A genome-wide strategy for the identification of essential genes in Staphylococcus aureus
A genome-wide strategy for the identification of essential genes in Staphylococcus aureus
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DOI：
10.1046/j.1365-2958.2002.02832.x
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发表时间：
2002-03-01
影响因子：
3.6
通讯作者：
Zyskind, JW
中科院分区：
文献类型：
--
作者：
Forsyth, RA;Haselbeck, RJ;Zyskind, JW
为了解决抗生素药物开发新方法的需求，我们使用快速猎枪反义RNA方法鉴定了细菌病原体金黄色葡萄球菌的大量必需基因。将金黄色葡萄球菌染色体DNA片段克隆到木糖诱导表达载体中，转化金黄色葡萄球菌。在这个特殊的反义筛选中确定的658个金黄色葡萄球菌基因与生殖支原体基因组(总共包含517个基因)进行同源性比较，得出168个保守基因，其中许多似乎是生殖支原体和其他细菌所必需的。给出了反义RNA的表达特异性地降低其同源mRNA的例子。还描述了一种基于细胞的药物筛选试验，其中反义RNA的表达赋予了针对该基因产物的化合物的特异性敏感性。这种方法使任何必要基因的高通量筛选能够简便地进行开发，而不受其生化功能的影响，从而极大地促进了寻找新的抗生素。
To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.