6-methylpurine (6mp) is a toxic analog of adenine that inhibits RNA and protein synthesis and interferes with adenine salvage mediated by adenine phosphoribosyltransferase (APRTase). Mutants of the ciliated protist Tetrahymena thermophila that are resistant to 6mp were isolated in 1974, but the mechanism of resistance has remained unknown. To investigate 6mp resistance in T. thermophila, we created 6mp-resistant strains and identified a mutation in the APRTase genomic locus (APRT1) that is responsible for 6mp resistance. While overexpression of the mutated APRT1 allele in 6mp-sensitive cells did not confer resistance to 6mp, reduced wild-type APRT1 expression resulted in a significant decrease in sensitivity to 6mp. Knocking out or reducing the expression of APRT1 by RNA interference (RNAi) did not affect robust cell growth, which indicates that adenine salvage is redundant or that de novo synthesis pathways provide sufficient adenosine monophosphate for viability. We also explored whether 6mp resistance could be used as a novel inducible selection marker by generating 6mp- and paromomycin-resistant double mutants. While 6mp- and paromomycin-resistant double mutants did express fluorescent proteins in an RNAi-based system, the system requires optimization before 6mp resistance can be used as an effective inducible selection marker.

During sexual reproduction/conjugation of the ciliate Tetrahymena thermophila, the germinal micronucleus undergoes meiosis resulting in four haploid micronuclei (hMICs). All hMICs undergo post-meiotic DNA double-strand break (PM-DSB) formation, cleaving their genome. DNA lesions are subsequently repaired in only one ‘selected’ hMIC, which eventually produces gametic pronuclei. DNA repair in the selected hMIC involves chromatin remodeling by switching from the heterochromatic to the euchromatic state of its genome. Here, we demonstrate that, among the 15 Tetrahymena Snf2 family proteins, a core of the ATP-dependent chromatin remodeling complex in Tetrahymena, the germline nucleus specific Iswi in Tetrahymena IswiG^Tt and Rad5^Tt is crucial for the generation of gametic pronuclei. In either gene knockout, the selected hMIC which shows euchromatin markers such as lysine-acetylated histone H3 does not appear, but all hMICs in which markers for DNA lesions persist are degraded, indicating that both IswiG^Tt and Rad5^Tt have important roles in repairing PM-DSB DNA lesions and remodeling chromatin for the euchromatic state in the selected hMIC.

Toxoplasma gondii is an intracellular parasitic protozoan with a high infection rate in mammals, including humans, and birds. There is no effective vaccine, and treatment relies on antiparasitic drugs. However, existing antiprotozoal drugs have strong side effects and other problems; therefore, new treatment approaches are needed. Metal nanoparticles have attracted increased interest in the biomedical community in recent years because of their extremely high surface area to volume ratio and their unique reactivity that could be exploited for medicinal purposes. Previously, we confirmed the anti-Toxoplasma effects of gold, silver, and platinum nanoparticles, in a growth inhibition test. Here, we asked whether the anti-Toxoplasma effect could be confirmed with less expensive metal nanoparticles, specifically iron oxide nanoparticles (goethite and hematite). To improve the selective action of the nanoparticles, we modified the surface with l-tryptophan as our previous findings showed that the bio-modification of nanoparticles enhances their selectivity against T. gondii. Fourier-Transform Infrared Spectroscopy (FTIR) analysis confirmed the successful coating of the iron oxide nanoparticles with l-tryptophan. Subsequently, cytotoxicity and growth inhibition assays were performed. L-tryptophan-modified nanoparticles showed superior anti-Toxoplasma action compared to their naked nanoparticle counterparts. L-tryptophan enhanced the selective toxicity of the iron oxide nanoparticles toward T. gondii. The bio-modified nanoparticles did not exhibit detectable host cell toxicity in the effective anti-Toxoplasma doses. To elucidate whether reactive oxygen species contribute to the anti-Toxoplasma action of the bio-modified nanoparticles, we added Trolox antioxidant to the assay medium and found that Trolox appreciably reduced the nanoparticle-induced growth inhibition.

Serine/arginine-rich splicing factor 1 (SR1), an orthologue of hypothetical RNA-binding protein (HRB1) in yeast, is essential to the asexual development of

Jeong Min Chung, Toshinobu Suzaki, Yasuhiro Fukuda, and Chihong Song. J. Microbiol. Biotechnol. 2026;36:1-8. https://doi.org/10.4014/jmb.2608.08034