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研究生:陳瑋琪
研究生(外文):Wei Chi Chen
論文名稱:犬類pinin之啟動子分析與基因結構的分析
論文名稱(外文):Promoter analysis and gene structure determination of canine pinin
指導教授:歐陽品
指導教授(外文):Pin Ouyang
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
中文關鍵詞:啟動子引子延伸轉錄因子基因結構
外文關鍵詞:promoterSP1Etsprimer extension5'' race
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pinin為本實驗室於上皮細胞找到新蛋白質,目前已知其是屬於moonlight protein,在細胞中存在有兩種形式,一個是位於胞橋小體圓盤(desmosome plaque)與中間絲(intermeamediate filament)的交界處,其功能與細胞與細胞間的接合有關。而另一個則是位於細胞核內的核斑,與mRNA的剪接(mRNA splicing)相關,除了參與mRNA剪接與運送的功能,近來的研究也顯示pinin可調節基因的表現與轉錄作用,此外,亦有研究發現pinin與許多癌症的發生有關係,因此pinin具有許多的功能,而為了能更加明白pinin的功能,及調節其基因表現的機轉,因此,本實驗將分成三個大方向來進行研究。一、研究人類pinin 5’序列的結構,並找出人類pinin基因上的啟動子,二、本實驗室之前的研究發現,犬類pinin基因有兩種的基因產物 [1],而目前本實驗室pinin 1 ( DG7 ) 及pinin 2 ( DG3 ) 的序列都已經定序完成,所以吾人將比較DG7與DG3這兩個基因株結構上的差別,三、找出調節DG7基因轉錄的啟動子區域。在人類的部分,經由搜尋EST database及5’race的實驗中,找到了pinin更為5’的cDNA序列,因此預測人類pinin可能具有10個exon,但此更為5’的cDNA序列尚未能由北方點墨法證實,此外,吾人也試圖找出調節人類pinin的啟動子區域。結果發現在位於新預測的exon1的上游約2.7 kb長的區域並不具啟動子的活性, 但因為利用EST database 所搜尋到的image 1733397,以及由5’race所得的5A-2,這兩個片段經過與pinin基因比對確實含有pinin更為5’端的cDNA序列,所以我們相信所預測到新的exon應是存在的,因此推測或許pinin的啟動子之位置與新exon 1 的距離可能大於2.7 kb,甚或在新exon1前其實還有另一個exon的存在。而在犬類pinin基因的部分,比較pinin 1及pinin 2可發現,這兩個基因上都有9個exon,第1個到第8個exon的序列與長度大致相同,僅只有幾個核苷酸不同,但在第9個exon上,DG7比DG3多了165 bp,pinin 1與pinin 2的intron序列差別很大,且在pinin 1的intron 6上具有一個SINE。藉由引子延伸實驗得知犬類pinin 1基因的轉錄起始點應是座落在ATG上游46 bp的位置,而進一步運用啟動子的分析發現位於轉譯起始點上游-145 ~ -133 bp之間的區域,具有一正向及一負向的Ets結合序列,而在- 100 ~ -77 bp的區域之間,則有SP1的結合序列。經由SP 1點突變片段冷光活性的分析,可進一步確認SP1序列具有啟動子活性,且經由EMSA的實驗也確認有特殊的轉錄因子會結合在轉譯起始點上游-145 ~ -133 bp之間的區域,而根據電腦分析結果,發現這個區域包含有Ets的結合序列,因此吾人推斷,pinin 1基因的啟動子區域可能是由ETS家族成員與SP1共同調控。但真正的調節pinin 1表現的詳細機轉尚需要更進一步的研究才能釐清。
Pinin is a dule location protein found in epithelial cells by our laboratory. It is a moonlighting protein, containing two forms in cells. One is localized to the cytosolic surface of the desmosomal plaque in the convergence of intermediate filament, the other is localized to nuclear speckled domain. The role of nucleus-form pinin is correlated with mRNA splicing and export. Recent experiments suggested that pinin in addition may be involved in regulation of gene expression. Moreover, researches discovered pinin is related to tumorigenesis of certain cancers. In order to get a better understanding of the function of pinin and the machanism of pinin gene regulation, I will divide my study into three parts. The first is to study the 5 ' sequence structure of the human pinin gene, and to identify the promoter of the human pinin gene. The second is to determinate the gene structure of the canine pinin gene. Last is to find the promoter region of the pinin1 gene.
In the part of the human pinin gene, we extended the pinin 5 ' DNA sequence via searching for EST database and the 5 ' race experiment. Thus we predict that human pinin gene has 10 exons, instead of 9 exons as expected before. However this more 5 ' DNA sequence can not be verified by northern blotting yet. In addition, we also attempt to identify the promoter region of human pinin gene by luciferase reporter assays. The luciferase assay revealed that the region located at 2.7kb upstream of the newly identified first exon does not have promoter activity. We therefore suggest the promoter region of the human pinin gene may be located beyond the 2.7 kb, or within the intron 1.
