Stable nuclear transformation of Pandorina morum
© Lerche and Hallmann; licensee BioMed Central Ltd. 2014
Received: 31 March 2014
Accepted: 11 July 2014
Published: 17 July 2014
Volvocine green algae like Pandorina morum represent one of the most recent inventions of multicellularity diverged from their unicellular relatives. The 8–16 celled P. morum alga and its close multicellular relatives constitute a model lineage for research into cellular differentiation, morphogenesis and epithelial folding, sexual reproduction and evolution of multicellularity. Pandorina is the largest and most complex organism in the volvocine lineage that still exhibits isogamous sexual reproduction. So far, molecular-biological investigations in P. morum were constricted due to the absence of methods for transformation of this species, which is a prerequisite for introduction of reporter genes and (modified) genes of interest.
Stable nuclear transformation of P. morum was achieved using chimeric constructs with a selectable marker, a reporter gene, promoters and upstream and downstream flanking sequences from heterologous sources. DNA was introduced into the cells by particle bombardment with plasmid-coated gold particles. The aminoglycoside 3′-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus under control of an artificial, heterologous promoter was used as the selectable marker. The artificial promoter contained a tandem arrangement of the promoter of both the heat shock protein 70A (hsp70A) and the ribulose-1,5-bisphosphat-carboxylase/-oxygenase S3 (rbcS3) gene of Volvox carteri. Due to the expression of aphVIII, transformants gained up to 333-fold higher resistance to paromomycin in comparison to the parent wild-type strain.
The heterologous luciferase (gluc) gene of Gaussia princeps, which was previously genetically engineered to match the nuclear codon usage of Chlamydomonas reinhardtii, was used as a co-transformed, unselectable reporter gene. The expression of the co-bombarded gluc gene in transformants and the induction of gluc by heat shock were demonstrated through bioluminescence assays.
Stable nuclear transformation of P. morum using the particle bombardment technique is now feasible. Functional expression of heterologous genes is achieved using heterologous flanking sequences from Volvox carteri and Chlamydomonas reinhardtii. The aphVIII gene of the actinobacterium S. rimosus can be used as a selectable marker for transformation experiments in the green alga P. morum. The gluc gene of the marine copepod G. princeps, expressed under control of heterologous promoter elements, represents a suitable reporter gene for monitoring gene expression or for other applications in P. morum.
KeywordsCo-transformation Gaussia princeps luciferase gene Genetic engineering Green algae Heterologous expression Reporter genes Selectable markers Streptomyces rimosus aphVIII gene Volvocaceae Volvocine algae
The cells of P. morum form an ellipsoid spheroid with a distinct anterior-posterior polarity. The way of vegetative reproduction and mode of daughter colony formation where the young colonies break out of the old parental layers reminded Bory of the legend of Pandora’s box; thus, he applied the French diminutive, Pandorina, to the genus he named . There are several distinctive features of Pandorina that warrant its inclusion in the series of volvocine algae to be analyzed molecularly in the future. Pandorina has a special morphology among the volvocine algae: the cells fill nearly the entire volume of the colony, as opposed to other, larger volvocine algae in which the cells are arranged at the periphery of the colony, with extracellular matrix filling the space between cells. The compact morphology characteristic of Pandorina (Figure 1) appears to be derived from ancestors with expanded ECM . Furthermore, Pandorina is the smallest volvocine alga to exhibit a ‘colonial boundary’, the tripartite boundary that surrounds the entire colony. This ‘colonial boundary’ is the most conserved morphological feature of the Volvocalean extracellular matrix . Pandorina is moreover the largest and most complex organism in the volvocine lineage that still exhibits isogamous sexual reproduction [2, 4]. Thus, the gametes of P. morum are almost identical in morphology, differing genetically only in allele expression in the mating-type locus (mating type plus or minus) . The molecular-biological investigation and the comparison of molecular characteristics between the isogamous sexual reproduction in P. morum and anisogamous species like Eudorina could reveal insights into the evolution of anisogamy, but such studies are hampered by the absence of methods for genetic engineering of P. morum. In contrast, the anisogamous species Eudorina is accessible for genetic manipulations . The technical feasibility of genetically manipulating a species of interest is a key factor for any detailed molecular analysis of gene or protein functions.
The nuclear genome of P. morum has not been sequenced, only its mitochondrial genome has been revealed . Actually, only the genomes of C. reinhardtii and V. carteri have been sequenced in the group of volvocine algae. For both species effective transformation protocols exist and they are accessible to molecular manipulations [15–19]. Nuclear transformation protocols also exist for the volvocine algae Gonium pectorale and Eudorina elegans. There is a single transformation technique that worked in all of the previously transformed volvocine species, namely particle bombardment.
Aside from the nuclear transformation protocol, selectable markers and reporter genes are required for genetic manipulations. In the volvocine algae, selectable markers and reporter genes have mainly been established for C. reinhardtii[5, 21–28] and, to a lesser extent, for V. carteri[28–31], G. pectorale and E. elegans. In the present study, we wanted to check whether the vectors and transformation methods that were developed earlier for volvocine algae — in particular those for G. pectorale and E. elegans — can be applied with equal success to Pandorina.
