Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast

Reporter genes have been successfully used in chloroplasts of higher plants, and high levels of recombinant protein expression have been reported. Reporter genes have also been used in the chloroplast of Chlamydomonas reinhardtii, but in most cases the amounts of protein produced appeared to be very low. We hypothesized that the inability to achieve high levels of recombinant protein expression in the C. reinhardtii chloroplast was due to the codon bias seen in the C. reinhardtii chloroplast genome. To test this hypothesis, we synthesized a gene encoding green fluorescent protein (GFP) de novo, optimizing its codon usage to reflect that of major C. reinhardtii chloroplast-encoded proteins. We monitored the accumulation of GFP in C. reinhardtii chloroplasts transformed with the codon-optimized GFP cassette (GFPct), under the control of the C. reinhardtii rbcL 5'- and 3'-UTRs. We compared this expression with the accumulation of GFP in C. reinhardtii transformed with a non-optimized GFP cassette (GFPncb), also under the control of the rbcL 5'- and 3'-UTRs. We demonstrate that C. reinhardtii chloroplasts transformed with the GFPct cassette accumulate approximately 80-fold more GFP than GFPncb-transformed strains. We further demonstrate that expression from the GFPct cassette, under control of the rbcL 5'- and 3'-UTRs, is sufficiently robust to report differences in protein synthesis based on subtle changes in environmental conditions, showing the utility of the GFPct gene as a reporter of C. reinhardtii chloroplast gene expression.     

The 5'' leader of a chloroplast mRNA mediates the translational requirements for two nucleus-encoded functions in Chlamydomonas reinhardtii.

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded function for psbC mRNA translation. The binding specificity of this protein appears to be for an AU-rich RNA sequence motif.     

N-terminal acetyltransferase 3 gene is essential for robust circadian rhythm of bioluminescence reporter in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a model species of algae for studies on the circadian clock. Previously, we isolated a series of mutants showing defects in the circadian rhythm of a luciferase reporter introduced into the chloroplast genome, and identified the genes responsible for the defective circadian rhythm. However, we were unable to identify the gene responsible for the defective circadian rhythm of the rhythm of chloroplast 97 (roc97) mutant because of a large genomic deletion. Here, we identified the gene responsible for the roc97 mutation through a genetic complementation study. This gene encodes a protein that is homologous to the subunit of N-terminal acetyltransferase (NAT) which catalyzes N-terminal acetylation of proteins. Our results provide the first example of involvement of the protein N-terminal acetyltransferase in the circadian rhythm.     

Real-time monitoring of chloroplast gene expression by a luciferase reporter: evidence for nuclear regulation of chloroplast circadian period

Chloroplast-encoded genes, like nucleus-encoded genes, exhibit circadian expression. How the circadian clock exerts its control over chloroplast gene expression, however, is poorly understood. To facilitate the study of chloroplast circadian gene expression, we developed a codon-optimized firefly luciferase gene for the chloroplast of Chlamydomonas reinhardtii as a real-time bioluminescence reporter and introduced it into the chloroplast genome. The bioluminescence of the reporter strain correlated well with the circadian expression pattern of the introduced gene and satisfied all three criteria for circadian rhythms. Moreover, the period of the rhythm was lengthened in per mutants, which are phototactic rhythm mutants carrying a long-period gene in their nuclear genome. These results demonstrate that chloroplast gene expression rhythm is a bona fide circadian rhythm and that the nucleus-encoded circadian oscillator determines the period length of the chloroplast rhythm. Our reporter strains can serve as a powerful tool not only for analysis of the circadian regulation mechanisms of chloroplast gene expression but also for a genetic approach to the molecular oscillator of the algal circadian clock.     

