Identification and Characterization of the Gene Responsible for the O³ Mating Type Substance in Paramecium caudatum

The process of sexual reproduction in eukaryotes starts when gametes from two different sexes encounter each other. Paramecium, a unicellular eukaryote, undergoes conjugation and uses a gametic nucleus to enter the sexual reproductive process. The molecules responsible for recognizing mating partners, hypothetically called mating-type substances, are still unclear. We have identified an O³-type mating substance polypeptide and its gene sequence using protein chemistry, molecular genetics, immunofluorescence, RNA interference, and microinjection. The O³-type substance is a polypeptide found in the ciliary membranes, located from the head to the ventral side of cells. The O³-type substance has a kinase-like domain in its N-terminal part located outside the cell and four EF-hand motifs that bind calcium ions in its C-terminal part located inside the cell. RNA interference and immunofluorescence revealed that this polypeptide positively correlated with the expression of mating reactivity. Microinjection of an expression vector incorporating the O³Pc-MSP gene (Oms3) induced additional O³ mating type in the recipient clones of different mating types or syngen. Phylogenetic analysis indicates that this gene is widely present in eukaryotes and exhibits high homology among closely related species. The O³Pc-MSP (Oms3) gene had nine silent mutations compared to the complementary mating type of the E³ homologue gene.