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標題: Vibrio fischeri 冷休克 cspA 基因的選殖與其調節機制分析
Cloning and charasteistic analysis of the cold shock cspA gene from Vibrio fischeri
作者: 陳志龍
Chen, Jyh-Long
關鍵字: cspA 基因;Vibrio fischeri
出版社: 生物化學研究所
藉由構築基因庫選殖得到 Vibrio fischeri ATCC 7744 cspA 基因;其基因的排列次序 -hvn-WT- R&R-5’UTR-cspA-WoT─ufoI/ufoII----. V. fischeri CspA 蛋白質由 69 個胺基酸組成,分子量為 7449 Da,具有 RNP1 (K15GFGFI20), RNP2 (V29FVHF33),2個 RNA binding motifs 顯示此蛋白可能為 RNA chaperone,此蛋白與 S. violacea CspG, CspA, E. coli CspA 有 82%, 80% 以及 64% 相同;序列分析顯示,V. fischeri cspA與 E. coli cspA 基因序列相似,包括 UP elements, 5’UTR, downstream box。利用 bioluminoassays in vivo 分析 cspA 基因 R&R, 5’UTR, cspA 三個區域對於基因表現之影響,並探討冷休克與基因調控之關係,結果顯示 (1) V. fischeri cspA promoter在 30oC 與 10 oC 下皆具有高的活性,與 E. coli 模式不同點為 V. fischeri cspA promoter 在 30oC 與 10oC 下受到負調控,而 negative element 位在 promoter 上游約 ~200 bp區域。(2) V. fischeri cspA 包含一段 173 nt 之 5’UTR,在 30oC 下完整的 5¢UTR 以及 cspA 區域對基因表現為負調控,其中 5¢UTR 113~160 nt 區域可能形成 hairpin loops 二級結構,可影響轉錄作用或其穩定度,並影響基因於 30 oC 表現。(3) RNA stability 分析,primer extension 與 northern blot 之結果顯示基因表現與螢光表現一致,而 V. fischeri cspA promoter negative element 之存在,只減低其活性,而不改變其轉錄機制。(4) mRNA 穩定度分析顯示,在 30oC、 20oC、 10oC 其半衰期約為 <45秒、<1分鐘、>8分鐘; mRNA 穩定度為影響基因在不同溫度表現之主因。(5) 利用細胞內互補性螢光表現分析以及 maxicell 分析 CspA 蛋白表現與功能分析,肯定有 CspA 蛋白質產生,且顯示蛋白質不影響基因表現; CspA 蛋白之功能仍不甚清楚。

The cspA gene of V. fischeri ATCC 7744 was cloned from the genome library. Nucleotide sequence of the cspA gene and the neighboring genes has been determined, and the encoded CspA was deduced. Sequence analysis revealed that the gene order of the relative genes is —hvn-WT- R&R-5'UTR-cspA- WoT─ufoI/ufoII---- ; whereas the regulatory region R&R and 5'UTR of the cspA gene included the UP elements, -35/-10 canonical promoter sequence and Shine-Dalgarno sequence and downsream box that regulates the cspA gene. The CspA has a calculated molecular weight 0000 and comprises 69 amino acid residues. The CspA has two RNA binding motifs, RNP1 (K15GFGFI20) and RNP2 (V29FVHF33), could be functioned as RNA chaperone. Protein assays confirm that the CspA protein encoded by the gene. Functional analysis elicits that the 5'UTR of the cspA gene is the major factor involved in the cold shock response. The result revealed that (1) V. fischeri cspA promoter is under negative regulation at 30 and 10oC, but its function is highly active, the negative element is located at upstream of promoter. (2) The 5'UTR is able to negative regulate the cspA gene expression at 30 oC. The 5'UTR 113~160 nt might form hairpin loop to maintain the mRNA stability and/or to function as attenuation-like transcriptional terminator. (3) Primer extension assays and northern blotting analysis confirms the transcription initiation site. (4) The mRNA stability analysis reveals that the cspA mRNA is extreamly unstabe at 30 and 20oC (half-life <45 sec. and <1 min.) and is stabe at 10oC (half-life >8 min). It elucidates that the mRNA's stability would affect the cspA gene expression at various temperatures. (5) In trans complementation bioluminoassays in vivo reveals the CspA does not affect it's gene expression at 30 and 10 oC.
Appears in Collections:生物化學研究所

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