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研究生:蔡淑娟
研究生(外文):SHU CHUAN TSAI
論文名稱:魚類CDC37基因結構與功能之研究
論文名稱(外文):Expression, characteriztion and genomic structure of fish CDC37
指導教授:黃 火 鍊 博 士黃 銓 珍 博 士
指導教授(外文):Fore-Lien HuangChang-Jen Huang
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:動物學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:123
中文關鍵詞:魚類基因結構生化方法胚胎發育期之表現顯微注射雙股RNA
外文關鍵詞:fishCDC37genomic structurebiochemical methodbindingdevelopmental expressionmicroinjectiondsRNA
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CDC37最早自酵母菌選殖得到,此基因突變會使酵母菌之細胞分裂週期停頓於G1階段。本論文則自圓斑河豚基因庫選殖到CDC37基因,經基因序列分析後得知此基因包含9個顯子 (exon)長度為3.5 kb。exon 1包含5’端不表現區域(5’ untranslated region)而exon 2 則包含了一個擬轉錄起始點(putative translation initiation site)。 藉由5’-RACE ( rapid amplication of cDNA ends) 快速放大篩選5’端序列,並經核酸序列分析後,演繹推論出其啟動子區域,並發現此區域之核酸序列並未包含典型之TATA或CCAAT box序列。
在擬轉錄起始點往上1.8 kb之DNA片段中,含有數個轉錄因子( transcription factors)之可能結合位置, 包含CREB、E2A、ETS-1、GATA、NF-IL6及PEA3。當將此段基因序列接在chloramphenicol acetyltransferase (CAT) reporter基因之前面,並將此重組基因感染至鯉魚的鰭細胞株中,會使CAT酵素之產量較未接上啟動子序列時提高四倍以上。
由基因序列分析得知圓斑河豚CDC37基因位於TYK2基因exon 1前292 bp處,以tail-to-head方式相連。為探討Cdc37與JAK家族之酪胺酸蛋白激間是否存在交互作用之現象,故以蛋白質離體結合試驗方式觀察Cdc37與JAK家族之激部位(JH1 domain)結合情形。結果Cdc37與GST-JAK2-JH1之結合量最高,與GST-JAK1-JH1之結合量次之,GST-JAK3-JH1再次之,與GST-Tyk2-JH1之結合量最低。
為辨別Cdc37與GST-JAK2-JH1之結合是否具專一性,故再以GST載體表現JAK家族之JH2部位及其他類型之酪胺酸蛋白激:Ret、Syk、Tie及Tec-2等之激部位(Kinase domain, KD)重組蛋白,再與Cdc37分別進行離體結合反應。結果各組Cdc37結合量均遠較GST-JAK2-JH1弱,其中以Ret-KD與Syk-KD之GST重組蛋白有略高之結合量。
Cdc37胺基酸序列上具有一擬酪胺酸磷酸化序列(WDDSQRY),為瞭解JAK2-JH1是否會對此序列進行磷酸化作用,故利用病毒表現系統表現Cdc37並純化後與JAK2-JH1於離體結合反應後,再進行離體磷酸化反應,以anti-phosphotyrosine之單株抗體進行西方墨點法分析,結果並未觀察到Cdc37被GST-JAK2-JH1磷酸化之現象。
Cdc37胺基酸序列上具有二個hyaluronic acid (HA)擬結合序列,且雞Cdc37會與HA結合,故利用160、32、16 μg/ml之HA先與Cdc37進行結合反應1小時後,再加入JAK2-JH1繼續進行結合反應,結果其GST-JAK2-JH1之Cdc37之結合量與未加入HA之對照組相當。 當以pQE蛋白質表現系統表現Cdc37,再與GST-JAK2-JH1於TNT離體表現蛋白質反應溶液存在與否之情況下,進行結合反應;結果二者之結合量相當,即二者之結合不受此混合溶液中之成分所影響。
於胚胎時期CDC37表現方面之研究,則先利用基因庫查詢得到與圓斑河豚CDC37序列具高相似度之斑馬魚CDC37序列,利用此序列合成專一性引子,再以RT-PCR之方法,觀察不同胚胎發育期CDC37基因之表現情形。 結果自受精後5至72小時之胚胎單股互補cDNA中均可觀察到CDC37之表現,其中以12至24小時之胚胎表現量較高,5小時與72小時發育期之胚胎表現量較微弱。 將離體轉錄合成之CDC37-dsRNA顯微注射入斑馬魚單細胞受精卵,此種類似基因剔除(gene knockout)之方式,抑制胚胎之CDC37表現時,可見到處理組之胚胎發育有異常情形,其表現型可概分為四類:1.體節發育嚴重異常2. 脊柱嚴重彎曲或局部發生皺折3. 眼睛發育異常4.血液系統異常。
The CDC37 gene was first isolated from yeast as a cell cycle division control gene. In the vertebrate, chick CDC37 was first isolated and organically analyzed the genome structure and function of CDC37. In this thesis, we have isolated and characterized the CDC37 gene from the round-spotted pufferfish. This gene is composed of 9 exons spanning 3.5 kb. Exon 1 contains the 5’-untranslated region and exon 2 contains the putative translation initiation site. By 5’-RACE and sequence analysis, we deduced the promoter region of CDC37 gene and found that the region does not contain typical TATA or CCAAT box. The 1.8 kb DNA fragment upstream of the putative transcription initiation site contains numerous potential binding sites for transcription factors including CREB, E2A, Ets-1, GATA, NF-IL6 and PEA3. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme 4 times more efficiently than the promoterless pCAT-Basic did. In addition, the CDC37 gene is linked to TYK2 gene in a tail-to-head manner with a small intergenic region of 292 bp between nearest exons of two genes.
