| Abstract: | Small G蛋白 Ras 家族包含 Ras、Rho、Ran、Arf、Rab 等次家族可以調控許多的生物功 能例如：細胞生長、細胞移動、細胞凋亡以及細胞分化。許多 Ras 次家族如: RhoA、Rac1 以及 CDC42 都被發現與神經發育有極密切關係。Rac1 及 CDC42 可以促進神經分化但 是 RhoA會抑制神經突觸產生。最近新的 Ras 次家族分支蛋白被發現稱為 Diras，Diras 蛋白主要表現於人類的心臟以及腦部組織中。不同於其他 Ras 次家族蛋白，Diras 蛋白 在人類的腦部腫瘤中會有表現亮下降的趨勢，過度表現 Diras 蛋白會造成人類腦部腫瘤 細胞生長減緩。之前研究亦顯示線蟲的 di-ras 基因只表現於神經組織並參與神經突觸功 能，之前我們的研究顯示斑馬魚的 di-ras 基因會表現在斑馬魚神經組織、但是 Diras 蛋 白在腦部胚胎發育所扮演的角色尚未清楚，在此研究中，我們將利用斑馬魚當作動物模 式來研究 diras 以及它的上下游基因在胚胎發育的角色。 (1) 在第一年的研究，我們將利用 DNA微陣列以及蛋白質二維電泳分析方法 去尋找 diras 下游基因，接著我們將利用即時定量 PCR 以及西方墨點法來 確認。最後，我們也將利用反譯股核酸將 diras 下游基因表現抑制後觀察斑 馬魚胚胎發育是否正常，我們也將利用不同的腦部標定基因進行全覆式定 位雜交觀察是否有腦部的缺失情形。 (2) 在第二年的研究，我們將利用啟動子區域的刪除方法或是生物資訊方法去 尋找調控 diras 蛋白表現的轉錄因子，接著我們將利用 leuciferase活性分 析，電泳偏移分析法以及染色體免疫沉澱法來確認此轉錄因子的作用。全 覆式定位雜交以及反譯股核酸的方法將利用來探討此轉錄因子的表現模式 以及在胚胎發育所扮演的角色。
The small GTPase Ras superfamily proteins which contain five subfamilies such as Ras, Rho, Ran, Rab, and Arf play crucial roles in a wide range of biological functions including cell proliferation, cell migration, apoptosis, and differentiation. Evidences have benn shown that Ras subfamily proteins such as RhoA, Rac1, and CDC42 played an important role in neuron development. Rac1 and CDC42 triggered neuron differentiation , in contrast, RhoA inhibited synapses formation. Now, a branch of Ras gene named as distinct Ras (Diras) has been identified to be restrictedly expressed in brain region and heart. Unlike like other Ras subfamily proteins, loss of Diras expression was commonly found in human neuron tumors. Overexpression of Diras decreased neuronal tumor cells growth. Similarly, di-ras identified from C. elegens also was shown to be restrictedly expressed in neurons and involved in synapses functions. Previous, our findings have shown that zebrafish di-ras also expressed in neuronal regions. However, the exact role of di-ras in neuronal development still remained to be unclear. In this study, we will use zebrafish as animal model to investigate the role of diras upstream and downstream targets in brain development. (1) During the first year, we will conduct DNA microarray and two-dimension gel electrophoresis analysis to identify the diras downstream targets. Real-time PCR and Western blot analysis will be also performed to confirm the results. Finally, morpholino microinjection for specific proteins will be performed to determine the biological role of these diras-target proteins. Specific brain markers will be selected for WISH in morphants. (2) During the second year, promoter deletion assay and bioinformatic analysis will be performed to determine which transcription factor could regulate the diras gene expression. Leuciferase assay, EMSA and chromatin inmmunoprecipitaion assay also be conducted to confirm the promoter analysis results. The expression pattern and developmental role of the specific gene will be investigated by whole mount in situ hybridization and morpholino microinjection. |
