Fukuma M, Hiraoka Y, Sakurai H, Fukasawa T
Division of Chemotherapy, Keio University School of Medicine, Tokyo, Japan.
Yeast. 1994 Mar;10(3):319-31. doi: 10.1002/yea.320100305.
We have developed a procedure to purify nucleosomal assembly-competent histones as a mixture of H2A, H2B, H3 and H4 from isolated nuclei of the yeast Saccharomyces cerevisiae with a purity of 70-80%. The mixture contained each of the histone subunits approximately at the equi-molar ratio. Plasmid pBR322 DNA was assembled into nucleosomes with the purified yeast histones in the presence of nucleoplasmin from unfertilized eggs of the frog Xenopus laevis. The efficiency of assembly of yeast histones was comparable to that of core histones purified from HeLa cells. The length of DNA fragment wrapping around a core histone particle and the molar ratio of histone components in an assembled nucleosome particle were estimated to be 150 +/- 10 bp long and H2A:H2B:H3:H4 = 1.0:0.9:0:9:1.0, respectively.
我们已开发出一种程序，可从酿酒酵母的分离细胞核中纯化出具有核小体组装能力的组蛋白，其为H2A、H2B、H3和H4的混合物，纯度达70 - 80%。该混合物中每个组蛋白亚基的含量大致为等摩尔比。在存在来自非洲爪蟾未受精卵的核质蛋白的情况下，将质粒pBR322 DNA与纯化的酵母组蛋白组装成核小体。酵母组蛋白的组装效率与从HeLa细胞纯化的核心组蛋白相当。包裹在核心组蛋白颗粒周围的DNA片段长度以及组装的核小体颗粒中组蛋白成分的摩尔比估计分别为150 +/- 10 bp长和H2A:H2B:H3:H4 = 1.0:0.9:0.9:1.0。
