Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21279
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dc.contributor.advisor賴美津zh_TW
dc.contributor.advisorMei-Chin Laien_US
dc.contributor.author余秉弘zh_TW
dc.contributor.authorYu, Ping-Hungen_US
dc.date2001zh_TW
dc.date.accessioned2014-06-06T07:15:32Z-
dc.date.available2014-06-06T07:15:32Z-
dc.identifier.urihttp://hdl.handle.net/11455/21279-
dc.description.abstract自嗜鹽甲烷古生菌Methanohalophilus mahii的質體pML (2163 bp)的迷你質體經序列分析顯示具有一與高鹽古生菌Halobacterium spp.的滾環型(rolling circle replication, RCR)質體pGRB1複製蛋白 (Rep) 有34 %序列相同的開放閱讀框架open reading frame (ORF1)。進一步比對後發現ORF1具有滾環型複製蛋白所特有的三個保留區域,分別為motif1, motif2, motif3。依據motif3的tyrosine殘基數目,可將pML歸類在RCR superfamily I。且orf1上游的反轉序列 (inverted repeat),具有RCR質體所特有的複製起始 (double strand origin, DSO) 切割位置TGATA,此反轉序列亦具有stem-loop 二級結構。亦預測出可能為single strand origin (SSO) 的位置。由序列分析結果顯示pML具有RCR質體的特徵,因此推測pML為一滾環型複製質體,亦是第一個發現具有滾環型複製特色的甲烷古生菌。同時利用膠體電泳及南方墨漬法分析M.mahii的全部核酸時亦發現質體單股DNA中間產物存在。此外,含pML質體的大腸桿菌及具有orf1基因在表現載體pET21b的大腸桿菌之全蛋白分析均具有一22 kD的蛋白條帶,此蛋白可能是滾環型複製蛋白。 目錄 頁 中文摘要………………………………………………………………….I 英文摘要…………………………………………………………………II 壹、前言…………………………………………………………………..1 貳、前人研究……………………………………………………………..2 一、太古生物 (古生菌) …………………………………………………2 二、嗜鹽甲烷古生菌…………………………………………………….7 三、古生菌質體………………………………………………………….9 四、質體複製機制………………………………………………………11 五、質體的滾環形複製機制與特性……………………………………14 六、古生菌質體的複製機制……………………………………………16 七、嗜鹽甲烷菌質體……………………………………………………17 參、材料與方法 一、菌種與質體…………………………………………………………20 二、培養基組成…………………………………………………………20 三、藥品…………………………………………………………………22 四、接菌及菌體生長……………………………………………………23 五、質體之抽取與純化…………………………………………………23 六、DNA回收與純化…………………………………………………26 七、核酸純度之鑑定與定量分析……………………………………..26 八、DNA黏合反應……………………………………………………27 九、質體的轉形作用…………………………………………………..27 十、核酸膠體電泳分析與紀錄………………………………………..28 十一、全細胞蛋白質製備……………………………………………..29 十二、蛋白質電泳……………………………………………………..29 十三、質體複製單股核酸中間物質的偵測…………………………..32 1.Toatal DNA之製備………………………………………………..32 2.DNA轉漬………………………………………………………….32 3.探針之製作……………………………………………….……….34 4.DNA雜合………………………………………………………….35 5.螢光偵測法……………………………………………….……….36 6.AP Based Chemilunimescene Substrate Incubation……………….38 十四、Rep 的酵素活性分析………………………………………..38 十五、Rep基因選殖與表現…………………………………………..38 1.引子的設計……………………………………………….………………38 2.聚合 連鎖反應…………………………………………………...39 3.PCR產物純化…………………………………………….………..40 4.表現質體pET21b-orf1 (rep)的構築……………………………….41 5.Rep蛋白的大量表現與製備細胞萃取液…………………………42 6.Ni2+樹脂回收純化Rep蛋白……………………………………….43 十六、核酸序列分析軟體之應用………………………………………43 1.Open reading frames ( orfs ) 之預測與相似蛋白產物之比對……44 2.滾環形複製蛋白胺基酸序列之全長比對………………………...44 3.Motifs alignment……………………………………………………44 4.Double strand origin ( DSO ) 預測與二級結構之建立…………..44 5.Singlestrand origin (SSO) 的預測…………………………………45 肆、結果與討論………………………………………………………..46 一、質體pML核酸序列分析………………………………………….46 二、質體pML的orf1可能轉錄RCR複製的蛋白…………………...47 三、質體pML具有滾環形複製的double strand origin ( DSO )……...49 四、質體pML具有滾環形複製的single strand origin ( SSO )……….50 五、帶抗藥基因之pML衍生質體的構築……………………………..50 六、在載體pGEM7Zf-的pML之基因表現……………………………51 七、orf1 (rep)基因之選殖大量表現與活性分析………………………52 伍、表與圖………………………………………………………………56 陸、參考文獻……………………………………………………………76 摘要 質體pML (2163 bp) 是自嗜鹽甲烷古生菌Methanohalophilus mahii中發現的迷你質體,經過序列分析顯示一open reading frame (ORF1) 的氨基酸序列,與高鹽古生菌Halobacterium spp.