小鼠心臟中新同源箱基因之分子選殖與分析
Molecular Cloning and Characterizing of Novel Murine Heart Expressed Homeobox Genes
中文摘要
同源箱基因群(homeobox gene superfamily)是一群會轉譯出同源區蛋白
(homeodomain protein)的基因群。同源區蛋白在生物發育的過程中往往扮演轉錄 調節因子(transcription factor)的角色以調節整個發育過程；同源區蛋白中的同源 區(homeodomain)包含 60 個高保留性的氨基酸，同源區蛋白即藉由其同源區去辨 認下游的目標基因的調控區並與之結合，以調節該基因的表現。在哺乳動物的胚 胎中，心臟是第一個形成並具有功能的器官。心臟的發育開始於胚胎的原腸期，
為許多調控因子參與的複雜過程。故本研究主要是選殖心臟表現的同源箱基因，
並初步分析基因的表現型態。
為了選殖表現於小鼠心臟的新同源箱基因，本研究根據同源區中高保留氨基酸序 列設計退化引子(degenerate primer)以進行 nested PCR；之後，再從電腦網路上進 行 mouse ESTs cDNA library 搜尋；藉由以上方法得到出一段長度為 663 bp 新同 源箱基因片段 Dux-3。Dux-3 基因片段可轉譯出一段包含 221 個氨基酸的序列，
其中包括兩個 paired-type 的同源區序列。從小鼠多組織北方墨點實驗分析結果顯 示，Dux-3 的 mRNA 主要表現在心臟，長度約 1.35 kb；進一步經由原位雜交實 驗證實 Dux-3 RNA 在成鼠心臟的心室外圍部位以及在小鼠胚胎第 13.5 及 15.5 天 的心臟有表現。另外，經比較 Dux-3 的同源區之氨基酸序列後發現，Dux-3 兩個 同源區序列與調節人類顏面上肩胛肌肉失養症(FSHD)可能有關的 D4Z4 tandem repeat 之中的兩個同源區序列(D4Z4 homeodomain 1， D4Z4 homeodomain 2)有 極高度相似性。
本研究成功地選殖出在心臟表現並含有兩個同源箱的新同源箱基因 Dux-3。實驗 證明，Dux-3 表現在成鼠心臟的心室外圍部位及第 13.5 和 15.5 天鼠胚的心臟，
故 Dux-3 基因的表現可能與心臟發育過程有關。另外從與調節人類調節顏面上肩 胛肌肉失養症(FSHD)有關的兩個同源區序列(D4Z4 homeodomain 1， D4Z4 homeodomain 2)有極高度的相似性結果推論，Dux-3 或許與肌肉功能的調節與維 持有關。
英文摘要
The genes of homeobox gene superfamily encode homeodomain proteins that act as transcription factors at critical points in development. The homeodomain region is a conserved 60 a.a. domain that recognizes and binds to regulatory region of the target gene. During the evolution, the homeobox gene superfamily is highly conserved in different species. The heart is the first organ that forms and functions within
mammalian embryo. Heart development begins during gastrulation and its process is very complex and may involve within many regulatory factors. Today, there are about 8 in a 1000 for live borns with cardiac structure abnormalities in human. In this study, we cloned the novel heart expressing homeobox gene Dux-3 and analyzed the
expression pattern of Dux-3.
A nested PCR and electronic screening of the mouse ESTs cDNA library were used to clone murine heart expressing homeobox genes. A 663-bp cDNA fragment of Dux-3 was cloned and encoded 221 amino acids with two paired-type homeodomains. A 1.35 kb transcript was found mostly in heart by the mouse multiple tissue northern blot analysis. Dux-3 mRNA was further detected in E15.5 heart and outer ventricle region of the adult heart by whole mount in situ and section in situ hybridization analyses.
FSHD is an autosomal dominant neuromuscular disorder and D4Z4 is FSHD associated 3.3kb tandem repeats with two homeodomains. Sequence comparison indicated that the sequence of Dux-3 homeodomain 1, 2 was highly homology to the two homeodomain of D4Z4/FSHD locus.
Based on these data, it has been shown that Dux-3 is a novel heart expressing homeobox gene and may be involved in the heart development. Sequence similarity implies that Dux-3 may also related to neuromuscular disorder.
