Mamoun C B, Gluzman I Y, Goyard S, Beverley S M, Goldberg D E
Department of Molecular Microbiology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8230, St. Louis, MO 63110, USA.
Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8716-20. doi: 10.1073/pnas.96.15.8716.
Genomic information is rapidly accumulating for the human malaria pathogen, Plasmodium falciparum. Our ability to perform genetic manipulations to understand Plasmodium gene function is limited. Dihydrofolate reductase is the only selectable marker presently available for transfection of P. falciparum. Additional markers are needed for complementation and for expression of mutated forms of essential genes. We tested parasite sensitivity to different drugs for which selectable markers are available. Two of these drugs that were very effective as antiplasmodial inhibitors in culture, blasticidin and geneticin (G418), were selected for further study. The genes BSD, encoding blasticidin S deaminase of Aspergillus terreus, and NEO, encoding neomycin phosphotransferase II from transposon Tn 5, were expressed under the histidine-rich protein III (HRPIII) gene promoter and tested for their ability to confer resistance to blasticidin or G418, respectively. After transfection, blasticidin and G418-resistant parasites tested positive for plasmid replication and BSD or NEO expression. Cross-resistance assays indicate that these markers are independent. The plasmid copy number and the enzymatic activity depended directly on the concentration of the drug used for selection. These markers set the stage for new methods of functional analysis of the P. falciparum genome.
人类疟原虫恶性疟原虫的基因组信息正在迅速积累。我们通过基因操作来了解疟原虫基因功能的能力有限。二氢叶酸还原酶是目前可用于恶性疟原虫转染的唯一选择标记。还需要其他标记来进行互补以及表达必需基因的突变形式。我们测试了疟原虫对有可用选择标记的不同药物的敏感性。选择了两种在培养中作为抗疟原虫抑制剂非常有效的药物，即杀稻瘟菌素和遗传霉素（G418）进行进一步研究。编码土曲霉杀稻瘟菌素S脱氨酶的BSD基因和编码转座子Tn 5的新霉素磷酸转移酶II的NEO基因，在富含组氨酸的蛋白III（HRPIII）基因启动子的控制下表达，并分别测试它们赋予对杀稻瘟菌素或G418抗性的能力。转染后，对杀稻瘟菌素和G418具有抗性的疟原虫对质粒复制以及BSD或NEO表达呈阳性。交叉抗性试验表明这些标记是独立的。质粒拷贝数和酶活性直接取决于用于选择的药物浓度。这些标记为恶性疟原虫基因组功能分析的新方法奠定了基础。
