Chlamydomonas reinhardtii proteomics

Proteomics, based on the expanding genomic resources, has begun to reveal new details of Chlamydomonas reinhardtii biology. In particular, analyses focusing on subproteomes have already provided new insight into the dynamics and composition of the photosynthetic apparatus, the chloroplast ribosome, the oxidative phosphorylation machinery of the mitochondria, and the flagellum. It assisted to discovered putative new components of the circadian clockwork as well as shed a light on thioredoxin protein-protein interactions. In the future, quantitative techniques may allow large scale comparison of protein expression levels. Advances in software algorithms will likely improve the use of genomic databases for mass spectrometry (MS) based protein identification and validation of gene models that have been predicted from the genomic DNA sequences. Although proteomics has only been recently applied for exploring C. reinhardtii biology, it will likely be utilized extensively in the near future due to the already existing genetic, genomic, and biochemical tools.     

Genome analysis and its significance in four unicellular algae, Cyanidioshyzon merolae, Ostreococcus tauri, Chlamydomonas reinhardtii, and Thalassiosira pseudonana

Algae play a more important role than land plants in the maintenance of the global environment and productivity. Progress in genome analyses of these organisms means that we can now obtain information on algal genomes, global annotation and gene expression. The full genome information for several algae has already been analyzed. Whole genomes of the red alga Cyanidioshyzon merolae, the green algae Ostreococcus tauri and Chlamydomonas reinhardtii, and the diatom Thalassiosira pseudonana have been sequenced. Genome composition and the features of cells among the four algae were compared. Each alga maintains basic genes as photosynthetic eukaryotes and possesses additional gene groups to represent their particular characteristics. This review discusses and introduces the latest research that makes the best use of the particular features of each organism and the significance of genome analysis to study biological phenomena. In particular, examples of post-genome studies of organelle multiplication in C. merolae based on analyzed genome information are presented.     

Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii

Background  

Photosynthetic organisms convert atmospheric carbon dioxide into numerous metabolites along the pathways to make new biomass. Aquatic photosynthetic organisms, which fix almost half of global inorganic carbon, have great potential: as a carbon dioxide fixation method, for the economical production of chemicals, or as a source for lipids and starch which can then be converted to biofuels. To harness this potential through metabolic engineering and to maximize production, a more thorough understanding of photosynthetic metabolism must first be achieved. A model algal species, C. reinhardtii, was chosen and the metabolic network reconstructed. Intracellular fluxes were then calculated using flux balance analysis (FBA).     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^– mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

Genes expressed during sexual differentiation of Chlamydomonas reinhardtii

Four genes specifically expressed during gametogenesis of Chlamydomonas reinhardtii have been cloned and their expression patterns analyzed. mRNAs encoded by these gamete-specific genes (gas) were absent or present only at very low levels in vegetative cells and mature zygotes. In young zygotes 2 h after gamete fusion, the mRNAs of three gas genes still persisted. The gas mRNAs accumulated during gametic differentiation. The temporal patterns of accumulation of individual mRNAs differed; some started to increase early during gametogenesis, others accumulated in the late phase. The accumulation of one of the late mRNAs (gas28) was stricly light-dependent. To illustrate the utility of the genes cloned in the analysis of sexual differentiation in Chlamydomonas reinhardtii we show that in a gametogenesis-defective mutant, the expression of late genes is prevented while that of early genes is normal.     

