Estévez A M, Kierszenbaum F, Wirtz E, Bringaud F, Grunstein J, Simpson L
Howard Hughes Medical Institute, UCLA School of Medicine, Los Angeles, CA 90095-1662, USA.
Mol Biochem Parasitol. 1999 May 15;100(1):5-17. doi: 10.1016/s0166-6851(99)00024-9.
Glutamate dehydrogenase (GDH) was shown previously to bind the 3' oligo[U] tail of the mitochondrial guide RNAs (gRNAs) of Leishmania tarentolae, apparently in the dinucleotide pocket (Bringaud F, Stripecke R, Frech GC, Freedland S, Turck C, Byrne EM, Simpson L. Mol. Cell. Biol. 1997; 17:3915-3923). Bloodstream Trypanosoma brucei cells in culture represent a good system to investigate the genetic effects of knocking out kinetoplastid nuclear genes to test a role in RNA editing, since editing of several mitochondrial genes occurs but is dispensable for viability (Corell RA, Myler P, Stuart K. Mol. Biochem. Parasitol. 1994; 64:65-74 and Stuart K. In: Benne R, editor. RNA editing--the alteration of protein coding sequences of RNA. New York: Ellis Horwood, 1993:25-52). Both GDH alleles of bloodstream T. brucei in culture were replaced by drug resistant markers without any effect on viability. The ratios of edited to unedited mRNAs for several cryptogenes were assayed by primer extension analysis. The steady state abundances of these edited RNAs were unaffected by the double knockout. This evidence suggests that GDH may not play a role in the editing reaction in bloodstream trypanosomes in culture, but this conclusion is tentative since there could be redundant genes for any biological function. We employed a double allelic replacement technique to generate a tetracycline inducible conditional expression of an ectopic copy of the deleted gene in bloodstream trypanosomes in culture. We used this strategy for genes encoding mitochondrial proteins which are not required during this stage of the life cycle, but as a general strategy it should be appropriate for generation of conditional null mutants for essential genes as well.
先前已表明，谷氨酸脱氢酶（GDH）可结合布氏锥虫线粒体引导RNA（gRNA）的3'寡聚[U]尾，显然是在二核苷酸口袋中（Bringaud F，Stripecke R，Frech GC，Freedland S，Turck C，Byrne EM，Simpson L. Mol. Cell. Biol. 1997；17：3915 - 3923）。培养的布氏锥虫血流型细胞是研究敲除动基体核基因对RNA编辑作用的遗传效应的良好系统，因为几种线粒体基因的编辑会发生，但对生存力并非必需（Corell RA，Myler P，Stuart K. Mol. Biochem. Parasitol. 1994；64：65 - 74以及Stuart K. 载于：Benne R主编。RNA编辑——RNA蛋白质编码序列的改变。纽约：Ellis Horwood，1993：25 - 52）。培养的布氏锥虫血流型细胞的两个GDH等位基因都被耐药标记取代，对生存力没有任何影响。通过引物延伸分析测定了几个隐蔽基因的编辑mRNA与未编辑mRNA的比例。这些编辑RNA的稳态丰度不受双敲除的影响。这一证据表明，GDH可能在培养的布氏锥虫血流型细胞的编辑反应中不发挥作用，但这一结论是暂时的，因为任何生物学功能可能都存在冗余基因。我们采用双等位基因替换技术，在培养的布氏锥虫血流型细胞中产生了缺失基因异位拷贝的四环素诱导型条件表达。我们将此策略用于编码生命周期此阶段不需要的线粒体蛋白的基因，但作为一种通用策略，它也应该适用于产生必需基因的条件性无效突变体。
