Project description:Estimates of the fitness effects of spontaneous mutations are important for understanding the adaptive potential of species. Here, we present the results of mutation accumulation experiments over 265-512 sequential generations in four species of marine unicellular green algae, Ostreococcus tauri RCC4221, Ostreococcus mediterraneus RCC2590, Micromonas pusilla RCC299, and Bathycoccus prasinos RCC1105. Cell division rates, taken as a proxy for fitness, systematically decline over the course of the experiment in O. tauri, but not in the three other species where the MA experiments were carried out over a smaller number of generations. However, evidence of mutation accumulation in 24 MA lines arises when they are exposed to stressful conditions, such as changes in osmolarity or exposure to herbicides. The selection coefficients, estimated from the number of cell divisions/day, varies significantly between the different environmental conditions tested in MA lines, providing evidence for advantageous and deleterious effects of spontaneous mutations. This suggests a common environmental dependence of the fitness effects of mutations and allows the minimum mutation/genome/generation rates to be inferred at 0.0037 in these species.
Project description:The trypanosomatid protozoan parasite Leishmania has a significant impact on human health globally. Understanding the pathways associated with virulence within this significant pathogen is critical for identifying novel vaccination and chemotherapy targets. Within this study we leverage an ultradeep proteomic approach to improve our understanding of two virulence associated genes in Leishmania; the Golgi Mannose/Arabinopyranose/Fucose nucleotide-sugar transporter LPG2, and the mitochondrial fucosyltransferase FUT1. Using deep peptide fractionation followed by complementary fragmentation approaches with higher energy collisional dissociation (HCD) and Electron-transfer dissociation (ETD) allowed the identification of over 6500 proteins, nearly doubling the experimentally observed Leishmania major proteome. This deep proteomic analysis revealed significant quantitative differences in both lpg2- and fut1s mutants with FUT1-dependent changes linked to marked alterations within mitochondrial associated proteins while LPG2-dependent changes impacted multiple aspects of the secretory pathway. While FUT1 has been shown to fucosylate peptides in vitro, no evidence for protein fucosylation was identified within our ultradeep analysis nor did we observe fucosylated glycans within Leishmania glycopeptides isolated using HILIC enrichment. Combined this work provides a critical proteomic resource for the community on the observable Leishmania proteome as well as highlights phenotypic changes associated with LPG2/FUT1 which may guide the development of future therapeutics.
Project description:Using the repeat finding algorithm FT-Rep, we have identified 154 pentatricopeptide repeat (PPR) proteins in nine fully sequenced genomes from green algae (with a total of 1201 repeats) and grouped them in 47 orthologous groups. All data are available in a database, PPRdb, accessible online at http://giavap-genomes.ibpc.fr/ppr. Based on phylogenetic trees generated from the repeats, we propose evolutionary scenarios for PPR proteins. Two PPRs are clearly conserved in the entire green lineage: MRL1 is a stabilization factor for the rbcL mRNA, while HCF152 binds in plants to the psbH-petB intergenic region. MCA1 (the stabilization factor for petA) and PPR7 (a short PPR also acting on chloroplast mRNAs) are conserved across the entire Chlorophyta. The other PPRs are clade-specific, with evidence for gene losses, duplications, and horizontal transfer. In some PPR proteins, an additional domain found at the C terminus provides clues as to possible functions. PPR19 and PPR26 possess a methyltransferase_4 domain suggesting involvement in RNA guanosine methylation. PPR18 contains a C-terminal CBS domain, similar to the CBSPPR1 protein found in nucleoids. PPR16, PPR29, PPR37, and PPR38 harbor a SmR (MutS-related) domain similar to that found in land plants pTAC2, GUN1, and SVR7. The PPR-cyclins PPR3, PPR4, and PPR6, in addition, contain a cyclin domain C-terminal to their SmR domain. PPR31 is an unusual PPR-cyclin containing at its N terminus an OctotricoPeptide Repeat (OPR) and a RAP domain. We consider the possibility that PPR proteins with a SmR domain can introduce single-stranded nicks in the plastid chromosome.
Project description:This data was generated to identify the molecular pathways responsible for nitrous oxide synthesis by the green algae Chlamydomonas reinhardtii, when supplied with nitrite under aerobic conditions (oxia). RNA samples were collected at three time points, 15 min, 3 hours, and 24 hours after the start of the experiment. The control and treatment groups were grown under the same conditions, except treatment group was supplied with 10mM nitrite at time 0. Illumina TruSeq stranded RNA libraries were synthesised from the resulting RNA before sequencing on a HiSeq2500 (125bp). The resulting sequence run generated 241,151,809 paired-end 125bp reads, of which 200,946,839 remained following quality filtering. The short data was mapped to the published genome and read counts were generated with HT-Seq count with the default settings. The raw read count data was analysed by DESeq2 in order to identify genes differentially expressed during nitrous oxide production.
