Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast

Luciferase reporter genes have been successfully used in a variety of organisms to examine gene expression in living cells, but are yet to be successfully developed for use in chloroplast. Green fluorescent protein (gfp) has been used as a reporter of chloroplast gene expression, but because of high auto-fluorescence, very high levels of GFP accumulation are required for visualization in vivo. We have developed a luciferase reporter for chloroplast by synthesizing the two-subunit bacterial luciferase (lux)AB, as a single fusion protein in Chlamydomonas reinhardtii chloroplast codon bias. We expressed a chloroplast luciferase gene, luxCt, in C. reinhardtii chloroplasts under the control of the ATPase alpha subunit (atpA) or psbA promoter and 5' untranslated regions (UTRs) and the rubisco large subunit (rbcL) 3' UTR. We show that luxCt is a sensitive reporter of chloroplast gene expression, and that luciferase activity can be measured in vivo using a charge coupled device (CCD) camera or in vitro using a luminometer. We further demonstrate that luxCt protein accumulation, as measured by Western blot analysis, is proportional to luminescence, as determined both in vivo and in vitro, and that luxCt is capable of reporting changes in chloroplast gene expression during a dark to light shift. These data demonstrate the utility of the luxCt gene as a versatile and sensitive reporter of chloroplast gene expression in living cells.     

Evaluation of the 5'-flanking regions of murine glutathione peroxidase five and cysteine-rich secretory protein-1 genes for directing transgene expression in mouse epididymis

Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice.     

Isolation and characterization of oxygen-evolving photosystem II complexes retaining the PsbO, P and Q proteins from Euglena gracilis

Oxygen-evolving photosystem II (PSII) complexes of Euglena gracilis were isolated and characterized. (1) The PSII complexes contained three extrinsic proteins of 33 kDa (PsbO), 23 kDa (PsbP) and 17 kDa (PsbQ), and showed oxygen-evolving activity of around 700 micromol O2 (mg Chl)(-1) h(-1) even in the absence of Cl- and Ca2+ ions. (2) NaCl-treatment removed not only PsbP and PsbQ but also a part of PsbO from Euglena PSII, indicating that PsbO binds to Euglena PSII more loosely than those of other organisms. Treatments by urea/NaCl, alkaline Tris or CaCl2 completely removed the three extrinsic proteins from Euglena PSII. (3) Each of the Euglena extrinsic proteins bound directly to PSII independent of the other extrinsic proteins, which is similar to the binding properties of the extrinsic proteins in a green alga, Chlamydomonas reinhardtii. (4) One of the significant features of Euglena PSII is that the oxygen evolution was not enhanced by Ca2+. When CaCl2-treated Euglena PSII was reconstituted with PsbO, the oxygen-evolving activity was stimulated by the addition of NaCl, but no further stimulation was observed by CaCl2. (5) Oxygen evolution of Euglena PSII reconstituted with PsbO from C. reinhardtii or spinach instead of that from Euglena also showed no enhancement by Ca2+, whereas a significant enhancement of oxygen evolution was observed by Ca2+ when the green algal or higher plant PSII was reconstituted with Euglena PsbO instead of their own PsbO. These results indicate that the PSII intrinsic proteins instead of the extrinsic PsbO protein, are responsible for the stimulation of oxygen evolution by Ca2+. Sequence comparison of major PSII intrinsic proteins revealed that PsbI of Euglena PSII is remarkably different from other organisms in that Euglena PsbI possesses extra 16-17 residues exposed to the luminal side. This may be related to the loss of enhancement of oxygen evolution by Ca2+ ion.     

A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii†

The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes. We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C. reinhardtii (cgfp). After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C. reinhardtii under control of the rbcS2 promoter and intron sequences. The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein. The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C. reinhardtii under control of the cop promoter. The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin. We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C. reinhardtii and possibly in related green algal species.     

