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- W1523505861 abstract "This chapter describes the methods for purifying CENP-A from HeLa cells. Because of its high hydrophobicity, CENP-A was difficult to solubilize and liable to be lost during purification. It is found that CENP-A can be purified under denaturing conditions. The mixture of purified CENP-A and histone H4 was partially reactivated by the renaturation procedure. The CENP-A/H4 complex was purified under native conditions using the baculovirus system. It is described that CENP-A can replace histone H3 and efficiently form nucleosomes together with histones H4, H2A, and H2B in vitro by using NAP-1. Structures of the reconstituted CENP-A nucleosomes are shown to be basically the same as those of H3 nucleosomes, but some differences are also suggested in nucleosome shape and length of the nucleosomal DNA unit. Although the efficiency of nucleosome formation is high by the NAP-1 method, spaces between nucleosomes are heterogeneous. CENP-A nucleosomes are formed selectively on I-type α-satellite arrays in vivo. Competitive CENP-A nucleosome formations performed between pUC119 DNA and pUC119-α-satellite 11-mer DNA and found no preference for I-type α-satellite DNA under the present conditions. This method for CENP-A nucleosome formation represents a useful tool in searching for remodeling factors; the final goal of the reconstitution system will be to reconstitute CENP-A nucleosomes that reflect in vivo events." @default.
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- W1523505861 date "2003-01-01" @default.
- W1523505861 modified "2023-09-24" @default.
- W1523505861 title "Histone Variant CENP-A Purification, Nucleosome Reconstitution" @default.
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- W1523505861 doi "https://doi.org/10.1016/s0076-6879(03)75017-4" @default.
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