Telomeres are essential for chromosome stability, but their functions at specific cell-cycle stages are unknown. Telomeres are now shown to have a role in chromosome separation during mitosis. In telomeric DNA mutants of Tetrahymena thermophila, created by expression of a telomerase RNA with an altered template sequence, division of the germline nucleus was severely delayed or blocked in anaphase. The mutant chromatids failed to separate completely at the midzone, becoming stretched to up to twice their normal length. These results suggest a physical block in mutant telomere separation.

The filamentous fungus Aspergillus nidulans has two genes encoding a-tubulin, tubA and tubB. Mutational analysis of tubA has demonstrated that the tubA gene is essential for mitosis and nuclear migration. In this study we have deleted the tubB gene by replacing it with a selectable marker and have named this new allele tubBA. The results demonstrate that the tubB gene is not required for vegetative growth or asexual reproduction, nor is it required for the initiation or early stages of sexual differentiation. Deletion of tubB, however, completely prevents ascosporogenesis, because tubBA strains produce no sexual spores when self-crossed. These strains produce viable ascospores when outcrossed to tubB + strains, indicating that the tubBA mutation is recessive. We have studied the cytology of sexual development in wild-type strains and in the tubB mutant and have observed that tubBA, strains develop normally to the stage of ascus formation. However, only a single nuclear mass is observed in the tubBA ascus, indicating that either the two zygotic haploid nuclei are blocked in karyogamy or that karyogamy occurs but the resulting diploid nucleus is subsequently blocked in meiosis I.

Processing of telomeric DNA is required to generate the 3' G strand overhangs necessary for capping chromosome ends. We have investigated the steps involved in telomere processing by examining G overhang structure in Tetrahymena cells that lack telomerase or have altered telomeric sequences. We show that overhangs are generated by two precise cleavage steps involving nucleases that are robust but lack sequence specificity. Our data suggest that a G overhang binding protein delineates the boundaries for G and C strand cleavage. We also show that telomerase is not the nuclease responsible for G strand cleavage, although telomerase depletion alters the precision of processing. This change in processing indicates that telomerase affects multiple transactions at the telomere and provides a physical footprint for the continued association of telomerase with the telomere after repeat addition is complete.

Telomeres are the nucleoprotein complexes at eukaryotic chromosomal ends. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which comprises a telomerase reverse transcriptase (TERT) and a telomerase RNA (TER). TER contains a template for telomeric DNA synthesis. Filamentous fungi possess extremely short and tightly regulated telomeres. Although TERT is well conserved between most organisms, TER is highly divergent and thus difficult to identify. In order to identify the TER sequence, we used the unusually long telomeric repeat sequence of Aspergillus oryzae together with reverse-transcription-PCR and identified a transcribed sequence that contains the potential template within a region predicted to be single stranded. We report the discovery of TERs from twelve other related filamentous fungi using comparative genomic analysis. These TERs exhibited strong conservation with the vertebrate template sequence, and two of these potentially use the identical template as humans. We demonstrate the existence of important processing elements required for the maturation of yeast TERs such as an Sm site, a 5′ splice site and a branch point, within the newly identified TER sequences. RNA folding programs applied to the TER sequences show the presence of secondary structures necessary for telomerase activity, such as a yeast-like template boundary, pseudoknot, and a vertebrate-like three-way junction. These telomerase RNAs identified from filamentous fungi display conserved structural elements from both yeast and vertebrate TERs. These findings not only provide insights into the structure and evolution of a complex RNA but also provide molecular tools to further study telomere dynamics in filamentous fungi.