Please use this identifier to cite or link to this item: http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/111387
Title: 基因表現對細菌基因位點的動態行為及相鄰核醣核酸聚合酶空間分布的影響
The motions of chromosomal loci and the vicinal distributions of RNA polymerase influenced by gene expression in E.coli
Authors: 張宜仁
Chang, Yi-Ren
鄧安祐
Teng, An-Yu
Keywords: 染色體分布
RNA聚合酶
基因表現
轉錄工廠
chromosome organization
RNA polymerase
gene expression
transcription factories
Issue Date: 2020
Abstract: 染色體的組織和基因表現之間存在著一定的關聯性,然而,對於原核細胞來說,基因表現具體是如何影響染色體組織,依舊是一個需要進行更多實驗驗證與討論的問題。其中,轉錄作用對染色體的組織更是扮演了一個相當重要的角色。轉錄工廠(transcription factories)便是在探討轉錄作用與染色體組織的課題之一。轉錄工廠的現象是指旺盛的轉錄行為可能會發生在細胞內部幾個離散的區域當中,RNA聚合酶(RNA polymerase)和其他的轉錄因子(transcription factors),以及被旺盛表達的基因會一同聚集在「工廠」,藉此達到最高的轉錄效率。然而在原核細胞當中我們無法以現有的螢光顯微術,直接觀察到這麼微小的結構。只能以各種旁敲側擊的實驗來印證轉錄工廠的存在。而從前人的驗證實驗當中,卻也一直都缺乏同時記錄基因與RNA聚合酶的位置資訊。   為此在本次實驗中,會以TetR-YFP/tetO的螢光抑制操作系統標定目標基因,並用單分子定位顯微(single-molecule localization microscopy)觀察由PAmCherry標定的RNA聚合酶,藉此同時得到基因位點與RNA聚合酶在細胞中的位置。之後再進一步分析RNA聚合酶的群聚行為,並探討基因表現旺盛與否,基因位點周圍RNA聚合酶的群聚現象。也藉由TetR-YFP/tetO和LacI-mScarlet/lacO標定目標基因的上下游,觀察基因表現對於染色體運動行為產生的影響。   而從結果可以看出,RNA聚合酶的分布,相較於基因表現的旺盛程度,可能更容易受到染色體的結構而影響。從軌跡追蹤實驗中,發現基因的表現的確會對基因位點的運動造成差異,然而這些差異似乎還是主要源自於類核自身的結構,這個結果與前一部分的實驗結果相互印證。而我們從擬合出軌跡互相關函數得到的DNA表觀尺寸也可以初步知道,致使基因位點關聯性下降的原因,有可能是因為染色體的聚集,使DNA產生糾纏,因此增加了傳遞的困難。
There is a certain correlation between the organization of chromosomes and gene expression. However, for prokaryotic cells, how gene expression specifically affects chromosome organization is still a problem that needs more experimental verification and discussion. Transcription plays a important role in the organization of chromosome. “Transcription factories” is one of the topics that discusses transcription and chromosome organization. The phenomenon of transcription factories means that vigorous transcription may occur in several discrete areas inside the cell. RNA polymerase and transcription factors, as well as the genes that need to be expressed vigorously, will gather together in the "factories" to achieve the highest transcription efficiency. However, we cannot directly observe such tiny structures in prokaryotic cells with the existing fluorescence microscopy. In previous verification experiments, there has been a lack of simultaneous recording of gene and RNA polymerase location information. For this reason, in our experiment, the target gene tagged with the fluorescent repressor-operator system of TetR-YFP/tetO, and single-molecule localization microscopy used to observe the RNA polymerase tagged by PAmCherry. Afterwards, we further analyzed the aggregation behavior of RNA polymerase, and the clustering phenomenon of RNA polymerase around the gene locus. The TetR-YFP/tetO and the LacI-mScarlet/lacO pairs had been applied as the loci tags at the upstream and downstream of the target gene. The influence of gene expression on chromosome movement had also been observed. It can be seen from the results that the distribution of RNA polymerase may be more susceptible to the structure of chromosomes than the exuberance of gene expression. In the single molecule trajectory tracking experiment, it is found that the performance of the gene does cause slight differences in the movement of the gene locus. However, these differences seem to be mainly due to the structure of the nucleoid itself, which is mutually corroborated by the experimental results in the previous part. From the “apparent size” of the DNA obtained by fitting the cross-correlation function of the trajectory, we can also initially know, the reason for the decline in the relevance of gene loci may be that the aggregation of chromosomes makes DNA entangled, thus increasing the difficulty of transmission.
URI: http://etds.lib.ntnu.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=id=%22G060741007S%22.&
http://rportal.lib.ntnu.edu.tw:80/handle/20.500.12235/111387
Other Identifiers: G060741007S
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