The Transmembrane Protein Semi1 Positions Gamete Nuclei for Reciprocal Fertilization in Tetrahymena

Abstract

SummaryDuring sexual reproduction in the ciliate, Tetrahymena thermophila, cells of complementary mating type pair (“conjugate”) undergo simultaneous meiosis and fertilize each other. In both mating partners only one of the four meiotic products is “selected” to escape autophagy, and this nucleus divides mitotically to produce two pronuclei. The migrating pronucleus of one cell translocates to the mating partner and fuses with its stationary pronucleus and vice versa. Selection of the designated gametic nucleus was thought to depend on its position within the cell because it always attaches to the junction with the partner cell. Here we show that a transmembrane protein, Semi1, is crucial for attachment. Loss of Semi1 causes failure to attach and consequent infertility. However, a nucleus is selected and gives rise to pronuclei regardless of Semi1 expression, indicating that attachment of a nucleus to the junction is not a precondition for selection but follows the selection process.

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue in Tetrahymena thermophila

“…RNAi vectors were constructed essentially as described ( 87 ). RebL1 target sequence used in hairpin RNA constructs was amplified from CU428 genomic DNA using PrimeSTAR Max DNA polymerase with primers as listed in Supplemental Table S13 .…”

“…To create the hairpin cassette, amplified target fragments were cloned into the BamHI–BamHI and PstI–PstI sites, respectively, of pAkRNAi-NEO5, which was kindly provided by Dr Takahiko Akematsu, with the NEBuilder HF DNA Assembly kit. In this system, the NEO5 cassette of the backbone plasmid, which confers paromomycin resistance, has been replaced by a puromycin resistance marker (PAC) under the MTT2 copper-inducible promoter ( 87 ). The resulting plasmid was linearized with SacI and KpnI before biolistic transformation.…”

Novel requirements for HAP2/GCS1-mediated gamete fusion in Tetrahymena

“…Using a reverse genetics approach designed to identify genes involved in conjugation, 41 we found two instances (Tetrahymena Genome Database: TTHERM_000161578, TTHERM_00569470) in which the deletion of a gene that is upregulated during this process ( Figures S2 A and S2B) resulted in unstable pairing between complementary mating types lacking the same gene. In each case, mutant strains recognized and adhered to one another at the conjugation junction ( Figures 1 A–1D) but could be separated by vigorous pipetting.…”

Functional characterization of RebL1 highlights the evolutionary conservation of oncogenic activities of the RBBP4/7 orthologue inTetrahymena thermophila

“…RNAi vectors were constructed essentially as described (102). Briefly, RebL1 target sequence used in hairpin RNA constructs was amplified from CU428 genomic DNA using PrimeSTAR Max DNA polymerase with primers as listed in Supplemental Table S8.…”

“…To create the hairpin cassette, amplified target fragments were cloned into the BamHI–BamHI and PstI–PstI sites, respectively, of pAkRNAi-NEO5, which was kindly provided by Dr. Takahiko Akematsu, with the NEBuilder HF DNA Assembly kit. In this system, the NEO5 cassette of the backbone plasmid, which confers paromomycin resistance, has been replaced by a puromycin resistance marker (PAC) under the MTT2 copper-inducible promoter (102). The resulting plasmid was linearized with SacI and KpnI before biolistic transformation.…”