During development of the somatic macronucleus from the germline micronucleus in ciliates, chromosome rearrangements occur in which specific regions of DNA are eliminated and flanking regions are healed, either by religation or construction of telomeres. We identified a gene, TWI1, in Tetrahymena thermophila that is homologous to piwi and is required for DNA elimination. We also found that small RNAs were specifically expressed prior to chromosome rearrangement during conjugation. These RNAs were not observed in TWI1 knockout cells and required PDD1, another gene required for rearrangement, for expression. We propose that these small RNAs function to specify sequences to be eliminated by a mechanism similar to RNA-mediated gene silencing.

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.

Phosphorylation of histone H3 at serine 10 occurs during mitosis in diverse eukaryotes and correlates closely with mitotic and meiotic chromosome condensation. To better understand the function of H3 phosphorylation in vivo, we created strains of Tetrahymena in which a mutant H3 gene (S10A) was the only gene encoding the major H3 protein. Although both micronuclei and macronuclei contain H3 in typical nucleosomal structures, defects in nuclear divisions were restricted to mitotically dividing micronuclei; macronuclei, which are amitotic, showed no defects. Strains lacking phosphorylated H3 showed abnormal chromosome segregation, resulting in extensive chromosome loss during mitosis. During meiosis, micronuclei underwent abnormal chromosome condensation and failed to faithfully transmit chromosomes. These results demonstrate that H3 serine 10 phosphorylation is causally linked to chromosome condensation and segregation in vivo and is required for proper chromosome dynamics.

H3 phosphorylation has been correlated with mitosis temporally in mammalian cells and spatially in ciliated protozoa. In logarithmically growing Tetrahymena thermophila cells, for example, H3 phosphorylation can be detected in germline micronuclei that divide mitotically but not in somatic macronuclei that divide amitotically. Here, we demonstrate that micronuclear H3 phosphorylation occurs at a single site (Ser-10) in the amino-terminal domain of histone H3, the same site phosphorylated during mitosis in mammalian cells. Using an antibody specific for Ser-10 phosphorylated H3, we show that, in Tetrahymena, this modification is correlated with mitotic and meiotic divisions of micronuclei in a fashion that closely coincides with chromosome condensation. Our data suggest that H3 phosphorylation at Ser-10 is a highly conserved event among eukaryotes and is likely involved in both mitotic and meiotic chromosome condensation.In eukaryotic cells, DNA is closely associated with histone proteins in the form of chromatin, packaging DNA in a way that remains only partially understood. Although the structure of the nucleosome core is now known in considerable detail (1), how higher order chromatin structures are folded and unfolded to accommodate processes such as transcription, replication, and chromosome segregation remains unclear. During mitosis, DNA is compacted nearly 10,000-fold to ensure proper segregation of the genetic material to daughter cells. Faithful segregation of sister chromatids requires proper condensation of the chromatin during entry into mitosis and decondensation of the fiber during exit from mitosis and is essential for the viability of the cells.Specific posttranslational modifications of histones, particularly acetylation and phosphorylation, correlate well with dynamic aspects of the folding and unfolding of the chromatin fiber (2). For example, hyperphosphorylation of linker histone H1 is temporally coupled with entry into mitosis and has often been presumed to function in mitotic chromatin condensation (3, 4). However, recent experiments show that chromatin condensation can occur in vivo (5) or in vitro (6, 7) in the absence of H1. Furthermore, H1 hyperphosphorylation does not occur in premature chromatin condensation induced by fostriecin (8) or okadaic acid (9). Therefore, the exact function of H1 hyperphosphorylation in mitosis remains unclear.In contrast to H1 hyperphosphorylation, site-specific phosphorylation of core histone H3 at serine 10 seems to occur exclusively during mitosis in mammalian cells (10, 11). Moreover, fostriecin and okadaic acid, which initiate premature chromatin condensation in cell cultures, also induce H3 phosphorylation (8, 9). Similarly, vanadate-induced dephosphorylation of H3 correlates with chromatin decondensation and the rescue of a mitotic mutant that otherwise fails to initiate postmitotic chromatin decondensation (12). Recent studies, using an antibody selective for the Ser-10 phosphorylated H3 amino terminus, have documented a tight correlation b...