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DC 欄位 | 值 | 語言 |
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dc.contributor.author | 李文志 | zh_TW |
dc.date.accessioned | 2021-07-01T08:12:01Z | - |
dc.date.available | 2021-07-01T08:12:01Z | - |
dc.date.issued | 2000 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75156 | - |
dc.description.abstract | 肌肉細胞的分化過程主要受到統稱為 muscle regulatory factors ( MRF )的肌肉特異性轉錄因數的調控,而肌肉組織的發育過程中 MRFs 基因之間的表現及機制可能在不同物種間有所不同。本實驗,我們以斑馬魚( Danio rerio )為對象,研究其 MRFs 之間表現的動態變化與調控機制,籍以進一步瞭解低等脊椎動物肌肉組織發育的過程。 利用 RT-PCR 及 RACE 的方法,得到斑馬魚myf-5 的 cDNA 全長。myf-5 cDNA 的全長約 1438 bp ,可轉譯出 236 個氨基酸序列;此序列中帶有與已知肌肉特異性調控因數相同的 basic Helix -Loop - Helix 結構特性。並利用 whole mount in situ hybridization 證明myf-5的表現時期約在受精後 7 .5 到 26 小時,其表現位置則集中於 segmental plate 與早期的體節( somite )。同時,我們也由 genomic library中, subcloning到長度約 6 . 3 kb 的myf-5上游調控區及 2 . 7 kb 的部份基因結構。將不同長度之上游調控區 DNA 片段與綠螢光蛋白 cDNA (報導基因)相連,以顯微注射之方式將之注射至單細胞期之斑馬魚胚胎;藉此我們找出了myf-5調控區中重要之 cis-acting elements 。結果證明長度約 82 bp(-82 ~-l )之調控區即具有組織特異性及發育時期特異性表現的能力,而近端-290 至- 154 bp 之調控區具有加強基因表現之功能。 | zh_TW |
dc.description.abstract | The myf-5 is the one of muscle regulatory genes which is involved in the proliferation of myoblasts and differentiation of myogenic cells. Although myf-5 cDNAs were cloned and studied in carp, Xenopus, chicken, mouse and human, the cis-acting elements of myf-5 gene are still no available. Using reverse transcriptase-polymerase chain reaction, we isolated a cDNA fragment with 1438 bp which encoded a Myf-5 myogenic factor from zebrafish (Danio rerio) myf-5 embryonic cDNAs. The deduced amino acid sequence contained 236 residues including a basic helix-loop-helix domain that is conserved in all known Myf-5 proteins. To study the temporal and spatial distribution of the zebrafish myf-5 (myf5) transcripts, whole-mount in situ hybridization demonstrated that the myf-5 transcripts were first detectable at 7.5 hpf, sustaintially increased at 16 hpf and then gradually reduced to an undetectable level after 26 hpf. During somitogenesis, myf-5 transcripts distributed in adaxial cells, segmental plates and somites. To define the cis-acting elements required for specific expression of myf-5, various length of regulatory sequences were fused with green fluorescent protein cDNA, which served as a reporter gene. By transgenic analysis, we defined a 82 bp (-82~-1) minimal promoter, which was sufficient to control the tissue-and stage-specific expression of myf-5 in the early development of embryos. Moreover, the proximal enhance element(s) located at nucleotide position from -290 to -154 was also identified. | en |
dc.description.provenance | Made available in DSpace on 2021-07-01T08:12:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2000 | en |
dc.description.tableofcontents | Abstract . . . . . . . . . . . .1 Lecture review . . . . . . . . . . . . .3 A single gene, myoD, can activate myogenesis . . . . . . . . . . . .3 The discovery of myoD gene family . . . . . . . . . . . . 4 The structural characters of MRF family . . . . . . . . . . . .5 Transcriptional activation of muscle-specific genes by MRFs . . . . . . . . . . . .8 Hierarchical Relationship between Members of MRFs . . . . . . . . . . . .9 Introduction . . . . . . . . . . . .12 Material and Methods . . . . . . . . . . . .16 Nucleotide sequences of the zebrafish myf-5 (myf-5) cDNA . . . . . . . . . . . .16 Temporal and spatial distribution of myf-5 transcripts . . . . . . . . . . . .20 Screening of the myf-5 genomic sequence . . . . . . . . . . . .21 Subcloning of the myf-5 genomic sequence . . . . . . . . . . . .24 Construction of the myf-5 promoter fused with EGFP cDNA constructs . . . . . . . . . . . .26 Generation of transgenic fish via microinjection . . . . . . . . . . . . 28 Results . . . . . . . . . . . .31 Sequences of nucleotide and deduced amino acid of zebrafish myf-5 (myf-5) cDNA . . . . . . .31 Temporal and spatial distribution of the myf-5 transcripts . . . . . . . . . . . .32 Molecular structures of the myf-5 gene . . . . . . . . . . . .33 Expression patterns of EGFP cDNA driven by myf-5 upstream regions in the transgenic embryos . . 34 Minimal tissue-specific regulatory sequence of the myf-5 promoter . . . . . . . . . . . . 35 Proximal enhance element(s) of the myf-5 promoter . . . . . . . . . . . .36 Discussion . . . . . . . . . . . .39 The sequence completeness of the zebrafish myf-5 (myf-5) cDNA . . . . . . . . . . . . 39 The stability of the myf-5 mRNA . . . . . . . . . . . .39 Putative phosphorylation site in the zebrafish Myf-5 . . . . . . . . . . . . 40 Spatial distribution of myf-5 transcripts . . . . . . . . . . . .40 Temporal expression of myf-5 transcripts . . . . . . . . . . . . 42 Sequential expression among zebrafish MRFs . . . . . . . . . . . .43 Cis-element(s) controlling the specific expression of the myf-5 promoter . . . . . . . . . . . . 44 Proximal and distal enhance elements of the myf-5 promoter . . . . . . . . . . . . 45 References . . . . . . . . . . . .47 Figures . . . . . . . . . . . . 55 Tables . . . . . . . . . . . .69 Appendixes . . . . . . . . . . . .76 | |
dc.language.iso | zh-TW | |
dc.title | 斑馬魚肌肉特異性調控因數Myf-5 之分子結構、表現型態與啟動子分析 | zh_TW |
dc.title | Molecular Structure, Dynamic Expression and Promoter Analysis of Zebrafish (Danio rerio) Muscle Regulatory Factor, Myf-5 | en |
dc.date.schoolyear | 88-2 | |
dc.description.degree | 碩士 | |
dc.relation.page | 89 | |
dc.rights.note | 未授權 | |
dc.contributor.author-dept | 生命科學院 | zh_TW |
dc.contributor.author-dept | 漁業科學研究所 | zh_TW |
顯示於系所單位: | 漁業科學研究所 |
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