tRNA modification profiles in obligate and moderate thermophilic bacilli

Transfer RNAs (tRNAs) are the most ancient RNA molecules in the cell, modification pattern of which is linked to phylogeny. The aim of this study was to determine the tRNA modification profiles of obligate (Anoxybacillus, Geobacillus, Paragebacillus) and moderate (Bacillus, Brevibacillus, Ureibacillus, Paenibacillus) thermophilic aerobic bacilli strains to find out its linkage to phylogenetic variations between species. LC-MS was applied for the quantification of modified nucleosides using both natural and isotopically labeled standards. The presence of m(2)A and m(7)G modifications at high levels was determined in all species. Relatively high level of i(6)A and m(5)C modification was observed for Paenibacillus and Ureibacillus, respectively. The lowest level of Cm modification was found in Bacillus. The modification ms(2)i(6)A and m(1)G were absent in Brevibacillus and Ureibacillus, respectively, while modifications Am and m(2)(2)G were observed only for Ureibacillus. While both obligate and moderate thermophilic species contain Gm, m(1)G and ms(2)i(6)A modifications, large quantities of them (especially Gm and ms(2)i(6)A modification) were detected in obligate thermophilic ones (Geobacillus, Paragebacillus and Anoxybacillus). The collective set of modified tRNA bases is genus-specific and linked to the phylogeny of bacilli. In addition, the dataset could be applied to distinguish obligate thermophilic bacilli from moderate ones

Synthesis of 5‑Hydroxymethyl‑, 5‑Formyl‑, and 5‑Carboxycytidine-triphosphates and Their Incorporation into Oligonucleotides by Polymerase Chain Reaction

The synthesis of the triphosphates of 5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine and the incorporation of these building blocks into long DNA fragments using the polymerase chain reaction (PCR) are reported. In this way DNA fragments containing multiple hmC, fC, and caC nucleobases are readily accessible

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair