The complete chloroplast and mitochondrial DNA sequence of Ostreococcus tauri: organelle genomes of the smallest eukaryote are examples of compaction

The complete nucleotide sequence of the mt (mitochondrial) and cp (chloroplast) genomes of the unicellular green alga Ostreococcus tauri has been determined. The mt genome assembles as a circle of 44,237 bp and contains 65 genes. With an overall average length of only 42 bp for the intergenic regions, this is the most gene-dense mt genome of all Chlorophyta. Furthermore, it is characterized by a unique segmental duplication, encompassing 22 genes and covering 44% of the genome. Such a duplication has not been observed before in green algae, although it is also present in the mt genomes of higher plants. The quadripartite cp genome forms a circle of 71,666 bp, containing 86 genes divided over a larger and a smaller single-copy region, separated by 2 inverted repeat sequences. Based on genome size and number of genes, the Ostreococcus cp genome is the smallest known among the green algae. Phylogenetic analyses based on a concatenated alignment of cp, mt, and nuclear genes confirm the position of O. tauri within the Prasinophyceae, an early branch of the Chlorophyta.     

Transformation of nuclear and plastomic plant genomes by biolistic particle bombardment

Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues. For plants, the biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported. In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique.     

Photosynthetic functions and thylakoid membrane polypeptide composition in light-sensitive mutants of Chlamydomonas reinhardii

In this paper we compared the pigment composition, photochemical activity, chloroplast ultrastructure, thylakoid membrane polypeptide composition and ribosomal content of wild-type and seven light-sensitive mutants of Chlamydomonas reinhardii.All the mutants had low chlorophyll and carotenoid content compared to wild-type. Mutants lts-30 and lts-135 were also characterized by a complete absence of visible carotenoids, while mutant lts-19 was fully deficient in chlorophylls.In most mutants, the chloroplast fragment could not carry out any DCIP photoreduction and O₂ evolution was also blocked. The PSI/P₇₀₀/activity was decreased in most cases.The mutant strains contained mostly single lamellae in their plastids, that is the stacking capacity of the thylakoid membranes was very decreased or fully absent. In most cases the number of lamellae was also very low.The relative amounts of 70 S ribosomes were decreased in all of the mutants. The thylakoid membranes showed anomalies in the region of 24 000–30 000 dalton polypeptides. The common characteristic for them was the relatively higher amount of the 30 000 dalton polypeptide and considerably decreased level of the 27 000 and 24 000 dalton polypeptides relative to the wild-type. These polypeptides were probably constituents of the chlorophyll-protein complex II which has been suggested to be the light harvesting pigment complex for PSII. The polypeptide of 30 000 daltons is the precursor for the LHCP apoprotein (24 000 dalton protein). It may be that the lighstimulated conversion of this precursor into LHCP apoprotein was blocked in our pigment-deficient mutants.Abbreviations CPI Chlorophyll-protein complex I - PSI Photosystem I - PSII Photosystem II - LHCP Light-harvesting pigment complex - DCIP 2,6-dichlorophenolindophenol - RuDPC-ase Ribulose-1,5-biphosphate-carboxylase - SDS Sodium dodecyl sulfate - LIDS Lithium dodecyl sulfate - PAG Polyacrylamide gel - TKM buffer 25 mM Tris-HCl, pH 7.S; 25 mM KCl; 25 mM Mg acetate     

Conserved gene clusters in the highly rearranged chloroplast genomes of Chlamydomonas moewusii and Chlamydomonas reinhardtii

