| Summary: | 碩士 === 國立陽明大學 === 微生物暨免疫學研究所 === 83 === IS2是存在於大腸桿菌及腸內菌基因體上一種簡單的轉位因子，計含1332bp，並於末端各有一長42-bp的反向重複序列。IS2的第一開放編閱區(ORF1)會表現一個14-kDa的蛋白質InsA，此蛋白可以和一段包含IS2右端的反向重複序列以及insA啟動子部分的DNA結合，並有自我調控蛋白產生及抑制IS2轉位之功能。由於此insA基因只佔IS2全長的三分之一，又不能促進IS2之轉位，故推測IS2除insA外，應速有其他的基因產物存在。
由IS2之核酸序列分析，可見insA基因末端有一段A6G並於其下游處可形成一RNA之二次結構。由於此為-1 frameshift之象徵，故推測IS2可能在A6G進行-1 frameshift 而將ORF1及ORF2融合產生一46kDa的蛋白。然而此蛋白在以迷你細胞表現IS2蛋白時並無法觀察得到，故懷疑此現象乃因其啟動子太弱而frameshift 之頻率不高所致。本研究乃以T7啟動子置於ORF1上游，在誘導T7啟動子表現的情況下，確可看見除InsA外尚有一大小約46-kDa之蛋白質出現。為瞭解是否插入一個A確可產生46-kDa大小的蛋白，乃於A6G處插入一個A，直接構築一ORFl、ORF2融合之突變基因，置於T7啟動子下游予以表現，結果確能產生一46kDa的蛋白質，顯示此46kDa蛋白質很可能由-1 frameshift所產生，本實驗室將其命名為InsAB'。
為了解InsAB'與IS2兩端反向重複序列結合的能力，乃進行電泳阻滯實驗。結果發現InsAB'可和一段長89bp包含IS2右端的反向重複序列(RIR)，以及另一段長110bp包含IS2左端的反向重複序列(LIR)結合。再經由去氧核醣核酸足印實驗確認，其保護部位涵蓋大部分之反向重複序列，如RIP內端(大寫字母)的5'-AATCTCCAGACAAcCAATA-ACTAAataa-3',及LTR內端的5'gTTAAGT--TAACAGATGTCTCGAAAT-3'。InsAB'所保護的RIR內端部位，正好與InsA之保護部位(5'-TAAataa-3')重疊。本人曾由引子延長作用之實驗，找出insA基因之轉錄起點(+1)及啟動子。此InsA之保護部位正好位於啟動子的-10上游4bp處。因此，InsA及InsAB'結合於此區城，正可阻止下游基因之表現，達到基因自我調控的目的。此外分析InsAB'之胺基酸序列，可找到類似其他轉位子的轉位酵素之motif。由此轉位酵素之motif的發現及InsAB'能保護IS2兩端之反向重複序列的事實，推論InsAB'很可能為IS2之轉位酵素。
Insertion sequence IS2 is a transposable element which was found in the genome of Escherichia coli aiKi other members of Enterobacteriaceae. The entire IS2 consists of 1332 base pairs with two 42-bp inverted repeats at each end. The ORF1 of IS2 encodes a 14-kDa protein. InsA, which binds to a DNA segment containing the RIR of IS2 and the promoter region of insA gene. InsA is autoregulated and can repress the transposition of IS2. Because insA occupies only one third of IS2 and InsA repress the transposition of IS2, IS2 may encode another gene product for the transposition of the sequence.
The presence of a frameshift signal A6G near the end of insA gene and a downstream RNA secondary structure near ORF2 suggested that IS2 may encode a 46-kDa protein resulted from a fusion between ORF1 and ORF2 via a -1 frameshifling. However, this 46-kDa protein was not detected in minicell expression system, probably due to low-level expression of insA and the low frequency of frameshifling. To investigate whether this protein is indeed produced, the insA was cloned downstream from a T7 promoter. With IPTG induction, a protein of 14-kDa and a fusion protein of 46-kDa were observed . The 46-kDa protein was designated as InsAB'.
Gel retardation assay was carried out to elucidate the binding of InsAB' to the inverted repeals of IS2. Result revealed that InsAB' binds to a 89-bp DNA fragment containing RIR, and a 110-bp DNA fragment containing LIR. The protection sequences were determined by footprinling experiment. These sequences were 5'-AATCTCCAGACAACCAATA-ACTTAAATAA-3', located at the inner end and flanking region fo RIR, and 5'-GTTAAGT--TAACAGATGTCTGGAAAT-3'. locked at the inner end of LIR. The protection sequence of lnsA was also determined, which overlaps with the InsAB' protection sequence within RIR and flnanking region. The transcription start site and the promoler region of insA was delermined by primer extension. ResulLs showed that InsA and InsAB' are able lo bind lo the -10 region of insA promoter, indicating that the expression of InsA and InsAB' are antoregulated. Sequenrice analysis revealed that a transposase motil is also present at the C-terminus of InsAB'. This sequence characteristic of InsAB' and its binding ability to the inverted repeats of IS2 suggested that InsAB' is the transposase of IS2.
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