A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes.
A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes.
复制标题
DOI：
10.1111/j.1574-6968.2012.02503.x
复制
发表时间：
2012-04
影响因子：
2.1
通讯作者：
Xu HH
中科院分区：
文献类型：
--
作者：
Meng J;Kanzaki G;Meas D;Lam CK;Crummer H;Tain J;Xu HH
调控反义RNA （asRNA）表达已成功应用于革兰氏阳性菌的全基因组必需基因鉴定和药物靶点确定。然而，目前尚未有报道描述asRNA基因沉默在革兰氏阴性菌必需基因的综合分析中的应用。在这项研究中，我们报道了大肠杆菌必需基因的asRNA结构的第一个全基因组鉴定。我们使用配对末端表达载体（pHN678）从大肠杆菌的两个基因库中筛选出25万个文库转化子，用于条件生长抑制重组克隆。在对675个已确认的诱导剂敏感细胞克隆的质粒插入物进行测序后，我们鉴定出152个独立的asRNA结构，其中134个插入物来自必需基因，18个来自非必需基因（但与必需基因共享操纵子）。在这些asRNA结构沉默的79个个体必需基因中，61个基因（77%）参与与蛋白质合成相关的过程。以fusA（编码伸长因子G）为靶点的asRNA克隆的细胞实验表明，诱导细胞对已知特异性抑制剂富西地酸敏感12倍。我们的研究结果证明了配对末端表达载体的实用性，以及利用调控asRNA表达在大肠杆菌中大规模基因沉默的可行性。
Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published reports describing the application of asRNA gene silencing for comprehensive analyses of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250,000 library transformants for conditional growth-inhibitory recombinant clones from two shot-gun genomic libraries of E. coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer-sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes while 18 originated from non-essential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12 fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E. coli using regulated asRNA expression.
登录
查看更多内容
影响因子：
4.9
作者：
Young, K;Jayasuriya, H;Wang, J
通讯作者：
Wang, J
影响因子：
11.8
作者：
Boucher, Helen W.;Talbot, George H.;Bartlett, John
通讯作者：
Bartlett, John
影响因子：
14.9
作者：
Nakashima N;Tamura T;Good L
通讯作者：
Good L
影响因子：
14.9
作者：
Nakashima N;Tamura T
通讯作者：
Tamura T
DOI：
10.1016/j.bbrc.2006.08.166
发表时间：
2006-11-03
影响因子：
3.1
作者：
Xu, H. Howard;Real, Lilian;Bailey, Melissa Wu
通讯作者：
Bailey, Melissa Wu