- 學位論文
探討不同的末端蛋白和端粒連結蛋白結合的活性
The study of the activity of various combinations of TapC and Tpg
摘要
鏈黴菌線形染色體和線形質體的末端不但含有豐富的迴文序列且5’端以共價鍵鍵結末端蛋白，此末端蛋白藉由與端粒結合蛋白交互作用來參與線形複製體的末端補齊機制，且它们的基因位於一個4.5kb的操縱子上，經由基因序列的分析，發現某些線形質體具有單獨的tpg同源體，如: S.lividans中的SLP2、S.avermitilis MA-4680中的pSAP1、S. violaceoruber中的pSV2和Streptomyces. sp. FR1中的pFRL1，在這些tpg同源體的研究中，tpgSLP2和迷你染色體前身pLUS980中的tpgC置換後，再以酵素AseI線性化，轉形到MR04 (為一種S.Lividans ZX7 染色體末端刪除而環化的變形株) ，可以得到線形複製體，由此證實TpgSLP2具有末端補齊機制的功能。利用相同的方法，本論文研究其它的tpg同源體，結果顯示，構築的迷你質體中，pCY77和pCY79，分別含有tpgpSAP1_11及tpgpFRL1.6c基因，均可在MR04中得到線形複製體，而含有tpgpSV2.102 基因的pCY78和pCY78b則無法找到線形複製體。 另外，在SLP2中，雖然tpgSLP2放在4.5kb的操縱子上，具有末端蛋白的功能。但在SLP2中，它是如何被轉錄的尚不清楚，因而建構迷你SLP2包含tpgSLP2和上游的片段，企圖得到一個有功能的TpgSLP2，結果顯示迷你SLP2必須包含tpgSLP2直到mtap的序列，才可在CK03(為一種MR04含有tapC的變形株)得到預期的結果。 本論文也對4.5kb的操縱子中，兩個未定義的編碼序列分別是8D11.22和8D11.23加入探討，實驗發現8D11.23的基因不參與線形複製體維持。而且為了純化蛋白的便利性，在末端蛋白TpgC接上(His)6或SNAP的標緻，實驗的結果發現仍具有引子的功能，參與末端空隙的補齊。
並列摘要
Abstract The terminal protein binds to the 5’end of the linear chromosome and the linear plasmids of the streptomyces by the covalent bond. The terminal protein participates the mechanism of end patching of the linear replicons along with telomere-associated protein, also their gene are closely to form an operon. In virtue of DNA sequence analysis, certain linear plasmids contain alone tpg homologues, for example: the SLP2 of the S.lividans, the pSAP1 of the S.avermitilis MA-4680, the pSV2 of S.violaceoruber and the pFRL1of the Streptomyces sp. FR1.For the functional study of these alone tpg homologues , tpgSLP2 has already been to replace tpgC in mini-chromosome pLUS980.The resulted pCY72 linearized by the enzyme AseI, and transferred into MR04 (as the derivatives of the S.Lividans ZX7 with circular chromosome), was founed to be in linear from.So it is confirmed that TpgSLP2 could function as TpgC at the mechanism of end patching. To study other tpg homologues by using the same method .we construct mini-plasmids, pCY77 and pCY79, containing tpgpSAP1_11 and the tpgpFRL1.6c gene respectively.Both of them form linear plasmid in MR04.Instence,pCY78 and pCY78b containing tpgpSV2.102 gene were founed only in circular forms. The tpgSLP2 has the terminal protein function as it locates in the tap operon, but how it is expressed in SLP2 is not clear. Therefore, we construct mini-SLP2 containing tpgSLP2 along the upstream segments to get a functional TpgSLP2. We had not successed until mtap is included. In the 4.5kb block, there are two unidentified coding sequence, 8D11.22 and 8D11.23. Here, we discovered that 8D11.23 gene does not invole in the maintenance of linear replicons in MR04. For the convenience of purifying terminal proteins, we added (His)6or SNAP to TpgC,and discovered that still both of them work as primers.