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.

SummaryHeterologous genes introduced into the nuclear genome of Chlamydomonas reinhardtii are often poorly expressed. To understand the molecular mechanisms underlying this effect, we examined the influence of various factors on the expression of a chimeric transgene that confers resistance to zeomycin. This marker comprises the bacterial ble gene flanked by 5Ј and 3Ј sequences from the Chlamydomonas RBCS2 gene. We found that the frequency with which transformants are recovered is significantly increased when ble is fused to shorter versions of the RBCS2 promoter and when Chlamydomonas introns are introduced into the coding region of ble. The latter effect is particularly evident in the case of the first intron of RBCS2, which dramatically stimulates the transformation frequency and the level of ble expression. We found that this improvement is mediated in part by an enhancer element within the intron sequence, and that this element acts in an orientation-independent manner and is effective when placed either upstream or downstream of the promoter. Our results demonstrate that stable high-level expression of a foreign gene in Chlamydomonas is possible, and highlight a potential role of introns as modulators of gene expression in this alga.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous inherited disorder arising from dysmotility of motile cilia and sperm. This is associated with a variety of ultrastructural defects of the cilia and sperm axoneme that affect movement, leading to clinical consequences on respiratory-tract mucociliary clearance and lung function, fertility, and left-right body-axis determination. We performed whole-genome SNP-based linkage analysis in seven consanguineous families with PCD and central-microtubular-pair abnormalities. This identified two loci, in two families with intermittent absence of the central-pair structure (chromosome 6p21.1, Zmax 6.7) and in five families with complete absence of the central pair (chromosome 6q22.1, Zmax 7.0). Mutations were subsequently identified in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect RSPH4A mutation present in UK-Pakistani pedigrees. Both RSPH9 and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 shows gene expression restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicate that radial spoke head proteins are important in maintaining normal movement in motile, "9+2"-structure cilia and flagella. This effect is rescued by reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in function of these genes was not associated with defects in left-right axis determination in humans or zebrafish.

The argininosuccinate lyase (ASL) gene of Chlamydomonas reinhardtii has been cloned using four oligonucleotide probes corresponding to highly conserved regions of the ASL polypeptide sequence. The identity of the gene was confirmed by partial sequencing. It is unique, contains several introns and spans a region < 7.8 kb that includes highly repetitive sequences. Using a particle gun, a reliable nuclear transformation system has been established by complementing three mutants deficient in ASL activity with the wild-type ASL gene. Analysis of the transformants reveals variable patterns of integration of the transforming DNA into the nuclear genome. Previous work has mapped the mutations in the mutants arg2 and arg7 to either end of the ARG7 locus 1.0 to 1.6 recombination map units apart. Our transformation results show that these two mutations are located within a region of 7.8 kb. This allows for the first correlation of the recombination map and the molecular map at the ARG7 locus and indicates a high recombination frequency in this region of the nuclear genome.

A chimeric gene composed of the coding sequence of the ble gene from Streptoalloteichus hindustanus fused to the 5' and 3' untranslated regions of the Chlamydomonas reinhardtii nuclear gene RBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome of C. reinhardtii by co-transformation with the ARG7 marker yields Arg+ transformants of which approximately 80% possess the ble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (PmR) phenotype. Western blot analysis using antibodies against the ble gene product confirms the presence of the protein in the PmR transformants and genetic analysis demonstrates the co-segregation of the ble gene with the phenotype in progeny arising from the mating of a PmR transformant to wild-type strains. Direct selection of PmR transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome of C. reinhardtii and provides a useful dominant marker for nuclear transformation.

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The spin-correlated radical pair [P(700)(+)A(1)(-)] gives rise to a characteristic "out-of-phase" electron spin-echo signal. The electron spin-echo envelope modulation (ESEEM) of these signals has been studied in thylakoids prepared from the wild-type strain of Chlamydomonas reinhardtii and in two site-directed mutants, in which the methionine residue which acts as the axial ligand to the chlorin electron acceptor A(0) has been substituted with a histidine either on the PsaA (PsaA-M684H) or the PsaB (PsaB-M664H) reaction center subunits. The analysis of the time domain ESEEM provides information about the spin-spin interaction in the [P(700)(+)A(1)(-)] radical pair, and the values of the dipolar (D) and the exchange (J) interaction can be extracted. From the distance dependence of the dipolar coupling term, the distance between the unpaired electron spin density clouds of the primary donor P(700)(+) and the phyllosemiquinone A(1)(-) can be determined. The [P(700)(+)A(1)(-)] ESEEM spectrum obtained in wild-type thylakoids can be reconstructed using a linear combination of the spectra measured in the PsaA and PsaB A(0) mutants, demonstrating that electron transfer resulting in charge separation is occurring on both the PsaA and PsaB branches. The [P(700)(+)A(1B)(-)] distance in the point dipole approximation in the PsaA-M684H mutant is 24.27 +/- 0.02 A, and the [P(700)(+)A(1A)(-)] distance in the PsaB-M664H mutant is 25.43 +/- 0.01 A. An intermediate value of 25.01 +/- 0.02 A is obtained in the wild-type membranes which exhibit both spin-polarized pairs.

Microalgae already serve as a major natural source of valuable macromolecules including carotenoids, long-chain polyunsaturated fatty acids and phycocolloids. As photoautotrophs, their simple growth requirements make these primitive plants potentially attractive bioreactor systems for the production of high-value heterologous proteins. The difficulty of producing stable transformants has meant that the field of transgenic microalgae is still in its infancy. Nonetheless, several species can now be routinely transformed and algal biotechnology companies have begun to explore the possibilities of synthesizing recombinant therapeutic proteins in microalgae and the engineering of metabolic pathways to produce increased levels of desirable compounds. In this review, we compare the current commercially viable bioreactor systems, outline recent progress in microalgal biotechnology and transformation, and discuss the potential of microalgae as bioreactors for the production of heterologous proteins.