Identification and Characterization of Cis-Encoded Antisense

发布时间：2023-05-05 19:13

　　背景： 细菌在遇到环境变化,尤其是在应激条件下,其非编码RNA在蛋白表达重编程过程中起到关键作用。这些非编码RNA参与细菌的各种胞内过程,包括耐酸、铁代谢、群体感应、镁和钙离子运输、毒力、ABC转运系统,外膜蛋白合成的抑制及转录因子的表达调控。在已知的非编码RNA中,对反式编码RNA的研究最为广泛。在细菌基因组内,虽然顺式编码的反式转录已被证明是广泛存在的,但它并未引起较多关注。这些反义RNA的合成、生理作用和作用机制至今仍是未知的。 目的： 细菌的复制体是由大量的酶组成的,这些酶相互间的协调作用得以完成染色体复制。通过对伤寒沙门菌转录组的高通量测序分析,发现一系列非编码RNA,其基因位于与细菌复制过程有关基因的互补链。本论文旨在研究这些反义RNA在细菌复制过程中的调节作用。 方法： 1.反义RNA转录起始及终止位点的确定：利用5’和3’RACE (末端快速扩增技术)确定各个反义RNA的转录起始及终止位点。RACE分析技术和Northern blot用以确定反义RNA的全长。 2.菌株构建：构建了相关反义RNA的高表达菌株,其带有可被阿拉伯糖诱导的启动子。将反义RNA的基因全长片段通过...

【文章页数】：105 页

【学位级别】：博士

【文章目录】：
ABSTRACT
摘要
ABBREVIATIONS
CHAPTER 1 Introduction
    1.1 Background
    1.2 Salmonella
    1.3 Bacterial non-coding RNAs
    1.4 Bacterial antisense RNAs
        1.4.1 Characteristics of cis-encoded antisense RNAs
        1.4.2 Mechanisms of action of cis-encoded antisense RNAs in bacteria
        1.4.3 Methods to find asRNAs
    1.5 DNA Replication in Bacteria
        1.5.1 Replication origin architecture
        1.5.2 Overview of bacterial replication initiation
        1.5.3 Elongation of DNA
        1.5.4 Termination of DNA replication
    1.6 Objectives
    1.7 Relevance of the study
    1.8 Experimental design
    1.9 References
CHAPTER 2 An antisense RNA AsdA increases the mRNA stability of the gene encoding the replicationinitiation protein of S. Typhi
    2.1 Introduction
    2.2 Materials and Methods
        2.2.1 Bacterial strains,plasmids,and growth conditions
        2.2.2 Construction of strains overexpressing the asRNAs
        2.2.3 Construction of strains overexpressing the asRNAs in RNase Ⅲ and RNase E mutants
        2.2.4 Construction of fur mutant
        2.2.5 5'-RACE
        2.2.6 3'-RACE
        2.2.7 Growth kinetic analysis
        2.2.8 Expression analysis of asRNA under stress conditions
        2.2.9 Overexpression analysis
        2.2.10 RNA extraction
        2.2.11 Quantitative RT-PCR
        2.2.12 Northern blot analysis
        2.2.13 Growth curves
    2.3 Results
        2.3.1 Identification and mapping of the 5' and 3' ends of AsdA
        2.3.2 Analysis of AsdA expression under different growth conditions
        2.3.3 Strain constructions
        2.3.4 Effect of overexpression of asdA on dnaA mRNA level
        2.3.5 Effect of overexpression of asdA on the growth of S. Typhi
    2.4 Discussion
    2.5 References
CHAPTER 3 Identification and characterization of a cis antisense RNA of parC gene encoding DNAtopoisomerase Ⅳ of S. Typhi
    3.1 Introduction
    3.2 Materials and Methods
        3.2.1 Bacterial strains and culture conditions
        3.2.2 Strain and plasmid construction
        3.2.3 5'-and 3'-RACE
        3.2.4 RNA extraction
        3.2.5 Northern blot analysis
        3.2.6 Quantitative RT-PCR
        3.2.7 Growth curves
        3.2.8 Motility assay
    3.3 Results
        3.3.1 Identification of antisense RNA complementary to parC mRNA
        3.3.2 Expression of AspC under different growth conditions
        3.3.3 Strain constructions
        3.3.4 Effect of overexpression of AspC on parC mRNA level
        3.3.5 Effect of overexpression of AspC on the growth of S. Typhi
    3.4 Discussion
    3.5 References
GENERAL CONCLUSIONS
ACKNOWLEDGEMENTS
LIST OF PUBLICATIONS
APPENDICES



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