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 chloroplast calcium sensor CAS is required for photoacclimation in Chlamydomonas reinhardtii

The plant-specific calcium binding protein CAS (calcium sensor) has been localized in chloroplast thylakoid membranes of vascular plants and green algae. To elucidate the function of CAS in Chlamydomonas reinhardtii, we generated and analyzed eight independent CAS knockdown C. reinhardtii lines (cas-kd). Upon transfer to high-light (HL) growth conditions, cas-kd lines were unable to properly induce the expression of LHCSR3 protein that is crucial for nonphotochemical quenching. Prolonged exposure to HL revealed a severe light sensitivity of cas-kd lines and caused diminished activity and recovery of photosystem II (PSII). Remarkably, the induction of LHCSR3, the growth of cas-kd lines under HL, and the performance of PSII were fully rescued by increasing the calcium concentration in the growth media. Moreover, perturbing cellular Ca(2+) homeostasis by application of the calmodulin antagonist W7 or the G-protein activator mastoparan impaired the induction of LHCSR3 expression in a concentration-dependent manner. Our findings demonstrate that CAS and Ca(2+) are critically involved in the regulation of the HL response and particularly in the control of LHCSR3 expression.     

REP27, a tetratricopeptide repeat nuclear-encoded and chloroplast-localized protein, functions in D1/32-kD reaction center protein turnover and photosystem II repair from photodamage

The goal of this research is elucidation of the molecular mechanism for the unique photosystem II (PSII) damage and repair cycle in chloroplasts. A frequently occurring, irreversible photooxidative damage inhibits the PSII charge separation reaction and stops photosynthesis. The chloroplast PSII repair process rectifies this adverse effect by selectively removing and replacing the photoinactivated D1/32-kD reaction center protein (the chloroplast-encoded psbA gene product) from the massive (>1,000 kD) water-oxidizing and O2-evolving PSII holocomplex. DNA insertional mutagenesis in the model organism Chlamydomonas reinhardtii was applied for the isolation and characterization of rep27, a repair-aberrant mutant. Gene cloning and biochemical analyses in this mutant resulted in the identification of REP27, a nuclear gene encoding a putative chloroplast-targeted protein, which is specifically required for the completion of the D1 turnover process but is not essential for the de novo biogenesis and assembly of the PSII holocomplex in this model green alga. The REP27 protein contains two highly conserved tetratricopeptide repeats, postulated to facilitate the psbA mRNA cotranslational insertion of the nascent D1 protein in the existing PSII core template. Elucidation of the PSII repair mechanism may reveal the occurrence of hitherto unknown regulatory and catalytic reactions for the selective in situ replacement of specific proteins from within multiprotein complexes.     

Stable expression of antibiotic-resistant gene ble from Streptoalloteichus hindustanus in the mitochondria of Chlamydomonas reinhardtii

The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii.     

Chloroplast-encoded PsbT is required for efficient biogenesis of photosystem II complex in the green alga Chlamydomonas reinhardtii

The small hydrophobic polypeptide PsbT is associated with the photosystem II (PSII) reaction center (D1/D2 heterodimer). Here, we report the effect of the deletion of PsbT on the biogenesis of PSII complex during light-induced greening of y-1 mutants of the green alga Chlamydomonas reinhardtii. The y-1 is unable to synthesize chlorophylls in the dark but do so in the light. The dark-grown y-1 cells accumulated no major PSII proteins but a small amount of PsbT. Upon illumination, PsbT was immediately synthesized while chlorophylls, major PSII proteins, and O(2)-evolving activity increased after a 1-h lag. The y-1 cells without PsbT accumulated chlorophylls and PSI protein at a similar rate, whereas the accumulation of PSII complex was specifically retarded during greening. The absence of PsbT did not affect the synthesis of PSII proteins. These results indicate that PsbT is required for the efficient biogenesis of PSII complex.     

