[Show abstract][Hide abstract] ABSTRACT: We performed whole-genome resequencing of 12 field isolates and eight commonly studied laboratory strains of the model organism Chlamydomonas reinhardtii to characterize genomic diversity and provide a resource for studies of natural variation. Our data support previous observations that Chlamydomonas is among the most diverse eukaryotic species. Nucleotide diversity is ∼3% and is geographically structured in North America with some evidence of admixture among sampling locales. Examination of predicted loss-of-function mutations in field isolates indicates conservation of genes associated with core cellular functions, while genes in large gene families and poorly characterized genes show a greater incidence of major effect mutations. De novo assembly of unmapped reads recovered genes in the field isolates that are absent from the CC-503 assembly. The laboratory reference strains show a genomic pattern of polymorphism consistent with their origin as the recombinant progeny of a diploid zygospore. Large duplications or amplifications are a prominent feature of laboratory strains and appear to have originated under laboratory culture. Extensive natural variation offers a new source of genetic diversity for studies of Chlamydomonas, including naturally occurring alleles that may prove useful in studies of gene function and the dissection of quantitative genetic traits.
[Show abstract][Hide abstract] ABSTRACT: The green alga Chlamydomonas reinhardtii evolves H2 gas under anaerobic conditions in reactions catalyzed by hydrogenase enzymes. Expression of the HYDA genes and related genes is regulated by environmental conditions including anoxia. To study the pathways of gene regulation, we utilized the reporter gene RSP3, encoding a radial spoke protein required for flagellar motility. Promoter/5′UTR sequences of HYDA1 and HYDA2 genes were fused to the RSP3 coding sequence and the constructs were transformed into immotile cells lacking a functional RSP3 gene. The resulting transformants express the RSP3 reporter under the transcriptional control of the HYDA1 or HYDA2 promoter/5′UTR sequences. They are paralyzed in the presence of O2, but motile in anoxic conditions. To identify cis-elements or trans-acting factors that regulate gene expression in response to hypoxia, the conditionally swimming transformant strains were mutagenized and constitutively swimming strains were selected under aerobic conditions. Molecular and genetic analyses showed that mutations in both cis-elements and trans-acting factors lead to constitutive motility. Some mutant strains contain transposable elements inserted into the HYDA promoter/5′UTR sequences. These interrupted regions might indicate regulatory sequences involved in the response to hypoxia or perhaps the presence of sequences within transposable elements that stimulate transcription of the reporter gene.
[Show abstract][Hide abstract] ABSTRACT: Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa.
Full-text · Article · May 2013 · Genes & development
[Show abstract][Hide abstract] ABSTRACT: A yellow-green alga, Heterococcus sp. DN1, was isolated from the snows
of the Rocky Mountains in Colorado. Heterococcus sp. DN1 displays
properties such as abundant intracellular lipid accumulation and cold
tolerance. Here we describe a number of genes discovered from a draft
genome constructed from Illumina GAIIx short reads. The genome of
Heterococcus sp. DN1 is 170 Mb, with 29,080 genes giving hits within
NCBI using Tera-BLASTx. We show that Heterococcus sp. DN1 has a
large number of putative genes involved in lipid metabolism and contains
the required genes for the biosynthesis of eicosapentaenoic acid, a lipid
required for nutrition in humans. A number of putative cold-tolerance genes
are present in the genome as well.
[Show abstract][Hide abstract] ABSTRACT: The length of Chlamydomonas flagella is tightly regulated. Mutations in four genes, LF1, LF2, LF3 and LF4, cause cells to assemble flagella up to three times wild-type length. LF2 and LF4 encode protein kinases. Here we describe a new gene, LF5, in which null mutations cause cells to assemble flagella of excess length. The LF5 gene encodes a protein kinase very similar in sequence to the protein kinase CDKL5. In humans, mutations in this kinase cause a severe form of juvenile epilepsy. The LF5 protein localizes to a unique location--the proximal 1 μm of the flagella. The proximal localization of the LF5 protein is lost when genes that make up the proteins in the cytoplasmic length regulatory complex (LRC), LF1, LF2 and LF3, are mutated. In these mutants LF5p becomes localized either at the distal tip of the flagella or along the flagellar length, indicating that length regulation involves, at least in part, control of LF5p localization by the LRC.
Full-text · Article · Jan 2013 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: Single nucleotide polymorphisms (SNPs) are typically studied to characterize adaptation in organisms. However, copy number variation has also been known to play an important role in evolution. With the advent of genome technologies such as microarrays and high throughput sequencing, duplications and deletions of genomic segments known as copy number variants (CNVs) are being studied more thoroughly. It is thought that the duplication of genes and regions of the genome specifically leads to the generation of material contributing to species adaptation. Here we present the use of deep sequencing data to identify copy number variants in lab and wild strains of the algal species Chlamydomonas reinhardtii with the hypothesis that genes which are adaptive to growth environments are likely to undergo duplication events.
