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研究生:曾怡潔
研究生(外文):I-Chieh Tseng
論文名稱:果蠅homothorax基因含有一個三鹼基的表現子
論文名稱(外文):A 3bp mino-exon in homothorax gene in Drosophila melanogaster
指導教授:孫以瀚孫以瀚引用關係
指導教授(外文):Y. Henry Sun
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:遺傳學研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:46
中文關鍵詞:果蠅表現子homothorax
外文關鍵詞:homothorax
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在果蠅發育中,許多方面都受到homothorax (hth)基因的調控。本實驗室陳政澤取一hth基因片段,利用雜合的方法篩選出十一個cDNA clones,hth基因與cDNA序列進行比較發現cDNA可分成兩類。差異在於是否擁有一個45 bp片段,此片段就對應在第八表現子(exon 8)中,帶有完整48 bp第八表現子的稱為long (L) isoform,只有3 bp (ATG)表現子稱為short (S) isoform。在第八表現子的基因序列上,開始的三個鹼基(ATG)後接著剪接機制(splicing mechanism)中的5端剪接點GT,hth RNA可能在剪接過程中利用不同剪接點的選擇而生成這兩類的產物。本論文排除其他的可能性:(1)果蠅基因組完整序列中,只發現一個hth基因存在,所以並非來自不同基因。(2)分析六株不同來源的果蠅,確認並非hth基因有多樣性而產生不同的產物。(3)在第八表現子的前後介子(intron)中並未發現還有另一個3 bp小表現子存在。利用RT-PCR方法證明在發育過程中hth確實在個體內可產生這兩種產物。建立一個活體中剪接系統,我將一段含有第七至第九表現子的基因片段送入果蠅中,測試此轉殖片段是否能進行剪接作用,同時也可檢測S isoform是利用選擇第八表現子中的GT剪接點剪接黏合而生成的。我也針對這兩種RNA的差異處設計dsRNA,在RNA interference (RNAi)作用機制下探討個別蛋白質的功能。
homothorax (hth) encodes a homeodomain protein and plays multiple roles in Drosophila development. Two classes of hth cDNA clones were isolated from a Drosophila imaginal discs cDNA library and defined two isoforms that differ by the presence or absence of a 45 bp stretch. The long isoform (L) contains a 48 bp exon 8 and the short isoform (S) contains only the first three bps (ATG) of exon 8. Genomic sequence showed that these three bps are flanked by consensus splice junction sequences (AGATGGT). No AGATGGT sequence can be found in intron 7 (13,255 bps) and intron 8 (14,602 bps). The 3 bps may represent an alternatively spliced mini-exon.
An alternative explanation is that the two isoforms may be derived from two alleles in the population. We analyzed six fly strains of independent origin, which were isogenic for the third chromosome (where hth is located). All six strains have only the full length exon 8 and the 3 bps is not alternatively linked to the 3’ end of exon 7 nor the 5’ end of exon 9. RT-PCR data also showed that both isoforms were expressed during development. My results showed that hth indeed generates two transcripts by alternative splicing.
I am in the process of making an in vivo mini-splice system. The hth genomic fragment from exon 7 to exon 9 will be tested in transgenic flies to see whether it can reproduce the putative alternative splicing. The putative GT splice site for the 3 bp mini-exon will be mutated to see whether it is used for the alternative splicing. Works have also been initiated to study whether the two isoforms have distinct expression patterns or functions.
中文摘要 1
Abstract 2
Introduction 3-8
Results 9-16
Discussion 17-20
Material and Methods 21-23
References 24-28
Figures 29-46
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