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Alga Chlamydomonas Reinhardtii (alga + chlamydomonas_reinhardtii)
Kinds of Alga Chlamydomonas Reinhardtii Selected AbstractsModeling and Optimization of Photosynthetic Hydrogen Gas Production by Green Alga Chlamydomonas reinhardtii in Sulfur-Deprived CircumstanceBIOTECHNOLOGY PROGRESS, Issue 2 2006Ji Hye Jo Biological hydrogen production by the green alga Chlamydomonas reinhardtii under sulfur-deprived conditions has attracted great interest due to the fundamental and practical importance of the process. The photosynthetic hydrogen production rate is dependent on various factors such as strain type, nutrient composition, temperature, pH, and light intensity. In this study, physicochemical factors affecting biological hydrogen production by C. reinhardtii were evaluated with response surface methodology (RSM). First, the maximum specific growth rate of the alga associated with simultaneous changes of ammonium, phosphate, and sulfate concentrations in the culture medium were investigated. The optimum conditions were determined as NH4+ 8.00 mM, PO43, 1.11 mM, and SO42, 0.79 mM in Tris-acetate-phosphate (TAP) medium. The maximum specific growth rate with the optimum nutrient concentrations was 0.0373 h,1. Then, the hydrogen production rate of C. reinhardtii under sulfur-deprivation conditions was investigated by simultaneously changing two nutrient concentrations and pH in the medium. The maximum hydrogen production was 2.152 mL of H2 for a 10-mL culture of alga with density of 6 × 106 cells mL,1 for 96 h under conditions of NH4+ 9.20 mM, PO43, 2.09 mM, and pH 7.00. The obtained hydrogen production rate was approximately 1.55 times higher than that with the typical TAP medium under sulfur deficiency. [source] Oda16/Wdr69 is essential for axonemal dynein assembly and ciliary motility during zebrafish embryogenesisDEVELOPMENTAL DYNAMICS, Issue 8 2010Chunlei Gao Abstract In the alga Chlamydomonas reinhardtii, Oda16 functions during ciliary assembly as an adaptor for intraflagellar transport of outer arm dynein. Oda16 orthologs only occur in genomes of organisms that use motile cilia; however, such cilia play multiple roles during vertebrate development and the contribution of Oda16 to their assembly remains unexplored. We demonstrate that the zebrafish Oda16 ortholog (Wdr69) is expressed in organs with motile cilia and retains a role in dynein assembly. Antisense morpholino knockdown of Wdr69 disrupts ciliary motility and results in multiple phenotypes associated with vertebrate ciliopathies. Affected cilia included those in Kupffer's vesicle, where Wdr69 plays a role in generation of asymmetric fluid flow and establishment of organ laterality, and otic vesicles, where Wdr69 is needed to develop normal numbers of otoliths. Analysis of cilium ultrastructure revealed loss of outer dynein arms in morphant embryos. These results support a remarkable level of functional conservation for Oda16/Wdr69. Developmental Dynamics 239:2190,2197, 2010. © 2010 Wiley-Liss, Inc. [source] Atmospheric molding of ionic copolymer MALDI-TOF/MS arrays: A new tool for protein identification/profilingELECTROPHORESIS, Issue 24 2006Alexander Muck Abstract An atmospheric molding protocol has been used to prepare an ionic methacrylate-based copolymer sample support chips for MALDI (pMALDI)-MS by targeting selected groups of various monomers copolymerized during molding, namely, carboxy, sulfo, dimethylalkyamino, and trimethylalkylammonium groups. The new disposable array chips provide analyte-oriented enhancement of protein adsorption to the modified substrates without requiring complicated surface coating or derivatization. The MALDI-MS performance of the new ionic copolymer chips was evaluated for lysozyme, ,-lactoglobulin,A, trypsinogen and carbonic anhydrase,I using washing with solutions prepared in pH or ionic strength steps. On cationic chips, the proteins are washed out at pH lower than their pI values, and on anionic chips at pH higher than their pI values. The ability of the microfabricated pMALDI chip set to selectively adsorb different proteins from real samples and to significantly increase their MS-signal was documented for the transmembrane photosystem,I protein complex from the green alga Chlamydomonas reinhardtii. The proteins were almost exclusively adsorbed according to calculated pI values and grand average of hydropathy (GRAVY) indexes. The new disposable chips