The Transformation of the Unicellular Alga Chlamydomonas reinhardtii by Electroporation

The cell wall–lacking mutant CW-15 of the unicellular green alga Chlamydomonas reinhardtii was transformed by electroporation using plasmid pCTVHyg, which was constructed with the hygromycin phosphotransferase genehpt as the selective marker and the Tn₅ transposon of Escherichia coli under the control of the virus SV40 early gene promoter. Under optimal conditions (10⁶ mid-exponential cells/ml; electric field strength 1 kV/cm; and pulse length 2 ms), the transformation yielded 10³ Hyg^R transformants per 10⁶ recipient cells. The exogenous DNA integrated into the nuclear genome of Ch. reinhardtii was persistently inherited through more than 350 cell generations. The advantages of this system for the transformation ofCh. reinhardtii with heterologous genes are discussed.     

Transformation of CW-15 Mutant Cells of Chlamydomonas reinhardtii Dang. with pCTVHyg Plasmid

A pCTVHyg plasmid was constructed in a unicellular green alga Chlamydomonas reinhardtii Dang. by using the hygromycin phosphotransferase gene (hpt) as a selectable marker and the Escherichia coli transposon Tn5 under the early SV40 viral gene promoter. CW-15 mutant cells devoid of cell walls were transformed by electroporation in an electric field of 1 kV/cm and a pulse duration of 2 ms. A suspension density of 10⁶ cell/ml and the mid-logarithmic growth phase were the optimum conditions for transformation, producing up to 10³ hygromycin-resistant (Hyg^R) clones per 10⁶ Hyg^R recipient cells. Exogenous DNA integrated in the nuclear genome of C. reinhardtii was steadily inherited in subsequent generations within at least a 8-month period; however, the Hyg^R trait manifestation was not stable. The comparative analysis of frequencies in codon usage in hpt and in the nuclear genes of C. reinhardtii significantly excluded the possibility that the bias in codon usage was the primary factor affecting foreign gene expression. The advantages of using theCW-15 mutant and the described selection system are discussed in the context of heterologous transformation of C. reinhardtii.     

Chloroplast genes encoding subunits of the H+-ATPase complex of Chlamydomonas reinhardtii are rearranged compared to higher plants: sequence of the atpE gene and location of the atpF and atpI genes

Summary The chloroplast gene for the epsilon subunit (atpE) of the CF₁/CF₀ ATPase in the green alga Chlamydomonas reinhardtii has been localized and sequenced. In contrast to higher plants, the atpE gene does not lie at the 3 end of the beta subunit (atpB) gene in the chloroplast genome of C. reinhardtii, but is located at a position 92 kb away in the other single copy region. The uninterrupted open reading frame for the atpE gene is 423 bp, and the epsilon subunit exhibits 43% derived amino acid homology to that from spinach. Codon usage for the atpE gene follows the restricted pattern seen in other C. reinhardtii chloroplast genes.The genes for the CF₀ subunits I (atpF) and IV (atpI) of the ATPase complex have also been mapped on the chloroplast genome of C. reinhardtii. The six chloroplast ATPase genes in C. reinhardtii are dispersed individually between the two single copy regions of the chloroplast genome, an organization strikingly different from the highly conserved arrangement in two operon-like units seen in chloroplast genomes of higher plants.Abbreviations bp base pairs - CF₁ chloroplast coupling factor 1 - CF₀ chloroplast coupling factor 0 - F₁ coupling factor 1 - F₀ coupling factor 0 - kb kilobase pairs     

Efficient transformation of mutant cells of Chlamydomonas reinhardtii by electroporation

Cells of CW-15 mutant of Chlamydomonas reinhardtii without a cell wall were transformed by electroporation. The hpt gene of hygromycin phosphotransferase was used as a selective marker. Optimal conditions of transformation were observed in the middle of the logarithmic growth phase at the density of suspension 10⁶ cells/ml, electric field intensity 1 kV/cm, and pulse duration 2 ms. Under these conditions up to 10³ hygromycin-resistant clones of trasformants per 10⁶ recipient cells were obtained that was 100 times higher than at the usage of wild-type cells. Exogenic DNA integrated into the genome of the nucleus C. reinhardtii was constantly inherited for more than 350 generations. The use of mutants without a cell wall and certain selective systems enable the efficiency of transformant yield to be doubled problems on unstable expression of geterologous genes to be investigated, and ways of obtaining super producers of foreign proteins using the alga C. reinhardtii investigated.     

