Avides M C, Sunkel C E
Centro de Citologia Experimental, Universidade do Porto, Portugal.
J Cell Biol. 1994 Dec;127(5):1159-71. doi: 10.1083/jcb.127.5.1159.
The molecular mechanism involved in packaging centromeric heterochromatin is still poorly understood. CENP-B, a centromeric protein present in human cells, is though to be involved in this process. This is a DNA-binding protein that localizes to the central domain of the centromere of human and mouse chromosomes due to its association with the 17-bp CENP-B box sequence. We have designed a biochemical approach to search for functional homologues of CENP-B in Drosophila melanogaster. This strategy relies upon the use of DNA fragments containing the CENP-B box to identify proteins that specifically bind this sequence. Three polypeptides were isolated by nuclear protein extraction, followed by sequential ion exchange columns and DNA affinity chromatography. All three proteins are present in the complex formed after gel retardation with the human alphoid satellite DNA that contains the CENP-B box. Footprinting analysis reveals that the complex occupies both strands of the CENP-B box, although it is still unclear which of the polypeptides actually makes contact with the DNA. Localization of fluorescein-labeled proteins after microinjection into early Drosophila embryos shows that they associate with condensed chromosomes. Immunostaining of embryos with a polyclonal serum made against all three polypeptides also shows chromosomal localization throughout mitosis. During metaphase and anaphase the antigens appear to localize preferentially to centromeric heterochromatin. Immunostaining of neuroblasts chromosome spreads confirmed these results, though some staining of chromosomal arms is also observed. The data strongly suggests that the polypeptides we have identified are chromosomal binding proteins that accumulate mainly at the centromeric heterochromatin. Furthermore, DNA binding assays clearly indicate that they have a high specific affinity for the human CENP-B box. This would suggest that at least one of the three proteins isolated might be a functional homologue of the human CENP-B.
着丝粒异染色质包装所涉及的分子机制仍知之甚少。CENP-B是一种存在于人类细胞中的着丝粒蛋白，被认为参与了这一过程。这是一种DNA结合蛋白，由于其与17bp的CENP-B框序列相关联，定位于人类和小鼠染色体着丝粒的中央结构域。我们设计了一种生化方法来寻找黑腹果蝇中CENP-B的功能同源物。该策略依赖于使用含有CENP-B框的DNA片段来鉴定特异性结合该序列的蛋白质。通过核蛋白提取，随后依次经过离子交换柱和DNA亲和层析，分离出了三种多肽。在用含有CENP-B框的人类α卫星DNA进行凝胶阻滞分析后形成的复合物中，这三种蛋白质均存在。足迹分析表明，该复合物占据了CENP-B框的两条链，尽管仍不清楚哪种多肽实际上与DNA发生了接触。将荧光素标记的蛋白质显微注射到早期果蝇胚胎后进行定位，结果显示它们与浓缩染色体相关联。用针对所有三种多肽制备的多克隆血清对胚胎进行免疫染色，也显示在整个有丝分裂过程中染色体定位。在中期和后期，抗原似乎优先定位于着丝粒异染色质。对神经母细胞染色体铺片的免疫染色证实了这些结果，不过也观察到了染色体臂的一些染色。数据强烈表明，我们鉴定出的多肽是主要在着丝粒异染色质处积累的染色体结合蛋白。此外，DNA结合分析清楚地表明，它们对人类CENP-B框具有高特异性亲和力。这表明分离出的三种蛋白质中至少有一种可能是人类CENP-B的功能同源物。
