Calcium-binding tyrosine phosphorylation-regulated protein (CABYR) is classified as a cancer-testis antigen, a category of cancer antigens whose expression in normal tissues is primarily restricted to male germ cells in the testis but not in adult somatic tissues. Recently, it has been suggested that this protein may play a role during brain development based on the observation that CABYR transcripts are differentially expressed in both fetal and adult brains. Studies on CABYR are very limited. Previous work of this laboratory found that CABYR transcripts are also detected in human leukocytes. To identify the cell type(s) expressing CABYR, RT-PCR and Western blot analysis were performed using total RNA and cell lysates, respectively, isolated from monocytes, lymphocytes and polymorphonuclear cells (PMN). The results showed that CABYR is expressed in lymphocytes and monocytes but not in the PMNs, suggesting that CABYR may play a role in acquired immune response. To underestand the mechanism responsible for the differential regulation of CABYR gene expression, the promoter activity of the 1-kb DNA fragment upstream of the start codon (designated +1) was analyzed in both CABYR positive and negative cells. For this purpose, a promoter-probing reporter plasmid, pEGFP-N1-CABYR-promoter, was constructed by cloning the above DNA fragment upstream of the EGFP coding region. pEGFP-N1-CABYR-promoter was transfected into lymphocytes, monocytes, and PMNs. Cells expressing GFP were found in all three transfected cell types indicating that the trans-factors required for the CABYR gene expression are present in these three cell types. Next, the effect of DNA methylation on the promoter activity was investigated because CpG islands were present in the 1-kb DNA fragment. Three luciferase expression plasmids, pGL3-luci1, pGL3-luci2 and pGL3-luci3 driven by the upstream regions encompassing -1 to -1075, -1 to -700, -1 to -300, respectively, relative to the start codon were constructed and subjected to in vitro methylation. 293T cells were transfected with either the methylated or unmethylated plasmids. The luciferase activities of cells transfected with the unmethylated plasmids were found to correlated with the length of the promoter regions. In contrast, very low activities were detected in the cells transfected with the methylated plasmids. Together, the results suggested that methylation on the promoter region may be one of the mechanisms that regulates the expression of CABYR. The methylation status of CABYR in lymphocyte and PMN in vivo deserves further investigation.Calcium-binding tyrosine phosphorylation-regulated protein (CABYR) 目前被認定為cancer-testis antigen，cancer-testis antigen主要表現在正常人睪丸的精細胞，而體細胞則不會表現。近來研究結果發現在成人腦細胞和嬰兒腦細胞中CABYR會表現不同的variants，因此認為CABYR可能與腦細胞的發育有關。目前為止對CABYR的相關研究有限，先前本實驗室發現在人體正常的白血球細胞中會表現CABYR。爲了觀察在哪一類的白血球細胞中會表現CABYR，將monocytes, lymphocytes和polymorphonuclear cells (PMN) 分離後，利用total RNA和cell lysates進行 RT-PCR以及Western blotting分析，結果顯示lymphocytes和monocytes會表現CABYR， PMN則不會表現CABYR，因此CABYR可能參與acquired immune response。為了瞭解CABYR gene之調控機制，分析CABYR positive 和 negative 細胞 之CABYR 基因轉錄起始點1-kb 之DNA片段。首先將此段約1-kb DNA構築於帶有螢光基因的質體，得pEGFP-N1-CABYR-promoter。再將pEGFP-N1-CABYR-promoter 轉染到monocytes、lymphocytes和PMNs，發現三種細胞都有螢光表現，顯示細胞內都含有調控CABYR 啟動子的調控因子。CABYR啟動子分析的結果得知其含有CpG islands，因此接著觀察DNA甲基化是否影響此1-kb 之CABYR啟動子的活性。構築三段帶有不同長度CABYR 啟動子序列的載體，分別為pGL3-luci1、pGL3-luci2、pGL3-luci3，長度分別取自轉錄起始點上游區域之 -1 to -1075、-1 to -700、-1 to -300，並將質體進行 in vitro甲基化。將甲基化和未甲基化的質體轉染到293T細胞內，細胞經轉染甲基化和未甲基化的質體所表現之Luciferase活性與啟動子之長度有相對關係。然而，DNA經過酵素甲基化後luciferase活性有顯著的降低。根據以上的實驗結果可得知CABYR基因在細胞中的調控可能受到甲基化的影響。對於lymphocytes和PMNs之CABYR 啟動子甲基化位置則留待未來進一步的探討。中文摘要……………………………………………………………………… i
英文摘要……………………………………………………………………… ii
壹、前言……………………………………………………………………… 1
貳、I材料………………………………………………....…………..……… 6
一、菌株及其培養條件.........................................................................6
二、引子 (primers) 與質體 (plasmids)……………………..……….6
三、培養基與溶液配方……………………………………………….6
II方法……………………………………………………...…………….11
(一) 血球細胞的分離...........................................................................11
(二) 劉氏染色 (Liu’s stain)……………………………….……...…..11
(三) 萃取RNA (extraction of RNA)…………………………….……11
(四) 反轉錄作用 (reverse transcription, RT)………………...……….12
(五) 聚合酶連鎖反應 (polymerase chain reaction, PCR)……..….….12
(六) 重組 DNA技術………………………………………………….12
(七) 轉型作用 (transformation)…………………………………..…..14
(八) DNA 洋菜膠體電泳分析 (agarose electrophoresis)……......…...14
(九) 細胞染色體之萃取 (extraction of genomic DNA)……….…..…15
(十) 細胞轉染 (Transfection)…………………………………….…...15
(十一) 核內核外蛋白質分離萃取…………………………………….15
(十二) 蛋白質樣品製備及定量分析………………………………….16
(十三) SDS 蛋白質膠體電泳分析 (SDS-PAGE)…………………….16
(十四) 西方墨點分析 (Western blot)……………………….………...17
(十五) 銀染 (sliver staining)………………………………………….18
(十六) 冷光報導基因分析 (Luciferase reporter gene assay)……..….18
(十七) gel retardation………………………………………………….19
(十八) Protein binding affinity assay……………………………..…….20
(十九) In-gel digestion………………………………………………….20
叁、結果...……………………………………………....………….………… 21
一、 以RT-PCR、Western blotting分析CABYR 在白血球內之轉錄及
轉譯之情形………………………………………………………….21
二、比較正常白血球細胞及癌症細胞株之CABYR promoter序列…..21
三、觀察CABYR promoter在monocyte、lymphocyte及PMN細胞內
之in vitro活性表現………………………………………………...22
四、觀察DNA甲基化in vitro是否會抑制CABYR promoter活性….23
五、初步搜尋與CABYR 基因轉錄有關之轉錄因子………………….24
肆、討論……………………………………………....…………….…………26
誌謝………………………………………...……………………….……...…. 29
圖表…………………………………………………………………………….30
參考文獻………………………………………………...………….……...…. 4
