Cdk1 and cks gene homologs are transcriptionally activated during induction of conjugating pairs in mating-type II cells of the ciliate Blepharisma japonicum

抄録

In Blepharisma japonicum, conjugation is induced by interaction between cells of complementary mating-types I and II. Cells of both mating-types produce and secrete mating pheromones (gamones). Cells that have received complementary gamones undergo morphological changes (rounding) and start to unite. Nuclear changes, including meiosis, gametic nuclei exchange, fertilization and development of new macro- and micronuclei occur in the conjugants. Although conjugation is such a striking phenomenon, the molecular mechanism of induction of conjugation remains unknown. In order to identify genes that are involved in formation of conjugating pairs, we isolated genes that were expressed specifically in conjugation-induced type II cells, using suppression subtractive hybridization. To induce conjugation, we treated type II cells for 4 hours with a cell-free fluid from type I cells which contained gamone1. During this period, type II cells formed pairs, although these homotypic pairs never entered meiosis. We then purified polyA⁺RNA and subjected it to cDNA synthesis. This cDNA was then subtracted with cDNA that was prepared from untreated cells. We obtained eight gene fragments. Homology searches revealed that three of these fragments showed significant homology to the cdk family (cdk1 and cdk2), 4-hydroxy-phenylpyruvate dioxygenase (4-HPPD) and cyclin dependent kinase regulatory subunit (cks). Northern hybridization demonstrated that these three genes were specifically transcribed in cells treated with gamone1. We also found that the transcripts had already appeared 2 hours after the onset of gamone1 treatment. Cdk1 and cks are generally involved in cell-cycle regulation, but are here specifically expressed during induction of homotypic type II pairs that undergo neither mitosis nor meiosis.