Plant Cell Rep (2006) 25: 582–591DOI 10.1007/s00299-006-0121-x

GENETIC TRANSFORMATION AND HYBRIDIZATION

A. Hallmann · S. Wodniok

Swapped green algal promoters: aphVIII-based gene constructswith Chlamydomonas flanking sequences work as dominantselectable markers in Volvox and vice versa

Received: 1 October 2005 / Revised: 11 December 2005 / Accepted: 14 January 2006 / Published online: 3 February 2006C© Springer-Verlag 2006

Abstract Production of transgenic organisms is a well-established, versatile course of action in molecular biology.Genetic engineering often requires heterologous, dominantantibiotic resistance genes that have been used as selectablemarkers in many species. However, as heterologous 5′ and3′ flanking sequences often result in very low expressionrates, endogenous flanking sequences, especially promot-ers, are mostly required and are easily obtained in modelorganisms, but it is much more complicated and time-consuming to get appropriate sequences from less commonorganisms. In this paper, we show that aminoglycoside 3′-phosphotransferase gene (aphVIII) based constructs with3′ and 5′ untranslated flanking sequences (including pro-moters) from the multicellular green alga Volvox work inthe unicellular green alga Chlamydomonas and flanking se-quences from Chlamydomonas work in Volvox, at least ifa low expression rate is compensated by an enforced highgene dosage. This strategy might be useful for all investi-gators that intend to transform species in which genomicsequences are not available, but sequences from relatedorganisms exist.

Keywords Green algae . Volvocales . Recombinant DNAtechnology . Transgenic expression . Genetictransformation . Paromomycin resistance

Abbreviations aphVIII: Aminoglycoside 3′-phosphotransferase (VIII) gene . hsp70A: Heat shock protein 70A gene . rbcS2: Ribulose-1,5-bisphosphat-carboxylase smallsubunit gene 2 from Chlamydomonas reinhardtii . rbcS3:Ribulose-1,5-bisphosphat-carboxylase small subunit gene3 from Volvox carteri . UTR: Untranslated region

Communicated by H. Ebinuma

A. Hallmann (�) · S. WodniokDepartment of Cellular and Developmental Biology of Plants,University of Bielefeld,Universitatsstr 25,33615 Bielefeld, Germanye-mail: armin.hallmann@gmx.deTel.: +49-521-1065592Fax: +49-1805-7511-1173-74

Introduction

The evolutionary transition from unicells towards multicel-lular forms of life represents one of the major achievementsin the evolution of complex organisms. Unfortunately, inmost multicellular lineages the branch points to multicel-lularity lie so deep in the past that it seems unlikely thatthese organisms retained within their genomes after thatextended period of genetic drift, making a molecular un-derstanding of this step unreachable. In contrast, there isa group of organisms, in which the transition to multicel-lularity was traversed much more recently and in whicheven intermediate stages exist: the green algae of the or-der Volvocales. The last common ancestor of the multi-cellular green alga Volvox carteri and the unicellular algaChlamydomonas reinhardtii was set at only 50–75 mil-lion years ago by molecular phylogenetic analysis (Rauschet al. 1989), while V. carteri diverged with other species ofChlamydomonas even more recently than that (Buchheimand Chapman 1991; Buchheim et al. 1994), so that themolecular reasons that permitted multicellularity are notobscured too much by time. Moreover, not only unicellularand multicellular forms exist within the order Volvocales,but also intermediate genera such as Gonium, Pandorina,Eudorina and Pleodorina. Thus, volvocine green algae canbe arranged in a row of progression in organismic com-plexity, which is expressed by an increase in the numberof cells, the amount of extracellular matrix, and the ex-tent to which cellular labour is divided between cell types(for review see Kirk 1998). Due to this unique situationvolvocine green algae offer the unrivalled opportunity toexplore the molecular changes that were necessary to bringabout the transition from unicellularity to multicellularityand cellular differentiation.

