溫和性噬菌體ΦAT3之基因組態及轉位子ISLC3在轉位時產生的環化結構之研究

Complete genomic sequence of the temperate bacteriophage ΦAT3 and functional assay of the circular structure in the transposition of insertion sequence ISLC3

羅大鈞(Ta-Chun Lo)

指導教授 ： 林志侯

國立清華大學/生命科學院/生命科學系/博士(2006年)

摘要

中文摘要:
一個分離自Lactobacillus casei ATCC 393 的溫和性噬菌體 ΦAT3 之genomic 順序已完成定序。在電子顯微鏡 (TEM) 的觀察下判定 ΦAT3 具有一直徑 53 nm 多面體頭部及 約 200 nm 的 不可伸縮的尾部，這些特徵可將 ΦAT3 歸入 Siphoviridae 類噬菌體家族。 ΦAT3的遺傳物質為 dsDNA，經定序判定其長度為 39,166 bp，涵蓋 53 個可判讀的 Open reading frames (Orfs)，這些 Orfs 所產生的蛋白大略可區分為噬菌體結構蛋白、遺傳物質嵌入或與寄主感染免疫、複製等功能相關的區域，我們也分離了ΦAT3 數種結構蛋白，並經過氨基酸 N端定序，已探討這些結構蛋白是否接受過post-translation。接著我們分離出 ΦAT3 在寄主 chromosome上的嵌入點 attachment site，在取得attB， attL， attR 和 attP 序列後，比對分析發現其中的 13 bp 為其嵌入點的 core sequence ，也發現位於寄主 chromosome上的 attB是在一 tRNAArg gene上， 而 13 bp 的 core sequence 位於 tRNAArg gene 的 amino acid acceptor stem 和 D arm 之間。另外，我們試圖將 ΦAT3 的嵌入脢(integrase Int) 基因及 attP 序列 建構為一嵌入載體 (integration vector)，南方墨點分析證明該載體能在乳酸菌 Lb. rhamnosus HN 001 或在其原本的寄主Lactobacillus casei ATCC 393體內有效的嵌入其 chromosome。
ISLC3，一個存在於ΦAT3 的轉位子，其長度為1,351 bp 的雙股DNA序列，經南方墨點分析發現此轉位子也分布在數種lactobacilli 中。在ISLC3的轉位研究中發現ISLC3在預備進行轉位時，當脫離寄主chromosome形成環化結構時常在左端逆向重複序列產生一個25 bp缺失，右端逆向重複序列則維持完整 (正常的環化結構會包括完整的左右兩端逆向重複序列和數個間隔在兩者的序列，此間隔序列常常就是IS的direct repeat)， 這種左端逆向重複序列的25 bp缺失的環化結構可以在數種lactobacilli 中被發現。我們在Escherichia coli系統內重覆ISLC3的轉位實驗時發現包括25 bp 缺失和完整的兩端逆向重複序列的環化結構都能被找到。 由於兩種環化結構都能造成完整的新啟動子(相較於原本在ISLC3 基因orfA上游的 promoter)。 我們利用 β-galactosidase 的產量分析系統來分析兩種環化結構所形成的啟動子的起動能力差異，發現 25 bp 缺失所形成的新啟動子能力遠弱於完整的兩端逆向重複序列所形成的新啟動子和原本在ISLC3 基因 orfA 上游的啟動子(後兩者的啟動能力相近)。由於 25 bp 缺失所形成環化結構不是正常的轉位子結構，可能不具備完成轉位的能力，故 25 bp 缺失的環化結構之所以存在，推測是為了能抑制ISLC3的轉位現象。

關鍵字

噬菌體 ； 轉位子 ； 乳酸菌 ； 環化結構

並列摘要

The complete genomic sequence of a temperate bacteriophage ΦAT3 from Lactobacillus casei ATCC 393 was reported. The newly isolated phage consisted of a linear dsDNA genome of 39,166 bp (accession no. AY605066), an isometric head of 53 nm in diameter, and a flexible noncontractile tail of approximately 200 nm in length. The morphology of ΦAT3 examined resembled that of Siphoviridae, the number of potential open reading frames determined on the phage genome is 53. The ΦAT3 genome was grouped into five distinct functional clusters: DNA packaging, morphogenesis, lysis, lysogenic/lytic switch, and replication. The amino acid sequences at the NH2-termini of some major proteins were determined. An in vivo integration assay for the ΦAT3 integrase (Int) protein in several lactobacilli was conducted by constructing an integration vector including ΦAT3 int gene and the attP (int-attP) region. It was found that the ΦAT3 genome integrated at the tRNAArg gene locus of Lb. rhamnosus HN 001 (also known as DR 20), similar to that observed in its native host, Lb. casei ATCC 393. An insertion sequence ISLC3 of 1,351 bp has been isolated from ΦAT3. The distribution of ISLC3 in several lactobacilli has been analyzed by Southern hybridization. The transposition of ISLC3 in all the lactobacilli studied produces a truncated junction of right and left inverse repeat where a direct repeat and 25 bp in the left inverse repeat are deleted. However, both the complete and truncated circles are detected in an Escherichia coli system studied. The activities of promoters formed by both the complete and truncated circles are analyzed using an assay system of β-galactosidase. That the promoter activity formed by the circle junction region of ISLC3 was nearly the same as the indigenous one and the activities of both are much stronger than that formed by the truncated one. However, the transposition will not occur in 25 bp truncated junction circles, indicating that formation of the truncated circles in the host is to suppress the effect of high frequent transposition which may be detrimental to the host.

並列關鍵字

bacteriophage ΦAT3 ； insertion sequence ISLC3 ； Lactobacillus ； circular structure

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