Cdc37 protein and Hsp90 function as molecular chaperones to regulate several protein kinase conformation and activity. Cdc37 can bind to Hsp90 as well as a variety of protein kinases including Cdk4, Cdk6, and Raf-1. In the previous results, the pufferfish CDC37 gene is linked to the TYK2 gene in a tail-to-head manner. The CDC37 gene and TYK2 gene might be coregulated like the IL-13 and IL-14 , that linked together also in tail-to-head manner. Such a linkage raises a question whether Cdc37 can interact with Tyk2 or other member of JAK family. To elucilate the interaction of Cdc37 and Tyk2 or other member of JAKs, we contructed GST fusion proteins of JAKs-JH1 or JAKs-JH2 or kinase domain of other tyrosine kinases; and in vitro transcripted/translated full length of Cdc37or Cdc37△N or Cdc37△C, to bind with the GST fusion protein in in vitro binding system.
In this investigation, our data indicate that Cdc37 indeed binds strongly to the catalytic JH1 domain of JAK2 through its C-terminal region, but weakly to the catalytic domain of a variety of tyrosine kinases including the other three JAKs and the JH2 domain of all JAKs. This is the first evidence to demonstrate that Cdc37 associates specifically with only one member of JAK kinase family.
Amino acid sequence comparison of zebrafish and pufferfish Cdc37 proteins indicated that they are highly conserved. To investigate the function of Cdc37 in the zebrafish embryo development, cDNA encoding zebrafish Cdc37 protein was amplified by reverse transcriptase from embryos at different developmental stage. Then CDC37 expression in developmental stages was detected by RT-PCR and its expression was detectable from 5 hours post fertilization (hpf) embryo; abundant at 12-24 hpf and diminished at 72 hpf.
The function of CDC37 at the embryonic development was characterized by RNA interference system. The dsRNA of zCDC37 was microinjected into single cell embryos and some of the treated zebrafish embryos had obvious phenotypic defects as described below:
1. greatly reduced tails and somites lacking the typical chevron shape.
2. severely or slightly curved or truncated tails.
3. mutation at eye development: lacking one eye or even two eyes.
4. defect at circulation system: lacking circulative flow or smaller heart size and slower heart beat.
封面
目錄
中文摘要
Abstract
文獻回顧
第一章圓斑河豚CDC37之基因結構與啟動子分析
摘要
緒言
材料與方法
結果
討論
第二章圓斑河豚Cdc37之功能研究
摘要
緒言
材料與方法
結果
討論
第三章斑馬魚CDC37基因功能之初步探討
摘要
緒言
材料與方法
結果
討論
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