Episomally driven antisense mRNA abrogates the hyperinducible expression and function of a unique cell surface class I nuclease in the primitive trypanosomatid parasite, Crithidia luciliae.
Episomally driven antisense mRNA abrogates the hyperinducible expression and function of a unique cell surface class I nuclease in the primitive trypanosomatid parasite, Crithidia luciliae.
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游离驱动的反义 mRNA 消除了原始锥虫寄生虫 Crithidia luciliae 中独特的细胞表面 I 类核酸酶的超诱导表达和功能。
DOI：
10.1016/j.jmb.2007.08.014
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发表时间：
2007
影响因子：
5.6
通讯作者：
Dwyer,DennisM
中科院分区：
文献类型：
--
作者：
Yamage,Mat;Joshi,ManjuB;Dwyer,DennisM
在这里，我们发现，Crithidia luciliae，一个原始的锥虫，嘌呤营养缺陷型，上调表达其独特的，双功能的，表面膜3′-核苷酸酶/核酸酶（Cl 3′NT/NU）活性的1000倍，响应嘌呤饥饿。还发现第二种表面膜磷酸单酯酶，即抗酒石酸酸性磷酸酶（Cl MAcP）在这种嘌呤饥饿的细胞中上调表达。在这里，我们使用同源附加表达的反义构建的Cl 3 ′NT/NU解剖这两个表面膜酶的功能表达。在反义转染的细胞中，产生了大量过量的反义转录本，并且没有检测到任何内源性Cl 3 'NT/NU有义信息的痕迹。此外，与对照组相比，这些转染细胞中嘌呤饥饿过度诱导的3′NT/NU酶活性水平完全消失。此外，这种反义转录完全消除了这些转染子在含有poly（A）的培养基中生长的能力，证明了3′NT/NU对于该寄生虫的生长/存活的本质。相反，反义转录对内源性或嘌呤饥饿诱导的MAcP酶活性水平、其稳态mRNA水平或这些转染子中管家基因（例如Cl α-微管蛋白）的组成型表达没有明显的有害影响。反义实验结果表明，Cl 3′NT/NU表面膜的功能性核酸酶活性对这些原始寄生虫的生长发育是至关重要的。
Here, we show that Crithidia luciliae, a primitive trypanosomatid, purine auxotroph, up-expressed its unique, bi-functional, surface membrane 3′-nucleotidase/nuclease (Cl 3′NT/NU) activity by ∼1000-fold in response to purine starvation. A second surface membrane phospho-monoesterase, i.e. a tartrate-resistant acid phosphatase (Cl MAcP) was also found to be up-expressed in such purine-starved cells. Here, we used homologous episomal-expression of an antisense construct of the Cl3′NT/NU to dissect the functional expression of these two surface membrane enzymes. In antisense transfected cells, a large excess of the antisense transcript was produced and no trace of any endogenous Cl3′NT/NU sense message was detected. Further, the purine-starvation hyper-induced levels of 3′NT/NU enzyme activity were completely abrogated in these transfected cells versus controls. Moreover, such antisense transcription completely abolished the ability of these transfectants to grow in poly(A)-containing medium demonstrating the essential nature of the 3′NT/NU for the growth/survival of this parasite. In contrast, antisense transcription had no apparent deleterious effects on either endogenous or purine-starvation-induced levels of MAcP enzyme activity, its steady-state mRNA levels, or the constitutive expression of house-keeping genes (e.g. Cl α-tubulin) in these transfectants. Cumulatively, results of our antisense experiments demonstrated that the functional nuclease activity of the surface membrane Cl 3′NT/NU was, in fact, critical/essential for the growth and development of these primitive parasites.