The most promising candidate for a selectable marker gene in Pandorina was the aphVIII gene of Streptomyces rimosus that has been used as a selectable marker in C. reinhardtii, V. carteri[30, 31], G. pectorale and E. elegans. The aphVIII gene codes for an aminoglycoside 3′-phosphotransferase enzyme that catalyzes the transfer of the γ-phosphate of ATP to the 3′-position of an aminoglycoside antibiotic like paromomycin; this transfer chemically modifies and inactivates the antibiotic molecule. Paromomycin otherwise inhibits cell growth by blocking the protein synthesis of ribosomes at the translocation stage .
The best candidate for a useful reporter gene in P. morum seemed to be the luciferase gene (gluc) of the deep sea copepod Gaussia princeps[33, 34], which was previously adapted to the nuclear codon usage of C. reinhardtii. The Gluc protein catalyzes the oxidation of the substrate coelenterazine into the excited form of coelenteramide that releases a photon with a wavelength of 480 nm upon electron transition to the ground state . The blue light produced by this reaction can be detected by various methods like luminometer measurements and assays on light sensitive films . The gluc reporter gene has successfully been introduced into C. reinhardtii, G. pectorale and E. elegans and it was functionally expressed in all three organisms.
The selectable markers and reporter genes also require appropriate promoters. Based on previous results, promising candidates were the C. reinhardtii promoter of the abundant protein of photosystem I complex gene (PSAD) , the C. reinhardtii promoter of the heat shock protein 70A (HSP70A) , and the V. carteri tandem promoter of the hsp70A and rbcS3 genes . The C. reinhardtii HSP70A promoter and the V. carteri hsp70A/rbcS3 tandem promoter have already proved useful for expression of the aphVIII gene in V. carteri, C. reinhardtii, G. pectorale and E. elegans[11, 20, 30].
Here we demonstrate the stable nuclear transformation of P. morum using chimeric constructs containing heterologous aphVIII and gluc genes under the control of heterologous 5′ and 3′ flanking sequences from V. carteri and C. reinhardtii. The enzyme activity of the heterologously expressed Gaussia luciferase was detectable by luminometer measurements and bioluminescence assays. The expression of the heterologous gluc gene was further increased in transformants containing the gluc gene under control of the heterologous tandem promoter including the heat shock promoter, if the transformants were subject to a temporary shift to higher temperatures.
Antibiotic tolerance of wild-type Pandorinaalgae
Transformation experiments using aphVIII as a selectable marker
Most successful combination of parameters for P. morum transformation using a PDS-1000/He biolistic device
Material of microprojectiles
Size of microprojectiles
0.6 μm in diameter
Selectable marker plasmid
Coating of microprojectiles
plasmid-DNA/microcarrier/CaCl2/spermidine/ ethanol -precipitation
immobilized on cellulose acetate membrane filter; almost free of liquid
Burst pressure of rupture disk
Rupture disk-macrocarrier distance
Macrocarrier-stopping screen distance
Stopping screen-target cell distance
27 inch Hg
Cultivation after particle bombardment
in liquid medium
Unselectable plasmids were co-transformed with the selectable marker plasmid pPmr3. For use as a reporter gene, the gluc gene was brought under control of different heterologous 5′ and 3′ flanking sequences from V. carteri and C. reinhardtii. The utilized plasmids were pPsaD-GLuc, pHsp70A-GLuc or pHRLucP (Figure 3B-D) [11, 20]. After the transformation procedure, the algae were incubated in liquid medium and allowed to recover for 24 h before paromomycin was added. Within 48 h all non-transformed algae died, which resulted in a temporary clarification of the medium. After 10–14 days of incubation, re-greening of the incubation flasks indicated the initial presence and reproduction of at least one paromomycin-resistant transformant per flask. Using this protocol, a total of 38 paromomycin resistant clones were obtained in 4 independent transformation experiments.
Paromomycin resistance of transformants
Results of paromomycin resistance assays*
Resistance up to x μg/ml paromomycin
Resistance up to x μg/ml paromomycin
Resistance up to x μg/ml paromomycin
Stable genomic integration and expression of the artificial aphVIII gene
In addition to the provided indirect evidence of aphVIII expression through the mediated antibiotic resistance in transformants, the transcription of the aphVIII gene was also examined directly by RT-PCR analysis. For it, RNA from paromomycin-resistant transformants was reverse transcribed, and products were amplified by PCR using aphVIII specific primers. A cDNA fragment with 324 bp was expected. Actually, a PCR fragment of the expected size was obtained from all investigated paromomycin-resistant transformants, but not from the wild-type (Figure 4D). The amplified fragments were cloned and sequenced (Figure 4E).
Calculation of transformation frequency
The transformation frequency was calculated as the ratio of transformants that exhibited both resistance against paromomycin and the correct aphVIII fragment following PCR and the total number of cells used for each transformation experiment (9 × 106 cells). Based on the results of 4 independent transformation experiments, a transformation frequency of 1.2 × 10-6 per cell was calculated. This corresponds to a transformation frequency of about 1.9 × 10-5 per organism.
