Effect of alkali on the size dispersity of mammalian DNA measured by filter elution

Abstract

DNA from unirradiated and irradiated cultured 9L rat brain tumor cells was held for varying times in low ionic strength solutions at pH 11.0, 12.3, or 12.9. The effect of this exposure to alkali on the DNA size distribution was determined by comparing the DNA filter elution profiles obtained experimentally with those theoretically predicted for monodispersed and random distributions. At pH 12.3 or 12.9, DNA from cells irradiated with 300 rad eluted with first-order kinetics corresponding to a random DNA size distribution. The median size of the distribution decreased if the irradiated DNA was exposed to pH 12.3 for 24 h. At pH 12.3 or 12.9, DNA from unirradiated cells eluted initially with complex kinetics that later became linear (18-21 h for pH 12.3 or 13-15 h for pH 12.9), characteristic of a monodispersed DNA size distribution. Holding either unirradiated or irradiated DNA at pH 11.0, below the critical unwinding pH, produced no effect on the elution profiles. Analysis of these filter elution data indicated that after sufficient exposure to pH 12.3 or 12.9, undamaged DNA molecules from mammalian cells elute as a single-stranded monodispersed size distribution of approximately 1 X 10(10) daltons. While the possibility cannot be completely eliminated that this monodispersed size represents an upper limit determined by physical forces, these results, in conjunction with those obtained using other techniques, lend credence to the existence of a nonrandom higher-order structure in mammalian chromosomal DNA.