Efficient expression of epidermal growth factor in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> CC400

The application of the green alga Chlamydomonas reinhardtii as a bioreactor is not adequate because of the difficulties caused by efficiency expressing foreign genes. To improve this efficiency a plasmid containing the epidermal growth factor (EGF) gene and a bleomycin resistance gene (ble) was constructed. We amplified the EGF gene according to the codon usage of C. reinhardtii. The vector carrying 2 expression cassettes for EGF gene and ble gene was constructed by adding rbc promoter and rbc terminator. Transformants, selected on Tris-acetate-phosphate medium containing 15 mg/L bleomycin, were screened by PCR and confirmed by Southern blotting, which showed that 3 transgenic C. reinhardtii cells contained only one copy of EGF gene integrated in different 3 sites of C. reinhardtii CC400 genome. Then EGF protein content of 3 transformants was determined by EGF precoated ELISA, indicating that EGF gene was first expressed, although at a low level, in algal cells. The presented study, as an example for expressing heterologous gene in green alga, provided feasibility to improve the efficiency of transformation of C. reinhardtii.     

Monitoring dynamic expression of nuclear genes in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> by using a synthetic luciferase reporter gene

For monitoring the expression profile of selected nuclear genes in Chlamydomonas reinhardtii in response to altered environmental parameters or during cell cycle, in the past many RNA or protein samples had to be taken and analyzed by RNA hybridization or protein immunoblotting. Here we report the synthesis of a gene that codes for the luciferase of Renilla reniformis (RLuc) and is adapted to the nuclear codon usage of C. reinhardtii. This crluc gene was expressed alone or as a fusion to the zeocin resistance gene ble under control of different promoter variants. Luciferase activity was monitored in living cells, increased with the promoter strength and paralleled the amount of expressed protein. Under control of the Lhcb-1 promoter the Luc-activity in synchronized cultures was dependent on the dark-light cycle. Additionally, crluc was placed under control of the Chop-2 promoter and activity was measured under different light conditions. Chop-2 promoter activity was found to be most pronouced under low-light and dark conditions, further supporting that channelrhodopsin-2 is most active in dark-adapted cells. We conclude that crluc is a reliable tool for convenient monitoring of nuclear gene expression in C. reinhardtii.     

Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation

Summary The respiratory deficient dum-1 mutant of Chlamydomonas reinhardtii fails to grow in the dark because of a terminal 1.5 kb deletion in the linear 15.8 kb mitochondrial genome, which affects the apocytochrome b (CYB) gene. In contrast to the wild type where only mitochondrial genomes of monomer length are observed, the dum-1 genomes are present as a mixture of monomer and dimer length molecules. The mutant dimers appear to result from head-to-head fusions of two deleted molecules. Furthermore, mitochondrial genomes of dum-1 were also found to be unstable, with the extent of the deletion varying among single cell clones from the original mutant population. The dum-1 mutant also segregates, at a frequency of ca. 4% per generation, lethal minute colonies in which the original deletion now extends at least into the adjacent gene encoding subunit four of NAD dehydrogenase (ND4). We have used the dum-1 mutant as a recipient to demonstrate stable mitochondrial transformation in C. reinhardtii employing the biolistic method. After 4 to 8 weeks dark incubation, a total of 22 respiratory competent colonies were isolated from plates of dum-1 cells bombarded with C. reinhardtii mitochondrial DNA (frequency 7.3 × 10^–7) and a single colony was isolated from plates bombarded with C. smithii mitochondrial DNA (frequency 0.8 × 10^–7). No colonies were seen on control plates (frequency < 0.96 × 10^–9). All transformants grew normally in the dark on acetate media; 22 transformants were homoplasmic for the wild-type mitochondrial genome typical of the C. reinhardtii donor. The single transformant obtained from the C. smithii donor had a recombinant mitochondrial genome containing the donor CYB gene and the diagnostic HpaI and XbaI restriction sites in the gene encoding subunit I of cytochrome oxidase (COI) from the C. reinhardtii recipient. The characteristic deletion fragments of the dum-1 recipient were not detected in any of the transformants.     

Successful Transient Expression of Cas9 and Single Guide RNA Genes in Chlamydomonas reinhardtii

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10⁹ cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing.     

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.     

Transformation of Chlamydomonas reinhardtii CW-15 with the hygromycin phosphotransferase gene as a selective marker

To transform Chlamydomonas reinhardtii Dang. Cells, plasmid pCTVHyg was constructed with the use of the Escherichia coli hygromycin phosphotransferase gene (hpt) controlled by the SV40 early promoter. Cells of the CW-15 mutant strain were transformed by electroporation, with the yield reaching 10(3) hygromycin-resistant (HygR) clones per 10(6) recipient cells. The exogenous DNA integrated in the Ch. reinhardtii nuclear genome showed stable transmission for approximately 350 cell generations, while hygromycin resistance was expressed as an unstable character. Codon usage was compared for the hpt gene and Ch. reinhardtii nuclear genes. The results testified that codon usage bias, which is characteristic of Ch. reinhardtii, is not the major factor affecting foreign gene expression. The advantages of the selective system for studying Ch. reinhardtii transformation with heterologous genes are discussed.     

