Detection of long-range sequence order in Drosophila melanogaster satellite DNA IV by a photochemical crosslinking reaction and denaturation microscopy

Abstract

Satellite DNA IV of Drosophila melanogaster has been isolated in an actinomycin/CsCl gradient and allowed to react with tritium-labeled 4,5′,8-trimethylpsoralen (trioxsalen) in the presence of long wavelength ultraviolet light. Saturation experiments showed a limiting covalent binding of approximately one trioxsalen molecule per three base-pairs for the photochemical reaction. The buoyant density change of satellite IV (ρ = 1·705 g/cm3) after photoreaction was found to be −0·182 (g/cm3)/(trioxsalen/base-pair). To visualize directly the interstrand crosslinks, the photochemically reacted satellite DNA was denatured and spread for electron microscopy. From 85% to 90% of the molecules examined have an appearance of long stretches of tandem loops with few observable double-stranded regions. The histogram of loop sizes in these denatured molecules shows a regular distribution of crosslinkable sites along satellite DNA IV with an interval of approximately 250 base-pairs, suggesting that a long-range periodicity of sequences exists in this satellite DNA. After extensive crosslinking, the observed loops still have sizes that are multiples of 250 base-pairs. These results are discussed in terms of the sequence of satellite DNA IV, the characteristics of the photochemical reaction, and theories of evolution of satellite DNA sequences.