However, in modern molecular, cellular and develop-mental biology the accessibility of the test organism togenetic manipulation is a conditio sine qua non, and sofar, only C. reinhardtii and V. carteri meet these require-ments within the order Volvocales. No genetic tools andhardly any gene sequence data are available in Gonium,

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Pandorina, Eudorina or Pleodorina. A precondition forgenetic transformation of these genera would be a reli-able, dominant selectable marker gene. Foreign selectablemarker genes like the ble gene, which mediates resistance tozeocin, or the aminoglycoside 3′-phosphotransferase (VIII)gene (aphVIII), have been shown to work both in V. carteriand C. reinhardtii by using endogenous 5′ and 3′ untrans-lated regions (UTRs) (Lumbreras et al. 1998; Hallmannand Rappel 1999; Sizova et al. 2001; Jakobiak et al. 2004).In contrast to the Ble protein, which involves a 1:1 bind-ing of zeocin, the aminoglycoside 3′-phosphotransferaseinactivates aminoglycoside antibiotics like paromomycinby transfer of ATP γ-phosphate to its hydroxyl groups witha high turnover rate. Wild-type volvocine algae are highlysensitive towards paromomycin, which is known to inhibitcell growth by blocking protein synthesis at the translo-cation step (Davies and Wright 1997). The aphVIII geneof Streptomyces rimosus (Danilenko et al. 1997; Sizovaet al. 2001) has a GC content of 68.9%, which is as highas the GC content of known genes from volvocine algae,and the codon preferences are almost matching (Schmittet al. 1992; Sizova et al. 2001). Transformation systemswhich comprise genes that code for toxin-modifying en-zymes, like the aminoglycoside 3′-phosphotransferase, areindependent from auxotrophic recipient strains. Thereforethese genes have been called dominant resistance markersand often they allow the genetic transformation of manydifferent strains or even species. So these conditions makethe aphVIII gene to a candidate selectable marker for allvolvocine algae. But as stated above, expression of foreigngenes normally requires also an endogenous 5′ UTR includ-ing the promoter and an endogenous 3′ UTR. Such DNAelements can easily be obtained by PCR if the genomeof the target organism is sequenced and annotated or atleast some strongly expressed genes are known. But if use-ful genomic sequences are not available in a less commonorganism it is much more time-consuming to get such se-quences, since 5′ and 3′ UTRs are weakly conserved evenbetween closely related organisms, making genomic PCRswith degenerated primers based on flanking sequences oforthologous genes de facto senseless. To bypass the lengthyprocedure of repeated cloning of flanking sequences fromdifferent less common green algal genomes in future exper-iments, we intended to check whether already cloned 5′ and3′ UTRs, which are well proven as endogenous flanking se-quences, also work in related organisms, i.e. we intendedto use 5′ and 3′ UTRs from V. carteri to generate transgenicC. reinhardtii strains and vice versa. As our group is alsointerested in evolution of complex extracellular structures,we planned to transform algae with wild-type phenotypeby particle gun transformation instead of transforming cellwall deficient strains by using the glass-bead method, whichis common in Chlamydomonas research (Kindle 1990).Starting plasmids were aphVIII based constructs contain-ing a combination of a heat-shock inducible hsp70A pro-moter, a constitutive ribulose-1,5-bisphosphat-carboxylasesmall subunit gene (rbcS) promoter, and a rbcS 3′ UTReach from C. reinhardtii (Schroda et al. 2000; Sizova et al.2001) or V. carteri (Jakobiak et al. 2004).

Here we show that flanking sequences from the unicellu-lar alga Chlamydomonas work in multicellular Volvox andvice versa if a low expression rate is compensated by ahigh gene dosage. By particle gun transformation, the geneconstructs can be introduced also into the genomes of cellwall- or extracellular matrix-sufficient wild-type algae andallow a paromomycin-based selection. This strategy mightalso be applicable to other species inside and outside theVolvocales if these species are more or less closely related.

Materials and methods

Strains and culture conditions

The wild-type Volvox carteri f. nagariensis strain EVE(female) was obtained by D. L. Kirk (Washington Univer-sity, St. Louis, MO) and was described previously (Adamset al. 1990). Cultures were grown in Volvox medium(Provasoli and Pintner 1959) at 28◦C in a 8 h dark/16 hlight (10,000 lux) cycle (Starr and Jaenicke 1974).

The wild-type C. reinhardtii strain CC-125 (mt + ) andthe mutant strain cw10, a cell wall assembly defectivestrain which is also called CC-849, were obtained from theChlamydomonas Genetics Center, Department of Botany,Duke University (Durham, NC). Cultures were maintainedat 20◦C in continuous light in tris-acetatephosphate (TAP)medium (Harris 1989) or on TAP plates containing 1%Bacto-Agar (Difco).