A marine Chlamydomonas sp. emerging as an algal model

The freshwater microalga Chlamydomonas reinhardtii, which lives in wet soil, has served for decades as a model for numerous biological processes, and many tools have been introduced for this organism. Here, we have established a stable nuclear transformation for its marine counterpart, Chlamydomonas sp. SAG25.89, by fusing specific cis‐acting elements from its Actin gene with the gene providing hygromycin resistance and using an elaborated electroporation protocol. Like C. reinhardtii, Chlamydomonas sp. has a high GC content, allowing reporter genes and selection markers to be applicable in both organisms. Chlamydomonas sp. grows purely photoautotrophically and requires ammonia as a nitrogen source because its nuclear genome lacks some of the genes required for nitrogen metabolism. Interestingly, it can grow well under both low and very high salinities (up to 50 g · L^‐1) rendering it as a model for osmotolerance. We further show that Chlamydomonas sp. grows well from 15 to 28°C, but halts its growth at 32°C. The genome of Chlamydomonas sp. contains some gene homologs the expression of which is regulated according to the ambient temperatures and/or confer thermal acclimation in C. reinhardtii. Thus, knowledge of temperature acclimation can now be compared to the marine species. Furthermore, Chlamydomonas sp. can serve as a model for studying marine microbial interactions and for comparing mechanisms in freshwater and marine environments. Chlamydomonas sp. was previously shown to be immobilized rapidly by a cyclic lipopeptide secreted from the antagonistic bacterium Pseudomonas protegens PF‐5, which deflagellates C. reinhardtii.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5 and 3 untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R 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 ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

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.     

DNA-Binding Proteins and Structural Characteristics of Chloroplast Nucleoids

A comparative analysis of proteins from chloroplast nucleoids was performed in two higher-plant species (Pisum sativumL. andArabidopsis thalianaL.) and a green alga Chlamydomonas reinhardtiiDang. In the nucleoids of the higher plants and the alga, 26–27 proteins were detected with their mol wts ranging from 10 to more than 94 kD. In all the species tested, the distribution of nucleoid proteins by their mol wts was similar, especially between the predominant proteins with mol wts of 10 to 40 kD. Six DNA-binding proteins (12–18 kD) were detected in nucleoids fromCh. reinhardtiichloroplasts after in vivocovalent cross-linking between chloroplast DNA and proteins. Under an electron miscroscope, some regular structures resembling nucleosome-like particles of bacterial cells were revealed.     

Effect of amplification ofdhfr andlac Z genes on growth and β-galactosidase expression in suspension cultures of recombinant CHO cells

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10⁻⁷, and 10⁻⁶ M methotrexate (MTX), and the β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10⁻⁷ M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10⁻⁶ M MTX was 20% lower than that of recombinant CHO cells at 10⁻⁷ M MTX. There was no effect of 10⁻⁵ M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect ofdhfr and foreign gene amplification and increased β-galactosidase expression.     

MOLYBDENUM DEPENDENCE,NITRATE UPTAKE AND PHOTOSYNTHESIS OF FRESHWATER PLANKTON ALGAE1,2

The effects of molybdenum on photosynthesis and nitrate uptake by two species of freshwater algae, Navicula pelliculosa (Bréd.) Hilse and Chlamydomonas reinhardtii Dang. Were examined. Photosynthetic rates in cells of N. Pelliculosa deprived of Mo for 48 h were significantly lower than in nondeprived cells; the rates were 2.6 and 4.5 μg C/10⁶ cells/h, respectively. There was no significant reduction in photosynthetic rates of C. reinhardtii under the 2 treatments, the rates were both ca. 6.0 μg C/10⁶ cells/h. These observed rates in the 2 species were not altered by added Mo concentrations as high as 1.0 ppm. The chlorophyll a values in 48 h Mo-deprived cells were 1.15 μg/10⁶ cells compared to 1.57 μg/10⁶ cells provided with this metal. Molybdenum deficiency did not affect the chlorophyll a concentration in Chlamydomonas; the chlorophyll levels were 2.44μg/10⁶ cells. Nitrate uptake by Mo-deprived cells of N. Pelliculosa was significantly lower than in cells cultured in Mo; the rates were 0.087 and 0.238 μM NO_a⁻/10⁶ cells/h, respectively. Uptake rates by C. reinhardtii were similar with or deprived of Mo. The K_m values for No₃⁻ uptake were 14.9 and 148.0 μM for N. Pelliculosa and C. reinhardtii, respectively.     

Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library

We report the rescue of an arginine-requiring mutant (arg7-8) of Chlamydomonas reinhardtii by complementation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were obtained from 3×10⁹ treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene (ARG7) encoding argininosuccinate lyase (ASL). The arginine-independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones. We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach.