected for further analysis. The E10 cDNA detected four transcripts of 9.5, 6.9, 4.7 and 2.1 kb in size, which were found to be transcribed from the same DNA strand. The DNA sequence of this E10 cDNA was determined and shown to contain 3223 nucleotides, however it lacked a polyadenylation signal and a poly A tract at the 3' end. The missing 3' terminus, designated as E10-A, was isolated using the polymerase chain reaction (PCJR) method and its DNA sequence of 1422 nucleotides was also determined. The combined sequence of E10 and E10-A (total of 4606 nucleotides) was designated as E10-C and is presented in this thesis. The E10-C cDNA (4.6kbp) most likely represents the 4.7 kb transcript. The E10-C cDNA sequence has one minor and one major open reading frame (ORF). The minor ORF is initiated by the first ATG triplet (nucleotide position 114) while the major ORF is initiated by the second triplet (nucleotide position 155). Since the sequence preceeding the second ATG triplet is in "good context" with regard to the translation initiation consensus sequence, it is most likely that the major ORF is translated. The major ORF (3600 bases) encodes a 1200 amino acid polypeptide, the putative E10 protein of approximately 135 kd in size. A protein close to that size was detected in one of the experiments in which RNAs, that were hybrid-selected by the E10 cDNA and eluted, were translated in vitro. The putative E10 protein lacks homology with any other protein in the data banks (SWISSPRT and GENPEPT). Portions of the viral genomic fragments Hindlll I and J were also sequenced to reveal the orientation of the gene coding for the E10 cDNA and its related transcripts. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Rhodobacter capsulatus photosynthesis gene was isolated by creating in-frame fusions in a lacZ transcriptional/translational vector, and selecting for those that directed oxygen-regulated levels of β-galactosidase in R. capsulatus. One lacZ fusion isolate was used to identify an open reading frame (ORF) of unknown function and flanking sequences that promoted initiation of transcription. Interposon mutagenesis experiments identified the ORF as the bchC gene, which encodes an enzyme that catalyses the penultimate step in the biosynthesis of bacteriochlorophyll a, and also showed that the bchC gene formed an operon with the bchA gene. The nucleotide sequence of this bchC gene and its 5' regulatory region were determined. The deduced amino acid sequence showed that the bchC gene encodes a 33 kDA protein that has hydrophobic segments that could interact with a lipid membrane, and that this putative BchC protein contains a potential bacteriochlorophyll a binding site. Deletion analysis, S1-nuclease protection, and primer extension experiments showed that promoter activity was associated with sequences to which a 5' end mapped, and that these sequences had significant similarity to the proposed promoter regions of several other R. capsulatus photosynthesis genes. RNA blotting and S1-nuclease protection end-mapping experiments using bipartite probes provided direct evidence that the mRNA transcripts of the bchCA operon overlap those of the two flanking operons, the crtEF and the puf operons, such that the crtEF, bchCA, and puf operons may be cotranscribable, and that RNA polymerase may initiate transcription at one of several