膜蛋白轉錄轉譯以及細胞膜的嵌入對染色體運動的影響

原核生物的DNA位在沒有膜的類核中，這使得其染色體能有機會和細胞的內膜產生互動。關於染色體跟細胞膜之間互動的研究，轉嵌現象（transertion）是其中一個重要的課題。此現象意指原核生物的膜蛋白基因在表達時，RNA的轉錄、蛋白質的轉譯、以及肽鏈會嵌進細胞膜中折疊成膜上的蛋白質等三個事件是會同時發生的。然此現象目前尚未被完全證實，僅有間接的現象來支持這個假說，無法肯定染色體和細胞膜之間是不是有物理上的連結。為此，我們提出在膜蛋白基因上下游標記螢光的方式，藉由單分子追蹤的方法觀察該上下游染色體位點的運動，藉以觀察細胞膜、膜蛋白基因表達以及染色體之間的關係，進而對膜蛋白基因在表達時是否會有轉嵌現象來提出證據。我們選擇用乳糖操縱子中的lacY膜蛋白基因來當作我們欲觀察的膜蛋白基因，並利用ParB-mCherry/parC2標記系統以及TetR-YFP/tetO標記系統，在lacY基因的上下游標記螢光。此外觀察基因在表現時，大量RNA聚合酶的聚集以及觀察膜蛋白基因表達使得染色體稍微離開類核下，對染色體運動造成的影響。我們的結果發現染色體的運動為碎形布朗運動。關於膜蛋白基因表達對染色體的影響，就染色體位置的分布而言，我們發現 lacY基因的表達會使得周遭的染色體往細胞膜的方向移動；細胞質蛋白基因的表達則會使得遠處的染色體往類核的內部移動。而就染色體運動的狀態而言，我們發現lacY基因的表達會使得周遭染色體的運動增加相關性；而細胞質蛋白基因的表達則會使得周遭染色體的運動相關性下降。我們推論當lacY基因在表達時，附近團繞在一起的染色體會增加一起移動的傾向，並且往細胞膜的方向移動。此結果可以支持轉嵌假說中，在表達的DNA、RNA聚合酶、mRNA、核醣體、肽鏈會組成大型複合物並且肽鏈會嵌進細胞膜中的說法。

關鍵字

轉嵌假說； lacY 膜蛋白基因表達；染色體和細胞膜的互動；單分子追蹤

並列摘要

The defining property of prokaryotes—the absence of a nucleus—opens the possibility for interactions between the chromosome and the cytoplasmic membrane. The phenomenon of transertion is one of the important topics in the study of the interaction between chromosome and cell membrane. Transertion means the coupled transcription, translation and insertion of nascent proteins into and through membrane. Despite early evidence in its favor, this hypothesis lacked further evidence to support it for long time. Here we used single-particle tracking method to study the correlations of movement between two fluorescently labeled gene loci on both sides of the E.coli gene during which gene is expressing or not. By study chromatin motion in live cells, we hope to provide further evidence for whether the membrane protein gene will be inserted during expression. The lacY membrane protein gene had been chosen as in the lactose operon as the target membrane protein gene, and the ParB-mCherry/parC2 and the TetR-YFP/tetO pairs had been applied as the loci tags at the upstream and downstream of the lacY gene. The effect of aggregation of large amounts of RNA polymerase and off–nucleus behaviors of chromosome had also been observed during gene expression. Our results show that the chromosome loci behave fractal Brownian motions. In terms of the distribution of chromosomal locations, we found that the expression of lacY gene causes the surrounding chromosome to move in the direction of cell membrane; the expression of the cytoplasmic protein gene causes the distant chromosome to move inside the nucleus. In terms of the state of chromosome movement, it has been found that the expression of the lacY gene increases the correlation between the movements of the surrounding chromosome; the expression of the cytoplasmic protein gene causes a decrease in the motion correlation of the surrounding chromosome. We conclude that when the lacY gene is expressed, the chromosome that are clustered together will increase the tendency to move together and move toward the cell membrane. These results agree with the transertion hypothesis.