IMPROVED METHODS FOR THE ISOLATION OF INDIVIDUAL AND CLUSTERED MITOTIC CHROMOSOMES
IMPROVED METHODS FOR THE ISOLATION OF INDIVIDUAL AND CLUSTERED MITOTIC CHROMOSOMES
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DOI：
10.1016/0014-4827(87)90334-x
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发表时间：
1987-11-01
影响因子：
3.7
通讯作者：
LAEMMLI, UK
中科院分区：
文献类型：
--
作者：
GASSER, SM;LAEMMLI, UK
我们优化了从组织培养细胞中分离有丝分裂染色体的程序。第一个程序是一种快速的方法，用于获得适合于电子显微镜分析的单个结构完整的染色体。在Percoll梯度上进一步纯化这些导致染色体无细胞质污染，允许有丝分裂染色体固有的结构蛋白和酶活性的生物化学表征。第三种方法允许有效的、大规模的纯化聚集在一起的染色体，称为染色体簇。使用含EDTA的多胺缓冲液可最大限度地减少分离过程中蛋白质和DNA的修饰，并保持染色体结构的完整性。分析了导致染色体簇而不是单个染色体分离的条件。与簇相比，来自单个染色体的蛋白质的凝胶模式的比较鉴定了后一模式中的另外的蛋白质。这些蛋白质可能参与维持染色体间的组织或定位在中期细胞。
We have optimized procedures for the isolation of mitotic chromosomes from tissue culture cells. The first procedure is a rapid method for obtaining individual, structurally intact chromosomes suitable for analysis by electron microscopy. Further purification of these on Percoll gradients results in chromosomes free of cytoplasmic contamination, allowing biochemical characterization of the structural proteins and enzymatic activities intrinsic to mitotic chromosomes. A third procedure permits efficient, large-scale purification of chromosomes clustered together, referred to as a chromosomal cluster. The use of EDTA-containing polyamine buffers minimizes modifications of proteins and DNA during isolation and maintains the integrity of the chromosomal structure. The conditions which lead to the isolation of chromosomal clusters, as opposed to individual chromosomes, have been analyzed. Comparison of the gel patterns of proteins derived from individual chromosomes, as compared to clusters, identifies additional proteins in the latter pattern. These proteins could be involved in maintaining interchromosomal organization or positioning in the metaphase cell.