Inaccessibility of the Euplotes telomere binding protein.
The telomere binding protein (TP) from the macronucleus of the ciliate Euplotes eurystomus was purified by removal of tenaciously bound DNA with hydroxylapatite, and the purified TP partially sequenced. Rabbit antiserum was generated against a synthetic peptide of 14 amino acids at the amino-terminus of the TP. This antiserum was employed to examine the accessibility of TP antigenic determinants in nuclei and chromatin. Immunofluorescent staining of isolated macronuclei revealed only weak reactivity with specific antiserum. Reactivity within replication bands was demonstrated, and could be augmented by preparation of nuclear scaffolds. Employing a dot immunoblot analysis, the amino-terminal antigenic determinants of TP were revealed after extraction of histone H1 (and some nonhistones). A different aspect of TP inaccessibility was demonstrated by immunoblot analysis of trypsin-treated macronuclei and chromatin; TP was considerably less susceptible to digestion by trypsin than were histones H1 and H3. The relative inaccessibility of TP was not a consequence of chromatin higher-order structure, since soluble macronuclear chromatin in low salt exhibited the same burying of antigenic determinants by dot blot analysis, and the same decreased susceptibility to trypsin, as did isolated nuclei. Electron microscopy of soluble macronuclear chromatin spread in low salt revealed that most telomeres appear unfolded, without stable higher-order structure. The mechanisms for the relative inaccessibility of TP are not yet known, but probably arise as a consequence of the strong interactions of TP with the telomere nucleotide sequence and additional interactions of TP with various chromatin proteins, perhaps including histone H1.
Olins
A L
AL
University of Tennessee Graduate School of Biomedical Sciences, Oak Ridge 37831-8077.
Cacheiro
L H
LH
Herrmann
A L
AL
Dhar
M S
MS
Olins
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis* / Q000276:immunology; D000939:Epitopes / Q000032:analysis*; D016812:Euplotes / Q000737:chemistry* / Q000276:immunology; D006657:Histones / Q000032:analysis* / Q000276:immunology; D008969:Molecular Sequence Data; D016615:Telomere / Q000737:chemistry* / Q000276:immunology; D014357:Trypsin
D002352:Carrier Proteins; D002868:Chromosomal Proteins, Non-Histone; D000939:Epitopes; D006657:Histones; D014357:Trypsin