Evidence for alkali-sensitive linkers in DNA of African green monkey kidney cells

Abstract

MITOCHONDRIAL DNA possesses ribonucleotide linkers^1–3 and although double-stranded eukaryotic chromosomal DNA seems to be continuous throughout the length of the chromosome⁴ it is possible that single-stranded DNA possesses physical (nicks) or chemical (non-deoxynucleotide linkers) discontinuities. To distinguish between an in vivo nick and a nick introduced by shear forces during DNA extraction is very difficult with the presently available techniques. The detection of non-deoxynucleotide linkers is, however feasible, providing that denatured DNA can be sedimented in a chemical environment in which the linker is not destroyed. In these conditions, the comparison between sedimentation profiles of DNA, previously treated or not with a reagent that attacks the linker, may reveal the discontinuity. An obvious such reagent is a strong base, thus precluding the use of alkaline gradients in these experiments, and so we have attempted to use sucrose gradients in non-aqueous formamide. In addition, the DNA had to be extracted by a very gentle method in order to keep its average molecular weight in the range of 50–80 × 10⁶. Using these conditions, we were able to detect alkali-sensitive linkers in chromosomal DNA of green monkey kidney cells.