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研究生:潘信全
研究生(外文):Shin-Chyuan Pan
論文名稱:睪丸鋅指蛋白基因TZF-376之分子特性探討
論文名稱(外文):Molecular Characterization of Testis Zinc Finger Protein Gene—TZF376
指導教授:謝秀梅謝秀梅引用關係
指導教授(外文):Hsiu-Mei Hsieh
學位類別:碩士
校院名稱:台北醫學院
系所名稱: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:2001
畢業學年度:89
語文別:中文
論文頁數:70
中文關鍵詞:鋅指蛋白表現序列標示原位雜交法
外文關鍵詞:testis zinc fingerESTin situ hybridization
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目前約有六分之一的夫婦可能面臨不孕症的困擾,而其中有
一半問題是出自於男性。主要造成男性不孕的原因,又以在精子
熟成(spermatogenesis)過程中發生障礙所造成的原因為最主要。
已知有許多鋅指蛋白(zinc finger proteins)會在精子的發育過程
表現,可能影響精子發育及其功能。鋅指蛋白是一種很常見的
DNA結合蛋白,一般是以扮演轉錄因子(transcription factor)的
角色,調節許多其它基因之表現。為了尋找精子熟成過程中新的
鋅指蛋白,我們利用網路上的基因庫進行人類表現序列標示
(expressing sequence tags)(簡稱EST)clones的搜尋,輸入關
鍵字testis及zinc finger,找到一些表現在睪丸的鋅指蛋白類EST
clones,並針對這些EST clones之序列來設計PCR引子,起
初進行分析的EST clones有AI376558、AI003931、AI014681、
AA417107四個,進行各個組織(例如:腦、肝、腎等組織)
的反轉錄PCR(RT-PCR)實驗,以便瞭解此基因在各組織的表
現,並製備riboprobes。結果發現AI376558在許多組織皆有表
現,但是以睪丸表現量最高,此外在睪丸有兩個不同之PCR產
物,我們猜測其有alternative splicing的情形,因此我們目前
較專注於AI376558之進一步分析,並將其命名為TZF376。由定
序結果發現這是一個具有四個C2H2鋅指區的鋅指蛋白,基因位
在人類第一號染色體上。另外我們亦定序幾個相關的EST
clones,結果顯示這個鋅指蛋白之alternative splicing相當地複
雜,至少有四種不同的alternative splicing,其中有一個clone
BE551217缺少一個20 bp之核酸片段,結果造成三個鋅指區之
缺損,這個蛋白質有可能對具鋅指區之蛋白產生負調節作用。此
外,我們根據第一號染色體上之基因組序列設計三組橫跨
TZF376 exons之primers,以檢測180例不孕症之男性是否有
TZF376基因之缺損情形。而更進一步原位雜交分析將有助於瞭
解這個鋅指蛋白在睪丸中精細胞(germ cells)及體細胞(Sertoli
and Leydig cells)的表現情形,如此可對基因之功能作一初步預
測。

About one-sixth of the couples have the problem with infertility
nowadays, and half of the problems come from male. Most of male
infertility results from the impairment of spermatogenesis. We searched
EST Genebank and identified several testis zinc finger protein genes. We
would like to know whether these genes could influence the development
and function of testis. Zinc finger is an extremely common protein domain
of DNA binding proteins, and zinc finger proteins usually regulate other
gene expression via their roles as transcription factors. The sequences of
these zinc finger EST clones was used to design the PCR primers. RT-PCR
analysis indicated that one of the EST clones, AI376558 might have some
alternative splicing processing. We now focus on this EST clone and name
this gene TZF376. Sequence analysis of several related EST clones showed
that the alternative splicing of TZF376 is complicated. We have found at
least 5 different alternative spliced EST clones, BE551217, with a 20 nt
deletion encodes a truncated protein without zinc finger domain. In situ
hybridization analysis was performed to characterize the expression pattern
of TZF376. The localization in germ cells, Sertoli or Leydig cells of testis
will imply the function of the genes. Intensive PCR screening of the
TZF376 coding region on human chromosome 1 were performed to see if
any deletion occur in cellular DNA of 180 infertile patients.These results
will imply the correlation between TZF376 gene infertility.