A luciferase reporter assay to investigate the differential selenium-dependent stability of selenoprotein mRNAs

The mechanisms regulating the differential selenium (Se)-dependent stability of selenoprotein mRNAs are partially characterized. To further study the Se-dependent regulation of selenoproteins, we developed a novel chemiluminescent reporter to monitor the steady-state mRNA level of an artificial selenoprotein. Our reporter is a fusion of the Renilla luciferase gene and of the β-globin gene, but contains features required for incorporation of selenocysteine (SEC), namely, a UGA-SEC codon and a 3' untranslated region RNA stem loop called a SEC incorporation sequence (SECIS). At various levels of Se, the activity of reporters containing GPX1 or GPX4 SECIS elements is proportional to the steady-state mRNA level of the reporter construct and reflects the level of the corresponding endogenous mRNA. In a reporter containing a UGA codon and a functional GPX1 SECIS, Se-dependent nonsense-mediated decay (NMD) occurred in the cytoplasm, as opposed to the more typical nuclear location. To validate the reporter system, we used genetic and pharmacologic approaches to inhibit or promote NMD. Modulation of UPF1 by siRNA, overexpression, or by inhibition of SMG1 altered NMD in this system. Our reporter is derived from a Renilla luciferase reporter gene fused to an intron containing B-globin gene and is subject to degradation by NMD when a stop codon is inserted before the second intron.     

Identification of Lhcb gene family encoding the light-harvesting chlorophyll-a/b proteins of photosystem II in Chlamydomonas reinhardtii

The Lhcb gene family in green plants encodes several light-harvesting Chl a/b-binding (LHC) proteins that collect and transfer light energy to the reaction centers of PSII. We comprehensively characterized the Lhcb gene family in the unicellular green alga, Chlamydomonas reinhardtii, using the expressed sequence tag (EST) databases. A total of 699 among over 15,000 ESTs related to the Lhcb genes were assigned to eight, including four new, genes that we isolated and sequenced here. A sequence comparison revealed that six of the Lhcb genes from C. reinhardtii correspond to the major LHC (LHCII) proteins from higher plants, and that the other two genes (Lhcb4 and Lhcb5) correspond to the minor LHC proteins (CP29 and CP26). No ESTs corresponding to another minor LHC protein (CP24) were found. The six LHCII proteins in C. reinhardtii cannot be assigned to any of the three types proposed for higher plants (Lhcb1-Lhcb3), but were classified as follows: Type I is encoded by LhcII-1.1, LhcII-1.2 and LhcII-1.3, and Types II, III and IV are encoded by LhcII-2, LhcII-3 and LhcII-4, respectively. These findings suggest that the ancestral LHC protein diverged into LHCII, CP29 and CP26 before, and that LHCII diverged into multiple types after the phylogenetic separation of green algae and higher plants.     

Cell fusion activity of hepatitis C virus envelope proteins

To examine the cell fusion activity of hepatitis C virus (HCV) envelope proteins (E1 and E2), we have established a sensitive cell fusion assay based on the activation of a reporter gene as described previously (O. Nussbaum, C. C. Broder, and E. A. Berger, J. Virol. 68:5411-5422, 1994). The chimeric HCV E1 and E2 proteins, each consisting of the ectodomain of the E1 and E2 envelope protein and the transmembrane and cytoplasmic domains of the vesicular stomatitis virus G glycoprotein, were expressed on the cell surface. Cells expressing the chimeric envelope proteins and T7 RNA polymerase were cocultured with the various target cell lines transfected with a reporter plasmid encoding the luciferase gene under the control of the T7 promoter. After cocultivation, the cell fusion activity was determined by the expression of luciferase in the cocultured cells. The induction of cell fusion requires both the chimeric E1 and E2 proteins and occurs in a low-pH-dependent manner. Although it has been shown that HCV E2 protein binds human CD81 (P. Pileri, Y. Uematsu, S. Campagnoli, G. Galli, F. Falugi, R. Petracca, A. J. Weiner, M. Houghton, D. Rosa, G. Grandi, and S. Abrignani, Science 282:938-941, 1998), the expression of human CD81 alone is not sufficient to confer susceptibility to cell fusion in the mouse cell line. Treatment of the target cells with pronase, heparinase, or heparitinase reduced the cell fusion activity induced by the chimeric envelope proteins. These results suggest (i) that both HCV E1 and E2 proteins are responsible for fusion with the endosomal membrane after endocytosis and (ii) that certain protein molecules other than human CD81 and some glycosaminoglycans on the cell surface are also involved in the cell fusion induced by HCV.     

