Jan K Y, Su P F, Lee T C
Exp Cell Res. 1985 Apr;157(2):307-14. doi: 10.1016/0014-4827(85)90115-6.
A differential Giemsa staining between sister chromatids was obtained by treating chromosomes replicated twice in medium containing 5-bromodeoxyuridine (BrdU) with Hoechst 33258 plus black light at 55 degrees C (HB pretreatment) and deoxyribonuclease (DNase) I, II, or micrococcal nuclease. In this staining pattern the BrdU bifilarly substituted chromatids were darkly and the unifilarly substituted chromatids lightly stained. This staining pattern was obtained only by staining the HB-DNase I-treated chromosomes with Giemsa and methylene blue, not by several other dyes tested. Relatively more DNA labelling was removed from the non-BrdU-substituted than the BrdU-substituted chromosomes, when the HB-pretreated chromosomes were digested with DNase I. But the protein labelling was not removed appreciably in the same treatment. The differential DNase I sensitivity between the non-BrdU-substituted and BrdU-substituted chromosomes disappeared when the HB-pretreated chromosomes were incubated with proteinase K before The DNase I digestion. Moreover, no differential DNase I sensitivity was found between the HB-pretreated isolated DNA containing and not containing BrdU. We propose that during the HB pretreatment, more DNA-protein cross-linkings are induced in BrdU bifilarly substituted than the unifilarly substituted chromatids. This structure protects the chromosomal DNA against the DNase I digestion. Thus, a reverse differential Giemsa staining between sister chromatids is obtained by the HB-DNase I treatment.
通过在含有5-溴脱氧尿苷（BrdU）的培养基中使染色体复制两次，然后用Hoechst 33258加55摄氏度的黑光（HB预处理）以及脱氧核糖核酸酶（DNase）I、II或微球菌核酸酶处理，获得了姐妹染色单体之间的差别吉姆萨染色。在这种染色模式中，BrdU双股取代的染色单体被染成深色，而单股取代的染色单体被染成浅色。这种染色模式仅通过用吉姆萨和亚甲蓝对经HB-DNase I处理的染色体进行染色获得，而不是通过测试的其他几种染料获得。当用DNase I消化经HB预处理的染色体时，非BrdU取代的染色体比BrdU取代的染色体去除的DNA标记相对更多。但在相同处理中蛋白质标记没有明显去除。当经HB预处理的染色体在DNase I消化前用蛋白酶K孵育时，非BrdU取代和BrdU取代的染色体之间的DNase I敏感性差异消失。此外，在经HB预处理的含有和不含有BrdU的分离DNA之间未发现DNase I敏感性差异。我们提出，在HB预处理期间，BrdU双股取代的染色单体比单股取代的染色单体诱导了更多的DNA-蛋白质交联。这种结构保护染色体DNA免受DNase I消化。因此，通过HB-DNase I处理获得了姐妹染色单体之间的反向差别吉姆萨染色。
