Abstract
In this study, the level of DNA modifications was investigated in three developmental stages of Drosophila melanogaster (larvae, pupae, imago) and in an in vitro model (Schneider 2 cells). Analysis was carried out using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry. Our method made it possible, for the first time, to analyze a broad spectrum of DNA modifications in the three stages of Drosophila. Each stage was characterized by a specific modification pattern, and the levels of these compounds fluctuated throughout the D. melanogaster life cycle. The level of DNA modification was also compared between insects bred at 25 °C (optimal temperature) and at 18 °C, and the groups differed significantly. The profound changes in N6-methyladenine and 5-hydroxymethyluracil levels during the Drosophila life cycle and as a result of breeding temperature changes indicate that these DNA modifications can play important regulatory roles in response to environmental changes and/or biological conditions. Moreover, the supplementation of Schneider 2 cells with 1 mM L-ascorbic acid caused a time-dependent increase in the level of 5-(hydroxymethyl)-2'-deoxyuridine. These data suggest that a certain pool of this compound may arise from the enzymatic activity of the dTET protein.