小鼠心臟中新同源箱基因之分子選殖與分析

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 Mu rine Heart Expressed Homeobox Genes

 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 (d egenerate 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 D ux-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 ho meodomain 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 ho meodomain 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。

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The genes of homeobox gene superfamily encode homeodomain proteins that act as transcripti on factors at critical points in development. The homeodomain region is a conserved 60 a.a. d omain that recognizes and binds to regulatory region of the target gene. During the evolution, t he homeobox gene superfamily is highly conserved in different species. The heart is the first o rgan that forms and functions within mammalian embryo. Heart development begins during ga strulation and its process is very complex and may involve within many regulatory factors. To day, there are about 8 in a 1000 for live borns with cardiac structure abnormalities in human. I n this study, we cloned the novel heart expressing homeobox gene Dux-3 and analyzed the ex pression pattern of Dux-3.

A nested PCR and electronic screening of the mouse ESTs cDNA library were used to clone m urine heart expressing homeobox genes. A 663-bp cDNA fragment of Dux-3 was cloned and e ncoded 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 an d 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 locu

s. 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 al

so related to neuromuscular disorder.