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

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。

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.