Whole Genome Sequencing Identifies a Novel Factor Required for Secretory Granule Maturation in Tetrahymena thermophila.
Whole Genome Sequencing Identifies a Novel Factor Required for Secretory Granule Maturation in Tetrahymena thermophila.
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DOI：
10.1534/g3.116.028878
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发表时间：
2016-08-09
期刊：
影响因子：
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通讯作者：
Turkewitz AP
中科院分区：
文献类型：
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作者：
Kontur C;Kumar S;Lan X;Pritchard JK;Turkewitz AP
无偏遗传方法具有识别与特定生物学途径相关的新基因的独特能力。由于下一代测序，正向遗传策略可以扩展到更广泛的模式生物。在纤毛虫嗜热四膜虫中，分泌颗粒（称为粘液囊）的形成部分依赖于祖先的溶酶体分选机制，但也可能涉及新的因素。在之前的工作中，通过亚硝基胍诱变产生了粘液囊生物发生缺陷的多个菌株，并使用遗传学和细胞生物学方法对其进行了表征，但遗传病变本身是未知的。在这里，我们展示了通过全基因组测序分析一个这样的突变揭示了粘液囊肿形成的一个新因素。菌株UC620在粘液囊成熟过程中存在形态和生化缺陷，这一过程类似于动物致密核颗粒的成熟过程。对一组UC620 F2克隆进行Illumina测序，发现了一个名为MMA1 （Mucocyst mature）的新基因的错义突变。UC620的缺陷通过表达MMA1的野生型拷贝和破坏其他野生型菌株表型UC620中的MMA1来修复。MMA1的产物，以cfp标记的拷贝为特征，编码一个大的可溶性细胞质蛋白。一小部分Mma1p-CFP呈颗粒状，这可能反映了与核内体的关联。除了在其他四膜虫物种中，该基因没有可识别的同源物，因此代表了颗粒成熟所需的进化上的最新创新。
Unbiased genetic approaches have a unique ability to identify novel genes associated with specific biological pathways. Thanks to next generation sequencing, forward genetic strategies can be expanded to a wider range of model organisms. The formation of secretory granules, called mucocysts, in the ciliate Tetrahymena thermophila relies, in part, on ancestral lysosomal sorting machinery, but is also likely to involve novel factors. In prior work, multiple strains with defects in mucocyst biogenesis were generated by nitrosoguanidine mutagenesis, and characterized using genetic and cell biological approaches, but the genetic lesions themselves were unknown. Here, we show that analyzing one such mutant by whole genome sequencing reveals a novel factor in mucocyst formation. Strain UC620 has both morphological and biochemical defects in mucocyst maturation—a process analogous to dense core granule maturation in animals. Illumina sequencing of a pool of UC620 F2 clones identified a missense mutation in a novel gene called MMA1 (Mucocyst maturation). The defects in UC620 were rescued by expression of a wild-type copy of MMA1, and disrupting MMA1 in an otherwise wild-type strain phenocopies UC620. The product of MMA1, characterized as a CFP-tagged copy, encodes a large soluble cytosolic protein. A small fraction of Mma1p-CFP is pelletable, which may reflect association with endosomes. The gene has no identifiable homologs except in other Tetrahymena species, and therefore represents an evolutionarily recent innovation that is required for granule maturation.