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Telomeres during macronuclear development in Euplotes crassus
Abstract
In order to learn more about telomere length regulation and de novo telomere synthesis, we have analyzed telomeres during the macronuclear development of Euplotes crassus. Euplotes is particularly useful for studying telomeres because (1) literally millions of telomeres are made de novo and (2) telomeres are trimmed from an oversized to a shorter, mature size at well defined times during the sexual stage of the life cycle. The parameters that influence the mating efficiency of Euplotes were optimized and the morphological changes occurring during development were described. A novel procedure for isolating developing macronuclei devoid of old macronuclear DNA was developed. This procedure made it possible to directly analyze the telomeres of anlagen DNA molecules. We have shown that the newly synthesized telomeres are not only longer, but also more heterogeneous in length than mature macronuclear telomeres. The G-rich strand exhibits variability both in the total number of G$\sb4$T$\sb4$ repeats and in the identity of the terminal nucleotide and extends beyond the C-rich strand to leave a 3$\sp\prime$ overhang. While the length of this overhang is variable, the median length is 10 nucleotides. Both the G-rich and C-rich strands show a bimodal length distribution. This research provides the first detailed picture of a newly synthesized telomere and has allowed us to formulate a model describing the various steps involved in de novo telomere synthesis. During the final stages of macronuclear development, the oversized telomeres are shortened to their mature size. We have shown that telomere processing occurs in the round anlagen prior to anlagen elongation. An active mechanism is involved in the processing since telomere shortening is replication independent. Studies of the telomere-binding protein indicated that this protein remains present in the old macronuclear fragments throughout development. The telomere-binding protein transcript levels increase at the time of de novo telomere synthesis, suggesting a role for this protein during de novo telomere synthesis. However, the protein could not be detected in the anlagen at this time.
Subject Area
Molecular biology|Biochemistry|Cellular biology
Recommended Citation
Vermeesch, Joris Robert, "Telomeres during macronuclear development in Euplotes crassus" (1993). ETD collection for University of Nebraska-Lincoln. AAI9402405.
https://digitalcommons.unl.edu/dissertations/AAI9402405