reduce manipulation times and increase measurement sensitivity for real-world proteomic samples. The simple atmospheric molding procedure enables additional proteomic operations to be incorporated on disposable MALDI-MS integrated platforms. [source] Analysis of glutathione endpoints for measuring copper stress in Chlamydomonas reinhardthENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2007Tasha L. Stoiber Abstract Glutathione (GSH) is the most abundant nonprotein thiol in eukaryotic cells and it protects cells by functioning as an antioxidant and a metal-binding ligand. Because glutathione readily undergoes oxidation-reduction reactions to combat oxidative stress, intracellular ratios of the reduced (GSH) to the oxidized (GSSG) forms of glutathione may serve as an important biomarker of exposure and effect of trace metals in eukaryotic cells. We compared sensitivity of glutathione ratios in the freshwater alga Chlamydomonas reinhardtii to the traditional endpoints of cell growth rates and chlorophyll a following exposure to Cu for periods of 6 and 24 h. A response of the GSH:GSSG ratio to Cu concentration was observed at Cu levels of 40 and 80 nM after exposure for both 6 and 24 h. The concentration of total GSH at 24 h was roughly half the value at 6 h after exposure to either 40 or 80 nM Cu. A response for cell growth rate was observed only at 24 h, whereby the average specific growth rate decreased from about 1.1 to 0.4 d,1. The total Cu concentrations eliciting a cell response of 50%, effect concentrations (EC50s), after 24 h of exposure were similar (49.2, 49.8, and 38.2 nM Cu) and not significantly different for GSH:GSSG ratio, GSH levels, and specific growth, respectively. Total cell-associated Cu concentrations after exposure for 24 h were calculated from the EC50 endpoints and ranged from 13.3 to 17.0 fg/cell. Overall, thiol ratios were indicative of toxicity resulting from exposure to Cu, but precision may be greater for the cell growth rate endpoints. [source] Glu 87 of Channelrhodopsin-1 Causes pH-dependent Color Tuning and Fast Photocurrent Inactivation,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2009Satoshi P. Tsunoda Channelrhodopsins (ChR1 and ChR2) are directly light-gated ion channels acting as sensory photoreceptors in the green alga Chlamydomonas reinhardtii. These channels open rapidly after light absorption and both become permeable for cations such as H+, Li+, Na+, K+ and Ca2+. Km for Ca2+ is 16.6 mm in ChR1 and 18.3 mm in ChR2 whereas the Km values for Na+ are higher than 100 mm for both ChRs. Action spectra of ChR1 peak between 470 and 500 nm depending on the pH conditions, whereas ChR2 peaks at 470 nm regardless of the pH value. Now we created two chimeric ChRs possessing helix 1,5 of ChR1 and 6, 7 of ChR2 (ChR1/25/2), or 1, 2 from ChR1 and 3,7 from ChR2 (ChR1/22/5). Both ChR-chimera still showed pH-dependent action spectra shifts. Finally, a mutant ChR1E87Q was generated that inactivated only slowly in the light and showed no spectral shift upon pH change. The results indicate that protonation/deprotonation of E87 in helix 1 alters the chromophore polarity, which shifts the absorption and modifies channel inactivation accordingly. We propose a trimodal counter ion complex for ChR1 but only a bimodal complex for ChR2. [source] Application of quantitative immunoprecipitation combined with knockdown and cross-linking to Chlamydomonas reveals the presence of vesicle-inducing protein in plastids 1 in a common complex with chloroplast HSP90CPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 11 2009Heinrich Heide Abstract Knowledge of the interaction partners of a protein of interest may provide important information on its function. Common to currently available tools for the identification of protein,protein interactions, however, is their high rates of false positives. Only recently an assay was reported that allowed for the unequivocal identification of protein,protein interactions in mammalian cells in a single experiment. This assay, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture, immunoprecipitation, and quantitative MS. We are using the unicellular green alga Chlamydomonas reinhardtii to understand the roles of chaperones in chloroplast biogenesis. The goal of this work was to apply QUICK to Chlamydomonas for the identification of novel interaction partners of vesicle-inducing protein in plastids 1 (VIPP1), a protein required for the