Differential rates of DNA denaturation and renaturation in situ in relation to the C-banding of Allium cepa chromosomes.

Telomeric heterochromatin can be demonstrated in Allium cepa chromosomes when root tip squashes are subjected to a C-banding procedure (treatment with saturated barium hydroxide for 10 min, followed by 1 h in phosphate buffer at 60 degrees C). Acridine orange (A0) staining indicated that the chromosomal DNA was denatured by the alkaline treatment and that it renatured within the first 3-7 min in the hot buffer. The DNA of the telomeres reannealed somewhat faster than the rest of the chromosomal DNA, but the AO staining suggested that all chromosomal DNA was double stranded after 7 min in buffer. Digestion of the chromosomes with a single strand specific nuclease, DNase S1, followed by Feulgen staining, demonstrated that the AO staining gives a somewhat misleading picture of the extent of DNA denaturation and renaturation. The S1 nuclease results showed that the chromosomal DNA was completely denatured by the alkaline treatment, but that a fraction of the DNA reannealed during the deionized water wash that preceded the incubation in hot buffer. Neither controls nor chromosomes subjected to the complete C-banding procedure were affected by S1 nuclease digestion, demonstrating that virtually all of the chromosomal DNA was double stranded both before and after the C-banding process. These results, along with the fact that the appearance of the bands was unaffected when the buffer incubation was performed at high (80 degrees C) or low (40 degrees C) temperature, indicated that differential DNA denaturation and renaturation is unlikely to be responsible for C-banding in this species.