promoters. The significance of these overlapping mRNAs was evaluated using two interposon mutant strains, one that prevented crtEF transcripts from overlapping those the bchCA and puf operons, and the other that prevented both crtEF and bchCA transcripts from overlapping those of the puf operon. The results suggested that transcriptional readthrough stimulates promoter activity. Moreover, a pufB::lac'Z fusion could be expressed from the bchCA promoter equally as well as from the puf promoter, suggesting that these overlapping transcripts are functionally significant in the chromosomal context. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Tsim Ting Yuk. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 119-135). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iii / 英漢譯名對照 --- p.v / ABBREVIATIONS --- p.vi / ACKNOWLEDGMENTS --- p.viii / Chapter 1. --- Introduction / Chapter 1.1. --- Olfactory system --- p.1 / Chapter 1.1.1. --- Olfactory bulb (OB) --- p.1 / Chapter 1.1.2. --- Accessory olfactory bulb (AOB) --- p.3 / Chapter 1.2. --- Stem cells --- p.5 / Chapter 1.3. --- Sexual differentiation --- p.8 / Chapter 1.3.1. --- Sexual dimorphic olfactory system --- p.8 / Chapter 1.3.2. --- Androgen receptor (AR) & estrogen receptor beta (ERβ) --- p.13 / Chapter 1.3.3. --- Aromatase --- p.15 / Chapter 1.3.4. --- Oligomycin sensitivity-conferringrotein (OSCP) --- p.18 / Chapter 1.4. --- rogrammed cell death (PCD) --- p.18 / Chapter 1.4.1. --- CD in the olfactory development --- p.18 / Chapter 1.4.2. --- Caspase 3 --- p.22 / Chapter 1.4.3. --- B cell leukemia/ Lymphoma 2 (Bcl-2) --- p.23 / Chapter 1.5. --- Axon guidance molecules --- p.25 / Chapter 1.5.1. --- Growth cone --- p.25 / Chapter 1.5.2. --- Mechanisms of growth cone advance --- p.26 / Chapter 1.5.3. --- Semaphorins --- p.28 / Chapter 1.5.4. --- Neuropilin --- p.31 / Chapter 1.5.5. --- lexin --- p.32 / Chapter 1.5.6. --- Collapsin response mediatorroteins (CRMPs) --- p.32 / Chapter 1.6. --- Olfactory markerroteins --- p.33 / Chapter 1.6.1. --- Markerroteins in ORNs --- p.33 / Chapter 1.6.2. --- Growth associatedrotein (GAP-43) --- p.34 / Chapter 1.6.3. --- Is the expression of GAP-43 in rat OB sexually dimorphic? --- p.36 / Chapter 1.6.4. --- Olfactory markerrotein (OMP) --- p.38 / Chapter 1.6.5. --- Golf --- p.39 / Chapter 1.7. --- Miscellaneous genes --- p.40 / Chapter 1.7.1. --- Substance (SP) --- p.40 / Chapter 1.7.2. --- Gonadotropin releasing hormone (GnRH) --- p.41 / Chapter 1.7.3. --- Metabotropic glutamate receptor 2 (mGluR2) --- p.42 / Chapter 1.7.4. --- Insulin-like growth factor binding protein-2 (IGFBP2) --- p.43 / Chapter 2. --- Materials and methods / Chapter 2.1. --- Animal study --- p.46 / Chapter 2.2. --- RNA extraction --- p.46 / Chapter 2.3. --- Quantitation of total RNA --- p.49 / Chapter 2.4. --- Reverse Transcription (RT) --- p.50 / Chapter 2.5. --- olymerase Chain Reaction (PCR) --- p.51 / Chapter 2.6. --- urification ofCRroducts --- p.55 / Chapter 2.7. --- Confirmation ofCRroducts --- p.56 / Chapter 2.8. --- Quantitation of cDNA --- p.57 / Chapter 2.9. --- Radioactive labeledCR --- p.58 / Chapter 2.10. --- Electrophoresis ofCRroducts --- p.59 / Chapter 2.11. --- Statistical analysis --- p.60 / Chapter 3. --- Results / Chapter 3.1. --- Standard curve construction --- p.61 / Chapter 3.2. --- β-actin --- p.62 / Chapter 3.3. --- Sexual