biosynthesis/maintenance of thylakoid membranes and known substrate of chloroplast HSP70B. We report here a robust QUICK protocol for Chlamydomonas that has been improved (i) by introducing a cross-linking step (-X) to improve protein complex stability and (ii) by including a control for the correction of unequal immunoprecipitation and/or labeling efficiencies. Using QUICK and cross-linking we could verify that HSP70B and CGE1 form a complex with VIPP1 and could also demonstrate that chloroplast HSP90C is part of this complex. Moreover, we could show that the chaperones interact with VIPP1 also in membrane fractions. [source] Mass spectrometric genomic data mining: Novel insights into bioenergetic pathways in Chlamydomonas reinhardtiiPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 23 2006Jens Allmer Abstract A new high-throughput computational strategy was established that improves genomic data mining from MS experiments. The MS/MS data were analyzed by the SEQUEST search algorithm and a combination of de novo amino acid sequencing in conjunction with an error-tolerant database search tool, operating on a 256 processor computer cluster. The error-tolerant search tool, previously established as GenomicPeptideFinder (GPF), enables detection of intron-split and/or alternatively spliced peptides from MS/MS data when deduced from genomic DNA. Isolated thylakoid membranes from the eukaryotic green alga Chlamydomonas reinhardtii were separated by 1-D SDS gel electrophoresis, protein bands were excised from the gel, digested in-gel with trypsin and analyzed by coupling nano-flow LC with MS/MS. The concerted action of SEQUEST and GPF allowed identification of 2622 distinct peptides. In total 448 peptides were identified by GPF analysis alone, including 98 intron-split peptides, resulting in the identification of novel proteins, improved annotation of gene models, and evidence of alternative splicing. [source] CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtiiPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2006Maria V. Turkina Abstract Acclimation of the green alga Chlamydomonas reinhardtii to limiting environmental CO2 induced specific protein phosphorylation at the surface of photosynthetic thylakoid membranes. Four phosphopeptides were identified and sequenced by nanospray quadrupole TOF MS from the cells acclimating to limiting CO2. One phosphopeptide originated from a protein that has not been annotated. We found that this unknown expressed protein (UEP) was encoded in the genome of C.,reinhardtii. Three other phosphorylated peptides belonged to Lci5 protein encoded by the low-CO2 -inducible gene,5 (lci5). The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein. Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes. Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light. Both proteins were phosphorylated in the low-CO2 -exposed algal mutant deficient in the light-activated protein kinase,Stt7. Phosphorylation of previously unknown basic proteins UEP and Lci5 by specific redox-dependent protein kinase(s) in the photosynthetic membranes reveals the early response of green algae to limitation in the environmental inorganic carbon. [source] Trans -splicing of organelle introns , a detour to continuous RNAsBIOESSAYS, Issue 9 2009Stephanie Glanz Abstract In eukaryotes, RNA trans -splicing is an important RNA-processing form for the end-to-end ligation of primary transcripts that are derived from separately transcribed exons. So far, three different categories of RNA trans -splicing have been found in organisms as diverse as algae to man. Here, we review one of these categories: the trans -splicing of discontinuous group II introns, which occurs in chloroplasts and mitochondria of lower eukaryotes and plants. Trans -spliced exons can be predicted from DNA sequences derived from a large number of sequenced organelle genomes. Further molecular genetic analysis of mutants has unravelled proteins, some of which being part of high-molecular-weight complexes that promote the splicing process. Based on data derived from the alga Chlamydomonas reinhardtii, a model is provided which defines the composition of an organelle spliceosome. This will have a general relevance for understanding the function of RNA-processing machineries in eukaryotic organelles. [source] Modeling and Optimization of Photosynthetic Hydrogen Gas Production by Green Alga Chlamydomonas reinhardtii in Sulfur-Deprived CircumstanceBIOTECHNOLOGY