Optimized expression of dual reporter genes in transient transfection of purified Toxoplasma gondii using different promoters

Fluorescent protein and luciferase genes are valuable reporter genes and have been widely used for noninvasive monitoring of gene expression in living tissues and cells. We tested expression of the dual reporter genes in transient transfection of purified Toxoplasma gondii tachyzoites. Two copies of the enhanced yellow fluorescent protein (EYFP) gene were put under the control of 3 representative T.?gondii promoters (GRA1, SAG1, and DHFR). Fluorescence from each EYFP reporter was significantly higher than that from a green fluorescent protein (GFP) reporter. The GRA1-EYFP reporter gave the highest fluorescence. Although both fluorescence and luciferase were expressed in the dual reporter system, the luciferase reporter was more efficient than either the EYFP or GFP reporters, and it required fewer parasites to be successfully used.     

Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5' untranslated region optimization

Microalgae have the potential to be a valuable biotechnological platform for the production of recombinant proteins. However, because of the complex regulatory network that tightly controls chloroplast gene expression, heterologous protein accumulation in a wild-type, photosynthetic-competent algal chloroplast remains low. High levels of heterologous protein accumulation have been achieved using the psbA promoter/5' untranslated region (UTR), but only in a psbA-deficient genetic background, because of psbA/D1-dependent auto-attenuation. Here, we examine the effect of fusing the strong 16S rRNA promoter to the 5' UTR of the psbA and atpA genes on transgene expression in the chloroplast of Chlamydomonas reinhardtii. We show that fusion of the 16S promoter had little impact on protein accumulation from the psbA 5' UTR in a psbA-deficient genetic background. Furthermore, the 16S/psbA promoter/UTR fusion was silenced in the presence of wild-type levels of D1 protein, confirming that the psbA 5' UTR is the primary target for D1-dependent auto-repression. However, fusion of the 16S promoter to the atpA 5' UTR significantly boosts mRNA levels and supports high levels of heterologous protein accumulation in photosynthetic-competent cells. The 16S/atpA promoter/UTR drove LUXCT protein accumulation to levels close to that of psbA in a psbA- background, and drove expression of a human therapeutic protein to levels only twofold lower than the psbA 5' UTR. The 16S/atpA promoter/UTR combination should have utility for heterologous protein production when expression from a photosynthetic-competent microalgal strain is required.     

Development of a destabilized firefly luciferase enzyme for measurement of gene expression

Firefly luciferase is used widely as a reporter enzyme for studies of gene regulation and expression. The recent development of new technologies that combine luciferase reporter technology and digital imaging microscopy has enabled multiple measurements of gene expression in the same living cell. Although this approach has already provided new insights about expression dynamics, its future utility is limited by the three- to four-hour half-life of firefly luciferase in mammalian cells. Because of this, rapid increases or decreases in gene expression may not be detected, owing to the accumulation of residual luciferase. Accordingly, the goal of the present study was to develop a luciferase reporter with a reduced functional half-life. This was accomplished by adding a synthetic fragment to the firefly luciferase-coding sequence that encoded the proteolytic "PEST" signal from mouse ornithine decarboxylase. When placed under the control of estrogen response elements and expressed in human breast cancer T-47D cells, the modified luciferase protein (LUCODC-DA) displayed a functional half-life of 0.84 h compared to 3.68 h for the wild-type enzyme. As anticipated, the overall rate of photonic emissions in cells expressing the destabilized luciferase was about sevenfold lower than that of their wild-type counterparts, presumably because of the reduction of steady-state luciferase accumulation. Even so, the photonic activity derived from LUCODC-DA was still sufficient to enable real-time measurements of gene expression in single living cells.     