differentiation related genes --- p.64 / Chapter 3.3.1. --- AR --- p.64 / Chapter 3.3.2. --- ERβ --- p.65 / Chapter 3.3.3. --- Aromatase --- p.65 / Chapter 3.3.4. --- OSCP --- p.66 / Chapter 3.4. --- CD related genes --- p.66 / Chapter 3.4.1. --- Bcl-2α --- p.66 / Chapter 3.4.2. --- Caspase 3 --- p.67 / Chapter 3.5. --- Axon guidance molecules and related genes --- p.67 / Chapter 3.5.1. --- SemaIII --- p.67 / Chapter 3.5.2. --- Neuropilin-1 --- p.68 / Chapter 3.5.3. --- lexin-1 --- p.68 / Chapter 3.5.4. --- CRMP-1 --- p.69 / Chapter 3.5.5. --- CRMP-2 --- p.70 / Chapter 3.5.6. --- CRMP-3 --- p.70 / Chapter 3.5.7. --- CRMP-4 --- p.71 / Chapter 3.6. --- Olfactory markerrotein genes --- p.71 / Chapter 3.6.1. --- GAP-43 --- p.71 / Chapter 3.6.2. --- OMP --- p.72 / Chapter 3.6.3. --- Golf --- p.72 / Chapter 3.7. --- Miscellaneous genes --- p.73 / Chapter 3.7.1. --- SubstanceP --- p.73 / Chapter 3.7.2. --- GnRH --- p.73 / Chapter 3.7.3. --- mGluR2 --- p.74 / Chapter 3.7.4. --- IGFBP-2 --- p.74 / Chapter 3.8. --- Graphs and tables --- p.75 / Chapter 4. --- Discussion / Chapter 4.1. --- Quantitation of cDNA and normalization of CR results --- p.97 / Chapter 4.2. --- Sexual differentiation related genes --- p.98 / Chapter 4.3. --- CD related genes --- p.100 / Chapter 4.4. --- Axon guidance molecule and related genes --- p.103 / Chapter 4.5. --- Olfactory markerrotein genes --- p.109 / Chapter 4.6. --- Miscellaneous genes --- p.112 / Chapter 5. --- References --- p.119

Shiu Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 176-182). / Abstract also in Chinese. / Acknowledgments --- p.i / Presentations Derived from the Present Thesis Work --- p.ii / Chinese Abstract --- p.iii / English Abstract --- p.iv / List of Abbreviations --- p.v / Abbreviation for Amino Acids and Nucleotides --- p.vii / List of Figures --- p.viii / List of Tables --- p.vi / Contents / Chapter Chapter.1 --- Literature Review --- p.1 / Chapter 1.1 --- Transcriptional Regulation of Gene Expression --- p.1 / Chapter 1.2 --- MT: A Brief Review --- p.4 / Chapter 1.3 --- Transcriptional Regulation of MT --- p.15 / Chapter 1.4 --- MT Promoter Organization and Function --- p.18 / Chapter 1.5 --- Fish MT Genes --- p.29 / Chapter 1.6 --- Aim and Rationale of Present Studies --- p.32 / Chapter Chapter 2 --- PCR Cloning of Common Carp MT Gene --- p.34 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.1.1 --- The Biology of Common Carp --- p.34 / Chapter 2.1.2 --- The Study of Common Carp MT --- p.35 / Chapter 2.2 --- Materials and Methods --- p.39 / Chapter 2.2.1 --- Materials --- p.39 / Chapter 2.2.1.1 --- Polymerase Chain Reaction (PCR) --- p.39 / Chapter 2.2.1.2 --- Agarose Gel Electrophoresis --- p.39 / Chapter 2.2.1.3 --- Gene Clean by Sephaglas´ёØ BandPrep Kit (Pharmacia) --- p.40 / Chapter 2.2.1.4 --- TA Cloning --- p.40 / Chapter 2.2.1.5 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.41 / Chapter 2.2.1.6 --- Preparation of Plasmid DNA --- p.41 / Chapter 2.2.1.7 --- DNA Sequencing --- p.42 / Chapter 2.2.1.7.1 --- Template Denaturation and Primer Annealing --- p.42 / Chapter 2.2.1.7.2 --- Labeling and Termination Reaction --- p.42 / Chapter 2.2.1.7.3 --- DNA Sequencing Electrophoresis --- p.43 / Chapter 2.2.1.8 --- Total RNA Extraction --- p.43 / Chapter 2.2.1.9 --- PolyA RNA Extraction --- p.44 / Chapter 2.2.1.10 --- Micro Bio-Spin Chromatography --- p.44 / Chapter 2.2.1.11 --- Analysis of the Transcription Start Site --- p.45 / Chapter 2.2.2 --- Methods --- p.46 / Chapter 2.2.2.1 --- Polymerase Chain Reaction (PCR) --- p.46 / Chapter 2.2.2.2 --- Gene Clean by Sephaglas ´ёØ BandPrep Kit (Pharmacia) --- p.46 / Chapter 2.2.2.3 --- TA Cloning --- p.47 / Chapter 2.2.2.4 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.47 / Chapter 2.2.2.5 --- Transformation of Plasmid Vector into Competent Cell (Heat Shock Method) --- p.48 / Chapter 2.2.2.6 --- Preparation of Plasmid DNA --- p.48 / Chapter 2.2.2.6.1 --- Small Scale Alkali Preparation of Plasmid DNA --- p.48 / Chapter 2.2.2.6.2 --- Large Scale Preparation of Plasmid DNA using Wizard Maxiprep Kit (Promega) --- p.49 / Chapter 2.2.7 --- DNA Sequencing --- p.50 / Chapter 2.2.2.7.1 --- Template Denaturation and Primer Annealing --- p.50 / Chapter 2.2.2.7.2 --- Labeling and Termination Reaction --- p.51 / Chapter 2.2.2.7.3 --- DNA Sequencing Electrophoresis --- p.51 / Chapter 2.2.2.8 --- Total RNA Extraction --- p.52 / Chapter 2.2.2.9 --- PolyA RNA Extraction --- p.53 / Chapter 2.2.2.10 --- Analysis of the Transcription Start Site --- p.55 / Chapter 2.3 --- Results --- p.56 / Chapter 2.3.1 --- PCR Cloning of the MT Gene --- p.56 / Chapter 2.3.2 --- Identification of the Transcriptional Start Site --- p.57 / Chapter 2.4 --- Discussion --- p.60 / Chapter 2.4.1 --- PCR Cloning of the MT Gene --- p.60 / Chapter 2.4.2 --- Comparison of Common Carp MT Promoter with Other --- p.60 / Chapter 2.4.3 --- Identification of the Transcriptional Start Site --- p.62 / Chapter 2.5 --- Conclusion --- p.63 / Chapter Chapter 3. --- Functional Assay of Common Carp MT Promoter --- p.64 / Chapter 3.1 --- Introduction --- p.64 / Chapter 3.1.1 --- Fish MT Promoters --- p.64 / Chapter 3.2 --- Materials and Methods --- p.68 / Chapter 3.2.1 --- Materials --- p.68 / Chapter 3.2.1.2 --- Micro Bio-Spin Chromatography --- p.68 / Chapter 3.2.1.3 --- Construction of Deletion Mutants --- p.68 / Chapter 3.2.1.4 --- Isolation of Hepatocytes --- p.69 / Chapter 3.2.1.5 --- Determination of LC50 Values for Common Carp Hepatocytes --- p.69 / Chapter 3.2.1.6 --- Transfection by LipofectAMINE´ёØ (Gibco) --- p.70 / Chapter 3.2.1.9 --- Determination of the Amount of Protein by BCA Protein Assay --- p.70 / Chapter 3.2.1.10 --- β-galactosidase Analysis --- p.71 / Chapter 3.2.2 --- Methods --- p.72 / Chapter 3.2.2.1 --- Subcloning of 5' Flanking Region of Common Carp MT Gene into Reporter Gene --- p.72 / Chapter 3.2.2.2 --- Micro Bio-Spin Chromatography (Bio-rad) --- p.72 / Chapter 3.2.2.3 --- Creating Deletion Mutants --- p.73 / Chapter 3.2.2.4 --- Isolation of Hepatocytes --- p.73 / Chapter 3.2.2.5 --- Determination ofLC50 Values for Common Carp Hepatocytes --- p.74 / Chapter 3.2.2.6 --- Transfection with LipofectAMINE´ёØ (Gibco BRL) --- p.75 / Chapter 3.2.2.7 --- Optimization of Incubation Time of Cells with LipofectAMINE´ёØ --- p.75 / Chapter 3.2.2.8 --- Optimization of Amount of DNA for Transfection --- p.76 / Chapter 3.2.2.9 --- Determination of Protein Concentration by --- p.76 / Chapter 3 2.2.10 --- β-galactosidase Analysis --- p.77 / Chapter 3.2.2.11 --- Fluorescence Measurement --- p.77 / Chapter 3.2.2.12 --- Dose-Response Curve of Different Metals on Transfected Cells --- p.77 / Chapter 3.2.2.13 --- "Fold-Induction of Different Metals, LPS and H202" --- p.78 / Chapter 