Hydrogen peroxide sensitivity of catechol-2,3-dioxygenase: a cautionary note on use of xylE reporter fusions under aerobic conditions

Catechol-2,3-dioxygenase (C23O) of Pseudomonas putida, encoded by the xylE gene, was found to be sensitive to hydrogen peroxide (H(2)O(2)) when used as a reporter in gene fusion constructs. Exposure of Pseudomonas aeruginosa katA or katA katB mutants harboring katA- or katB-lacZ (encoding beta-galactosidase) or -xylE fusion plasmids to H(2)O(2) stimulated beta-galactosidase activity, while there was little or no detectable C23O activity in these strains. More than 95% of C23O activity was lost after a 5-min exposure to equimolar H(2)O(2), while a 10,000-fold excess was required for similar inhibition of beta-galactosidase. Electron paramagnetic resonance spectra of the nitrosyl complexes of C23O showed that H(2)O(2) nearly stoichiometrically oxidized the essential active-site ferrous ion, thus accounting for the loss of activity. Our results suggest using caution in interpreting data derived from xylE reporter fusions under aerobic conditions, especially where oxidative stress is present or when catalase-deficient strains are used.     

Differing involvement of sulfoquinovosyl diacylglycerol in photosystem II in two species of unicellular cyanobacteria.

Sulfoquinovosyl diacylglycerol (SQDG) is involved in the maintenance of photosystem II (PSII) activity in Chlamydomonas reinhardtii[Minoda, A., Sato, N., Nozaki, H., Okada, K., Takahashi, H., Sonoike, K. & Tsuzuki, M. et al. (2002) Eur. J. Biochem.269, 2353-2358]. To understand the spread of the taxa in which PSII interacts with SQDG, especially in cyanobacteria, we produced a mutant defective in the putative sqdB gene responsible for SQDG synthesis from two cyanobacteria, Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942. The mutant of PCC6803, designated SD1, lacked SQDG synthetic ability and required SQDG supplementation for its growth. After transfer from SQDG-supplemented to SQDG-free conditions, SD1 showed decreased net photosynthetic and PSII activities on a chlorophyll (Chl) basis with a decrease in the SQDG content. Moreover, the sensitivity of PSII activity to 3-(3,4-dichlorophenyl)-1,1-dimethylurea and atrazine was increased in SD1. However, SD1 maintained normal amounts of cytochrome b559 and D1 protein (the subunits comprising the PSII complex) on a Chl basis, indicating that the PSII complex content changed little, irrespective of a decrease in the SQDG content. These results suggest that the role of SQDG is the conservation of the PSII properties in PCC6803, consistent with the results obtained with C. reinhardtii. In contrast, the SQDG-null mutant of PCC7942 showed the normal level of PSII activity with little effect on its sensitivity to PSII herbicides. Therefore, the difference in the SQDG requirement for PSII is species-specific in cyanobacteria; this could be of use when investigating the molecular evolution of the PSII complex.     