We have extended to about 75 the number of genes mapped on the Chlamydomonas moewusii and Chlamydomonas reinhardtii chloroplast DNAs (cpDNAs) by partial sequencing of the very closely related C. eugametos and C. moewusii cpDNAs and by hybridizations with Chlamydomonas chloroplast gene-specific sequences. Only four of these genes (tscA and three reading frames) have not been identified in any other algal cpDNAs and thus may be specific to Chlamydomonas. Although the C. moewusii and C. reinhardtii cpDNAs differ by complex sequence rearrangements, 38 genes scattered throughout the genome define 12 conserved clusters of closely linked loci. Aside from the rRNA operon, four of these gene clusters share similarity to evolutionarily primitive operons found in other cpDNAs, representing in fact remnants of these operons. Our results thus indicate that most of the ancestral bacterial operons that characterize the chloroplast genome organization of land plants and early-diverging photosynthetic eukaryotes have been disrupted before the emergence of the polyphyletic genus Chlamydomonas. All gene rearrangements between the C. moewusii and C. reinhardtii cpDNAs, with the exception of those accounting for the relocations of atpA, psbI and rbcL, occurred within corresponding regions of the genome. One of these rearrangements seems to have led to disruption of the ancestral region containing rpl23, rpl2, rps19, rpl16, rpl14, rpl5, rps8 and the psaA exon 1. This gene cluster, which bears striking similarity to the Escherichia coli S10 and spc operons, spans a continuous DNA segment in C. reinhardtii, while it maps to two separate fragments in C. moewusii.     

衣藻叶绿体遗传转化的研究

叶绿体遗传转人是近几年发展起来的新领域。本文主要介绍了叶绿体遗传转化的特点、基本原理和衣藻叶绿体遗传转化的方法与技术;     

The molecular anatomy and function of thylakoid proteins

Abstract. The structure of chloroplast membrane proteins and their organization into photosynthetically-active multimeric complexes is described. Extensive use has been made of information derived from gene sequencing and other biochemical studies to predict likely protein conformations. These predictions have been assimilated into structural models of the various thylakoid complexes. The enzymatic activities of the complexes have also been described and where possible related to individual polypeptides.     

Promiscuous DNA in the nuclear genomes of hemiascomycetous yeasts

Transfer of fragments of mtDNA to the nuclear genome is a general phenomenon that gives rise to NUMTs (NUclear sequences of MiTochondrial origin). We present here the first comparative analysis of the NUMT content of entirely sequenced species belonging to a monophyletic group, the hemiascomycetous yeasts ( Candida glabrata, Kluyveromyces lactis, Kluyveromyces thermotolerans, Debaryomyces hansenii and Yarrowia lipolytica , along with the updated NUMT content of Saccharomyces cerevisiae ). This study revealed a huge diversity in NUMT number and organization across the six species. Debaryomyces hansenii harbors the highest number of NUMTs (145), half of which are distributed in numerous large mosaics of up to eight NUMTs arising from multiple noncontiguous mtDNA fragments inserted at the same chromosomal locus. Most NUMTs, in all species, are found within intergenic regions including seven NUMTs in pseudogenes. However, five NUMTs overlap a gene, suggesting a positive impact of NUMTs on protein evolution. Contrary to the other species, K. lactis and K. thermotolerans harbor only a few diverged NUMTs, suggesting that mitochondrial transfer to the nuclear genome has decreased or ceased in these phylogenetic branches. The dynamics of NUMT acquisition and loss are illustrated here by their species-specific distribution.     

Isolation of biochemically active chloroplasts from chlamydomonas

Chloroplasts from the cell wall mutant cw-15-2 of Chlamydomonas reinhardii were isolated by disruption of the cells in the Yeda press and fractionation through step gradients of Percoll. The resulting chloroplast fraction contained 80–85% intact chloroplasts. Electron micrographs of thin sections of the chloroplast fraction showed some cytoplasmic impurities, although almost no cytoplasmic ribosomes were detected by analysis of the ribosomal subunits.The isolated chloroplasts are active in photosynthetic O₂-evolution and CO₂-fixation, with the highest rates obtained in the presence of ATP.The chloroplast fraction also showed high rates of light-dependent in organello protein synthesis, with labelling of discrete chloroplast proteins known to be synthesized in the chloroplasts.     