Plasmids for co-transformation with non-selectable reporter gene constructs
To investigate the efficiency of co-transformation in P. morum, three different plasmids were used containing the gluc gene of G. princeps, which was previously engineered to match the codon usage of C. reinhardtii.
Plasmid pPsaD-GLuc contains the gluc gene under control of 5′ and 3′ control elements of the PSAD gene of C. reinhardtii[27, 36] (Figure 3B). In three volvocine species, i.e., C. reinhardtii, G. pectorale and E. elegans, the PSAD promoter was shown to mediate strong expression [11, 20, 27].
Plasmid pHsp70A-GLuc [11, 20, 27] (Figure 3C) contains the gluc gene flanked by the C. reinhardtii HSP70A 5′UTR and the RBCS2 3′UTR of C. reinhardtii. The HSP70A promoter was shown to mediate not only strong but also heat-inducible expression. The plasmid also contains the first three exons and two introns of the C. reinhardtii HSP70B gene that codes for the chloroplast transit peptide of the HSP70B protein [27, 38, 39].
Except for flanking sequences of C. reinhardtii, we also tested flanking sequences of V. carteri. Plasmid pHRLucP contains the coding region of the gluc gene fused to a V. carteri hsp70A/rbcS3 tandem promoter and a C. reinhardtii PSAD 3′UTR (Figure 3D). Like in plasmid pHsp70A-GLuc, the tandem promoter should mediate not only strong but also heat-inducible expression.
Stable integration of non-selectable reporter gene constructs into the genome
Functional expression of co-transformed reporter genes
In contrast, luciferase activity was detected in several transformants generated by co-transformation with the plasmid pHRLucP (Figure 6B). In this DNA construct the gluc gene is under control of the artificial tandem promoter from the hsp70A and rbcS3 genes of V. carteri. Extracts of ten out of sixteen transformants clearly showed emission of light and, thus, functional expression of luciferase. The amount of detected relative light units (rlu) is significantly higher than in the wild-type strain (853 ± 11 rlu). The transformants PanHR-9 and PanHR-11 exhibited the highest Gluc activity with 407,500 ± 45,000 rlu and 378,500 ± 57,000 rlu, respectively.
Extracts of transformants generated by co-transformation with the plasmid pHsp70A-GLuc showed a significant but mostly moderate luciferase activity when cultivated under standard conditions at 29°C (Figure 6C). In this DNA construct the gluc gene is under the control of the C. reinhardtii HSP70A promoter; there is also the genomic sequence of the C. reinhardtii HSP70B chloroplast transit peptide containing two introns that precedes the gluc coding sequence. Only transformant PanHsp-4 exhibited a high Gluc activity of 786,000 ± 20,000 rlu under these conditions, while the wild-type strain showed only 966 ± 50 rlu.
The DNA constructs in pHRLucP and pHsp70A-GLuc are driven by heat-shock promoters that show inducibility by heat in their donor organisms V. carteri and C. reinhardtii, respectively.
The results from the in-gel activity assays substantiate the heat-inducibility of heat-shock promoters from heterologous sources in P. morum.
Long term stability of DNA integration and gene expression
For any biotechnological approach with transgenic organisms, the stability of the transgenes is of fundamental importance. Therefore, the stability and expression of the aphVIII and gluc genes introduced into P. morum transformants was investigated after twelve months of cultivation or, in other words, after about 300 generations. Cultivation was without selective pressure during the twelve months. The presence of the heterologous genes was rechecked by PCR and the functionality of the expression products was reexamined by paromomycin resistance assays and luminometer assays. In all transformants, PCR fragments of the expected sizes were amplified and, thus, the genomic integration of the artificial gene constructs could be confirmed by PCR. All transformants exhibited about the same antibiotic resistance compared to the values determined right after transformation; the range of fluctuations was only +/-6%. Likewise, luciferase activity in the transformants was stable. The comparison of the values after twelve months of cultivation with the values determined right after transformation gave a fluctuation range of only +/-7%. Thus, both integration and expression of the heterologous aphVIII and gluc genes are stable in P. morum.
Here we demonstrate the stable nuclear transformation of the green alga P. morum by particle bombardment with plasmid-coated gold particles. Our approach only uses plasmids containing chimeric constructs from heterologous sources. Particle bombardment also was efficient for transformation of C. reinhardtii, V. carteri, G. pectorale and E. elegans. The capability for stable genetical modification of an organism of interest is an important requirement in modern molecular biology, because this technique facilitates the identification of gene functions and allows for the introduction of additional characteristics and abilities.
For stable nuclear transformation of P. morum the aphVIII gene of S. rimosus driven by a V. carteri hsp70A/rbcS3 tandem promoter proved to be a suitable selectable marker, even though all parts of the DNA construct came from heterologous sources. Transformants generated with the aphVIII construct show an up to 333-fold higher tolerance to the aminoglycoside antibiotic paromomycin compared to the parent wild-type strain. When the aphVIII gene was used as a selectable marker in other volvocine algae, the factor for the increase in antibiotic tolerance in transformants was similar or lower. For V. carteri the increase in antibiotic tolerance was about 100-fold [30, 31], it was 100 to 200 fold in C. reinhardtii[25, 30], about 330-fold in E. elegans and 15 to 40 fold in G. pectorale. Even if the antibiotic tolerance of transformants varies between the investigated volvocine species, the aphVIII gene allowed for selection of transformants in all these species.
