An investigation of the mechanism of the reciprocal differential staining of BUdR-substituted and unsubstituted chromosome regions.

Abstract

Abstract After treatment with hot NaH 2 PO 4 at pH 9, BUdR-substituted and unsubstituted chromosome regions are palely and intensely stained with Giemsa, respectively; however, after treatment with the same solution at pH 4, the reciprocal staining patterns are produced, i.e. these chromosome regions are intensely and palely stained, respectively. The nature of the mechanisms responsible for this reciprocal differential Giemsa staining of BUdR-substituted and unsubstituted chromosome regions has been investigated by Feulgen staining, electron microscopy, and radioisotope analyses involving scintillation counting and autoradiography. The results indicate that different mechanisms are responsible for the two types of staining effect. The high pH NaH 2 PO 4 treatment preferentially extracts BUdR-substituted DNA into the treatment solution, relative to unsubstituted DNA. The collective evidence from this and other work suggests that BUdR-substituted DNA in the chromosomes is partially photolysed by exposure to daylight during the harvesting procedure, and the degraded DNA is subsequently solubilized and extracted during the high pH treatment. This quantitative reduction of DNA in the BUdR-substituted chromosome regions results in pale Giemsa staining of these regions. The low pH NaH 2 PO 4 treatment does not produce a significant extraction of either BUdR-substituted or unsubstituted DNA into the treatment solution; rather, there may be a redistribution of the unsubstituted DNA relative to the BUdR-substituted DNA such that the unsubstituted DNA is preferentially dispersed outside the boundaries of the chromosomes onto the surrounding area of the slide. It is suggested that the BUdR-substituted chromosome regions stain relatively intensely with Giemsa after the low pH treatment because the DNA in these regions is less dispersed than that in the unsubstituted regions.