PROGRESS, Issue 2 2006Ji Hye Jo Biological hydrogen production by the green alga Chlamydomonas reinhardtii under sulfur-deprived conditions has attracted great interest due to the fundamental and practical importance of the process. The photosynthetic hydrogen production rate is dependent on various factors such as strain type, nutrient composition, temperature, pH, and light intensity. In this study, physicochemical factors affecting biological hydrogen production by C. reinhardtii were evaluated with response surface methodology (RSM). First, the maximum specific growth rate of the alga associated with simultaneous changes of ammonium, phosphate, and sulfate concentrations in the culture medium were investigated. The optimum conditions were determined as NH4+ 8.00 mM, PO43, 1.11 mM, and SO42, 0.79 mM in Tris-acetate-phosphate (TAP) medium. The maximum specific growth rate with the optimum nutrient concentrations was 0.0373 h,1. Then, the hydrogen production rate of C. reinhardtii under sulfur-deprivation conditions was investigated by simultaneously changing two nutrient concentrations and pH in the medium. The maximum hydrogen production was 2.152 mL of H2 for a 10-mL culture of alga with density of 6 × 106 cells mL,1 for 96 h under conditions of NH4+ 9.20 mM, PO43, 2.09 mM, and pH 7.00. The obtained hydrogen production rate was approximately 1.55 times higher than that with the typical TAP medium under sulfur deficiency. [source] Analysis of force generation during flagellar assembly through optical trapping of free-swimming Chlamydomonas reinhardtiiCYTOSKELETON, Issue 3 2005Rachel Patton McCord Abstract Many studies have used velocity measurements, waveform analyses, and theoretical flagella models to investigate the establishment, maintenance, and function of flagella of the biflagellate green algae Chlamydomonas reinhardtii. We report the first direct measurement of Chlamydomonas flagellar swimming force. Using an optical trap ("optical tweezers") we detect a 75% decrease in swimming force between wild type (CC124) cells and mutants lacking outer flagellar dynein arms (oda1). This difference is consistent with previous estimates and validates the force measurement approach. To examine mechanisms underlying flagella organization and function, we deflagellated cells and examined force generation during flagellar regeneration. As expected, fully regenerated flagella are functionally equivalent to flagella of untreated wild type cells. However, analysis of swimming force vs. flagella length and the increase in force over regeneration time reveals intriguing patterns where increases in force do not always correspond with increases in length. These investigations of flagellar force, therefore, contribute to the understanding of Chlamydomonas motility, describe phenomena surrounding flagella regeneration, and demonstrate the advantages of the optical trapping technique in studies of cell motility. Cell Motil. Cytoskeleton 61:137,144, 2005. © 2005 Wiley-Liss, Inc. [source] Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplastsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Miller Tran Abstract Monoclonal antibodies can be effective therapeutics against a variety of human diseases, but currently marketed antibody-based drugs are very expensive compared to other therapeutic options. Here, we show that the eukaryotic green algae Chlamydomonas reinhardtii is capable of synthesizing and assembling a full-length IgG1 human monoclonal antibody (mAb) in transgenic chloroplasts. This antibody, 83K7C, is derived from a human IgG1 directed against anthrax protective antigen 83 (PA83), and has been shown to block the effects of anthrax toxin in animal models. Here we show that 83K7C heavy and light chain proteins expressed in the chloroplast accumulate as soluble proteins that assemble into complexes containing two heavy and two light chain proteins. The algal-expressed 83K7C binds PA83 in vitro with similar affinity to the mammalian-expressed 83K7C antibody. In addition, a second human IgG1 and a mouse IgG1 were also expressed and shown to properly assemble in algal chloroplast. These results show that chloroplasts have the ability to fold and assemble full-length human mAbs, and suggest the potential of algae as a platform for the cost effective production of complex human therapeutic proteins. Biotechnol. Bioeng. 2009; 104: 663,673 © 2009 Wiley Periodicals, Inc. [source] | |||||||||||||||||||