The frequency of transformation was calculated to be 1.2 × 10-6 per cell in P. morum. The transformation frequency in V. carteri was estimated to be 2.5 × 10-5. In C. reinhardtii the frequency of transformation was 1.3 - 1.9 × 10-7; it was 1.1 – 6.6 × 10-7 in G. pectorale and 3.7 × 10-7 in E. elegans. Thus, the transformation frequency in P. morum is about 20 times lower than in V. carteri but about 2 to 10 times higher than in C. reinhardtii, G. pectorale and E. elegans. On an overall basis, the transformation frequencies in volvocine algae vary by about two orders of magnitude (1.1 × 10-7 to 2.5 × 10-5). There is limited information about transformation frequencies in other algae with the exception of some diatom species. The reported frequencies are 1 × 10-7 - 1 × 10-6 per cell in Phaeodactylum tricornutum and 3 × 10-8 - 3 × 10-7 per cell in Cyclotella cryptica and Navicula saprophila. Therefore, in diatoms the frequency of transformation is similar or somewhat lower compared to that in volvocine algae.
Co-transformation of a selectable marker plasmid and the gene of interest on a separate plasmid is a common strategy especially when large genes and complex gene constructs have to be integrated into the genome [16, 20, 24, 26, 29, 31, 44–46]. Another advantage of co-transformation is that the same selectable marker plasmid can be used for all transformation experiments. In P. morum the co-transformation rate was calculated to be at about 80%. For V. carteri co-transformation rates of 10% - 60%  or 40% - 80%  have been reported. The co-transformation rate was about 80% in C. reinhardtii, 30% - 50% in G. pectorale and 50% - 100% in E. elegans. The co-transformation rates in volvocine algae are high in all volvocine species and particularly in P. morum. Therefore, transformation of P. morum does not require construction of vectors that contain all required genes and regulatory elements on a single plasmid.
Expression of heterologous genes using heterologous promoters
Some promoters from C. reinhardtii have been shown to work in V. carteri and vice versa . Likewise, some promoter elements from C. reinhardtii and V. carteri also work in G. pectorale and E. elegans[11, 20]. Here we show for P. morum that both the heterologous hsp70A/rbcS3 tandem promoter of V. carteri and the heterologous HSP70A promoter of C. reinhardtii work in this species. Three out of the four constructs clearly lead to functional expression of the heterologous Gluc luciferase and the heterologous aminoglycoside 3′-phosphotransferase VIII (AphVIII) enzymes. It is unclear why transformants produced with pPsaD-GLuc (PanTJ…) didn’t produce significant luciferase activity.
The heterologous heat shock promoters retained their inducibility by heat when expressed in P. morum. The expression was up to 555 times higher in heat-shocked versus non-heat-shocked cultures.
The introduction of foreign genes into the nuclear genome of P. morum by particle bombardment is now feasible. Both the heterologous hsp70A/rbcS3 tandem promoter of V. carteri and the heterologous HSP70A promoter of C. reinhardtii drive the expression of heterologous genes in P. morum. Both promoters retained their heat-inducibility in P. morum. Furthermore, the heterologous aphVIII gene of S. rimosus was established as a selectable marker and the gluc luciferase gene was found to be a useful reporter gene in P. morum. It is thus to be emphasized that genetic engineering of a species is possible even without the availability of any endogenous genes and promoters.
Strains and culture conditions
The wild-type P. morum strain SAG 32.96 was obtained from the Culture Collection of Algae at Goettingen University (SAG), Germany. Cultures were maintained in Jaworski medium (JM)  at 29°C in an 8 h dark/16 h light (~10,000 lux) cycle [11, 20]. Cultures were grown in 10 ml glass tubes, 50 or 300 ml Erlenmeyer flasks, or 1,000 ml Fernbach flasks. The glass tubes had caps that allow for gas exchange, and Erlenmeyer and Fernbach flasks were aerated via Pasteur pipettes with approximately 50 cm3 sterile air/min [11, 20]. Transgenic strains that express the aphVIII gene were grown in JM in the presence of 1 μg paromomycin/ml (paromomycin sulfate, Sigma-Aldrich, St. Louis, MO).
The pPmr3 plasmid (Figure 3A) contains the 0.8 kb coding region of the S. rimosus aphVIII selectable marker gene, a V. carteri hsp70A-rbcS3 tandem promoter (0.5 kb of V. carteri hsp70A and 0.27 kb of V. carteri rbcS3 sequences), the 3′ UTR from the V. carteri rbcS3 gene (0.53 kb of downstream sequence), and the pBluescript II vector backbone [GenBank: AY429514] . The total size of the pPmr3 plasmid is 5.1 kb.