的滾環型(rolling circle replication, RCR)質體pGRB1複製蛋白 (Rep) 有34 %序列相同。進一步比對後發現ORF1具有滾環型複製蛋白所特有的三個保留區域,分別為motif1, motif2, motif3。依據motif3的tyrosine殘基數目,可將pML歸類在RCR superfamily I。在orf1的上游發現到一個反轉序列 (inverted repeat),此序列包含有RCR質體所特有的複製起始 (double strand origin, DSO) 切割位置TGATA,利用PC / GENE模擬出此反轉序列之二級結構 (stem-loop structure),亦預測出single strand origin (SSO)。經由序列分析所得之結果符合RCR質體所具備之特徵,因此,推測pML為一滾環形複製質體,此亦是甲烷古生菌首次發現具此特性的質體。同時以膠體電泳分析質體pML的total DNA發現到可能有單股DNA中間產物存在,更進一步利用南方墨漬法發現在超螺旋的下方有一可能為單股DNA中間產物的訊號。另外,更進一步將orf1基因接入表現載體pET21b進行大量表現後,利用Ni2+親和性管柱純化蛋白進行in vitro活性測試,以確認ORF1蛋白是否具有滾環形複製蛋白 (Rep) 的功能與活性。zh_TW
dc.description.abstractA small cryptic plasmid (2.16-kb), designated pML, was isolated from the moderately halophilic methanogen Methanohalophilus mahii SLP. Sequence analysis indicated the possibility of the two open reading frames (ORF 1, ORF 2). The predicted protein of ORF1 showed 34 % sequence identity to a putative replication protein (Rep) of pGRB1 from Halobacterium spp. Both putative proteins contained three sequence motifs (motif1, motif 2 and motif 3) that conserved in Rep proteins of rolling circle replication (RCR) mechanism. Based on the numbers of Tyr residues in the motif 3, pML was belonged to superfamily I of RCR. Moreover, two inverted repeated sequences around the nick site (TGATA) within the double strand origin (DSO) of plasmids and phages that replicate via a RCR mechanism.And the nicking site of pML was situated at loop region of IRI stem-loop structure. we also find single strand origin (SSO) near orf1. ss DNA intermediates were confirmed by Southern analysis. Based on above analyses, plasmid pML from the halophilic methanogen was proposed to replicate via a RCR mechanism and is the first RC plasmid discussed in methanogenic archaea. Predicted ORF1 encoded a putative 20.8 kDa protein consists of 176 amino acids. Further step, the orf1 was amplified with PCR technique with the additional restriction enzyme cut sites of Nde I and Xho I. The orf1 was further subcloned into the expression vector pET21b. After IPTG induction of the T7 lac promoter of pET21b, a 22 kDa polypeptide was heavily accumulated only at strain with orf1 gene. Next, study the activities of its encoded polypeptide in vitro will help us to understand RCR more.en_US
