?郭魚的兩型c-ski基因

Abstract

本論文對於兩型吳郭魚c-ski基因(tski1, tski2)的研究內容包括有解析c-ski基因的序列及結構；分析c-ski mRNA在吳郭魚不同組織及發育過程中的表現，還有初步分析這兩型基因?動子的性質三部份。 在基因結構的部份；由16公分長的吳郭魚所萃取出的mRNA中定義出兩型的c-ski cDNA，命名為tski1、tski2，分別可轉譯出687及714個氨基酸序列，和在雞、人、爪蟾所發表過的序列各有60％、57％、57% (tski1)和百分之67％、63％、61% (tski2)的相似度。從genomic DNA library選殖出的tski1、tski2基因在染色體中至少包含16kb與20kb的genomic DNA序列，其中分別具有3kb及2kb的?動子區域，1.5 kb及1.3 kb的exon1，並有大於10 kb及2 kb的序列介於exon1、exon2之間，之後exon2到exon7兩種型的tski都一樣是約2kb大小的genomic DNA，而最後的exon7，前段是可轉譯區域(coding region)後接著0.4kb (tski1)、0.8 kb(tski2)的非轉譯區。 在基因表現的部份；對吳郭魚成魚的各種組織進行mRNA表現的觀察發現tski1和tski2廣泛的表現在各組織，其中腦表現最多，骨骼肌則表現最少。而tski1在卵巢及成熟卵細胞的表現量較tski2高出許多；tski2在精巢的表現量較tski1高出許多。發育早期過程中，不同時期tski1的表現是母體遺傳(maternally-inherited)，從未授精的卵開始到四公分小魚，皆有表現。tski2則是從胚胎到四公分小魚，皆有表現。而tski1和tski2表現程度的比值有逐漸下降的趨勢。在?動子分析的部份；將tski1現有的3 kb起動子區域與tski2現有的2kb起動子區域進行EGFP的斑馬魚顯微注射，tski1起動子區域在斑馬魚授精後58小時可清楚觀察到脊索(notochord)、骨骼肌、心肌、尾鰭、眼部細胞的螢光；而相同時期所觀察到的tski2起動子所驅動的表現位置大致與tski1相同但螢光表現是零星分佈且微弱很多。

Two classes of tilapia c-ski cDNA (Accession number: AJ012011, AJ012012), designated as tski1 and tski2, respectively encoded a 687 and a 714 AA protein and shared a 57% AA identity. Comparison with the Ski proteins of chickens, humans and Xenopus, tilapia TSki polypeptides shared a 60, 57 and 57% (TSki1) and 67, 63 and 61% (TSki2) AA identity, respectively. The tski1 and tski2 gene extend a minimum of 16 kb and 20kb in the tilapia genome. The exon 1 comprising 1.5 kb (tski1) and 1.3kb (tski2) of cDNA sequence have been mapped with screening tilopia genomic DNA library. Then exons 1 and 2 are separated by more than 10 kb (tski1) and 2kb (tski2) of genomic sequence. Using SSP-PCR (single specific primer-polymerase chain reaction) and PCR with genomic DNA templates, it is clear that tski1 and tski2 cDNA are encoded seven exons. In this two genes, the exons 2 through 7 are closely linked within 2 kb in the tilopia genome. The long 3’ untranslated region is contiguous in the genome with the distal portion of the open reading frame such that the exon 7 is composed of both coding and noncoding sequence. The most and the least abundant c-ski mRNAs are located in the brain and the skeletal muscle, respectively. Both tski1 and tski2 were widely expressed in the adult tissues examined, but tski2 transcripts were at higher levels except in the ovary and oocytes: tski1 transcripts were predominant in the ovary, whereas tski2 transcripts were predominant in the testes. In the oocytes, the tski1 mRNA was a maternally-inherited stockpile that subsequently was degraded, so that the expression ratio of tski1 to tski2 transcripts declined gradually as the fish developed from oocyte to 4-cm fry. The notochord, eye, muscle fiber, tail fin and heart transient expression pattern showed that the 3 kb tski1 promoter promotes tski1 gene wildly in zebrafish. As to the pT2EGFP, had the same EGFP expression pattern with pT1EGFP but weak and mosaic extremely.