碩士 / 國立臺灣大學 / 分子與細胞生物學研究所 / 94 / Myf5 is one of myogenesis regulatory factors (MRFs), which plays roles in the specification and differentiation of muscular cells during myogenesis. The expression of myf5 is in a somite- and stage-specific manner under a fine-tuned control mechanism. However, the molecular mechanism of repression of myf5 is still unknown, although repressive element within intron I (+502/+835, I300) of zebrafish myf5 was reported. In this study, we microinjected the upstream region (-6300 / -1) of zebrafish myf5 fused to a luciferase reporter gene and a sense RNA corresponding for I300 into the fertilized embryos, resulting the luciferase activity was down-regulated by sense I300 RNA, but was not affected by anti-sense I300 RNA. In addition, the I300-mediated repression was promoter-specific because only myf5 promoter activity was repressed, but not for other promoters, such as myod, cytomegalovirus and thymidine kinase. These evidences demonstrate that the zebrafish myf5 repression modulated by I300 was controlled at RNA level. Using bioinformatics and Northern blotting, we identified an intronic microRNA motif (miR-In, +610/+632) within I300 fragment. In order to study the function of miR-In, we microinjected a DNA construct in which the luciferase reporter fused with 5 copies of miR-In complementary sequences at its 3’UTR region, resulting the target mRNA expression was down-regulated by sense I300 RNA: the luciferase activity was reduced down to 25 % of control. Over-expression of I300 resulted in the morphological defect of brain and somites, which photocopied the embryos injected with myf5 morpholino, a myf5-specific translation inhibitor. In 24-hpf zebrafish embryos, the myf5 transcripts were abolished at the older formed somites, but miR-In was detected prominently at these somites, indicating that miR-In played an important role in repression of myf5 during somitogenesis. Based on these evidences, we propose that miR-In is involved in muscle development through repressing the expression of myf5.
| Identifer | oai:union.ndltd.org:TW/094NTU05061006 |
| Date | January 2006 |
| Creators | Hao-Seong Hoi, 許巧雙 |
| Contributors | Huai-Jen Tsai, 蔡懷楨 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 52 |
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