鏈黴菌線形質體SLP2上的末端蛋白基因的活性探討

Investigation of the priming activity of the tpg gene of Streptomyces linear plasmid SLP2

呂芳錫(Fang-Shyi Lue)

指導教授 ： 楊千金

中原大學/理學院/化學研究所/碩士(2005年)

https://doi.org/10.6840/cycu200500755

摘要

鏈黴菌末端蛋白基因，被發現並分別命名為tpgC (from S. coelicolor)、tpgL (from S. lividand)和tpgR (from S. rochi)。其中tpgC和tpgL的胺基酸序列完全相同，DNA序列只有兩個不同。在鏈黴菌S. lividans的線形質體SLP2上另外還有兩個類似末端蛋白的基因，tpgSLP2 和pseudotpg。tpgSLP2位於SLP2左端6 kb，DNA序列與tpgL(C)有74 % 相似。pseudo-tpg 位於右端8 kb，DNA序列與tpgL(C)有54 % 相似。鏈黴菌線形質體的複製和噬菌體ψ29同樣是以末端蛋白(TP)作為引子，雖已知端點DNA是與末端蛋白TpgC上的蘇氨酸鍵結，但引子與DNA複製時鍵結的位置尚未確定。
本論文主要是在探討ㄧ系列的末端蛋白基因的引子活性，使用不含末端蛋白tpgL基因且只能維持環形質體的S. lividand 菌種MR04作為宿主，將包含已改變或突變的tpg基因之迷你質體pLUS981；以限制酶AseI線形化後再送入MR04菌株中，檢測迷你線形質體在MR04轉形株中的穩定性。實驗結果顯示:(1) 迷你線形質體pLUS981含有原生種tpgSLP2的 MR04的轉形株中存在線形質體，顯示原生種的tpgSLP2具有引子活性；(2) 迷你線形質體pLUS981含有原生種pseudo-tpg的MR04的轉形株中均無線形質體，表示原生種pseudo-tpg無引子活性。 (3) 在TpgSLP2 上第114位置由蘇氨酸突變成絲氨酸MR04的轉形株中有線形質體存在，顯示其有引子活性。TpgC上第101、108、114、123和176位置由蘇氨酸突變成絲氨酸仍具有引子活性；但TpgC上第114位置由蘇氨酸突變成胱氨酸則失去引子活性，推論TpgC上第114位置可能是與鏈黴菌末端DNA的鍵結位置。(4) tpgC fused egfp在MR04存在線形質體，顯示與TpgC融合的綠色螢光蛋白具有引子活性。(5)TpgC融合TapC蛋白，則不具有引子活性。

關鍵字

末端蛋白基因 ； 引子活性 ； 鏈黴菌線形質體

並列摘要

The termenial protein gene of streptomyces had been found and named tpgC ( from S. coelicolor), tpgL (from S. lividans) and tpgR (from S. rochi). The amino acid sequence of tpgC is the same as tpgL, but the DNA sequence owned two different nucleotides. Two tpg homologues, tpgSLP2 and pseudo-otpg are founded on the linear plasmid SLP2 of Streptomyces lividans. The tpgSLP2 located at 6 kb from the left end, has the homology of tpgC is 74 percentage. The pseudo-tpg located at the right end 8 kb, has the homology of tpgC is 54 percentage. Streptomyces termenial protein as primer for replication of linear plasmid is similar to that of phageψ29. Although it was suggested that the DNA end is binding with threonine of TpgC, the accurate site is not determined.
To investigate a series of Tp’s primer activity, we use the Streptomyces lividans stain MR04 as host which is tpgL- and only maintains circular plasmids. Transfering the linearized plasmids containing various form of tpg gene to MR04 and examination it’s stability of linear plasmids to determine the primer activitying of Tp. The results display: (1)The capability of wild type tpgSLP2 to maintain linear plasmids in MR04 indicateds that tpgSLP2 is functional as tpgC. (2)The incapability of wild type pseudo-tpg to maintain linear plasmids in MR04 displays that pseudo-tpg is non functional. (3)The mutated of TpgSLP2 at the 114th threonine to serine, can maintain linear plasmid in MR04. So do mutated TpgC at the 101th、108th、114th、123th and 176th threonine to serine. But the mutated of TpgC at the 114th threonine to cysteine is non functional, this result suggested that the linkage site might be at the 114th. (4)The C-terminal egfp-fused tpgC can maintain linear plasmid in the MR04, indicateds that is functional. (5)The tpgC fused to tapC can’t maintain linear plasmid.

並列關鍵字

tpgC ； termenial protein gene ； SLP2 ； pseudotpg ； tpgSLP2

參考文獻

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