微RNA Cre-miR914参与调控衣藻对高温胁迫的适应过程

高温胁迫是生物所面临的常见环境胁迫,因此在长期进化中生物逐渐进化出了对高温胁迫的高效适应能力.目前,有关藻类对高温胁迫的适应机制研究主要集中在生理调控及其相关的编码基因调控方面,而有关非编码基因对高温适应的调控尚无报道.在前期研究中,我们通过对衣藻细胞的定量PCR筛选和生物信息学分析发现,在多种胁迫处理后Cre-miR914表达下调且其靶基因有可能是RPL18,但对它们的作用却不清楚.本研究中利用生物信息学结合降解组测序确定了Cre-miR914的靶基因是RPL18,接着利用定量PCR验证Cre-miR914及其靶基因的表达情况,发现Cre-miR914表达在高温处理后明显下调,而RPL18表达明显上调,同时通过构建Cre-miR914过表达株和靶基因RPL18过表达株,结合高温胁迫处理和抗性表型研究,发现Cre-miR914过表达明显降低衣藻对抗高温胁迫能力,而靶基因RPL18过表达提高了衣藻对抗高温胁迫能力.本研究发现了一个microRNA参与调控藻类高温适应过程的分子机制,即衣藻通过调控Cre-miR914及其靶基因RPL18表达参与了的高温胁迫适应过程.     

Complementation analysis of the inorganic carbon concentrating mechanism of Chlamydomonas reinhardtii

Summary Six independently isolated mutants of Chlamydomonas reinhardtii that require elevated CO₂ for photoautotrophic growth were tested by complementation analysis. These mutants are likely to be defective in some aspect of the algal concentrating mechanism for inorganic carbon as they exhibit CO₂ fixation and inorganic carbon accumulation properties different from the wild-type. Four of the six mutants defined a single complementation group and appear to be defective in an intracellular carbonic anhydrase. The other two mutations represent two additional complementation groups.Abbreviations HS high salt medium which has 13 mM phosphate at pH 6.8 - HSA high salt plus 36 mM acetate medium - YA high salt medium with 4 g yeast extract per L and 36mM acetate - Arg arginine - cia^- CO₂ accumulation mutants that cannot grow on low CO₂ - C_i inorganic carbon (CO₂+HCO _- ³ ) - CA carbonic anhydrase - mt mating type Supported in part by the McKnight Foundation and by NSF grant PCM 8005917 and published as journal article 11924 from the Michigan State Agriculatural Experiment Station     

Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii

Two new nitrate assimilation-related genes, Nrt2;3 and Nar5, have been identified in Chlamydomonas reinhardtii. The Nrt2;3 gene is a new member of the Nrt2 family, encoding high-affinity nitrate (nitrite) transporters. Like that of the nitrate assimilation genes, expression of the Nrt2;3 gene is down-regulated by ammonium and positively controlled by Nit2, a regulatory locus specific for the pathway. The three Nrt2 genes of C. reinhardtii are differentially regulated by the nitrogen source. Expression of Nrt2;3 and of Nrt2;1, a nitrate/nitrite-bispecific transporter gene, was induced by nitrate and more efficiently by nitrite. Accumulation of mRNA of Nrt2;2, the nitrate-specific transporter gene, was only induced efficiently by nitrate. The Nar5 gene is located upstream of the Nrt2;3 genomic region and expression of its mRNA is down-regulated by ammonium. The Nrt2;3 and Nar5 genes are overexpressed in a deletion mutant that lacks nitrate assimilation loci. Received: 6 October 1997 / Accepted: 30 December 1997     

Knock-down of the COX3 and COX17 gene expression of cytochrome c oxidase in the unicellular green alga Chlamydomonas reinhardtii

The COX3 gene encodes a core subunit of mitochondrial cytochrome c oxidase (complex IV) whereas the COX17 gene encodes a chaperone delivering copper to the enzyme. Mutants of these two genes were isolated by RNA interference in the microalga Chlamydomonas. The COX3 mRNA was completely lacking in the cox3-RNAi mutant and no activity and assembly of complex IV were detected. The cox17-RNAi mutant presented a reduced level of COX17 mRNA, a reduced activity of the cytochrome c oxidase but no modification of its amount. The cox3-RNAi mutant had only 40% of the wild-type rate of dark respiration which was cyanide-insensitive. The mutant presented a 60% decrease of H₂O₂ production in the dark compared to wild type, which probably accounts for a reduced electron leakage by respiratory complexes III and IV. In contrast, the cox17-RNAi mutant showed no modification of respiration and of H₂O₂ production in the dark but a two to threefold increase of H₂O₂ in the light compared to wild type and the cox3-RNAi mutant. The cox17-RNAi mutant was more sensitive to cadmium than the wild-type and cox3-RNAi strains. This suggested that besides its role in complex IV assembly, Cox17 could have additional functions in the cell such as metal detoxification or Reactive Oxygen Species protection or signaling. Concerning Cox3, its role in Chlamydomonas complex IV is similar to that of other eukaryotes although this subunit is encoded in the nuclear genome in the alga contrary to the situation found in all other organisms.     

