Abstract
本研究﹐利用 quantitative RT-PCR 的方法來分析神經生長抑制因子(金屬硫蛋白III) 在豬腦中表現的情形。由於金屬硫蛋白基因之間﹐其核酸序列有很高的相似性﹐所以﹐必須找到一組專一於金屬硫蛋白III 的primer 組合﹐配合上適當的 PCR amplify 條件﹐再進一步以此條件來定此基因的表現量。我們利用此法來比較金屬硫蛋白III 在豬腦的不同部位(包括大腦､小腦､橋腦及延腦) 的表現情形。由實驗結果可以看到﹐金屬硫蛋白III 基因在豬腦中以大腦部位的表現量最多﹐而其它三個區域的表現量則較低。除了觀察金屬硫蛋白III 基因的表現情形﹐我們也嘗試去分析此基因的基因結構與調控的機制。首先﹐抽取出豬的 genomic DNA 並以genomicSouthern hybridization 的方法觀察此基因的 copy number﹐發現金屬硫蛋白III 基因是一個 single copy 的基因。之後﹐我們利用兩個步驟的 PCR 從 genomic DNA 中 amplify 出金屬硫蛋白III 基因﹐再將所得到的 PCR 產物選殖入 pGEMT 的載體並以核酸序列分析確定其為金屬硫蛋白III 基因。比對小老鼠及人類的金屬硫蛋白III 基因﹐發現豬的金屬硫蛋白III 基因同樣具有三個exons 和兩個introns。我們打算利用這一段DNA來篩選豬的基因庫以便得到包含啟動子部分的金屬硫蛋白III基因。而我們也試著利用Primer extension的方法決定此基因的轉錄起始位置﹐結果顯示豬的金屬硫蛋白III基因的轉錄起始位置是位在轉譯起始位置前約250 bp 的地方。A quantitative Reverse Transcriptase-Polymerase ChainReaction (RT-PCR) technique was employed to assess the level ofneuron growth inhibitory factor (metallothionein III) mRNA inbrain. Due to the high sequence homology among MT gene families,a specific primer set was used to define the appropriateconditions that can be used to quantitate the MT-III genetranscript by PCR. This method was used to compare the quantityof MT-III mRNA in various regions of porcine brain, includingcerebrum,cerebellum,pons and medulla oblongata. The resultsshowed that the MT-III expression is higher in cerebrum thanthat in other three regions. In addition to quantitate MT-IIItranscripts, we attempt to analyze the gene structure andregulation of gene expression of porcine MT-III. To achieve thisend, porcine genomic DNA was extracted and genomic Southernhybridization was conducted. The results indicated that MT-IIIgene is present ina single-copy form. Then, we amplified MT-IIIgene by two step PCR. The amplified product was cloned andconfirmed to be MT-III gene by sequence analysis. Similar tomouse and human MT-III gene, the porcine MT-III gene consists ofthree exons and two introns. This DNA fragment was used as aprobe to screen porcine genomic DNA library. However, theattempt is not successful at present. We also tried to determinethe transcription start point (tsp) by primer extension. Theresults revealed that tsp locates at 250 nucleotides 5' upstreamof thetranslation initiation site.