Isolation of centromeric DNA by utilizing the DNA binding activity of recombinant centromere proteins.

着丝粒/着丝粒被定义为染色体初级收缩处的微管附着部位。电子显微镜研究表明，哺乳动物的动粒具有三层结构。然而，着丝粒/着丝粒结构的分子基础还没有得到广泛的表征。它在细胞分裂周期中由着丝粒异染色质组装而成。到目前为止，三种主要的着丝粒自身抗原(着丝粒蛋白：CENP-A、CENP-B和CENP-C)以及果蝇异染色质相关蛋白1的人类同源物HP1HSalpha，已经被证明在整个细胞周期中定位于着丝粒。我们已经证明它们是着丝粒/着丝粒异染色质的结构成分，在体外具有DNA结合活性，并确定了DNA结合域。在这个项目中，我们进一步研究了DNA和CENPs之间的分子相互作用，并通过分子生物学和生化方法在间期和中期染色体中鉴定了与这些CENPs相关的新的着丝粒/动粒蛋白。

Centromere/kinetochore is defined as the microtubule attachment site at the primary constriction of chromosomes. Electron microscopy studies have revealed that mammalian kinetochore has the trilaminar structure. However, the molecular basis of centromere/kinetochore structure has not been characterized extensively. It is assembled from the underlying centromere heterochromatin during the cell division cycle. Thus far, three major centromere autoantigens (CENtromere proteins ; CENP-A,CENP-B and CENP-C) together with HP1HSalpha, human homolog of Drosophila heterochromatin-associated protein 1, have been shown to be localized to centromere throughout the cell cycle. We have shown that they are structural components of centromere/kinetochre heterochromatin which possess DNA-binding activity in vitro and determined the DNA-binding domains. In this project, we further characterize the molecular interactions between DNA and CENPs further and identify novel centromere/kinetochore proteins that are associated with these CENPs in interphase and metaphase chromosomes by molecular biological and biochemical procedures.