Construction of plasmids

Plasmid pSI103 (Sizova et al. 2001) carries the aminogly-coside 3′-phosphotransferase (AphVIII)-encoding gene,aphVIII, from Streptomyces rimosus (Danilenko et al. 1997;Sizova et al. 2001), which confers resistance to paro-momycin. The aphVIII gene is also called aphH gene(Jakobiak et al. 2004). The aphVIII sequence within plas-mid pSI103 (Sizova et al. 2001) is identical with the se-quence of GenBank Acc. No. AF182845 (the correspond-ing protein ID is AAG11411.2), but there is also an olderGenBank entry with Acc. No. U24442 (the correspondingprotein ID is AAB03856.1) (Danilenko et al. 1997) whichshows 98% identity with AF182845. pSI103 contains theaphVIII gene under control of a C. reinhardtii hsp70A-rbcS2 hybrid promoter. pSI103 also contains intron 1 ofthe C. reinhardtii rbcS2 gene at position − 42 bp upstreamof the translation start codon and a 3′ UTR from the C.reinhardtii rbcS2 gene. To construct paphA, an XbaI re-striction enzyme cleavage site (T↓CTAGA) was insertedupstream of the promoter region of the gene construct anda SpeI site (A↓CTAGT) downstream of the 5′ UTR. Thiswas done by recombinant PCR using the oligonucleotideprimers 5′-TGTCTAGAGAGGCTTGACATGATTGGTG(sense primer) and 5′-GCACTAGTATGGAGAAAGAGGCCAAAATC (antisense primer), which did not match attheir 5′ends, but carried the restriction sites (underlined).The PCR fragment was cloned into the unique SmaI site of

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the E. coli pBluescript II vector through blunt end ligationby looking out for such an orientation in which the endof the PCR fragment that carries the antisense primer, ison the same side as the EcoRI, HindIII or SalI restrictionsites of the pBluescript II multiple cloning site. The result-ing plasmid, paphA, has two SpeI sites and two XbaI sites,whereas one SpeI and one XbaI site come from the multiplecloning site, so there is an XbaI–SpeI–XbaI constellationat that end of the insert that carries the sense primer of thepreceded PCR. Plasmid paphA was digested with XbaI andreligated, which results in loss of the SpeI site between theXbaI sites and reduction of the number of XbaI sites fromtwo to one. The resulting plasmid is paphB, which car-ries unique XbaI and SpeI sites on both ends of the insertand a unique EcoRI site, which comes from the multiplecloning site, close to the SpeI site. Plasmids paphC, pa-phD, paphE and paphG are derived from plasmid paphBby repeated duplication of the respective chimeric aphVIIIgene. For duplication of the aphVIII cassettes the ‘cassettemultiplication technique’ was applied as described in thesection ‘Results’. Constructs were confirmed by restrictionanalysis and sequencing.

Stable nuclear transformation of Volvox by particle gun

All nuclear transformation experiments with V. carteri wereperformed by using a particle gun (biolistic bombardment)as described (Schiedlmeier et al. 1994), but with severalmodifications with respect to this reference. Circular plas-mid DNAs for transformation of V. carteri were purifiedusing anion exchange columns (Qiagen, Hilden, Germany).In case of very large plasmids (e.g. paphG) a freshlytransformed E. coli colony has been used to inoculatean overnight bacterial culture, which was harvested dur-ing exponential growth for plasmid preparation. Gold mi-croprojectiles (1 µm in diameter, Bio-Rad, Hercules, CA,USA) were coated with the required targeting plasmids andstable transformation was performed by using a BiolisticPDS-1000/He (Bio-Rad, Hercules, CA, USA) particle gun,600 µg microprojectiles and 1 µg DNA per shot, 900 psirupture disks, and a target distance of 16.5 cm. Isolated re-productive cells (gonidia) of wild-type V. carteri strain EVEwere used as a target and the bombardment chamber wasevacuated to 27 in. Hg. After transformation the algae wereincubated under standard conditions in petri dishes (9 cmdiameter) filled with ∼ 35 ml Volvox liquid medium. Thestarting concentration was 10–15 Volvox colonies/ml. Toselect for paromomycin resistance in transgenic V. carteri5.0 µg paromomycin/ml (paromomycin sulphate, Sigma-Aldrich, USA) was added to the medium at the fourth dayand an additional 5.0 µg paromomycin/ml at the eighthday after transformation. From the fifth day onwards, afterthe first paromomycin addition, bombarded cultures wereexamined by dark-field stereomicroscopy (MZ16A; Leica;Germany) and each green alga in a background of bleach-ing spheroids was transferred into a microtiter well (24-wellculture plates; working volume 2 ml; Costar, Cambridge,MA, USA) containing fresh Volvox medium with 5.0 µg

paromomycin/ml. After incubation in the presence of theantibiotic for at least 6 days, the microtiter wells were in-spected for green and living transformants.

