Olins A L, Cacheiro L H, Herrmann A L, Dhar M S, Olins D E
University of Tennessee Graduate School of Biomedical Sciences, Oak Ridge 37831-8077.
Chromosoma. 1993 Dec;102(10):700-11. doi: 10.1007/BF00650896.
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.
通过用羟基磷灰石去除紧密结合的DNA，从纤毛虫多态喇叭虫的大核中纯化出端粒结合蛋白（TP），并对纯化后的TP进行了部分测序。针对TP氨基末端的14个氨基酸的合成肽制备了兔抗血清。该抗血清用于检测细胞核和染色质中端粒结合蛋白抗原决定簇的可及性。对分离出的大核进行免疫荧光染色，结果显示其与特异性抗血清的反应较弱。在复制带内检测到了反应性，并且通过制备核支架可以增强这种反应性。采用斑点免疫印迹分析，在提取组蛋白H1（和一些非组蛋白）后，揭示了TP的氨基末端抗原决定簇。通过对胰蛋白酶处理的大核和染色质进行免疫印迹分析，证明了端粒结合蛋白不可及性的另一个方面；与组蛋白H1和H3相比，端粒结合蛋白对胰蛋白酶消化的敏感性要低得多。端粒结合蛋白相对不可及性并非染色质高级结构的结果，因为低盐条件下的可溶性大核染色质通过斑点印迹分析显示出相同的抗原决定簇埋藏情况，并且对胰蛋白酶的敏感性降低情况与分离的细胞核相同。低盐条件下展开的可溶性大核染色质的电子显微镜观察表明，大多数端粒看起来是展开的，没有稳定的高级结构。端粒结合蛋白相对不可及性的机制尚不清楚，但可能是由于端粒结合蛋白与端粒核苷酸序列的强相互作用以及端粒结合蛋白与各种染色质蛋白（可能包括组蛋白H1）的额外相互作用所致。
