Project description:Primary objectives: The primary objective is to investigate circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Primary endpoints: circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Project description:Diatoms are important primary producers in the world’s oceans, yet their growth is constrained in large regions by low bioavailable iron (Fe). Low Fe-induced limitation of primary production is due to requirements for Fe in components of essential metabolic pathways including photosynthesis and other chloroplast plastid functions. Studies have shown that under Fe-limited stress, diatoms alter plastid-specific processes, including components of electron transport. These physiological changes suggest changes of protein content and their abundance within the diatom plastid. While in-silico predictions provide putative information on plastid-localized proteins, knowledge of diatom plastid proteins remains limited in comparison to model photosynthetic organisms. To characterize proteins enriched in diatom plastids we have used shotgun proteomics to assess the proteome of subcellular plastid-enriched fractions from Thalassiosira pseudonana. To improve our understanding of how the plastid proteome is remodeled in response to Fe limitation, proteome sequencing has been performed on T. pseudonana grown under Fe replete and limited conditions. These analyses have shown that Fe limitation regulates major metabolic pathways in the plastid, including the Calvin cycle, as well as changes in light harvesting protein expression. In-silico localization predictions of proteins identified in this plastid-enriched proteome allowed for an in-depth comparison of theoretical vs observed plastid-localization, providing evidence for the potential of additional protein import pathways into the diatom plastid.
Project description:Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare L). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identifications of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions, as well as opposite to annotated genes demonstrating the existence of numerous non-coding RNA candidates.
Project description:Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare L). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identifications of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions, as well as opposite to annotated genes demonstrating the existence of numerous non-coding RNA candidates. dRNA-seq analysis of total RNA from green and white plastids of the barley mutant line albostrians
Project description:We report that phosphatidylglycerol (PG) biosynthesis in plastid is required for plastid gene expression mediated by plastid-encoded RNA polymerase and light-induced expression of nuclear-encoded photosynthesis-associated genes. A transcription factor GOLDEN-LIKE1 was also found to be involved in the downregulation of nuclear photosynthesis genes in responce to PG deficiency.
Project description:The unicellular, free-living, nonphotosynthetic chlorophycean alga Polytomella parva, closely related to Chlamydomonas reinhardtii and Volvox carteri, contains colorless, starch-storing plastids. The P. parva plastids lack all light-dependent processes but maintain crucial metabolic pathways. The colorless alga also lacks a plastid genome, meaning no transcription or translation should occur inside the organelle. Here, using an algal fraction enriched in plastids as well as publicly available transcriptome data, we provide a proteomic characterization of the P. parva plastid, ultimately identifying several plastid proteins, both by mass spectrometry and bioinformatic analyses. Altogether these results led us to propose a plastid proteome for P. parva, i.e., a set of proteins that participate in carbohydrate metabolism; in the synthesis and degradation of starch, amino acids and lipids; in the biosynthesis of terpenoids and tetrapyrroles; in solute transport and protein translocation; and in redox homeostasis. This is the first detailed plastid proteome from a unicellular, free-living colorless alga.