Mutation of the telomeric repeat sequence has severe cellular consequences in a variety of systems. A Tetrahymena thermophila telomerase template mutant, ter1-43AA, displays an acute mitotic chromosome segregation defect. In the study described here we investigated the molecular basis for this lethality. Although cloned ter1-43AA macronuclear telomeres had long tracts of wild-type G4T2 repeats, they were capped by a mixture of G4T3 repeats, shown previously to be non-lethal, and G4T4 repeats, the telomeric sequence normally found in hypotrichous ciliates such as Oxytricha. To test further the functionality of the G4T4 repeat sequence in T. thermophila, we devised a new template mutation, ter1-44+AA, that resulted in more uniform synthesis of this sequence at telomere caps in vivo. The ter1-44+AA mutant displayed the most severe mitotic defect reported to date, with up to 85% of the population having micronuclei in anaphase, providing firm evidence that the hypotrich repeat sequence is not functional in Tetrahymena. Surprisingly, in spite of the telomeric sequence mutation, neither the ter1-43AA nor ter1-44+AA mutant displayed any significant loss of telomere length regulation. These results demonstrate that loss of telomere cap integrity, rather than length regulation, leads to the anaphase defect.
Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cell's oral apparatus. We suggest that micronuclear telomere loss leads to a "dynamic pause" in the program of cortical development, which may reveal an unusual cell cycle checkpoint.Telomeres have been implicated in a number of diverse cellular processes. Normal telomeres serve as the means by which chromosomes can be replicated completely, and they function as a cap, thereby protecting the natural ends from inappropriate fusion. Telomeric mutants can be generated by making predictable changes in the RNA template of the enzyme telomerase, which synthesizes telomeres, such that the corresponding complementary mutation is made at the telomeres. Although these types of telomeric DNA mutants in complex eukaryotes are poorly studied, particularly with respect to the downstream developmental consequences, those in simplified yeasts and the ciliated protozoa are better understood.Ciliated protozoa, such as Tetrahymena thermophila, provide excellent model systems for studying telomerase, telomeric DNA mutants, and the downstream developmental consequences to changing the telomeric repeat sequence. This is because the components of telomeres and telomerase have been exceptionally well documented (5) and the cells are large enough (roughly 60 to 70 m) to allow a glimpse of their unique developmental processes.Interestingly, T. thermophila, like other ciliates, bears two nuclei distinct in function and mode of division (reviewed in reference 25). The micronucleus represents the germ line nucleus and is transcriptionally silent, whereas the macronucleus serves as the vegetative nucleus of the cell. The micronucleus contains five pairs of chromosomes, which are passed on through mitosis and meiosis. In contrast, the macronucleus contains many subchromosomal fragments, each of which is amplified to ϳ45 copies, excluding the ribosomal DNA, which has thousands of copies. The macronuclear chromosomes are not faithfully inherited, as the macronucleus divides amitotically.The telomeres of the two T. thermophila nuclei are dramatically different. The macronuclear telomeres, present at a copy number of roughly 40,000 per cell, contain about 0.3 to 0.5 kb of G4T2 repeats (3). Mic...
The ends of eukaryotic chromosomes are protected by DNA-protein structures called telomeres. Telomeric DNA is highly conserved, usually consisting of long tracts of a repeating G-rich sequence. Tetrahymena thermophila telomeric DNA consists of alternating blocks of GGGG and TT sequences (i.e. a G4T2 repeat sequence). We examined the relative importance of the guanine and thymine elements of the repeat sequence in promoting in vitro binding by T. thermophila proteins. We identified single- and, for the first time, double-stranded telomere binding activities from a crude T. thermophila protein extract and tested the binding of these activities to altered telomere repeat sequences. All deletions or substitutions made to the guanine element virtually abolished binding, indicating that four G's are essential for recognition by the binding activity. However, G's alone are not sufficient for efficient binding, as elimination of the thymine element dramatically reduced binding. By contrast, substantial expansion of the thymine element was well tolerated, even though one such change, G4T4, is lethal in vivo. We tested up to a four-fold expansion of the thymine element and found that highly efficient binding was still achieved. These results suggest a minimal recognition sequence for T. thermophila proteins, with the T element providing an important spacer between essential G elements.
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