Stable nuclear transformation of Chlamydomonasby glass beads

The cell wall assembly defective C. reinhardtii strain cw10was transformed by a modified protocol of the glass-beadmethod (Kindle 1990). To this end cells were grown todensities of (1–2) × 106 cells/ml in TAP medium (Harris1989). Cells from 50 ml of this culture were harvested bycentrifugation (2,000 × g, 5 min), resuspended in 300 µlTAP medium, and transferred to a 1.5 ml tube. 100 µlpolyethylene glycol 6,000 20% (v/v), 1-2 µg circular plas-mid DNA and 300 mg of 0.45–0.50 mm glass beads wereadded. The tube was agitated vigorously for 4 × 15 s with30 s breaks in between. Cells were diluted in 50 ml TAPmedium and incubated under constant light for 18 h withgentle shaking. Cells were harvested by centrifugation(2,000 × g, 5 min), resuspended in 400 µl TAP mediumand plated on TAP-agar plates containing 10.0 µg/mlparomomycin. Plates were incubated under reduced con-stant illumination at 20◦C. Colonies were visible after 7–14 days.

Stable nuclear transformation of Chlamydomonasby particle gun

Stable nuclear transformation of wild-type C. reinhardtiiwith intact cell walls by particle gun was as described(Sanford et al. 1993; Sodeinde and Kindle 1993), but withmodifications as to this references: Stable transformationwas performed by using a Biolistic PDS-1000/He particlegun, 600 µg microprojectiles 0.6 µm in diameter (Bio-Rad)and 1 µg DNA per shot, 1,100 psi rupture disks, and a targetdistance of 16.5 cm. C. reinhardtii cells ( ∼ 5 × 107), platedon TAP-agar plates, were used as a target and the bombard-ment chamber was evacuated to 27 in. Hg. Thereafter, thealgae were removed from the plates by washing with TAPmedium, kept in this non-selective growth medium for18 h at 20◦C in continuous light, and then plated on TAPagar plates with paromomycin (10.0 µg/ml) for selection.

Isolation of genomic DNA from Chlamydomonasor Volvox

One to four millilitres of C. reinhardtii or V. carteri cellswere harvested by centrifugation (5,700 × g, 3 min).The pellet was washed twice with water, centrifuged(2 × ), resuspended in water and frozen in liquid nitrogen.Frozen samples were pulverized in a mortar. Afterhomogenization, the sample was warmed to 65◦C in awater bath and lysis buffer (Qiagen) containing RNase A1was added. Genomic DNA was isolated using the DNeasyspin columns of the DNeasy Plant Mini Kit (Qiagen).

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Lysates of Chlamydomonas cellsfor genomic PCR

About 1.5 × 106 C. reinhardtii cells were harvested bycentrifugation (2,000 × g, 10 min), washed with 300 µl10 mM phosphate buffer, pH 7.4, containing 150 mMNaCl, centrifuged (2,000 × g, 10 min), and resuspendedin 15 µl sterile lysis buffer (0.1 M NaOH, 2.0 M NaCl,0.5% SDS). After 5 min at 95◦C 300 µl of 50 mMTris/HCl pH 7.5 were added immediately. 1 µl of the re-sulting lysate was used for genomic PCR (see the nextsection).

Lysates of Volvox spheroids for genomic PCR

Fifty Volvox spheroids were selected under the stereomicro-scope and transferred into 10 µl sterile lysis buffer (0.1 MNaOH, 2.0 M NaCl, 0.5% SDS). After 5 min at 95◦C 200 µlof 50 mM Tris/HCl, pH 7.5, were added immediately. Onemicrolitre of the resulting lysate was used for genomic PCR(see the next section).

Genomic PCR

Genomic PCR was in a total volume of 50 µl. 1 µlof the described extracts from whole algae or puri-fied genomic DNA was used as a template. A 776 bpDNA fragment was expected when the sense primer 5′-GTCGGTATCCCGGTTGTGAG and the antisense primer5′-TCAGAAGAACTCGTCCAACAG was used for PCR.Both primers hybridize to the aphVIII gene whichnormally is not present in wild-type Chlamydomonasor Volvox. In an alternative PCR reaction the senseprimer 5′-GTCGGTATCCCGGTTGTGAG and the anti-sense primer 5′-GTAATACGACTCACTATAGGGC wasused for PCR. The last-mentioned primer is vector-based(T7). Fourty cycles of PCR amplification (94◦C, 30 s;54◦C, 30 s; 72◦C, 2 min) were performed by standardprotocol. Products of PCR amplification were clonedinto the SmaI site of the pBluescript II SK vector andsequenced.

