Forsyth R Allyn, Haselbeck Robert J, Ohlsen Kari L, Yamamoto Robert T, Xu Howard, Trawick John D, Wall Daniel, Wang Liangsu, Brown-Driver Vickie, Froelich Jamie M, C Kedar G, King Paula, McCarthy Melissa, Malone Cheryl, Misiner Brian, Robbins David, Tan Zehui, Zhu Zy Zhan-yang, Carr Grant, Mosca Deborah A, Zamudio Carlos, Foulkes J Gordon, Zyskind Judith W
Elitra Pharmaceuticals, San Diego, CA 92121, USA.
Mol Microbiol. 2002 Mar;43(6):1387-400. doi: 10.1046/j.1365-2958.2002.02832.x.
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.
为满足对抗生素药物研发新方法的需求，我们使用快速鸟枪法反义RNA方法，鉴定出了大量针对细菌病原体金黄色葡萄球菌的必需基因。将金黄色葡萄球菌染色体DNA片段克隆到木糖诱导型表达质粒中，并转化到金黄色葡萄球菌中。在这个特定的反义筛选中鉴定出的658个金黄色葡萄球菌基因与总共包含517个基因的生殖支原体基因组之间的同源性比较，产生了168个保守基因，其中许多基因在生殖支原体和其他细菌中似乎是必需的。文中给出了反义RNA的表达特异性降低其同源mRNA的实例。还描述了一种基于细胞的药物筛选试验，其中反义RNA的表达赋予了对靶向该基因产物的化合物的特异性敏感性。这种方法能够轻松开发用于高通量筛选任何必需基因的试验，而不依赖于其生化功能，从而极大地促进了新型抗生素的寻找。
