Dr. Vinogradov points out that propidium iodide (PI) binding data do not always reflect accurate genome size values. PI binding can be influenced by the chromatin conformation. In addition, PI is not DNA specific and pretreatment of samples with RNase can affect the fluorescence intensity in an unpredictable way. According to Dr. Vinogradov, these pitfalls would be particularly significant when small variations in the DNA content are being investigated, as we recently did (1). Here we explain how we tried, as far as possible, to avoid artefacts during our study.
We compared four different methods of cell fixation. The published paper does not contain these data. Since no difference was observed between unfixed cells and cells fixed with 70% ethanol for 15 min, fixation was omitted in the analysis of the samples. Thus, our flow cytometric studies (1) are certainly free of artefacts deriving from fixation.
We exercised great care also while treating samples with RNase. We consistently did not find differences between samples incubated with varying doses of RNase. More importantly, PI binding disappeared completely upon addition of DNase. In general, our best control against artefacts was the high reproducibility of the results (SD comprised between 0.002 and 0.009).
In principle, we cannot exclude that our data -rather than differences in DNA content -might reflect differences in chromatin conformation existing among mouse strains and causing variation in the access of the fluorochrome to DNA or in the binding of the fluorochrome to the chromatin. Given the complexity of chromatin structure (2), we do not know how this hypothesis could be tested critically. Inherited differences in chromatin conformation have not been reported so far, in the mouse or in any other species. In this context, the interpretation which we have proposed for our results seems the most convincing.
Dr. Vinogradov reminds us that PI is not DNA specific and stresses the need to use special techniques for accurate measurements of small genome size variations. Recently Dr. Vinogradov (3) has reported differences in genome size as small as -and even smaller than -those we (1) have reported. Dr. Vinogradov for these measurements relied on the use of olivomicin and Hoechst 33258 (3). This procedure cannot represent the special technique