Resistance assay

After transformation, identified paromomycin-resistantclones of C. reinhardtii or V. carteri were further ana-lyzed for their exact resistance level. To that end identicalquantities of each transformant population were transferredinto microtiter wells (24-well culture plates; working vol-ume 2 ml; Costar, Cambridge, MA, USA) containing therespective medium and increasing concentrations of paro-momycin. Incubation under standard conditions continuedfor 2 weeks. Subsequently, the wells with the highest antibi-otic concentration which still contained green and growingalgae were recorded.

Results and discussion

Stable transformation of C. reinhardtii using anaphVIII-based gene construct with Volvoxflanking sequences

Actually, plasmid pPmr3 was constructed for transforma-tion of the multicellular green alga Volvox carteri (Jakobiaket al. 2004), therefore the aphVIII gene within this plasmidis flanked by 5′ and 3′ untranslated regions from V. carteri;more precisely in this construct the aphVIII gene is undercontrol of a V. carteri hsp70A-rbcS3 hybrid promoter andthe 3′ UTR is also derived from the V. carteri rbcS3 gene(Fig. 1).

There are no detectable sequence similarities betweenthe V. carteri hsp70A 5′ UTR and the C. reinhardtii hsp70A5′ UTR, between the V. carteri rbcS3 5′ UTR and the C.reinhardtii rbcS2 5′ UTR, or between the V. carteri rbcS33′ UTR and the C. reinhardtii rbcS2 3′ UTR (see below).

We intended to transform the unicell C. reinhardtii withplasmid pPmr3 and therefore examined the target strainsCC-125 (wild-type) and cw10 (cell wall assembly defec-tive) for their sensitivity towards paromomycin when platedon TAP agar plates. At concentrations ≥ 5.0 µg/ml paro-momycin not a single colony of strains CC-125 or cw10emerged under these conditions. Just to be on the safer side,we decided to use the twofold concentration, 10.0 µg/ml,for the C. reinhardtii transformation experiments.

At first we used the cell wall assembly defective straincw10 and a modified glass-bead protocol (see ‘Materi-als and methods’ section) in which polyethylene glycol6,000 20% (v/v) and 0.45–0.50 mm glass beads were ap-plied. Cells were incubated for 18 h in liquid mediumunder non-selective conditions and then transferred ontoselective TAP-agar plates containing 10.0 µg/ml paro-momycin. About 100 paromomycin-resistant colonies weredetectable after 7–14 days. Re-analysis of transformantswas in a liquid medium and with increasing concentrationsof paromomycin (see ‘Materials and methods’ section).About 30% of the investigated transformants tolerated upto 200 µg/ml paromomycin.

Genetic and molecular analyses of many nuclear transfor-mants both in Chlamydomonas (Debuchy et al. 1989) andin Volvox (Hallmann et al. 1997) revealed that after trans-formation experiments integration of DNA into the genomeoccurs almost exclusively via non-homologous recombina-tion resulting in the introduction of the DNA at apparentlyrandom loci. Genomic PCR analysis was used to verifythe integration of plasmid pPmr3 into the C. reinhardtiigenome of transformants. In PCR1, both primers hybridizeto the aphVIII gene which normally is not present in C.reinhardtii strain cw10, in PCR2 one primer hybridizes tothe aphVIII gene and the other one to the flanking vectorsequence which becomes also integrated into the genomeduring transformation. PCR fragments of 776 or 1362 bp,respectively, in size were predicted in transformants. Theresult with a representative transformant and the parentstrain is shown in Fig. 2a. The PCR-fragments were also