Recombination and Heterologous Expression of Aliophycocyanin Gene in the Chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires theintroduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins ormetabolic pathways.In order to accomplish the expression of multiple genes in a single transformationevent,we inserted both large and small subunits of allophycocyanin gene (apcA and apcB) into Chlamydomonasreinhardtii chloroplast expression vector,resulting in papc-S.The constructed vector was then introducedinto the chloroplast of C.reinhardtii by micro-particle bombardment.Polymerase chain reaction and Southernblot analysis revealed that the two genes had integrated into the chloroplast genome.Western blot andenzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria couldbe correctly expressed in the chloroplasts of C.reinhardtii.The expressed foreign protein in transformantsaccounted for about 2%-3% of total soluble proteins.These findings pave the way to the reconstitution ofmulti-subunit proteins or metabolic pathways in transgenic C.reinhardtii chloroplasts in a single transformationevent.     

Cloning and characterization of a class II DNA photolyase from Chlamydomonas

Damage to DNA induced by ultraviolet light can be reversed by a blue light-dependent reaction catalyzed by enzymes called DNA photolyases. Chlamydomonas has been shown to have DNA photolyase activity in both the nucleus and the chloroplast. Here we report the cloning and sequencing of a gene, PHR2, from Chlamydomonas encoding a class II DNA photolyase. The PHR2 protein, when expressed in Escherichia coli, is able to complement a DNA photolyase deficiency. The previously described Chlamydomonas mutant, phr1, which is deficient in nuclear but not chloroplast photolyase activity was shown by RFLP analysis not to be linked to the PHR2 gene. Unlike the recently reported class II DNA photolyase from Arabidopsis, the protein encoded by PHR2 is predicted to contain a chloroplast targeting sequence. This result, together with the RFLP data, suggests that PHR2 encodes the chloroplast targeted DNA photolyase.     

Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii

Heterogeneous expression of multiple genes in the nucleus of transgenic plants requires the introduction of an individual gene and the subsequent backcross to reconstitute multi-subunit proteins or metabolic pathways. In order to accomplish the expression of multiple genes in a single transformation event, we inserted both large and small subunits of allophycocyanin gene （apcA and apcB） into Chlamydomonas reinhardtii chloroplast expression vector, resulting in papc-S. The constructed vector was then introduced into the chloroplast of C. reinhardtii by micro-particle bombardment. Polymerase chain reaction and Southern blot analysis revealed that the two genes had integrated into the chloroplast genome. Western blot and enzyme-linked immunosorbent assay showed that the two genes from the prokaryotic cyanobacteria could be correctly expressed in the chloroplasts of C. reinhardtii. The expressed foreign protein in transformants accounted for about 2%-3% of total soluble proteins. These findings pave the way to the reconstitution of multi-subunit proteins or metabolic pathways in transgenic C. reinhardtii chloroplasts in a single transformation event.     

Studies on the maintenance and expression of cloned DNA fragments in the nuclear genome of the green alga Chlamydomonas Reinhardtii

Using a biolistic device built here and based on the principle of the device described by Klein et al. (1987). we have reproducibly obtained transformants of Chlamydomonas reinhardtii . The reproducibility of the method has allowed us to examine the maintenance and expression of cloned DNA fragments introduced into C. Reinhardtii .     

Outlook in the application of Chlamydomonas reinhardtii chloroplast as a platform for recombinant protein production

Microalgae, also called microphytes, are a vast group of microscopic photosynthetic organisms living in aquatic ecosystems. Microalgae have attracted the attention of biotechnology industry as a platform for extracting natural products with high commercial value. During last decades, microalgae have been also used as cost-effective and easily scalable platform for the production of recombinant proteins with medical and industrial applications. Most progress in this field has been made with Chlamydomonas reinhardtii as a model organism mainly because of its simple life cycle, well-established genetics and ease of cultivation. However, due to the scarcity of existing infrastructure for commercial production and processing together with relatively low product yields, no recombinant products from C. reinhardtii have gained approval for commercial production and most of them are still in research and development. In this review, we focus on the chloroplast of C. reinhardtii as an algal recombinant expression platform and compare its advantages and disadvantages to other currently used expression systems. We then discuss the strategies for engineering the chloroplast of C. reinhardtii to produce recombinant cells and present a comprehensive overview of works that have used this platform for the expression of high-value products.     

