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This dissertation, Zebrafish Telomerase Reverse Transcriptase (TERT): Molecular Cloning, Characterization and Retinal Expression by Wui-man, Lau, 劉匯文, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Zebrafish Telomerase Reverse Transcriptase (TERT): Molecular Cloning, Characterization and Retinal expression Submitted by LAU Wui Man For the degree of Master of Philosophy At The University of Hong Kong In August 2005 Telomerase (telomere terminal transferase), composed of a catalytic subunit (Telomerase Reverse Transcriptase, TERT) and a RNA template (Telomerase RNA, TR), is an enzyme specifically elongates chromosomal ends (telomeres). Chromosomes are not completely replicated after cell division and thus lengths of telomeres decrease as a function with the number of times a cell has divided. When there are only several thousands of DNA bases left at the telomeres, Mortality stage 1 (M1) will occur and cause proliferative failure. Activation of telomerase causes elongation of telomeres by adding telomeric repeats (TTAGGG) to chromosomal ends and thus prevents cellular senescence. A large body of evidence suggests that telomerase is involved in different biological processes, such as development and carcinogenesis. Although telomerase activity and TERT mRNA can be detected in highly proliferative cells such as cancer IIcells, germ line cells and activated lymphocytes, they cannot be detected in most human normal somatic tissues, including central nervous system. In contrast to mammals, teleost somatic tissues were found to express telomerase activity ubiquitously, including central nervous system. Teleosts, or bony fish, are organisms with continuous growth throughout their lives and they have offered a unique opportunity to examine neurogenesis in adult vertebrates, since these animals grow throughout life by adding cells to all organs, including nervous system. The major objectives of my study were to reveal the zebrafish TERT cDNA sequence to provide a basis for further investigation of telomerase function in teleost central nervous system and of possible role of telomerase in neurogenesis. The first part of my study was to clone and characterize zebrafish TERT. Utilizing tBLASTn, a bioinformatics search tool, and Rapid amplification of cDNA Ends (RACE), the TERT coding sequence was revealed. The predicted amino acid sequence of zebrafish TERT protein contains 1091 amino-acid residues and has a molecular mass of 126 kDa. CLUSTAL W shows that the protein sequence contains the signature motifs and residues found in TERT of other species, indicating a particularly conservative phylogeny. RT- PCR and TRAP assay detect TERT mRNA expression and telomerase activity in all zebrafish tissues examined, including ovary, heart, liver, kidney, muscle, brain and retina. The second part of my study was to investigate the expression pattern of TERT in adult zebrafish retina. The zebrafish retina was used as the model of study since persistent neurogenesis could be found and it could be easily accessed and manipulated. Western IIIblot, RT-PCR and TRAP assay were used to detect the expression of telomerase at mRNA, protein and functional level, respectively in zebrafish retina. RT-PCR and western blot analysis showed that zebrafish TERT transcripts and proteins were synthesized in normal adult zebrafish retina. TRAP assay indicated that telomerase activity was present in zebrafish retina and this implied that a fully functional form of telomerase could be found there. Immunohistochemistry reve

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