目錄
中文摘要……...………………………………………………………….I
英文摘要…...…………………………………………………………….III
中英文對照表……………………………………………………………IV
目錄………………………………………………………………………..V
圖表目錄………………………………………………………………VIII
附錄………………………………………………………………………IX
第一章、緒論…...………………………………………………………….1
一、精子產生的過程.…….………………………………………….2
二、男性不孕症與基因的關係…….………………………………. 2
三、鋅指蛋白基因在精子發育過程中的表現情形.…….………….3
(一)發現.………………………………………………………3
(二)功能.………………………………………………………4
(三)鋅指蛋白基因與發育之相關研究….……………………4
(1)鋅指蛋白於泌尿系統之表現..……..………………….5
(2)基因位於性染色體之鋅指蛋白……………………….5
(3)影響精子發育過程之鋅指蛋白……………………….6
第二章、材料與方法……………...…………………………………..….10
一、表現序列標示(EST)clones search………....………….………10
二、睪丸組織的取樣及RNA的萃取……..………………………..10
(一) 包埋儲存……...…..………………………………….11
(二) RNA萃取……...….………………………………….11
三、反轉錄PCR(RT-PCR)……….…………………………….….13
四、16組human tissue cDNA PCR……...…………………………14
五、北方轉漬法(Northern blot analysis)……….….………………15
(一) Prehybridization……..………………..……………16
(二) 探針之標定…………....…….…………………….16
六、Screen mouse testis cDNA library………..……………………17
(一)製備cDNA library…...………….…………………..17
(二)Screen cDNA library……..…………………………17
(1) primary screening…….…………………………..18
(2) second screening……………………………...….19
(3)第三次 screening…...….…………………………..19
(三)刮菌抽plasmid DNA...………..………………….…20
七、探針(Riboprobe)之製備……….……….……………….…….21
八、原位雜交法(In situ hybridization)………..….………….…….23
(一) 組織切片……..….…...…………………………..…24
(二) H&E staining………...….……………………..…….24
(三) 原位雜交……..….…………………………………..25
(四) 呈色反應……………………………………………27
九、基因庫之比對………………………………………………….27
十、5’端延伸方法……….………………………………………….28
第三章、結果…………………………………………………………….29
一、EST clones的搜尋及PCR的結果……………………………...29
二、Multiple tissue screening的結果……………………….……….30
三、TZF376之cDNA及胺基酸序列………………..………………30
四、利用人類的基因尋找老鼠相似之基因………………………..31
五、cDNA library screening後之序列分析………………………...31
六、TZF376在第一號染色體之相對位置………………………….32
七、TZF376與相關EST clone DNA序列之比較…………………..32
八、利用北方轉漬法來看mRNA的表現情形……………………..33
九、利用原位雜交法來看mRNA在組織的表現情形……………..33
十、PCR篩檢180 位infertility……………………………………..33
第四章、討論…….……………………………………………………….34
一、多組織PCR篩檢………………………………………………..34
二、TZF376與相關EST clone進行比對…...………………………34
三、TZF376與blimp-1之相關性…………………………………...35
四、TZF376之序列分析…………………………………………….35
五、TZF376 mRNA的表現情形……………………………………37
六、TZF376之基因缺損與男性不孕症之相關性………………….38
第五章、未來研究方向………..………………………………………..39
參考文獻……………………………..……………………………..……60
圖表目錄
表1、根據TZF EST clones所設計出之引子對及所得到之最佳
PCR annealing condition………………..………..…...….40
表2、以EST clone引子對作PCR screening之結果……..……….……41
表3、Exon-Intron Junctions of TZF376…….…………………………….42
表4、利用PCR篩檢臨床180位不孕症病人之結果……………..……..43
表5、 Mouse cDNA library screening之結果………………...…………44
圖1、EST clones之direct PCR及RT-PCR結果…………...…………….45
圖2、Multiple tissue screening的結果……...…………………………..46
圖3、TZF376之cDNA及氨基酸之序列……………………………….47
圖4、TZF376 ZNF domain與相近ZNF蛋白之比較……...…………….48
圖5、TZF376在第一號染色體之相對位置……………..….……………49
圖6、TZF376相關EST clone DNA序列之比較…………..…………….50
圖7、Dot blot的表現情形……...…………………………………….…..51
圖8、Northern Blot的表現情形………………………...……….....…….52
圖9、In situ hybridization 之表現情形…………………..…………...…53
附錄目錄
附錄一、精子發育的形成過程………………………………………..54
附錄二、鋅指區示意圖………………………………………………..55
附錄三、已知的鋅指蛋白與精子發育的關係………………….…….56
附錄四、所Screen之testis cDNA Library所用之 pCMV-SPORT2之
載體………………………………………………..………..57
附錄五、RNA探針所用之pGEM-T EasyTM 載體……………………58
附錄六、AI560530所用之 pCMV-SPORT6之載體…………………59

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