Project description:We study the behaviour of the green alga Chlamydomonas reinhardtii (CR) in the presence of neighbouring regions of different viscosity. We show that the velocity and angular diffusion of the algae decreases when the viscosity of the surrounding medium is increased. We report on a phenomenon occurring when the algae try to cross from a region of low viscosity to a highly viscous one, which causes CR to re-orient and scatter away from the interface if it is approached at a sufficiently small angle. We highlight that the effect does not occur for CR crossing from high to low viscosity regions. Lastly we show that algae do not concentrate in the region of high viscosity despite them swimming slower there. On the contrary, they concentrate in the region of low viscosity or maintain a uniform concentration profile, depending on the viscosity ratio between the two regions.
Project description:Polycistronic gene expression, common in prokaryotes, was thought to be extremely rare in eukaryotes. The development of long-read sequencing of full-length transcript isomers (Iso-Seq) has facilitated a reexamination of that dogma. Using Iso-Seq, we discovered hundreds of examples of polycistronic expression of nuclear genes in two divergent species of green algae: Chlamydomonas reinhardtii and Chromochloris zofingiensis Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis using trimethylation of lysine 4 on histone H3 marks confirmed that transcription begins exclusively at the upstream gene. Quantification of polyadenylated [poly(A)] tails and poly(A) signal sequences confirmed that transcription ends exclusively after the downstream gene. Coexpression analysis found nearly perfect correlation for open reading frames (ORFs) within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, terminal peptides from both ORFs were identified from proteomics datasets, consistent with independent translation. Synthetic polycistronic gene pairs were transcribed and translated in vitro to recapitulate the production of two distinct proteins from a common transcript. The relative abundance of these two proteins can be modified by altering the Kozak-like sequence of the upstream gene. Replacement of the ORFs with selectable markers or reporters allows production of such heterologous proteins, speaking to utility in synthetic biology approaches. Conservation of a significant number of polycistronic gene pairs between C. reinhardtii, C. zofingiensis, and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.
Project description:The green algal phylum Chlorophyta has six diverse classes, but the phylogenetic relationship of the classes within Chlorophyta remains uncertain. In order to better understand the ancient Chlorophyta evolution, we have applied a site pattern sorting method to study compositional heterogeneity and the model fit in the green algal chloroplast genomic data. We show that the fastest-evolving sites are significantly correlated with among-site compositional heterogeneity, and these sites have a much poorer fit to the evolutionary model. Our phylogenomic analyses suggest that the class Chlorophyceae is a monophyletic group, and the classes Ulvophyceae, Trebouxiophyceae and Prasinophyceae are non-monophyletic groups. Our proposed phylogenetic tree of Chlorophyta will offer new insights to investigate ancient green algae evolution, and our analytical framework will provide a useful approach for evaluating and mitigating the potential errors of phylogenomic inferences.
Project description:1. By means of (14)C tracer experiments and isotope competition experiments the roles of d-tyrosine, p-hydroxyphenylpyruvic acid, p-hydroxyphenylacetic acid, phenylacetic acid, homogentisic acid and homoarbutin (2-methylquinol 4-beta-d-glucoside) in the biosynthesis of plastoquinones, tocopherols and alpha-tocopherolquinone by maize shoots was investigated. It was established that d-tyrosine, p-hydroxyphenylpyruvic acid and homogentisic acid can all be utilized for this purpose, whereas p-hydroxyphenylacetic acid, phenylacetic acid and homoarbutin cannot. Studies on the mode of incorporation of d-tyrosine, p-hydroxyphenylpyruvic acid and homogentisic acid showed that their nuclear carbon atoms and the side-chain carbon atom adjacent to the nucleus give rise (as a C(6)-C(1) unit) to the p-benzoquinone rings and nuclear methyl groups (one in each case) of plastoquinone-9 and alpha-tocopherolquinone and the aromatic nuclei and nuclear methyl groups (one in each case) of gamma-tocopherol and alpha-tocopherol. 2. By using [(14)C]-homogentisic acid it has been shown that homogentisic acid is also a precursor of plastoquinone, tocopherols and alpha-tocopherolquinone in the higher plants Lactuca sativa and Rumex sanguineus, the green algae Chlorella pyrenoidosa and Euglena gracilis and the blue-green alga Anacystis nidulans.