Dynamic changes in genomic 5-hydroxymethyluracil and N6-methyladenine levels in the Drosophila melanogaster life cycle and in response to different temperature conditions

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.

Unprecedentedly High Level of Intracellular Vitamin C and DNA Epigenetic Marks in Prostate: Relevant for Male Fertility?

“…Determination of intracellular vitamin C was described in [11]. The samples were homogenised using an ultrasonic homogeniser (SONOPULS UW 2070, BANDELIN electronic GmbH & Co. KG), twice for 10 s on ice.…”

Breast cancer epigenetics: comprehensive investigation of genes and chemical compounds involved in active DNA demethylation

“…14) and Bacetonitrile, according to the following elution program: 0-0.1 min, isocratic, 0.1% B; 0.1-2 min, linear gradient 0.1% − 15% B; 2-3 min, linear gradient 15% − 50% B; 3-3.5 min, isocratic, 50% B; 3.5-3.51 min, linear gradient, 50% − 0.1% B. The chromatographic peak of thymine was monitored with a photodiode array detector at 254 nm and analysed with the MassLynx software.Determination of intracellular vitamin C in leukocytes by UPLC-MS, described in[54].Forty-ve microliters of the sample were mixed with 50 µL of 10% (m/v) trichloroacetic acid (Merck KGaA, Germany) and 5 µL of 100 µM stable isotope-labelled internal standard solution ([13C6] L-ascorbic acid-, Toronto Research Chemicals) and incubated for 20 min on ice. Then, the samples were vortexed and centrifuged at 24400 × g for 20 min at 4°C.…”