van Duijn P, van Prooijen-Knegt A C, van der Ploeg M
Histochemistry. 1985;82(4):363-76. doi: 10.1007/BF00494066.
A new hypothesis is proposed on the involvement of nucleosomes in Giemsa banding of chromosomes. Giemsa staining as well as the concomitant swelling can be explained as an insertion of the triple charged hydrophobic dye complex between the negatively-charged super-coiled helical DNA and the denatured histone cores of the nucleosomes still present in the fixed chromosomes. New cytochemical data and recent results from biochemical literature on nucleosomes are presented in support of this hypothesis. Chromosomes are stained by the Giemsa procedure in a purple (magenta) colour. Giemsa staining of DNA and histone (isolated or in a simple mixture) in model experiments results in different colours, indicating that a higher order configuration of these chromosomal components lies at the basis of the Giemsa method. Cytophotometry of Giemsa dye absorbance of chromosomes shows that the banding in the case of saline pretreatment is due to a relative absence of the complex in the faintly coloured bands (interbands). Pretreatment with trypsin results in an increase in Giemsa dye uptake in the stained bands. Cytophotometric measurements of free phosphate groups before and after pretreatment with saline, reveal a blocking of about half of the free phosphate groups indicating that a substantial number of free amino groups is still present in the fixed chromosomes. Glutaraldehyde treatment inhibited Giemsa-banding irreversibly while the formaldehyde-induced disappearance of the bands could be restored by a washing procedure. These results correlate with those of biochemical nucleosome studies using the same aldehydes. Based on these findings and on the known properties of nucleosomes, a mechanism is proposed that explains the collapse of the chromosome structure when fixed chromosomes are transferred to aqueous buffer solutions. During homogeneous Giemsa staining reswelling of the unpretreated chromosome is explained by insertion of the hydrophobic Giemsa complex between the hydrophobic nucleosome cores and the superhelix DNA. Selective Giemsa staining of the AT-enriched bands after saline pretreatment is thought to be due to the, biochemically well-documented, higher affinity of arginine-rich proteins present in the core histones for GC-enriched DNA, which prevents the insertion of the Giemsa complex in the interbands. Production of Giemsa bands by trypsin pretreatment can be related to the action of this enzyme on the H1 histones and subsequent charge rearrangements.(ABSTRACT TRUNCATED AT 400 WORDS)
提出了一个关于核小体参与染色体吉姆萨带型形成的新假说。吉姆萨染色以及伴随的膨胀现象可以解释为三价疏水染料复合物插入到固定染色体中仍然存在的带负电荷的超螺旋螺旋状DNA与核小体变性的组蛋白核心之间。本文展示了新的细胞化学数据以及生化文献中关于核小体的最新研究结果来支持这一假说。通过吉姆萨染色程序，染色体被染成紫色（品红色）。在模型实验中，吉姆萨对DNA和组蛋白（分离的或简单混合的）染色会产生不同颜色，这表明这些染色体成分的高级结构是吉姆萨染色方法的基础。对染色体吉姆萨染料吸光度进行细胞光度测定表明，在盐水预处理的情况下，带型是由于在浅色带（间带）中复合物相对缺乏所致。用胰蛋白酶预处理会导致染色带中吉姆萨染料摄取增加。对盐水预处理前后游离磷酸基团进行细胞光度测量，发现约一半的游离磷酸基团被封闭，这表明固定染色体中仍存在大量游离氨基。戊二醛处理不可逆地抑制吉姆萨带型，而甲醛导致的带型消失可通过洗涤程序恢复。这些结果与使用相同醛类进行生化核小体研究的结果相关。基于这些发现以及核小体的已知特性，提出了一种机制来解释当固定染色体转移到水性缓冲溶液中时染色体结构的崩溃。在均匀吉姆萨染色过程中，未预处理染色体的再膨胀被解释为疏水吉姆萨复合物插入疏水核小体核心与超螺旋DNA之间。盐水预处理后富含AT的带的选择性吉姆萨染色被认为是由于核心组蛋白中富含精氨酸的蛋白质对富含GC的DNA具有更高的亲和力，这在生化上已有充分记录，从而阻止了吉姆萨复合物插入间带。胰蛋白酶预处理产生吉姆萨带型可能与该酶对H1组蛋白的作用以及随后的电荷重排有关。（摘要截选至400字）