Three-dimensional structure of Chlamydomonas reinhardtii and Synechococcus elongatus photosystem II complexes allows for comparison of their oxygen-evolving complex organization

Electron microscopy and single-particle analyses have been carried out on negatively stained photosystem II (PSII) complexes isolated from the green alga Chlamydomonas reinhardtii and the thermophilic cyanobacterium Synechococcus elongatus. The analyses have yielded three-dimensional structures at 30-A resolution. Biochemical analysis of the C. reinhardtii particle suggested it to be very similar to the light-harvesting complex II (LHCII).PSII supercomplex of spinach, a conclusion borne out by its three-dimensional structure. Not only was the C. reinhardtii LHCII.PSII supercomplex dimeric and of comparable size and shape to that of spinach, but the structural features for the extrinsic OEC subunits bound to the lumenal surface were also similar thus allowing identification of the PsbO, PsbP, and PsbQ OEC proteins. The particle isolated from S. elongatus was also dimeric and retained its OEC proteins, PsbO, PsbU, and PsbV (cytochrome c(550)), which were again visualized as protrusions on the lumenal surface of the complex. The overall size and shape of the cyanobacterial particle was similar to that of a PSII dimeric core complex isolated from spinach for which higher resolution structural data are known from electron crystallography. By building the higher resolution structural model into the projection maps it has been possible to relate the positioning of the OEC proteins of C. reinhardtii and S. elongatus with the underlying transmembrane helices of other major intrinsic subunits of the core complex, D1, D2, CP47, and CP43 proteins. It is concluded that the PsbO protein is located over the CP47 and D2 side of the reaction center core complex, whereas the PsbP/PsbQ and PsbV/PsbU are positioned over the lumenal surface of the N-terminal region of the D1 protein. However, the mass attributed to PsbV/PsbU seems to bridge across to the PsbO, whereas the PsbP/PsbQ proteins protrude out more from the lumenal surface. Nevertheless, within the resolution and quality of the data, the relative positions of the center of masses for OEC proteins of C. reinhardtii and S. elongatus are similar and consistent with those determined previously for the OEC proteins of spinach.     

Binding and functional properties of the extrinsic proteins in oxygen-evolving photosystem II particle from a green alga,Chlamydomonas reinhardtii having his-tagged CP47

Oxygen-evolving photosystem II (PSII) particles were purified from Chlamydomonas reinhardtii having His-tag extension at the C terminus of the CP47 protein, by a single-step Ni(2+)-affinity column chromatography after solubilization of thylakoid membranes with sucrose monolaurate. The PSII particles consisted of, in addition to intrinsic proteins, three extrinsic proteins of 33, 23 and 17 kDa. The preparation showed a high oxygen-evolving activity of 2,300-2,500 micro mol O(2) (mg Chl)(-1) h(-1) in the presence of Ca(2+) using ferricyanide as the electron acceptor, while its activity was 680-720 micro mol O(2) (mg Chl)(-1) h(-1) in the absence of Ca(2+) and Cl(-) ions. The activity was 710-820 micro mol O(2) (mg Chl)(-1) h(-1) independent of the presence or absence of Ca(2+) and Cl(-) when 2,6-dichloro-p-benzoquinone was used as the acceptor. These activities were scarcely inhibited by DCMU. The kinetics of flash-induced fluorescence decay revealed that the electron transfer from Q(A)(-) to Q(B) was significantly inhibited, and the electron transfer from Q(A)(-) to ferricyanide was largely stimulated in the presence of Ca(2+). These results indicate that the acceptor side, Q(B) site, was altered in the PSII particles but its donor side remained intact. Release-reconstitution experiments revealed that the extrinsic 23 and 17 kDa proteins were released only partially by NaCl-wash, while most of the three extrinsic proteins were removed when treated with urea/NaCl, alkaline Tris or CaCl(2). The 23 and 17 kDa proteins directly bound to PSII independent of the other extrinsic proteins, and the 33 kDa protein functionally re-bound to CaCl(2)-treated PSII which had been reconstituted with the 23 and 17 kDa proteins. These binding properties were largely different from those of the extrinsic proteins in higher plant PSII, and suggest that each of the three extrinsic proteins has their own binding sites independent of the others in the green algal PSII.