串联抗菌肽Cecropin B基因在莱茵衣藻中的表达及其抗菌活性分析

为了应对各种抗生素在水产养殖业所带来的副作用,我们在本文中尝试利用微藻对一种抗菌肽进行表达的可行性研究．根据莱茵衣藻核基因组偏爱密码子对抗菌肽Cecropin B基因进行改造,并将4个经改造的Cecropin B基因依次串联起来,中间加上莱茵衣藻的自剪切连接肽序列LWMRFA,人工合成总长度为522 bp的串联Cecropin B基因．将串联Cecropin B基因克隆到含hsp70-RBCS2启动子和RBCS2终止子的pH105载体上,再与携带ble筛选基因的表达框架连接,构建重组表达载体pCB124．采用玻璃珠转化法将载体pCB124导入莱茵衣藻cc-849中,筛选得到能表达抗菌肽Cecropin B的转基因衣藻．经过6个月的保持培养后,进一步对转基因藻细胞提取液进行抗菌活性分析,发现转基因藻具有明显的抑制革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(枯草芽孢杆菌和溶壁微球菌)生长的特征．这一结果为具有抗菌活性的饵料藻的生产和应用提供了新的途径．     

Chloroplast gene transmission in Chlamydomonas reinhardtii: A random choice model

Chloroplast genes in Chlamydomonas reinhardtii are inherited uniparentally. In a laboratory cross the majority (>95%) of the zygotes transmit to the meiotic progeny only those chloroplast genes donated by the maternal parent. This occurs even though the parents are isogamous and the chloroplasts from the two parents fuse shortly after mating. Uniparental inheritance of the chloroplast genes can be altered by several methods. If maternal gametes are irradiated with ultraviolet light prior to mating, the proportion of zygotes transmitting chloroplast genes from the paternal parent rises dramatically. In this paper I examine in detail the effects of uv irradiation on both maternal and paternal gametes and the effect of photore-activation following the uv irradiation. The effect of uv irradiation can be largely reversed by photoreactivation, and both the starting time and the intensity of the photoreactivating light used are found to be critical. Examination of the frequencies of the different zygote types obtained with respect to chloroplast gene transmission following uv treatment of the maternal gametes shows that they fit a hypergeometric distribution, in which choices are made from a population without replacement. By rearranging the basic hypergeometric equation I was able to estimate that the choice is made from a population of 27 maternal and 2 paternal units of gene transmission. These units probably contain more than one genome each, since their number is much lower than the estimated number of genomes per cell. My model explains both the observed distribution of zygote types and the bias in favor of maternal alleles found in the progeny of a biparental zygote, and may have a wider application to other organelle genetic systems. I also suggest that the extreme degree of uniparental inheritance of chloroplast genes in Chlamydomonas found in the laboratory may not be seen in nature.     

The detection and characterization of G-proteins in the eyespot of Chlamydomonas reinhardtii

The presence of G-proteins in the eyespot fraction of Chlamydomonas reinhardtii is shown. This fraction is capable of binding (GTPγ[³⁵S], possesses the GTPase activity and interacts with antibodies raised against a highly conserved peptide of most G-proteins' -subunit. Cross-reaction with a 24-kDa protein is detected on immunoblots. Using an antiserum prepared from vertebrate β-subunit peptide, two additional proteins with apparent M_r 21 and 29 kDa could be revealed. The light-dependence of GTPase extraction from eyespot membranes is shown. The results make it possible to suggest the participation of G-proteins in the photosensory transduction chain of Ch. reinhardtii.     

