第二型Integron之Integrase引發特定部位重組作用之分析

Analysis of site-specific recombination mediated by the class 2 integron DNA integrase

林清玫(Ching-Mei Lin)

指導教授 ： 張瑞烽 ； 張仲羽

高雄醫學大學/醫學院/醫學研究所/碩士(2003年)

摘要

Integron是近幾年來被認為繼抗藥性質體與轉位子外，另一個與細菌獲得抗藥性基因以及抗藥性基因在細菌間散佈有關的機制。目前僅class 1與class 3 integron的intI基因得到實驗證實可主導integron與基因片匣間之特定部位重組作用。第二型integron主要是位在轉位子Tn7及Tn7相關轉位子上，而第二型integron之integrase基因( intI2 )，其open reading frame內有一個內部終止密碼（internal stop codon）－TAA存在，可能導致intI2基因無法產生有活性的integrase產物。本實驗主要在探討class 2 integron之integrase基因（ intI2 ）是否具有催化基因片匣進行特定部位重組作用進入integron之能力。利用傳導試驗（conduction assay）分析IntI2主導基因片匣重組部位與integron間發生特定部位重組進入到integron的頻率。實驗結果顯示，IntI2雖可能具有催化基因片匣與integron進行重組的能力，但頻率顯著低於IntI1所主導的重組結果。
此外，為分析intI2基因產物的活性是否可被活化，利用定點突變（site-directed mutagenesis）分別以alanine ( GCG )、glutamic acid ( GAG )、glutamine ( CAG ) 與 arginine ( CGC ) 取代終止密碼 ( TAA) 。結果發現，突變之intI2基因催化特定部位重組發生的頻率大都較原始intI2基因高，此結果顯示可能是因為原終止密碼的存在，造成integrase基因無法轉譯出完整的integrase酵素，以致影響重組作用的發生。
此外，分析cointegrate的結果，基因片匣與integron間特定重組部位的重組交換是發生在核心部位GTTRRRY之處，且發生特定部位重組形成cointegrate的位置大部份都是位在R388的orfA片匣下游的重組部位。顯示此特定部位之重組作用是在integrase的驅動之下，攜帶抗藥基因和attC site的基因片匣，利用其核心部位重組交換點與integron的特定部位發生重組。同時結果也發現突變後之intI2基因在重組作用上對於辨識結合同源的attI site具特異性。

關鍵字

第二型integron ； integrase酵素 ； 基因片匣 ； 特定部位重組

並列摘要

In recent years, integrons have been demonstrated to play a significant role in the dissemination and acquisition of antibiotic-resistant genes among bacteria. Integron-determined site-specific recombination systems of class 1 and class 3 integrons have been studied. Another class of integron found on transposon Tn7 and its relatives is designated as class 2. It has been mentioned that the integrase gene of class 2 integron, intI2, carries an internal stop codon TAA, which may make this gene inactive. In this study, by using a conduction assay, we have sought to establish the ability of IntI2-mediated site-specific recombination between integron and gene cassette recombination sites. In the event, the IntI2 integrase may appear to promote recombination between cassette recombination sites present in different plasmids, but at frequencies significantly lower than observed with IntI1, the class 1 integron integrase.
In addition, to determine whether the IntI2 integrase could be activated, we changed the internal stop codon ( TAA ) into a triplet coding for alanine ( GCG )、glutamic acid ( GAG )、glutamine ( CAG ) and arginine ( CGC ). In the event, mutagenesis of the stop codon restored activity to IntI2 and promoted site-specific recombination at different frequencies mostly higher than those with wild IntI2. These results indicated that the internal stop codon contained in the intI2 gene might to cause the integrase gene unable to encode the complete integrase enzyme.
In addition, the crossover sequences in recombination were localized to within the core site GTTRRRY by nucleotide sequencing across crossover point in cointegrates. Moreover, the most efficient recombination site of R388 is the attC site downstream of the orfA, and most cointegrates resulted from crossovers at this site. These results agree with previous studies that gene cassette can be integrated into or excised from their receptor element, the integron, via site-specific recombination catalyzed by an integron intI gene-encoded integrase. In cointegration assays, the mutagenic IntI2 recognized the attI2 site as a recombination substrate, this suggested that IntI2 exhibits specificity for its own cognate attI site.

並列關鍵字

Class 2 integron ； integrase ； gene cassette ； site-specific recombination

參考文獻

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