The pPsaD-GLuc plasmid (Figure 3B) contains the 0.57 kb coding region of the luciferase (gluc) gene from G. princeps, which was engineered to match the codon usage in C. reinhardtii, 0.8 kb of upstream sequence, which includes the C. reinhardtii PSAD promoter, the 3′-UTR of the C. reinhardtii psaD gene (0.56 kb of downstream sequence), and the pBluescript vector backbone [27, 36] [GenBank: EU372000, AF335592]. The pPsaD-GLuc plasmid is 5.0 kb in size.
The pHsp70A-GLuc plasmid (Figure 3C) contains the 0.57 kb coding region of the luciferase (gluc) gene from G. princeps (codon-optimized for C. reinhardtii)  fused downstream of a 0.8 kb genomic DNA fragment that contains the first three exons and two introns of the HSP70B gene of C. reinhardtii; this fragment of the HSP70B gene contains the sequence coding for the chloroplast transit peptide of HSP70B. The hybrid HSP70B/gluc gene is driven by the C. reinhardtii HSP70A promoter (0.26 kb of upstream sequence) and the 3′ UTR comes from the C. reinhardtii RBCS2 gene (0.22 kb of downstream sequence) . The total size of the pHsp70A-GLuc plasmid is 4.9 kb.
The pHRLucP plasmid (Figure 3D) contains the 0.57 kb coding region of the luciferase (gluc) gene of G. princeps, which was engineered to match the codon usage in C. reinhardtii, the V. carteri hsp70A-rbcS3 tandem promoter (0.5 kb of V. carteri hsp70A and 0.27 kb of V. carteri rbcS3 sequences), the C. reinhardtii PSAD 3′ UTR (0.55 kb of downstream sequence), and the pBluescript II vector backbone [11, 27, 36]. The pHRLucP plasmid is 5.0 kb in size.
Preparation of plasmid DNA
Plasmid DNA was purified using the Nucleospin® Plasmid Kit according to the manufacturers’ instructions (Macherey-Nagel, Düren, Germany).
Coating of microprojectiles
Gold microprojectiles (0.6 μm in diameter, Bio-Rad, Hercules, CA) were coated with the required plasmids for biolistic transformation as previously described [11, 20]. The DNA-coated microprojectiles were resuspended in 60 μl of ethanol and kept at 4°C; these microprojectiles were used within 3 h of preparation.
Determination of cell concentration
In P. morum the number of cells per colony varies. Therefore, we refer to “cells/ml” rather than “colonies/ml”. The concentration of cells was determined using a hemacytometer with Neubauer ruling.
Stable nuclear transformation by particle bombardment
The stable transformation of P. morum was performed using a Biolistic® PDS-1000/He (Bio-Rad) particle gun. To this end, a logarithmically growing P. morum culture (150 ml) at a cell concentration of ~7 × 104 cells/ml was harvested by centrifugation (1,000 g, 5 min, swing-out rotor) and resuspended in 6 ml of JM. One milliliter of the suspension was evenly spread on a cellulose acetate membrane filter and excess liquid was removed as previously described [11, 20]. One-sixth of the DNA-coated microprojectiles were evenly spread on a macrocarrier (Bio-Rad) that was placed in a macrocarrier holder (Bio-Rad). The transformation procedure was as previously described [11, 20] and the most successful combination of parameters is shown in Table 1. After the bombardment step, the algae were washed off from the membrane filter with JM. The procedure was repeated five times and the algae from six rounds of bombardment were pooled and then evenly distributed among twelve 50 ml Erlenmeyer flasks containing a volume of ~40 ml of JM. The bombarded colonies were incubated under standard conditions for 24 h to allow for regeneration and expression of the transgenes. Then, 2 μg of paromomycin/ml was added. The large fraction of non-transformed cells died within 48 h and the medium clarified. After 10–14 days of incubation in the presence of antibiotic, re-greening of flasks indicated the initial presence and reproduction of at least one paromomycin-resistant P. morum transformant that led to a population of transformants. No more than one transformant per flask was analyzed [11, 20].
Re-isolation of transformants
Transformants were re-isolated to ensure uniform genetic conditions for all further analyses. For this purpose, a serial dilution of an exponentially growing P. morum culture was performed in a Terasaki plate (Nunc™ MicroWell™ MiniTrays; Thermo Fisher Scientific, Langenselbold, Germany) [11, 20]. Each well of the Terasaki plate was filled with 10 μl of JM. A single P. morum colony was finally transferred into a standard glass tube with JM, containing 1 μg of paromomycin/ml. Further incubation was under standard conditions.
Cells of transformed or wild-type P. morum strains were transferred into the wells of a 24-well culture plate (Sarstedt, Nümbrecht, Germany) with a wide range of paromomycin concentrations from 0 to 100 μg/ml in 1 ml JM (concentrations are given in Figure 2). At the beginning of the assay, each well contained approximately 4,000 cells, which corresponds to 250–300 colonies. After incubation under standard conditions for 12 days, the wells were analyzed for viable green cells or white remains of lysed cells.
Oligonucleotide primers were designed using the primer analysis software Oligo 6 (Molecular Biology Insights, Cascade, CO), DNASIS™ (version 7.00; Hitachi Software Engineering, San Francisco, CA), and Primer Express® (Applied Biosystems, Foster City, CA).