dc.description.tableofcontents目錄 頁 中文摘要…………………………………………………………………I 英文摘要…………………………………………………………………II 表目錄……………………………………………………………………VII 圖目錄……………………………………………………………………VIII 壹、前言…………………………………………………………………1 貳、前人研究……………………………………………………………2 一、太古生物 (古生菌…………………………………………………2 二、嗜鹽甲烷古生菌……………………………………………………6 三、古生菌質體…………………………………………………………6 四、質體複製機制………………………………………………………11 五、質體的滾環形複製機制與特性……………………………………14 六、古生菌質體的複製機制……………………………………………16 七、嗜鹽古生菌質體……………………………………………………18 參、材料與方法…………………………………………………………19 一、菌種與質體…………………………………………………………19 二、培養基組成…………………………………………………………19 三、細菌之培養與保存…………………………………………………21 四、接菌及菌體生長……………………………………………………21 五、質體之抽取與純化…………………………………………………22 六、DNA回收與純化……………………………………………………24 七、核酸純度之鑑定與定量分析………………………………………25 八、DNA黏合反應………………………………………………………26 九、質體的轉形作用……………………………………………………27 十、核酸膠體電泳分析與紀錄…………………………………………27 十一、全細胞蛋白質製備………………………………………………28 十二、蛋白質電泳………………………………………………………28 十三、質體複製單股核酸中間物質的偵測……………………………31 十四、Rep基因選殖與表現………………………………………………35 1.引子的設計……………………………………………………………35 2.聚合脢連鎖反應………………………………………………………36 3.PCR產物純化…………………………………………………………37 4.表現質體pET21b-orf1(rep)的構築…………………………………38 5.Rep蛋白的大量表現與製備細胞萃取液……………………………39 6.Ni2+樹脂回收純化Rep蛋白…………………………………………40 7.濃縮蛋白質溶液………………………………………………………41 十五、核酸序列分析軟體之應用………………………………………42 1.Open reading frames(orfs) 之預測與相似蛋白產物之比對………42 2.胺基酸序列之全長比對………………………………………………42 3.滾環形複製蛋白Motifs之比對………………………………………42 4.double strand origin預測與二級結構之建立………………………43 5.single strand origin預測………………………………………………43 肆、結果與討論…………………………………………………………44 一、質體pML核酸序列分析…………………………………………44 二、質體pML的orf1可能轉錄RCR複製的蛋白…………………45 三、質體pML具有滾環形複製的double strand origin ( DSO )……47 四、質體pML具有滾環形複製的single strand origin ( SSO )………48 五、帶抗藥基因之pML衍生質體的構築……………………………48 六、在載體pGEM7Zf-與pET21b的pML之基因表現………………49 七、orf1 (rep) 基因之選殖大量表現…………………………………50 伍、結論…………………………………………………………………53 陸、表與圖………………………………………………………………54 柒、參考文獻……………………………………………………………73 摘要 迷你質體pML(2163 bp)是自嗜鹽甲烷古生菌Methanohalophilus mahii中發現的質體,也是高鹽厭氧生物至今所發現的唯一質體。其核酸序列與限制脢圖譜已全部定出,經過序列分析發現一open reading frame(ORF1)的氨基酸序列,與高鹽古生菌Halobacterium spp.的滾環型(rolling circle replication, RCR)質體pGRB1複製蛋白(Rep)有34 %序列相同。將預測的ORF1蛋白和目前已知的所有滾環型複製蛋白比對,發現ORF1具有滾環型複製蛋白所特有的三個保留區域,分別為motif1, motif2, motif3。依據motif3的tyrosine殘基數目,可將pML歸類在RCR superfamily I。 在orf1的上游發現到一個反轉序列(inverted repeat),此序列包含有RCR質體所特有的複製起始 (double strand origin)切割位置TGATA,並可利用PC/GENE模擬出此反轉序列之二級結構 (stem-loop structure)。在非常接近orf1的位置亦發現另一反轉序列,此序列上含有一single strand origin TAGCG保留序列,推測應為pML之single strand origin。 進一步分析以全細胞蛋白質圖譜,比較發現含有pML的DH5α( pMM3 )具有一大約21 kDa的蛋白條帶出現。此21 kDa的蛋白和預測的ORF1分子量相似。因此推測此一蛋白條帶可能是orf1所轉譯而成。接著將orf1基因接入表現載體pET21b進行大量表現後,發現亦具有此一大約21 kDa的蛋白質條帶。 綜合以上分析,推測pML為一滾環形複製質體,亦是甲烷古生菌首次發現具此特性的質體,並且將對在嗜鹽古生菌中所發現的ORF1複製蛋白進行功能活性之探討。 Abstract A small cryptic plasmid (2.16-kb), designated pML, was isolated from the moderately halophilic methanogen Methanohalophilus mahii SLP. Sequence analysis indicated the possibility of the two open reading frames (ORF 1, ORF 2). The predicted protein of ORF1 showed 34% sequence identity to a putative replication protein (Rep) of pGRB1 from Halobacterium spp. Both putative proteins contained three sequence motifs (motif1, motif 2 and motif 3) that conserved in Rep proteins of rolling circle