Isolation of (6-4)Photoproduct-Binding Proteins from Chlorella Pyrenoidosa and cDNA Cloning

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Project title

Code/計畫編號

NSC92-2311-B019-001

Translated Name/計畫中文名

小球藻6-4光合產物辨識蛋白分離與cDNA選殖

Project Coordinator/計畫主持人

Todd Hsu

Funding Organization/主管機關

National Science and Technology Council

Department/Unit

Department of Bioscience and Biotechnology

Website

https://www.grb.gov.tw/search/planDetail?id=859754

Year

2003

Start date/計畫起

01-08-2003

Expected Completion/計畫迄

01-07-2004

Bugetid/研究經費

950千元

ResearchField/研究領域

生物科學
生物技術(理)

本研究以帶有 6-4PP 之 DNA 藉親和性吸附、蛋白質與 DNA 交錯實驗與雙向蛋白質電泳法分離小球單胞藻中之核酸切割修補作用辨識蛋白群，結果發現確有些藻蛋白可結合 6-4PP，且交錯實驗顯示一分子量約 70-72 kDa 之蛋白似對 6-4PP 有較佳專一性，而吸附後蛋白質分布差異經雙向蛋白質電泳法已確認此 72kDa 蛋白等電點介於 4-6 間，以等電點 4.7 至 5.9 進行細部分離發現此 72kDa 蛋白係由數個分子量約 50 至 55kDa 且電點介於 5.2 至 5.6 間之蛋白所組成。初步質譜分析顯示這些蛋白質與資料庫中與已知蛋白均無明顯相似性。另外在本計劃經費補助下，本實驗室也對斑馬魚(Danio rerio) 中之 DNA 錯誤配對辨識蛋白 MutS homolog 2(MSH2)進行研究，結果選殖出全長 MSH2 cDNA，重組斑馬魚 MSH2 可辨識 G-T 錯誤配對，原位雜交分析顯示 MSH2 mRNA 在胚胎中主要表現於頭部及胃腸道，但在成熟後斑馬魚中此 mRNA 表現量明顯下降，故 MSH2 基因表現可能因成熟後斑馬魚 DNA 複製活動減緩而下降，本研究成果已投稿發表(Biochim. Biophys. Acta 1680:129-136, 2004)。The goal of this research was to isolate DNA damage-recognition proteins associated with NER systems operating in unicellular alga C. pyrenoidosa by affinity adsorption using 6-4PP-specific DNA as the ligand, protein-DNA crosslinking assay , 2-D gel electrophoresis and mass spectrometric analysis. Differential polypeptide patterns were observed after affinity adsorption with DNA probes carrying different types of lesion and one 70 to 72 kDa polypeptide seemed to bind more strongly to 6-4PP as shown by a UV crosslinking assay. The 72-kDa polypeptide was found to possess a pI between 4 to 6 based on two-dimensional gel electrophoresis. The 72-kDa spot was composed of several 50 to 55-kDa polypeptides having pIs between 5.2 and 5.6. The possible functions of these 50 to 55 kDa proteins are now explored by protein mass spectrometry. Under the financial support of this grant, a full-length cDNA encoding zebrafish(Danio rerio) Muts homolog2 (MSH2), a DNA mismatch 2 recognition protein, was cloned by RT-PCR and RACE. Recombinant zebrafish MSH2 was shown to bind preferentially to G-T mispair. A high expression of MSH2 mRNA in the head region of zebrafish embryos was detected by whole-mount in situ hybridization. Because of the gradual decrease in MSH2 mRNA expression in more mature zebrafish, the activity of MSH2 gene was thought to be down-regulated by the number of proliferating cells.(Published in B.B.A 1680:129-136,2004)

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Isolation of (6-4)Photoproduct-Binding Proteins from Chlorella Pyrenoidosa and cDNA Cloning

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