Replication slippage may cause parallel evolution in the secondary structures of mitochondrial transfer RNAs

Presence of the dihydrouridine (D) stem in the mitochondrial cysteine tRNA is unusually variable among lepidosaurian reptiles. Phylogenetic and comparative analyses of cysteine tRNA gene sequences identify eight parallel losses of the D-stem, resulting in D-arm replacement loops. Sampling within the monophyletic Acrodonta provides no evidence for reversal. Slipped-strand mispairing of noncontiguous repeated sequences during replication or direct replication slippage can explain repeats observed within cysteine tRNAs that contain a D-arm replacement loop. These two mechanisms involving replication slippage can account for the loss of the cysteine tRNA D-stem in several lepidosaurian lineages, and may represent general mechanisms by which the secondary structures of mitochondrial tRNAs are altered.      

Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues,Cell Lines,FFPE Tissues,Aspirates, Lavages,Effusions, Plasma,Serum, and Urine

DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.     

The target of rapamycin kinase affects biomass accumulation and cell cycle progression by altering carbon/nitrogen balance in synchronized Chlamydomonas reinhardtii cells

Several metabolic processes tightly regulate growth and biomass accumulation. A highly conserved protein complex containing the target of rapamycin (TOR) kinase is known to integrate intra‐ and extracellular stimuli controlling nutrient allocation and hence cellular growth. Although several functions of TOR have been described in various heterotrophic eukaryotes, our understanding lags far behind in photosynthetic organisms. In the present investigation, we used the model alga Chlamydomonas reinhardtii to conduct a time‐resolved analysis of molecular and physiological features throughout the diurnal cycle after TOR inhibition. Detailed examination of the cell cycle phases revealed that growth is not only repressed by 50%, but also that significant, non‐linear delays in the progression can be observed. By using metabolomics analysis, we elucidated that the growth repression was mainly driven by differential carbon partitioning between anabolic and catabolic processes. Accordingly, the time‐resolved analysis illustrated that metabolic processes including amino acid‐, starch‐ and triacylglycerol synthesis, as well RNA degradation, were redirected within minutes of TOR inhibition. Here especially the high accumulation of nitrogen‐containing compounds indicated that an active TOR kinase controls the carbon to nitrogen balance of the cell, which is responsible for biomass accumulation, growth and cell cycle progression.     

An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome

Agrobacterium transfer DNA (T-DNA) is an effective plant mutagen that has been used to create sequence-indexed T-DNA insertion lines in Arabidopsis thaliana as a tool to study gene function. Creating T-DNA insertion lines requires a dependable method for locating the site of insertion in the genome. In this protocol, we describe an adapter ligation-mediated PCR method that we have used to screen a mutant library and identify over 150,000 T-DNA insertional mutants; the method can also be applied to map individual mutants. The procedure consists of three steps: a restriction enzyme-mediated ligation of an adapter to the genomic DNA; a PCR amplification of the T-DNA/genomic DNA junction with primers specific to the adapter and T-DNA; and sequencing of the T-DNA/genomic junction to enable mapping to the reference genome. In most cases, the sequenced genomic region extends to the T-DNA border, enabling the exact location of the insert to be identified. The entire process takes 2 weeks to complete.