Isolation of genomic DNA
Ten milliliters of a logarithmically growing P. morum culture with a density of 6 × 106 cells/ml was harvested by centrifugation (3,500 g for 10 min). The resulting pellet, which had a wet weight of ~80 mg, was washed with water and resuspended in 40 μl water. Isolation of genomic DNA was as previously described [11, 20].
PCR reactions with genomic DNA as a template were carried out in a total volume of 50 μl that contained ~100 ng of genomic DNA, 300 nM of each primer, 0.2 mM dNTP, 1.5 mM MgCl2, and 2.6 units of Expand High Fidelity enzyme mix in 1× Expand High Fidelity buffer (Roche Applied Science, Basel, Switzerland). The PCR reactions were performed on a T3 Thermocycler PCR system (Biometra, Göttingen, Germany) using the following conditions: 40 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 45 s and a final extension was at 72°C for 10 min. The PCR products were cloned and sequenced (Eurofins, Ebersberg, Germany).
Isolation of total RNA
Total RNA was extracted from ~100 mg of concentrated, frozen P. morum algae using 1 ml of phenol-based TRI Reagent (Sigma-Aldrich, St. Louis, MO) and 300 μl trichloromethane. RNA precipitation and RNA purification was as previously described [11, 20].
Reverse Transcription (RT)-PCR
First strand cDNA synthesis was carried out using 1 μg of total RNA and Moloney murine leukemia virus (MMLV) reverse transcriptase lacking ribonuclease H activity (H minus) according to the manufacturer’s instructions (Promega). The subsequent PCR step was performed using the Expand High Fidelity PCR system (Roche Applied Science, Basel, Switzerland), a T3 Thermocycler PCR system (Biometra), and the following cycling conditions: 40 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 45 s and a final extension at 72°C for 10 min. The RT-PCR products were cloned and sequenced.
Induction of luciferase activity
Preliminary tests showed that the shift of a P. morum culture (3–6 × 105 cells/ml) from 29°C to 39°C for 1 h, followed by a recovery phase at 29°C for 15 min, leads to the strongest induction of luciferase activity in the temperature range investigated (27°C to 51°C).
For assays on light-sensitive films, P. morum cultures with a cell density of 3–6 × 105 cells/ml were divided into aliquots of 50 ml. One aliquot was incubated at the optimal temperature for heat stress-induced expression of luciferase (39°C) for 1 h, and another aliquot was incubated at 29°C as a reference control. Further treatment and execution of the luciferase enzyme assay was as previously described [11, 20]. The final exposure to a chemiluminescence sensitive film (Retina XBA; Fotochemische Werke) was for 2 h at 20°C [11, 20, 35].
The quantification of luciferase activity was conducted as previously described [20, 27] using a MiniLumat LB9506 luminometer (Berthold, Bad Wildbad, Germany). For this purpose, aliquots of a P. morum culture (3–6 × 105 cells/ml) were heat-stressed at 39°C or kept at 29°C (control), respectively. Further treatment was as previously described . Luminescence was recorded as relative light units (rlu). Induction factors were calculated by comparison of samples from heat-shocked versus non-heat-shocked cultures.
In-gel activity assay
One liter of logarithmically growing, heat-stressed or untreated cultures of transformed or wild-type P. morum cultures with cell densities of 4–7 × 105 cells/ml were harvested by filtration on a 10-μm mesh nylon screen. Cells passing the screen were harvested by centrifugation (4,000 g, 5 min) and added to cells collected on the screen. Cells lysates were produced as previously described . The protein concentration of the cell extracts was determined photometrically using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad). For gel electrophoresis, 50 μg of total protein was loaded onto a standard SDS-polyacrylamide gel. The gel electrophoresis, the in-gel renaturation, the execution of the luciferase enzyme assay and the light detection on a chemiluminescence sensitive film was performed as previously described .
- aphVIII S. rimosus:
aminoglycoside 3′-phosphotransferase VIII gene
Green fluorescent protein
- gluc G. princeps :
luciferase gene (adapted to C. reinhardtii nuclear codon usage)
Heat shock protein 70A gene
Moloney murine leukemia virus
Polymerase chain reaction
- PSAD :
Abundant protein of photosystem I complex gene
Ribulose bisphosphate carboxylase small chain gene 3
Relative light units
Sodium dodecyl sulfate
We are grateful to T. Jakobiak and R. Schmitt (University of Regensburg, Germany) for providing the aphVIII gene, to N. Shao and R. Bock (Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm, Germany) for providing the gluc gene, and to K. Puls for technical assistance. We acknowledge the support of the Deutsche Forschungsgemeinschaft and the Open Access Publication Funds of Bielefeld University, which funded the article processing charges.
- Herron MD, Hackett JD, Aylward FO, Michod RE: Triassic origin and early radiation of multicellular volvocine algae. Proc Natl Acad Sci U S A. 2009, 106 (9): 3254-3258.View ArticleGoogle Scholar
- Kirk DL: Volvox: Molecular-Genetic Origins of Multicellularity and Cellular Differentiation. 1998, Cambridge: Cambridge University Press, 1Google Scholar
- Höhn S, Hallmann A: There is more than one way to turn a spherical cellular monolayer inside out: type B embryo inversion in Volvox globator. BMC Biol. 2011, 9 (1): 89-View ArticleGoogle Scholar
- Hallmann A: Evolution of reproductive development in the volvocine algae. Sex Plant Reprod. 2011, 24: 97-112.View ArticleGoogle Scholar
- Harris EH, Stern DB, Witman GB: The Chlamydomonas Sourcebook (2nd Edition). 2009, San Diego, CA: Academic Press, 2Google Scholar
- Fulton AB: Colonial development in Pandorina morum. II. Colony morphogenesis and formation of the extracellular matrix. Dev Biol. 1978, 64 (2): 236-251.View ArticleGoogle Scholar
- Lamouroux JV, de Saint-Vincent JBGM B, Deslongschamps E: Encyclopédie méthodique ou par ordre de matières. Histoire naturelle des zoophytes, ou animaux rayonnés, faisant suite à l’histoire naturelle des vers de Bruguière. 1824, Paris: Chez Mme Veuve AgasseView ArticleGoogle Scholar
- Herron MD, Michod RE: Evolution of complexity in the volvocine algae: transitions in individuality through Darwin’s eye. Evolution. 2008, 62 (2): 436-451.View ArticleGoogle Scholar
- Nozaki H, Kuroiwa T: Ultrastructure of the extracellular matrix and taxonomy of Eudorina, Pleodorina and Yamagishiella gen. nov. (Volvocaceae, Chlorophyta). Phycologia. 1992, 31 (6): 529-541.View ArticleGoogle Scholar
- Nozaki H, Kazaki H: The sexual process of Japanese Pandorina morum Bory (Chlorophyta). J Jpn Bot. 1979, 54 (12): 363-370.Google Scholar
- Lerche K, Hallmann A: Stable nuclear transformation of Eudorina elegans. BMC Biotechnol. 2013, 13: 11-View ArticleGoogle Scholar
- Moore LJ, Coleman AW: The linear 20 kb mitochondrial genome of Pandorina morum (Volvocaceae, Chlorophyta). Plant Mol Biol. 1989, 13 (4): 459-465.View ArticleGoogle Scholar
- Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L, Marshall WF, Qu LH, Nelson DR, Sanderfoot AA, Spalding MH, Kapitonov VV, Ren Q, Ferris P, Lindquist E, Shapiro H, Lucas SM, Grimwood J, Schmutz J, Cardol P, Cerutti H, Chanfreau G, Chen CL, Cognat V, Croft MT, Dent R, et al: The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science. 2007, 318 (5848): 245-250.View ArticleGoogle Scholar
- Prochnik SE, Umen J, Nedelcu AM, Hallmann A, Miller SM, Nishii I, Ferris P, Kuo A, Mitros T, Fritz-Laylin LK, Hellsten U, Chapman J, Simakov O, Rensing SA, Terry A, Pangilinan J, Kapitonov V, Jurka J, Salamov A, Shapiro H, Schmutz J, Grimwood J, Lindquist E, Lucas S, Grigoriev IV, Schmitt R, Kirk D, Rokhsar DS, Hellsten U, Chapman J, et al: Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri. Science. 2010, 329 (5988): 223-226.View ArticleGoogle Scholar
- Schiedlmeier B, Schmitt R, Müller W, Kirk MM, Gruber H, Mages W, Kirk DL: Nuclear transformation of Volvox carteri. Proc Natl Acad Sci U S A. 1994, 91 (11): 5080-5084.View ArticleGoogle Scholar
- Kindle KL: High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1990, 87 (3): 1228-1232.View ArticleGoogle Scholar
- Klein RM, Wolf ED, Wu R, Sanford JC: High-velocity microprojectiles for delivering nucleic acids into living cells. 1987. Biotechnology. 1992, 24: 384-386.Google Scholar
- Brown LE, Sprecher SL, Keller LR: Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation. Mol Cell Biol. 1991, 11 (4): 2328-2332.View ArticleGoogle Scholar
- Kumar SV, Misquitta RW, Reddy VS, Rao BJ, Rajam MV: Genetic transformation of the green alga Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci. 2004, 166: 731-738.View ArticleGoogle Scholar
- Lerche K, Hallmann A: Stable nuclear transformation of Gonium pectorale. BMC Biotechnol. 2009, 9 (1): 64-View ArticleGoogle Scholar
- Nelson JA, Savereide PB, Lefebvre PA: The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation. Mol Cell Biol. 1994, 14 (6): 4011-4019.View ArticleGoogle Scholar
- Randolph-Anderson BL, Sato R, Johnson AM, Harris EH, Hauser CR, Oeda K, Ishige F, Nishio S, Gillham NW, Boynton JE: Isolation and characterization of a mutant protoporphyrinogen oxidase gene from Chlamydomonas reinhardtii conferring resistance to porphyric herbicides. Plant Mol Biol. 1998, 38 (5): 839-859.View ArticleGoogle Scholar
- Kovar JL, Zhang J, Funke RP, Weeks DP: Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation. Plant J. 2002, 29 (1): 109-117.View ArticleGoogle Scholar
- Berthold P, Schmitt R, Mages W: An engineered Streptomyces hygroscopicus aph 7″ gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist. 2002, 153 (4): 401-412.View ArticleGoogle Scholar
- Sizova I, Fuhrmann M, Hegemann P: A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene. 2001, 277 (1–2): 221-229.View ArticleGoogle Scholar
- Stevens DR, Rochaix JD, Purton S: The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol Gen Genet. 1996, 251 (1): 23-30.Google Scholar
- Shao N, Bock R: A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii. Curr Genet. 2008, 53 (6): 381-388.View ArticleGoogle Scholar
- Hallmann A: Algal transgenics and biotechnology. Transgenic Plant J. 2007, 1: 81-98.Google Scholar
- Hallmann A, Rappel A: Genetic engineering of the multicellular green alga Volvox: a modified and multiplied bacterial antibiotic resistance gene as a dominant selectable marker. Plant J. 1999, 17 (1): 99-109.View ArticleGoogle Scholar
- Hallmann A, Wodniok S: Swapped green algal promoters: aphVIII-based gene constructs with Chlamydomonas flanking sequences work as dominant selectable markers in Volvox and vice versa. Plant Cell Rep. 2006, 25 (6): 582-591.View ArticleGoogle Scholar
- Jakobiak T, Mages W, Scharf B, Babinger P, Stark K, Schmitt R: The bacterial paromomycin resistance gene, aphH, as a dominant selectable marker in Volvox carteri. Protist. 2004, 155 (4): 381-393.View ArticleGoogle Scholar
- Davies J, Wright GD: Bacterial resistance to aminoglycoside antibiotics. Trends Microbiol. 1997, 5 (6): 234-240.View ArticleGoogle Scholar
- Barnes AT, Case JF: Bioluminescence in the mesopelagic copepod, Gaussia princeps (T. Scott). J Exp Mar Biol Ecol. 1972, 8 (1): 53-71.View ArticleGoogle Scholar
- Szent-Gyorgyi C, Ballou BT, Dagmal E, Bryan B: Cloning and characterization of new bioluminescent proteins. Proc SPIE. 1999, 3600: 4-11.View ArticleGoogle Scholar
- Jacobsen I, Hennig-Pauka I, Baltes N, Trost M, Gerlach GF: Enzymes involved in anaerobic respiration appear to play a role in Actinobacillus pleuropneumoniae virulence. Infect Immun. 2005, 73 (1): 226-234.View ArticleGoogle Scholar
- Fischer N, Rochaix JD: The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Genet Genomics. 2001, 265 (5): 888-894.View ArticleGoogle Scholar
- Schroda M, Blöcker D, Beck CF: The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J. 2000, 21 (2): 121-131.View ArticleGoogle Scholar
- Shao N, Krieger-Liszkay A, Schroda M, Beck CF: A reporter system for the individual detection of hydrogen peroxide and singlet oxygen: its use for the assay of reactive oxygen species produced in vivo. Plant J. 2007, 50 (3): 475-487.View ArticleGoogle Scholar
- Drzymalla C, Schroda M, Beck CF: Light-inducible gene HSP70B encodes a chloroplast-localized heat shock protein in Chlamydomonas reinhardtii. Plant Mol Biol. 1996, 31 (6): 1185-1194.View ArticleGoogle Scholar
- Kindle KL, Schnell RA, Fernandez E, Lefebvre PA: Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol. 1989, 109 (6 Pt 1): 2589-2601.View ArticleGoogle Scholar
- Sizova IA, Lapina TV, Frolova ON, Alexandrova NN, Akopiants KE, Danilenko VN: Stable nuclear transformation of Chlamydomonas reinhardtii with a Streptomyces rimosus gene as the selective marker. Gene. 1996, 181 (1–2): 13-18.View ArticleGoogle Scholar
- Apt KE, Kroth-Pancic PG, Grossman AR: Stable nuclear transformation of the diatom Phaeodactylum tricornutum. Mol Gen Genet. 1996, 252 (5): 572-579.Google Scholar
- Dunahay TG, Jarvis EE, Roessler PG: Genetic transformation of the diatoms Cyclotella cryptica and Navicula saprophila. J Phycol. 1995, 31: 1004-1012.View ArticleGoogle Scholar
- Hallmann A, Sumper M: Reporter genes and highly regulated promoters as tools for transformation experiments in Volvox carteri. Proc Natl Acad Sci U S A. 1994, 91 (24): 11562-11566.View ArticleGoogle Scholar
- Hallmann A, Sumper M: The Chlorella hexose/H+ symporter is a useful selectable marker and biochemical reagent when expressed in Volvox. Proc Natl Acad Sci U S A. 1996, 93 (2): 669-673.View ArticleGoogle Scholar
- Hallmann A, Rappel A, Sumper M: Gene replacement by homologous recombination in the multicellular green alga Volvox carteri. Proc Natl Acad Sci U S A. 1997, 94 (14): 7469-7474.View ArticleGoogle Scholar
- Thompson AS, Rhodes JC, Pettman I: Culture Collection of Algae and Protozoa - Catalogue of Strains. 1988, Cumbria, UK: CCAP, 5Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.