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Inaccessibility of the Euplotes telomere binding protein
A L Olins1, L H Cacheiro, A L Herrmann
1University of Tennessee Graduate School of Biomedical Sciences, Oak Ridge 37831-8077.
Chromosoma
|December 1, 1993
Summary
Telomere binding protein (TP) in Euplotes eurystomus is relatively inaccessible within the nucleus. Its accessibility increases after histone removal and it shows resistance to trypsin digestion, suggesting complex interactions.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Telomere binding protein (TP) plays a crucial role in maintaining telomeres.
- Understanding TP's nuclear accessibility is key to elucidating its function.
- Euplotes eurystomus provides a model system for studying ciliate nuclear organization.
Purpose of the Study:
- To investigate the accessibility of telomere binding protein (TP) in the macronucleus of Euplotes eurystomus.
- To determine factors influencing TP's accessibility within the nucleus and chromatin.
- To explore the relationship between TP accessibility and chromatin structure.
Main Methods:
- Purification and partial sequencing of TP.
- Generation of rabbit antiserum against a synthetic TP peptide.
- Immunofluorescence staining of isolated macronuclei.
- Dot immunoblot and trypsin digestion assays on macronuclei and chromatin.
- Electron microscopy of spread macronuclear chromatin.
Main Results:
- TP shows weak reactivity in immunofluorescence, indicating limited accessibility.
- TP accessibility is enhanced after histone H1 extraction.
- TP is significantly more resistant to trypsin digestion than histones H1 and H3.
- Inaccessibility is observed in both isolated nuclei and soluble chromatin, independent of higher-order structure.
Conclusions:
- TP exhibits relative inaccessibility within the Euplotes macronucleus.
- This inaccessibility is not solely due to higher-order chromatin structure.
- Strong interactions of TP with telomeric DNA and other proteins likely contribute to its buried state.