Darzynkiewicz Z, Traganos F, Carter S P, Higgins P J
Sloan-Kettering Institute for Cancer Research, Walker Laboratory, Rye, New York 10580.
Exp Cell Res. 1987 Sep;172(1):168-79. doi: 10.1016/0014-4827(87)90103-0.
The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.
早期细胞化学研究的数据表明，完整中期染色体或G0期细胞浓缩染色质中的原位DNA比间期核解聚染色质中的DNA对热或酸诱导的变性更敏感。在这些研究中，异染荧光染料吖啶橙（AO）被用于对单链和双链DNA进行差异染色。目前的研究表明，事实上，与间期细胞相比，通透的中期细胞中的DNA在暴露于低pH（1.5 - 2.8）缓冲液时，可被单链特异性的S1或绿豆核酸酶消化。各种提取程序和酶处理提供了证据，表明染色质中组蛋白、HMG蛋白和二硫键的存在，以及染色质蛋白的磷酸化或聚（ADP）核糖基化，可以被排除是导致中期与间期DNA对变性敏感性差异的因素。用浓度为1.2 N及以上的NaCl处理细胞消除了间期和有丝分裂细胞之间的差异，使有丝分裂细胞中的DNA对变性的敏感性降低；这种处理还导致显微镜下可见的染色质解聚。目前的数据表明，用大于或等于1.2 N NaCl可提取的结构蛋白可能参与将DNA锚定到核基质或染色体支架上，并可能负责维持原位高度的染色质压缩，如在中期染色体或G0期细胞中观察到的那样。组蛋白解离后，带电荷的DNA聚合物的高空间密度可能会在双螺旋上诱导拓扑应变，从而降低其局部稳定性；这可以通过用AO对DNA进行异染染色或用单链特异性核酸酶消化来检测。