3.3. --- Result --- p.79 / Chapter 3.3.1 --- Deletion Mutants --- p.79 / Chapter 3.3.2 --- LC50 of Common Carp Hepatocytes --- p.80 / Chapter 3.3.3 --- Optimization of Transfection --- p.81 / Chapter 3.3.4 --- Dose Response Curve --- p.85 / Chapter 3.3.5 --- Deletion Mutants with Different Treatments --- p.95 / Chapter 3.4 --- Discussion --- p.109 / Chapter 3.4.1 --- LC50 Values of Metal Toxicity in Different in vitro Fish Cells Studies --- p.109 / Chapter 3.4.2 --- Dose Response Curve (Figure 3.9 to 3.16) --- p.110 / Chapter 3.4.3 --- Fold Induction in Deletion Mutants --- p.111 / Chapter 3.5 --- Conclusion --- p.128 / Chapter Chapter 4. --- MRE-Binding Proteins --- p.129 / Chapter 4.1 --- Introduction --- p.129 / Chapter 4.1.1 --- MTF-1 --- p.129 / Chapter 4.1.1.1 --- Structure of MTF-1 --- p.129 / Chapter 4.1.1.2 --- MTF-1 is a Zinc Dependent Factor --- p.130 / Chapter 4.1.1.3 --- Band-shift Assay of MTF-1 --- p.132 / Chapter 4.1.1.4 --- MTF-1 is Essential for Both Basal and Metal-Induced MT Transcription --- p.133 / Chapter 4.1.2 --- MBP-l --- p.134 / Chapter 4.1.3 --- MBF-l l --- p.35 / Chapter 4.1.4 --- Rat Zinc Activated Protein --- p.135 / Chapter 4.1.5 --- MREBF-1 and MREBF-2 --- p.136 / Chapter 4.1.6 --- Human Zinc Regulatory Factor --- p.136 / Chapter 4.1.7 --- MREBP --- p.137 / Chapter 4.1.8 --- Aim of This Chapter --- p.138 / Chapter 4.2 --- Materials and Methods --- p.139 / Chapter 4.2.1 --- Materials --- p.139 / Chapter 4.2.1.1 --- Preparation of Nuclear Extract from Common Carp Liver Tissue --- p.139 / Chapter 4.2.1.2 --- Preparation of the Double-Stranded Oligonucleotides --- p.139 / Chapter 4.2.1.3 --- Binding Reaction of Protein and DNA --- p.141 / Chapter 4.2.1.4 --- Gel-Shift Mobility Electrophoresis --- p.142 / Chapter 4.2.1.5 --- Screening of Expression Library --- p.142 / Chapter 4.2.1.5.1 --- Preparation of Labeled DNA Probe --- p.142 / Chapter 4.2.1.5.2 --- Plating of the Library --- p.142 / Chapter 4.2.1.6. --- Isolation of Positive Clones In Vivo Excision --- p.143 / Chapter 4.2.2 --- Methods --- p.144 / Chapter 4.2.2.1 --- Gel Mobility-Shift Assays --- p.144 / Chapter 4.2.2.1.1 --- Preparation of Nuclear Extract from Common Carp Liver Tissue --- p.145 / Chapter 4.2.2.1.2 --- Preparation of the Double-Stranded Oligonucleotides --- p.145 / Chapter 4.2.2.1.3 --- Binding Reaction of Protein and DNA --- p.146 / Chapter 4.2.2.1.4 --- Gel-Shift Mobility Electrophoresis --- p.146 / Chapter 4.2.2.2 --- Screening of Expression Library --- p.146 / Chapter 4.2.2.2.1 --- Preparation of Labeled DNA Probe --- p.147 / Chapter 4.2.2.2.2 --- Plating of the Library --- p.148 / Chapter 4.2.2.2.3 --- Isolation of Positive Clones --- p.150 / Chapter 4.3 --- Results --- p.150 / Chapter 4.3.1 --- Gel Mobility-Shift Assays --- p.150 / Chapter 4.3.2 --- Expression Library Screening --- p.163 / Chapter 4.4 --- Discussion --- p.166 / Chapter 4.4.1 --- Gel Mobility-Shift Assays --- p.166 / Chapter 4.4.2 --- Expression Library Screening --- p.171 / Chapter 4.5 --- Conclusion --- p.172 / Chapter Chapter 5 --- Conclusion --- p.173 / Chapter 5.1 --- Conclusion --- p.173 / Chapter 5 2 --- Model of MT Gene Transcription --- p.174 / Chapter 5.3 --- Future Direction --- p.175 / references --- p.176

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 114-136).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 141-164).