Identification and utilization of a mutated 60S ribosomal subunit coding gene as an effective and cost-efficient selection marker for Tetrahymena genetic manipulation.

Since the onset of molecular biology, the ciliate Tetrahymena thermophila has been one of the most convenient single-celled model eukaryotes for genetics, biochemistry, and cell biology. Particularly, thanks to the availability of several different selection markers, it is possible to knock out or knock in genes at multiple genetic loci simultaneously in Tetrahymena, which makes it an excellent model ciliate for tackling complex regulatory mechanisms. Despite these selection markers are efficient for genetic manipulation, the costly drugs used for selection have highlighted the urgent demand for an additional cost-efficient and effective selection marker. Here, we found that a mutated 60S ribosomal subunit component, RPL36A, confers Tetrahymena with cycloheximide resistance. On top of that, we developed a cycloheximide cassette and explored suitable transformation and selection conditions. Using the new cassette, we obtained both knock-out and knock-in strains successfully at a relatively low cost. This study also provided the first evidence that a cycloheximide resistance gene can be engineered as a selection marker to completely delete a gene from the highly-polyploid somatic nucleus in Tetrahymena.

Redescription and molecular characterization of two Trichodina species (Ciliophora, Peritrichia, Mobilida) from freshwater fish in China.

During an investigation of parasitic ciliates in northern China, two Trichodina species, T. acuta Lom, 1970 and T. nigra Lom, 1960, were isolated from the freshwater fish Cyprinus carpio Linnaeus, 1758. The morphology of each species was investigated based on dry silver nitrate-stained specimens. In addition, the molecular phylogeny of each was analyzed based on small subunit ribosomal DNA (SSU rDNA) sequence data. Trichodina acuta can be distinguished from its congeners by the undefined periphery of the central circle, the distinct gap between the rays and the central circle, and the distinctly sickle-shaped blades. Trichodina nigra is a cosmopolitan ciliate and is characterized by its densely linked denticles, broad, rounded spatula-shaped blades, robust central parts, and well developed rays. Phylogenetic analyses revealed that T. acuta and T. nigra nest within different clades, supporting the assertion that the GC content of SSU rDNA sequences could reflect evolutionary relationships among Trichodina species.