Chlamydomonas reinhardtii as a unicellular model for circadian rhythm analysis

Chlamydomonas reinhardtii has been used as an experimental model organism for circadian rhythm research for more than 30 yr. Some of the physiological rhythms of this alga are well established, and several clock mutants have been isolated. The cloning of clock genes from these mutant strains by positional cloning is under way and should give new insights into the mechanism of the circadian clock. In a spectacular space experiment, the question of the existence of an endogenous clock vs. an exogenous mechanism has been studied in this organism. With the emergence of molecular analysis of circadian rhythms in plants in 1985, a circadian gene expression pattern of several nuclear and chloroplast genes was detected. Evidence is now accumulating that shows circadian control at the translational level. In addition, the gating of the cell cycle by the circadian clock has been analyzed. This review focuses on the different aspects of circadian rhythm research in C. reinhardtii over the past 30 yr. The suitability of Chlamydomonas as a model system in chronobiology research and the adaptive significance of the observed rhythms will be discussed.     

Chlamydomonas reinhardtii as a unicellular model for circadian rhythm analysis

Chlamydomonas reinhardtii has been used as an experimental model organism for circadian rhythm research for more than 30 yr. Some of the physiological rhythms of this alga are well established, and several clock mutants have been isolated. The cloning of clock genes from these mutant strains by positional cloning is under way and should give new insights into the mechanism of the circadian clock. In a spectacular space experiment, the question of the existence of an endogenous clock vs. an exogenous mechanism has been studied in this organism. With the emergence of molecular analysis of circadian rhythms in plants in 1985, a circadian gene expression pattern of several nuclear and chloroplast genes was detected. Evidence is now accumulating that shows circadian control at the translational level. In addition, the gating of the cell cycle by the circadian clock has been analyzed. This review focuses on the different aspects of circadian rhythm research in C. reinhardtii over the past 30 yr. The suitability of Chlamydomonas as a model system in chronobiology research and the adaptive significance of the observed rhythms will be discussed.     

Genetic engineering of thylakoid protein complexes by chloroplast transformation in Chlamydomonas reinhardtii

Chloroplast transformation of Chlamydomonas reinhardtii has developed into a powerful tool for studying the structure, function and assembly of thylakoid protein complexes in a eukaryotic organism. In this article we review the progress that is being made in the development of procedures for efficient chloroplast transformation. This focuses on the development of selectable markers and the use of Chlamydomonas mutants, individually lacking thylakoid protein complexes, as recipients. Chloroplast transformation has now been used to engineer all four major thylakoid protein complexes, photosystem II, photosystem I, cytochrome b₆/f and ATP synthase. These results are discussed with an emphasis on new insights into assembly and function of these complexes in chloroplasts as compared with their prokaryotic counterparts.Abbreviations ENDOR electron nuclear double resonance - ESEEM electron spin echo envelope modulation - LHC light harvesting complex - PSI Photosystem I - PS II Photosystem II - P₆₈₀ primary electron donor in PS II - P₇₀₀ primary electron donor in PS I     

Comparative analysis of the complete mitochondrial genomes of three geographical topmouth culter (Culter alburnus) groups and implications for their phylogenetics

Topmouth culter (C. alburnus) is an important commercial fish in China. We compared the nucleotide variations in the mtDNA genomes among three geographical groups of Culter alburnus: Liangzi Lake, Hubei Province (referred to as LZH); Taihu Lake, Jiangsu Province (TH); and Poyang Lake, Jiangxi Province (PYH). The similarity of whole mtDNA genomes ranged from 0.992 to 0.999. The similarity among 13 protein-coding genes, 2 rRNA genes, and the D-loop sequences was found to range from 0.982 to 0.996. This is useful data for future designing work for making specific molecular marker for distinguishing individuals of C. alburnus from the three geographical groups. An extended termination-associated sequence (ETAS) and several conserved blocks (CSB-F, CSB-E, CSB-D, CSB1, CSB2, and CSB3) were identified in the mtDNA control regions. A phylogenetic analysis shows a monophyletic relationship of the LZF-female and the LZF-male. However, the analysis also showed paraphyletic relationships for the other two geological groups. This result will be useful for the future breeding work of C. alburnus.