replication (RCR) mechanism. The function of Motif1 is not clear, Motif 2 was thought to be the metal-binding domain of the Rep proteins and motif 3 contained the tyrosine (Tyr) residue involved in the nucleophilic attack to the plasmid DNA at initiation of replication. Based on the numbers of Tyr residues in the motif 3, pML was belonged to superfamily I of RCR. Moreover, two inverted repeated sequences around the nick site (TGATA) within the double strand origin of plasmids and phages that replicate via a RCR mechanism. Interestingly, the nicking site of pML was situated at loop region of IRI hairpin structure postulated by PC/GENE. A conserve six-nucleotide sequence, TAGCGt/a present in ssoA-type origins was shown to function as transcriptional terminator for the synthesis of an RNA primer. The sequence TAGCG, with a high level of secondary structure, lies in the upstream of the orf1 gene of pML, was predicted to be the putative sso region of pML. Predicted ORF1 encoded a putative 20.8 kDa protein consists of 176 amino acids. Throughing total cell protein analysis of pML containing E.coli DH5a revealed a significant 21 kDa polypeptide band in the sodium dodecyl sulfate polyacrylamide gel, but not in the E. coli DH5a containing pGEM7Zf only. Orf1 was amplified with PCR technique with the additional restriction enzyme cut sites of Nde I and Xho I. The orf1 was further subcloned into the expression vector pET21b. After IPTG induction of the T7 lac promoter of pET21b, a 21 kDa polypeptide was heavily accumulated only at strain with orf1 gene. Based on above analyses, plasmid pML from the halophilic methanogen was proposed to replicate via a RCR mechanism and is the first RC plasmid discussed in methanogenic archaea. In the future, study of ORF1 replication property will be an important job.zh_TW
dc.language.isoen_USzh_TW
dc.publisher植物學系zh_TW
dc.subject甲烷古生菌zh_TW
dc.subjectmethanogenic archaeaen_US
dc.subject質體zh_TW
dc.subject開放閱讀框架zh_TW
dc.subject滾環型複製zh_TW
dc.subject反轉序列zh_TW
dc.subject雙股複製起始zh_TW
dc.subject單股複製起始zh_TW
dc.subject單股中間產物 去氧核糖核酸zh_TW
dc.subjectplasmiden_US
dc.subjectopen reading frame (ORF)en_US
dc.subjectrolling circle replication (RCR)en_US
dc.subjectinverted repeaten_US
dc.subjectdouble strand origin (DSO)en_US
dc.subjectsingle strand origin (SSO)en_US
dc.subjectsingle strand DNA intermediates (ss DNA)en_US
dc.title嗜鹽甲烷古生菌質體pML序列分析與滾環形複製機制之探討zh_TW
dc.titleSequence analysis and rolling circle replication mechanism of plasmid pML in Methanohalophilus mahiien_US
dc.typeThesis and Dissertationzh_TW
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item.fulltextno fulltext-
item.cerifentitytypePublications-
item.languageiso639-1en_US-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
Appears in Collections:生命科學系所
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