The NCPs were prepared by the salt dialysis method with the palindromic 146 base-pair α-satellite DNA fragment, as described previously1 (link),65 (link). The DNA fragment containing one half of the α-satellite DNA fragment in pGEM-T Easy vector was amplified in the E. coli strain DH5α, and was excised from the plasmid DNA by EcoR V (Takara). The DNA fragment was then dephosphorylated by alkaline phosphatase (Takara), and was further cleaved by EcoR I. The DNA fragment was purified by DEAE-5PW anion-exchange column chromatography (TOSOH). The DNA fragment was self-ligated by T4 DNA ligase (NIPPON GENE), and the resulting DNA fragment was further purified by DEAE-5PW anion-exchange column chromatography (TOSOH).
The reconstituted NCPs were purified by non-denaturing gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad). For the H2A/H2A.B heterotypic NCP preparation, the 146 base-pair DNA, the H2A-H2B dimer, the H2A.B-H2B dimer, and the H3.1-H4 tetramer were mixed in a 1:0.85:2.55:1.7 molar ratio. The NCPs were reconstituted by the salt dialysis method. As a result, H2A/H2A, H2A/H2A.B, and H2A.B/H2A.B NCPs were reconstituted. The resulting three types of NCPs were separated by non-denaturing gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad), and the heterotypic NCP was selectively purified.
The reconstituted NCPs were purified by non-denaturing gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad). For the H2A/H2A.B heterotypic NCP preparation, the 146 base-pair DNA, the H2A-H2B dimer, the H2A.B-H2B dimer, and the H3.1-H4 tetramer were mixed in a 1:0.85:2.55:1.7 molar ratio. The NCPs were reconstituted by the salt dialysis method. As a result, H2A/H2A, H2A/H2A.B, and H2A.B/H2A.B NCPs were reconstituted. The resulting three types of NCPs were separated by non-denaturing gel electrophoresis, using a Prep Cell model 491 apparatus (Bio-Rad), and the heterotypic NCP was selectively purified.