Mutations Altering the Predicted Secondary Structure of a Chloroplast 5′ Untranslated Region Affect Its Physical and Biochemical Properties as Well as Its Ability To Promote Translation of Reporter mRNAs Both in the Chlamydomonas reinhardtii Chloroplast and in Escherichia coli

Random mutations were generated in the sequence for the 5' untranslated region (5'UTR) of the Chlamydomonas reinhardtii chloroplast rps7 mRNA by PCR, the coding sequence for the mutant leaders fused upstream of the lacZ' reporter in pUC18, and transformed into Escherichia coli, and white colonies were selected. Twelve single base pair changes were found at different positions in the rps7 5'UTR in 207 white colonies examined. Seven of the 12 mutant leaders allowed accumulation of abundant lacZ' message. These mutant rps7 leaders were ligated into an aadA expression cassette and transformed into the chloroplast of C. reinhardtii and into E. coli. In vivo spectinomycin-resistant growth rates and in vitro aminoglycoside adenyltransferase enzyme activity varied considerably between different mutants but were remarkably similar for a given mutant expressed in the Chlamydomonas chloroplast and in E. coli. The variable effect of the mutants on aadA reporter expression and their complete abolition of lacZ' reporter expression in E. coli suggests differences in the interaction between the 5'UTR of rps7 and aadA or lacZ' coding regions. Several rps7 5'UTR mutations affected the predicted folding pattern of the 5'UTR by weakening the stability of stem structures. Site-directed secondary mutations generated to restore these structures in the second stem suppressed the loss of reporter activity caused by the original mutations. Additional site-directed mutations that were predicted to further strengthen (A-U-->G-C) or weaken (G-C-->A-U) the second stem of the rps7 leader both resulted in reduced reporter expression. This genetic evidence combined with differences between mutant and wild-type UV melting profiles and RNase T1 protection gel shifts further indicate that the predicted wild-type folding pattern in the 5'UTR is likely to play an essential role in translation initiation.     

Chloroplast ribosomal protein S7 of Chlamydomonas binds to chloroplast mRNA leader sequences and may be involved in translation initiation

Certain mutations isolated in the 5' untranslated region (5'UTR) of the chloroplast rps7 gene in Chlamydomonas reduce expression of reporter genes. Second site suppressors in this 5'UTR sequence restore reporter expression. 5'UTR sequences with the original mutations fail to bind a 20-kD protein, one of five proteins that bind to leaders of several chloroplast genes. However, 5'UTRs from suppressed mutants restore binding to this protein but do not bind a 47-kD protein present on the wild type and the original mutant 5'UTRs. The 20-kD protein was shown to be the S7 protein of the chloroplast ribosomal small subunit encoded by rps7, whereas the 47-kD protein was shown to be RB47, a poly(A) binding protein. Our data are consistent with the hypothesis that the S7 protein plays either a general or a specific regulatory role in translation initiation in the chloroplast.     

The chloroplast ycf7 (petL) open reading frame of Chlamydomonas reinhardtii encodes a small functionally important subunit of the cytochrome b6f complex.

The small chloroplast open reading frame ORF43 (ycf7) of the green unicellular alga Chlamydomonas reinhardtii is cotranscribed with the psaC gene and ORF58. While ORF58 has been found only in the chloroplast genome of C.reinhardtii, ycf7 has been conserved in land plants and its sequence suggests that its product is a hydrophobic protein with a single transmembrane alpha helix. We have disrupted ORF58 and ycf7 with the aadA expression cassette by particle-gun mediated chloroplast transformation. While the ORF58::aadA transformants are indistinguishable from wild type, photoautotrophic growth of the ycf7::aadA transformants is considerably impaired. In these mutant cells, the amount of cytochrome b6f complex is reduced to 25-50% of wild-type level in mid-exponential phase, and the rate of transmembrane electron transfer per b6f complex measured in vivo under saturating light is three to four times slower than in wild type. Under subsaturating light conditions, the rate of the electron transfer reactions within the b6f complex is reduced more strongly in the mutant than in the wild type by the proton electrochemical gradient. The ycf7 product (Ycf7) is absent in mutants deficient in cytochrome b6f complex and present in highly purified b6f complex from the wild-type strain. Ycf7-less complexes appear more fragile than wild-type complexes and selectively lose the Rieske iron-sulfur protein during purification. These observations indicate that Ycf7 is an authentic subunit of the cytochrome b6f complex, which is required for its stability, accumulation and optimal efficiency. We therefore propose to rename the ycf7 gene petL.