Search Results (5)

Gaillardia pulchella is an important plant species with pharmacological and ornamental applications. It contains a wide array of phytocompounds which play roles against diseases. In vitro propagation requires callogenesis and differentiation of plant organs, which offers a sustainable, alternative synthesis of compounds. The morphogenetic processes and the underlying mechanisms are, however, known to be under genetic regulation and are little understood. The present study investigated these events by generating transcriptome data, with de novo assembly of sequences to describe shoot morphogenesis molecularly in G. pulchella. The RNA was extracted from the callus of pre- and post-shoot organogenesis time. The callus induction was optimal using leaf segments cultured onto MS medium containing α-naphthalene acetic acid (NAA; 2.0 mg/L) and 6-benzylaminopurine (BAP; 0.5 mg/L) and further exhibited a high shoot regeneration/caulogenesis ability. A total of 68,366 coding sequences were obtained using Illumina150bpPE sequencing and transcriptome assembly. Differences in gene expression patterns were noted in the studied samples, showing opposite morphogenetic responses. Out of 10,108 genes, 5374 (53%) were downregulated, and there were 4734 upregulated genes, representing 47% of the total genes. Through the heatmap, the top 100 up- and downregulating genes’ names were identified and presented. The up- and downregulated genes were identified using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Important pathways, operative during G. pulchella shoot organogenesis, were signal transduction (13.55%), carbohydrate metabolism (8.68%), amino acid metabolism (5.11%), lipid metabolism (3.75%), and energy metabolism (3.39%). The synthesized proteins displayed phosphorylation, defense response, translation, regulation of DNA-templated transcription, carbohydrate metabolic processes, and methylation activities. The genes’ product also exhibited ATP binding, DNA binding, metal ion binding, protein serine/threonine kinase -, ATP hydrolysis activity, RNA binding, protein kinase, heme and GTP binding, and DNA binding transcription factor activity. The most abundant proteins were located in the membrane, nucleus, cytoplasm, ribosome, ribonucleoprotein complex, chloroplast, endoplasmic reticulum membrane, mitochondrion, nucleosome, Golgi membrane, and other organellar membranes. These findings provide information for the concept of molecular triggers, regulating programming, differentiation and reprogramming of cells, and their uses. Full article

Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical eRF1, and isoform 2 is 33 amino acid residues shorter than isoform 1 and completely unstudied. Using a reconstituted mammalian in vitro translation system, we showed that the isoform 2 of human eRF1 is also involved in translation. We showed that eRF1iso2 can interact with the ribosomal subunits and pre-termination complex. However, its codon recognition and peptide release activities have decreased. Additionally, eRF1 isoform 2 exhibits unipotency to UGA. We found that eRF1 isoform 2 interacts with eRF3a but stimulated its GTPase activity significantly worse than the main isoform eRF1. Additionally, we studied the eRF1 isoform 2 effect on stop codon readthrough and translation in a cell-free translation system. We observed that eRF1 isoform 2 suppressed stop codon readthrough of the uORFs and decreased the efficiency of translation of long coding sequences. Based on these data, we assumed that human eRF1 isoform 2 can be involved in the regulation of translation termination. Moreover, our data support previously stated hypotheses that the GTS loop is important for the multipotency of eRF1 to all stop codons. Whereas helix α1 of the N-domain eRF1 is proposed to be involved in conformational rearrangements of eRF1 in the A-site of the ribosome that occur after GTP hydrolysis by eRF3, which ensure hydrolysis of peptidyl-tRNA at the P site of the ribosome. Full article

The RNA world is one of the principal hypotheses to explain the emergence of living systems on the prebiotic Earth. It posits that RNA oligonucleotides acted as both carriers of information as well as catalytic molecules, promoting their own replication. However, it does not explain the origin of the catalytic RNA molecules. How could the transition from a pre-RNA to an RNA world occur? A starting point to answer this question is to analyze the dynamics in sequence space on the lowest level, where mononucleotide and short oligonucleotides come together and collectively evolve into larger molecules. To this end, we study the sequence-dependent self-assembly of polymers from a random initial pool of short building blocks via templated ligation. Templated ligation requires two strands that are hybridized adjacently on a third strand. The thermodynamic stability of such a configuration crucially depends on the sequence context and, therefore, significantly influences the ligation probability. However, the sequence context also has a kinetic effect, since non-complementary nucleotide pairs in the vicinity of the ligation site stall the ligation reaction. These sequence-dependent thermodynamic and kinetic effects are explicitly included in our stochastic model. Using this model, we investigate the system-level dynamics inside a non-equilibrium ‘RNA reactor’ enabling a fast chemical activation of the termini of interacting oligomers. Moreover, the RNA reactor subjects the oligomer pool to periodic temperature changes inducing the reshuffling of the system. The binding stability of strands typically grows with the number of complementary nucleotides forming the hybridization site. While shorter strands unbind spontaneously during the cold phase, larger complexes only disassemble during the temperature peaks. Inside the RNA reactor, strand growth is balanced by cleavage via hydrolysis, such that the oligomer pool eventually reaches a non-equilibrium stationary state characterized by its length and sequence distribution. How do motif-dependent energy and stalling parameters affect the sequence composition of the pool of long strands? As a critical factor for self-enhancing sequence selection, we identify kinetic stalling due to non-complementary base pairs at the ligation site. Kinetic stalling enables cascades of self-amplification that result in a strong reduction of occupied states in sequence space. Moreover, we discuss the significance of the symmetry breaking for the transition from a pre-RNA to an RNA world. Full article

At the focus of abiotic chemical reactions is the synthesis of ribose. No satisfactory explanation was provided as to the missing link between the prebiotic synthesis of ribose and prebiotic RNA (preRNA). Hydrogen cyanide (HCN) is assumed to have been the principal precursor in the prebiotic formation of aldopentoses in the formose reaction and in the synthesis of ribose. Ribose as the best fitting aldopentose became the exclusive sugar component of RNA. The elevated yield of ribose synthesis at higher temperatures and its protection from decomposition could have driven the polymerization of the ribose-phosphate backbone and the coupling of nucleobases to the backbone. RNA could have come into being without the involvement of nucleotide precursors. The first nucleoside monophosphate is likely to have appeared upon the hydrolysis of preRNA contributed by the presence of reactive 2′-OH moieties in the preRNA chain. As a result of phosphorylation, nucleoside monophosphates became nucleoside triphosphates, substrates for the selective synthesis of genRNA. Full article

Consumption of dietary bioactives is an avenue to enhancing the effective healthiness of diets by attenuating the glycaemic response. The intestinal brush border enzyme sucrase-isomaltase (SI) is the sole enzyme hydrolysing consumed sucrose, and we previously showed the acute effects of olive leaf extract (OLE) on sucrase activity when given together with sugars both in vitro and in vivo. Here we tested whether OLE could affect sucrase expression when pre-incubated chronically, a “priming” effect not dependent on competitive interaction with SI, in both a cell model and a human intervention. Using differentiated Caco-2/TC7 cells, long-term pre-treatment with oleuropein-rich olive leaf extract (OLE) lowered SI mRNA, surface protein and activity, and attenuated subsequent sucrose hydrolysis. Based on these results, a randomised, double-blinded, placebo-controlled, crossover pilot study was conducted. OLE (50 mg oleuropein) was consumed in capsule form 3 times a day for 1 week by 11 healthy young women followed by an oral sucrose tolerance test in the absence of OLE. However this treatment, compared to placebo, did not induce a change in post-prandial blood glucose maximum concentration (Glc_max), time to reach Glc_max and incremental area under the curve. These results indicate that changes in SI mRNA, protein and activity in an intestinal cell model by OLE are not sufficient under these conditions to induce a functional effect in vivo in healthy volunteers. Full article