王作仁2006-07-262018-07-112006-07-262018-07-111998http://ntur.lib.ntu.edu.tw//handle/246246/22822FMR1是引起X染色體脆折症的基因。X 染色體脆折症是由染色體Xq27.3位置的脆 折點而命名。FMR1篩選到以後，只需要去 測量基因CGG重覆片段的長度及基因的甲 基化，就可以診斷患者、帶原者，及產前 診斷。可是由於FMR1是直接靠它在染色體 上的位置而被選殖到的，所以直到現在它 的功能仍不清楚，X染色體脆折症也缺乏有 效的治療。 為了能瞭解FMR1的功能，我們首先研 究其基因的調控。我們以診斷X染色體脆折 症的經驗為基礎，首先分析了FMR1啟動子 的構造，並証明FMR1啟動子會被甲基化抑 制(Hwu et al.BBRC 1993; 193:324-9)。 我們接著以遺傳學(突變株的研究)及生化 學(以合成的轉錄因子進行研究)上的証 據，指出FMR1啟動子可以被cAMP及CREB所 活化(Hwu et al. DNA & Cell Biology)。 在本研究中我們試圖用細胞內足印 (in vivo DNA footprinting)，去觀察FMR1 啟動子(包括MSE,methylation sensitive element)在細胞中和轉錄因子的結合，進 而瞭解FMR1 在細胞內的作用及調控。我們 並計畫發展RNase protection assay (RPA)，以便更精確的測定細胞在不同狀況 中FMR1 表現的變化。 很不幸的，我們無法突破細胞內足印 的技術，因為FMR1 啟動子GC 含量極高， 更有CGG 重複序列，導致細胞內足印分析 中primer extension 的部分極為困難。 RPA 實驗則無法看到FMR1 在細胞內的變 動，因為FMR1 的調控其實只局限在少數的 細胞，甚至細胞的一部分。所以我們目前 已改變研究方法，將直接以動物模式來研 究，預期會有突破性的成果。FMR1 is the gene responsible for the fragile X syndrome. The cloning of FMR1 make revolutionary changes in the diagnosis of fragile X syndrome. Both the diagnosis of patients and carriers, and even prenatal diagnosis, could be easily and confidently done by the analysis of CGG repeat and gene methylation. However, position cloning did not help the understanding of the function of FMR1. Till now, the function of FMR1 is unknown, and there is no effective treatment for the fragile X syndrome. We have studied the structure of FMR1 promoter, and proved its methylation sensitivity (Hwu et al. BBRC 1993; 193:324- 9). We also showed by genetic (study of mutants) and biochemical (study with recombinant transcription factor) evidences that, FMR1 promoter could be activated by cAMP and CREB (Hwu et al. DNA & Cell Biology). In this study, we want to use in vivo DNA footprinting to study the interactions between FMR1 promoter (including the methylation sensitive element, MSE) and nuclear proteins. These interactions will help the understanding of the regulation and function of FMR1 protein. Beside, we will develop an technique called RNase protection assay (RPA). RPA is a sensitive, stable and reliable method to quantitate RNA. It will be used to detect changes of FMR1 expression in cells under various conditions, to prove the presence of FMR1 regulation. 2 Unfortunately, we met a great difficulty in the in vivo DNA footprinting assay. The FMR1 promoter is extremely GC rich, and it also contains the CGG repeat. This made primer extension, the critical step in footprinting assay, very inefficiency. RPA assay could not detect any changes in the cellular FMR1 gene, since the regulation of FMR1 is very cell specific, and even subcellular specific. Now we have shifted our method into animal model study which goes smoothly recently.application/pdf97203 bytesapplication/pdfzh-TW國立臺灣大學醫學院小兒科FMR1基因調控X 染色體脆折症DNA 足印gene regulationfragile X syndromeDNA footprintingreporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/22822/1/872314B002126.pdf