Mapping of chloroplast genes involved in chloroplast ribosome biogenesis in Chlamydomonas reinhardtii

Summary Chloroplast gene mutations which confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii have been tested for allelism and mapped by recombination analysis of progeny from biparental zygote clones. Thirty-one independently isolated streptomycin resistant mutants have chloroplast ribosomes which are resistant to this drug in an assay based on misreading of isoleucine in response to a poly U template, and comprise one nuclear and four chloroplast gene loci. Four mutants resistant to spectinomycin, and three mutants resistant to neamine and kanamycin, which have chloroplast ribosomes resistant to their respective antibiotics in poly U directed phenylalanine incorporation, appear to map in a single chloroplast gene locus. Representative alleles of this nr/spr locus, the four streptomycin resistance loci, and two chloroplast gene loci for erythromycin resistance, have been analyzed in a series of parallel crosses to establish the following map order for these seven genes in the chloroplast genome: er-u-la-er-u-37-nr-u-2-1/spr-u-1-H-4-sr-u-2-23-sr-u-2-60-sr-u-sm3-sr-u-sm2. These seven genes may constitute a ribosomal region within the chloroplast genome of Chlamydomonas comparable to the ribosomal gene clusters in bacteria.     

A mutant of Chlamydomonas reinhardtii altered in the transport of ammonium and methylammonium

Summary A methylammonium-resistant mutant, named hereafter strain 2170 (ma-1), was isolated for the first time from a eukaryotic phototrophic organism. Mutant 2170 from Chlamydomonas reinhardtii carries a single mendelian mutation which results in a decreased rate of uptake of both ammonium and methylammonium without being affected either in uptake of nitrate or nitrite or any of the tested enzyme activities related to ammonium assimilation. Mutant cells could not use methylammonium as nitrogen source nor excrete ammonium into the medium but they had derepressed nitrate and nitrite reductases when growing in the presence of ammonium. Mutant 2170 also exhibited a diminished methylammonium transport rate in comparison with the wild-type cells. We conclude that mutant 2170 is affected in a transport system responsible for the entrance of both ammonium and methylammonium into the cells.Abbreviations CHES 2-(N-Cyclohexylamino)ethanesulphonic acid - MOPS 3(N-morpholine)propanesulphonic acid     

Structure, evolution and expression of the mitochondrial ADP/ATP translocator gene from Chlamydomonas reinhardtii

Summary The first AUG in the Chlamydomonas reinhardtii ADP/ATP translocator (CRANT) mRNA initiates an open reading frame (ORF) which is very similar (51–79% amino acid identity) to other ANT proteins. In contrast to higher plants, no evidence for a long amino-terminal extension was obtained. The 5 non-transcribed region of the single-copy CRANT gene contains sequence motifs present in other C. reinhardtii nuclear genes. Four introns, whose positions are not conserved in other ANT genes, interrupt the protein coding region. A short heat shock specifically reduces CRANT mRNA levels. CRANT mRNA levels were unaffected by a mutation in photosynthesis. In a dark/light regime CRANT mRNA levels are high in the dark phase and low in the early light phase. Data on translation initiation sites, splice junctions and the codon preferences of C. reinhardtii nuclear genes were compiled. With the exception of two rare codons, ACA and GGA, the CRANT gene exhibits the biased codon usage of C. reinhardtii nuclear genes that are highly expressed during normal vegetative growth.     

A uniparental mutant of Chlamydomonas reinhardtii deficient in the chlorophyll-protein complex CP1

Summary A mutant of C. reinhardtii was isolated which displays an abnormal fluorescence yield of chlorophyll in vivo. This mutant is deficient specifically in Photosystem I activity, and it contains only 10–15% of the normal amount of the chlorophyll-protein complex CP1 in its thylakoid membranes. Genetic analysis shows that the mutation is inherited in a uniparental manner.This is the first report of a cytoplasmic mutation in c. reinhardtii affecting a thylakoid membrane polypeptide whose function in the photosynthetic process is well established.