[Show abstract][Hide abstract] ABSTRACT: A process for isolation of three products (fatty acids, chars and nutrient-rich aqueous phases) from the hydrothermal carbonization of microalgae is described. Fatty acid products derived from hydrolysis of fatty acid ester groups in the microalgae were obtained in high yield and were found to be principally adsorbed onto the char also created in the process. With the highest lipid-containing microalga investigated, 92% of the fatty acids isolated were obtained by solvent extraction of the char product, with the remaining 8% obtained by extraction of the acidified filtrate. Obtaining the fatty acids principally by a solid–liquid extraction eliminates potential emulsification and phase separation problems commonly encountered in liquid–liquid extractions. The aqueous phase was investigated as a nutrient amendment to algal growth media, and a 20-fold dilution of the concentrate supported algal growth to a level of about half that of the optimal nutrient growth medium. Uses for the extracted char other than as a solid fuel are also discussed. Results of these studies indicate that fatty acids derived from hydrothermal carbonization of microalgae hold great promise for the production of liquid biofuels.
[Show abstract][Hide abstract] ABSTRACT: Mutations at the APM1 and APM2 loci in the green alga Chlamydomonas reinhardtii confer resistance to phosphorothioamidate and dinitroaniline herbicides. Genetic interactions between apm1 and apm2 mutations suggest an interaction between the gene products. We identified the APM1 and APM2 genes using a map-based cloning strategy. Genomic DNA fragments containing only the DNJ1 gene encoding a type I Hsp40 protein rescue apm1 mutant phenotypes, conferring sensitivity to the herbicides and rescuing a temperature-sensitive growth defect. Lesions at five apm1 alleles include missense mutations and nucleotide insertions and deletions that result in altered proteins or very low levels of gene expression. The HSP70A gene, encoding a cytosolic Hsp70 protein known to interact with Hsp40 proteins, maps near the APM2 locus. Missense mutations found in three apm2 alleles predict altered Hsp70 proteins. Genomic fragments containing the HSP70A gene rescue apm2 mutant phenotypes. The results suggest that a client of the Hsp70-Hsp40 chaperone complex may function to increase microtubule dynamics in Chlamydomonas cells. Failure of the chaperone system to recognize or fold the client protein(s) results in increased microtubule stability and resistance to the microtubule-destabilizing effect of the herbicides. The lack of redundancy of genes encoding cytosolic Hsp70 and Hsp40 type I proteins in Chlamydomonas makes it a uniquely valuable system for genetic analysis of the function of the Hsp70 chaperone complex.
[Show abstract][Hide abstract] ABSTRACT: The objective of this work was to quantify the kinetic behavior of Dunaliella primolecta (D. primolecta) subjected to controlled fluid flow under laboratory conditions. In situ velocities of D. primolecta were quantified by micron-resolution particle image velocimetry and particle tracking velocimetry. Experiments were performed under a range of velocity gradients and corresponding energy dissipation levels at microscopic scales similar to the energy dissipation levels of natural aquatic ecosystems. An average swimming velocity of D. primolecta in a stagnant fluid was 41 microm/s without a preferential flow direction. In a moving fluid, the sample population velocities of D. primolecta follow a log-normal distribution. The variability of sample population velocities was maximal at the highest fluid flow velocity in the channel. Local fluid velocity gradients inhibited the accrual of D. primolecta by twofold 5 days after the initiation of the experiment in comparison to the non-moving fluid control experiment.
Preview · Article · Sep 2010 · Biotechnology and Bioengineering
[Show abstract][Hide abstract] ABSTRACT: Hydrothermal carbonization is a process in which biomass is heated in water under pressure to create a char product. With higher plants, the chemistry of the process derives primarily from lignin, cellulose and hemicellulose components. In contrast, green and blue-green microalgae are not lignocellulosic in composition, and the chemistry is entirely different, involving proteins, lipids and carbohydrates (generally not cellulose). Employing relatively moderate conditions of temperature (ca. 200 °C), time (<1 h) and pressure (<2 MPa), microalgae can be converted in an energy efficient manner into an algal char product that is of bituminous coal quality. Potential uses for the product include creation of synthesis gas and conversion into industrial chemicals and gasoline; application as a soil nutrient amendment; and as a carbon neutral supplement to natural coal for generation of electrical power.
Full-text · Article · Jun 2010 · Biomass and Bioenergy
[Show abstract][Hide abstract] ABSTRACT: Chlamydomonas and other flagellated and ciliated organisms have evolved a specific and sophisticated mechanism for regulating flagellar length. The rate of regeneration of the growing flagellum is somehow coupled to the length of the resorbing flagellum. The strongest evidence that cells actively control flagellar length comes from experiments utilizing mutants with long flagella. Four genes, LF1, LF2, LF3 and LF4, have been identified in which mutation can lead to a long-flagella phenotype. Each of the genes has been cloned, and the gene product characterized. This is evidenced in this chapter with the mechanism of flagella length control. The LF1, LF2, and LF3 genes encode proteins (AAP83163, ABK34487, and AAO62545, respectively) that interact in the regulation of flagellar length. Although there may only be four genes that can produce a long-flagella phenotype upon mutagenesis, there are clearly many other genes involved in flagellar length control. Early models and balance point models are explained elaborately. Biochemical approaches to flagellar length control are likely to be the most important avenue for discovering additional proteins involved in the process.
