Ghirlando Rodolfo, Litt Michael D, Prioleau Marie-Noëlle, Recillas-Targa Félix, Felsenfeld Gary
Department of Health and Human Services, Laboratory of Molecular Biology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
J Mol Biol. 2004 Feb 20;336(3):597-605. doi: 10.1016/j.jmb.2003.12.049.
We have studied the physical properties of a segment of condensed chromatin that lies upstream of the chicken beta-globin locus. This segment can be excised from an avian erythroleukemia cell line by restriction enzyme digestion and released from the nucleus as an essentially homogeneous fragment about 15.5 kbp long. Because of this homogeneity we could measure its sedimentation coefficient quite accurately by a combination of sucrose gradient and analytical ultracentrifugation. By measuring additionally the buoyant density of the cross-linked particle in CsCl we could deduce the total mass of the particle, hence its frictional coefficient, f, directly related to its shape. The measured value of f is consistent with a rod-like particle of the approximate length and diameter proposed earlier for the 30 nm chromatin fiber. The method is generally applicable to homogeneous particles of unique sequence at genomic abundance.
我们研究了鸡β-珠蛋白基因座上游一段浓缩染色质的物理性质。该片段可通过限制性内切酶消化从禽红细胞白血病细胞系中切除,并作为一个长度约为15.5 kbp的基本均匀的片段从细胞核中释放出来。由于这种均匀性,我们可以通过蔗糖梯度和分析超速离心相结合的方法相当准确地测量其沉降系数。通过额外测量交联颗粒在CsCl中的浮力密度,我们可以推断出颗粒的总质量,从而得出其摩擦系数f,该系数与颗粒形状直接相关。f的测量值与先前为30 nm染色质纤维提出的近似长度和直径的棒状颗粒一致。该方法通常适用于基因组丰度下具有独特序列的均匀颗粒。