Canine pinin contain 2 genes: pinin1 and pinin2. Compare pinin 1 and pinin 2 we found that both pinin 1 and pinin 2 have nine exons, and both genes are almost the same in terms of nucleotide sequence and length. The exception is in 9th exon, the sequence of pinin1 is longer 165 bps in length than pinin2. Of note is the existence of a SINE among the pinin1 intron 6. Primer extension assays revealed the transcription start site of pinin1 is located at -46 relative to translation start site. Using promoter analysis, we identified that a set of Ets binding sequence located between nucleotide -145 ~ -133 and a SP1 binding sequence located between nucleotide -100 ~ -77 are response for pinin promoter regulation. The gel shift assay revealed that a unique transcription factor binding to the fragment between -150 and -129, and and the transcription factor may be a ETS family member. Furthermore SP1 binding site is essential for the pinin basal promoter activity. We surmise the members of Ets transcription factor family displayed synergistic stimulation to canine pinin 1 promoter along with SP1. Nevertheless, the mechanism underlying pinin1 gene regulation awaits in depth analysis.
中文摘要…………………………………...……………………………..i
英文摘要…………………………………..……………………………..ii
前言………………………………………………………………………1
1. Pinin屬於moonlighting protein…………………………………1
1.1 desmosome-form ( d-form ) pinin………………………...………………2
1.2 nucleus-form ( n-form ) pinin……………………………..…………...…4
2. pinin基因可能是與癌症相關的抑癌基因…………..………….6
3. pinin基因結構...……………………………………..…………..7
實驗方法與材料……………………………………………….………...9
1. 細胞培養 ( cell culture )……………………………….…….…9
2. 質體的篩選與基本克隆( cloning )….……………….……..…..9
2.1 轉型作用( transformation )………………………………….…….…….9
2.2 小量製備質體DNA ( Minipreparation of Plasmid DNA ) …….……..10
3. 北方墨點法(northern blot)…………………………….……….11
3.1 Total RNA的萃取 ( Extraction of Total RNA )……………………....11
3.2 電泳及轉印 ( Electrophoresis and Transfer ) …………………..…….12
3.3 探針之標定 ( probe labeling ) …………………..……………………12
3.4 雜合反應 ( Hybridization ) …………………..……………………….13
4. 5’ - Race ( Rapid amplification of cDNA ends )……………….14
5. 引子延伸 ( Primer Extension )………………………….…….15
6. DNA刪減反應 ( DNA Deletion Reaction )…………………..16
7. 點突變方式 ( Site - Directed Mutagenesis )…………….…….17
8. 啟動子分析 ( Pomoter Assay )………………………………..18
8.1 細胞轉染 ( Transfection )……………………………………………..18
8.1-1質體DNA的萃取 ( Plasmid Extraction )………………………..18
8.1-2 磷酸鈣轉染法 ( Calcium Phosphate Transfection ).……………..19
8.1-3 METAFECTENE轉染法 ( METAFECTENE Transfection )…….20
8.2 冷光活性分析法 ( Luciferase Assay )………………………………...21
9. 細胞核萃取物的製備 ( Preparation of Nuclear Extracts )……22
10. EMSA ( Electrophoretic Mobility Shift Assays )………………23
結果……………………………………….…………………………….25
1. 人類pinin基因的結構與啟動子的分析…………………….25
1.1 人類pinin基因的結構………..……………………………………25
1.2 人類pinin基因的啟動子活性分析………………………………..27
2. 狗pinin基因pinin 1 ( DG7 ) 及pinin 2 ( DG3 ) 結構上的比較……………………………………………………………...28
3. pinin 1 ( DG7 ) 的啟動子分析………………………………30
3.1 pinin 1 ( DG7 ) 基因的轉錄起始點位置…….…...…………...…….30
3.2 電腦分析可能結合在pinin 1基因啟動子區域上的轉錄因子..……31
3.3 pinin 1基因的啟動子區域分析……………………………………..32
討論……………………………………………………………………..36
1. 人類pinin基因的結構與啟動子的分析…………………….36
1.1 人類pinin基因的結構………..……………………………………36
1.2 人類pinin基因的啟動子活性分析………………………………..37
2. 狗pinin基因pinin 1 ( DG7 ) 及pinin 2 ( DG3 ) 結構上的比較……………………………………………………………...38
3. pinin 1 ( DG7 ) 的啟動子分析………………………………39
圖表及說明……………………………………………………………..43
附錄……………………………………………………………………..70
參考資料………………………………………………………………..72
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