[Show abstract][Hide abstract] ABSTRACT: Mutations in the UNI2 locus in Chlamydomonas reinhardtii result in a "uniflagellar" phenotype in which flagellar assembly occurs preferentially from the older basal body and ultrastructural defects reside in the transition zones. The UNI2 gene encodes a protein of 134 kDa that shares 20.5% homology with a human protein. Immunofluorescence microscopy localized the protein on both basal bodies and probasal bodies. The protein is present as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phosphorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Using the Uni2 protein as a cell cycle marker of basal bodies, we observed migration of basal bodies before flagellar resorption in some cells, indicating that flagellar resorption is not required for mitotic progression. We observed the sequential assembly of new probasal bodies beginning at prophase. The uni2 mutants may be defective in the pathways leading to flagellar assembly and to basal body maturation.
Full-text · Article · Feb 2008 · Molecular biology of the cell
[Show abstract][Hide abstract] ABSTRACT: Positive signaling by nitrate in its assimilation pathway has been studied in Chlamydomonas reinhardtii. Among >34,000 lines generated by plasmid insertion, 10 mutants were unable to activate nitrate reductase (NIA1) gene expression and had a Nit(-) (no growth in nitrate) phenotype. Each of these 10 lines was mutated in the nitrate assimilation-specific regulatory gene NIT2. The complete NIT2 cDNA sequence was obtained, and its deduced amino acid sequence revealed GAF, Gln-rich, Leu zipper, and RWP-RK domains typical of transcription factors and transcriptional coactivators associated with signaling pathways. The predicted Nit2 protein sequence is structurally related to the Nin (for nodule inception) proteins from plants but not to NirA/Nit4/Yna proteins from fungi and yeast. NIT2 expression is negatively regulated by ammonium and is optimal in N-free medium with no need for the presence of nitrate. However, intracellular nitrate is required to allow Nit2 to activate the NIA1 promoter activity. Nit2 protein was expressed in Escherichia coli and shown to bind to specific sequences at the NIA1 gene promoter. Our data indicate that NIT2 is a central regulatory gene required for nitrate signaling on the Chlamydomonas NIA1 gene promoter and that intracellular nitrate is needed for NIT2 function and to modulate NIA1 transcript levels.
[Show abstract][Hide abstract] ABSTRACT: Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying
chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were
inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the ∼120-megabase nuclear
genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated
with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic
and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.
[Show abstract][Hide abstract] ABSTRACT: Little is known about how cells regulate the size of their organelles. In this study, we find that proper flagellar length control in Chlamydomonas reinhardtii requires the activity of a new member of the cyclin-dependent kinase (CDK) family, which is encoded by the LF2 (long flagella 2) gene. This novel CDK contains all of the important residues that are essential for kinase activity but lacks the cyclin-binding motif PSTAIRE. Analysis of genetic lesions in a series of lf2 mutant alleles and site-directed mutagenesis of LF2p reveals that improper flagellar length and defective flagellar assembly correlate with the extent of disruption of conserved kinase structures or residues by mutations. LF2p appears to interact with both LF1p and LF3p in the cytoplasm, as indicated by immunofluorescence localization, sucrose density gradients, cell fractionation, and yeast two-hybrid experiments. We propose that LF2p is the catalytic subunit of a regulatory kinase complex that controls flagellar length and flagellar assembly.
Full-text · Article · Apr 2007 · The Journal of Cell Biology
[Show abstract][Hide abstract] ABSTRACT: Flagellar length is tightly regulated in the biflagellate alga Chlamydomonas reinhardtii. Several genes required for control of flagellar length have been identified, including LF1, a gene required to assemble normal-length flagella. The lf1 mutation causes cells to assemble extra-long flagella and to regenerate flagella very slowly after amputation. Here we describe the positional cloning and molecular characterization of the LF1 gene using a bacterial artificial chromosome (BAC) library. LF1 encodes a protein of 804 amino acids with no obvious sequence homologs in other organisms. The single LF1 mutant allele is caused by a transversion that produces an amber stop at codon 87. Rescue of the lf1 phenotype upon transformation was obtained with clones containing the complete LF1 gene as well as clones that lack the last two exons of the gene, indicating that only the amino-terminal portion of the LF1 gene product (LF1p) is required for function. Although LF1 helps regulate flagellar length, the LF1p localizes almost exclusively in the cell body, with <1% of total cellular LF1p localizing to the flagella.
[Show abstract][Hide abstract] ABSTRACT: Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that
lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive
signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and
that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3
was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3
correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase
in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation
level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.