果蠅端粒連結反轉錄子TART之反轉錄酶活性研究

大部分真核生物的端粒長度是由端粒酶 (telomerase) 所維持，端粒酶以自身的 RNA 作為模板進行反轉錄以延長染色體末端。然而，在已知的所有果蠅品種內皆不具有端粒酶活性，目前研究的結果發現，果蠅末端是由兩種非 LTR 反轉錄子 – HeT-A 及 TART 不間斷的形成。TART 會產生 ORF1p 及 ORF2p 兩個蛋白質。TRAT ORF2p 蛋白質序列與其他研究較廣的非 LTR 反轉錄子，如人類的 LINE 1 的蛋白質序列相比，具有內切酶 (endonucleaase) 與反轉錄酶 (reverse transcriptase) 兩種酵素的蛋白質序列相似性。雖然已有研究報導 TART 會移動到染色體末端，但截至目前為止，都沒有證據顯示這種移動是利用反轉錄移位 (retrotransposition) 還是重組機制 (recombination)。我的研究主要是利用 in vitro 與 in vivo 兩種系統釐清 TART 是否真具有反轉錄酶的活性。在 in vitro 方面，利用大腸桿菌表現 TRAT 反轉錄酶，並純化偵測是否有酵素活性，預測之活性點突變及去除此片段之蛋白質反轉錄酶活性偵測結果皆顯示目前測試過的 TART RT 片段可能不包含完整的反轉錄酶全長。in vivo 方面，在酵母菌系統內將反轉錄子 Ty1 的 TYB 反轉錄酶片段置換為 TART ORF2 ，在 in vivo RT assay 可測到 TART 反轉錄酶活性。南方墨點法顯示因 TART 反轉錄酶作用造成 marker 插入質體的頻率約為 4.5x10-9。將此具有 marker 插入之質體定序，結果發現 TART ORF2 進行反轉錄過程時發生模板轉換現象 (template switch)，且 TART ORF2 mRNA 部分可能有強烈二級結構 (secondary structure) 使得 TRAT ORF2p 進行 cDNA 合成時容易掉落。由我的研究證實果蠅端粒連結反轉錄子 TART 的確具有反轉錄酶活性。

關鍵字

果蠅；反轉錄酶

並列摘要

In most eukaryotes, telomeres are maintained by telomerase. Telomerase extends telomeres by reverse transcribing a segment of its internal RNA template onto the end of the chromosome. However, telomeres in all Drosophila species are composed of tandem arrays of repeated sequences formed by two non-LTR retrotransposons, HeT-A, TART. TART encodes two proteins, ORF1p and ORF2p. ORF2 shares homology to two enzyme activities, endonuclease (EN) and reverse transcriptase (RT) to other non-LTR retrotransposons. Sheen and Levis previously found TART can move to chromosome ends. But there is no evidence showing that this movement is through retrotransposition or recombination. My project focuses on identifying TART RT activity and the detail mechanism of TART. In the in vitro approach, I purified TART RT protein to detect its activity. However, both RT assay of point mutations at RT active motif YADD and the truncated protein showed that the construct may not have full length TART RT. In the in vivo assay , I set up a yeast retrotransposition system, as described previously (Teng et al., 1996), and Southern blot analysis was performed to obtain a insertion frequency, which is about 4.5x10-9. The sequencing data showed that the template switch may happen when TART ORF2 synthesized cDNA in VLPs. And TRAT ORF2p may prefer to fall out from mRNA because of the mRNA secondary structure in TART ORF2. My studies demonstrated that TART possesses RT activity.

並列關鍵字

Drosophila ； TART ； Reverse Transcriptase

參考文獻

Biessmann, H., Carter, S.B. and Mason, J.M. (1990) Chromosome ends in Drosophila without telomeric DNA sequences. Proc Natl Acad Sci U S A, 87, 1758-1761.

Blackburn, E.H. (1992) Telomerases. Annu Rev Biochem, 61, 113-129.

Cost, G.J., Feng, Q., Jacquier, A. and Boeke, J.D. (2002) Human L1 element target-primed reverse transcription in vitro. Embo J, 21, 5899-5910.

Curcio, M.J. and Garfinkel, D.J. (1991) Single-step selection for Ty1 element retrotransposition. Proc Natl Acad Sci U S A, 88, 936-940.

Danilevskaya, O.N., Traverse, K.L., Hogan, N.C., DeBaryshe, P.G. and Pardue, M.L. (1999) The two Drosophila telomeric transposable elements have very different patterns of transcription. Mol Cell Biol, 19, 873-881.

國際替代計量

果蠅端粒連結反轉錄子TART之反轉錄酶活性研究

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