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Whole genome sequencing identifies a novel factor required for secretory granule maturation in Tetrahymena thermophila

By Cassandra Kontur, Santosh Kumar, Xun Lan, Jonathan K Pritchard, Aaron P Turkewitz

Posted 01 Mar 2016
bioRxiv DOI: 10.1101/042085 (published DOI: 10.1534/g3.116.028878)

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 into 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 defect 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 wildtype copy of MMA1, and disruption of MMA1 in an otherwise wildtype strain generated a phenocopy of 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.

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