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Fig. 1 Schematic diagram of the chimeric selectable marker con-structs pPmr3, pSI103, paphA, paphB, paphC, and paphG. pPmr3contains the aphVIII gene with 5′ and 3′ UTRs from V. carteri; inpPmr3 the aphVIII gene is under control of an hsp70A-rbcS3 hybridpromoter. pSI103 contains the aphVIII gene with 5′ and 3′ UTRsfrom C. reinhardtii; in pSI103 the aphVIII gene is under control of ahsp70A-rbcS2 hybrid promoter, the construct also contains intron 1(int) of the rbcS2 gene at position − 42 bp upstream of the translationstart codon. Plasmid paphA is a precursor of pahB (see text). paphB

contains, in comparison to pSI103, three additional unique cleavagesites of the restriction enzymes XbaI, SpeI, and EcoRI, which wereinserted by recombinant DNA technology at both ends of what wecall the aphVIII cassette. paphC contains two aphVIII cassettes in thesame orientation constructed by the cassette multiplication technique(see text). paphG contains 16 repeated aphVIII cassettes in the sameorientation. In paphC and paphG the restriction enzyme cleavagesites of XbaI, SpeI, and EcoRI are still unique. All constructs arewithin the E. coli pBluescript II vector (pBS)

Fig. 2 Detection of the aphVIII gene originating from plasmidpPmr3 (containing 5′ and 3′ UTRs from V. carteri) in transformantsof C. reinhardtii cell wall assembly defective or wild-type strainstransformed by glass-beads or by particle gun. Genomic DNA wasextracted from untransformed C. reinhardtii algae (cw10 or wild-typestrain CC-125) or C. reinhardtii transformants (transf.) which inte-grated aphVIII genes originating from plasmid pPmr3. Only trans-

formants were expected to yield a 776 bp fragment in PCR1 and a1362 bp fragment in PCR2. a The cell wall assembly defective straincw10 was transformed by glass-beads. (Left side): parent strain inPCR1 and 2; (right side): transformant in PCR1 and 2. b The wild-type strain CC-125 (WT) with its intact cell wall was transformed byparticle gun. (Left side): parent strain in PCR1; (right side): transfor-mant in PCR1. Products of PCR were cloned and sequenced

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checked by sequencing. All paromomycin-resistant trans-formants were positive in this genomic PCR analysis. Asubset of paromomycin-resistant transformants was alsotested by an additional genomic PCR in which one primerhybridizes within the hsp70A-rbcS3 hybrid promoter (5′UTR) and the other one within the aphVIII gene; again,all tested paromomycin-resistant transformants were pos-itive (data not shown). Furthermore, these experimentswere repeated after a 3-month propagation period with-out selective pressure through paromomycin. After this pe-riod, which corresponds to ∼ 270 generations, the trans-formants still were positive in the genomic PCR and stillretained their paromomycin resistance. Thus, stable trans-formation of C. reinhardtii strain cw10 by glass-beads us-ing plasmid pPmr3 with its aphVIII gene under controlof an hsp70A-rbcS3 hybrid promoter from V. carteri wasdemonstrated.

As mentioned earlier transformation of cell wall as-sembly defective Chlamydomonas strains by glass-beadsis a widespread method (Kindle 1990). But since ourlaboratory is interested in cell wall- or extracellular matrix-structure and assembly, it is self-evident that cell wallassembly defective target strains are normally not suitablefor us. Therefore, we also tried to transform the wild-typeC. reinhardtii strain CC-125 using plasmid pPmr3. As theglass beads method only works with cell wall assemblydefective strains, we used the biolistic method for transfor-mation (Debuchy, Purton et al. 1989). To that end plasmidpPmr3-coated microprojectiles were introduced into C.reinhardtii cells by high-velocity bombardment. Here weused a modified protocol and a PDS-1000/He biolisticdevice, 0.6 µm gold microprojectiles, 1,100 psi rupturedisks, and a target distance of 16.5 cm (see ‘Materialsand methods’ section). Cells were incubated for 18 h innon-selective liquid medium and then transferred ontoselective TAP plates containing 10.0 µg/ml paromomycin.Again, paromomycin-resistant colonies were detectableafter 7–14 days. Re-analysis of transformants forparomomycin-resistance and genomic PCRs gave virtuallythe same results as with the glass bead transformed cellwall assembly defective target strain cw10 (see above).The only detectable difference between glass beadstransformations and particle gun transformations was thatparticle gun transformations gave on average 30–50% lesstransformants. In Fig. 2b the result with the genomic PCR1of a representative particle gun transformant and the parentwild-type strain CC-125 is shown. Like with glass beadtransformants, all results with particle gun transformantswere reproducible even after a 3-month propagation periodwithout selective pressure through paromomycin, showingthat plasmid pPmr3 with its 5′ and 3′ untranslated regionsfrom V. carteri is not only useful for stable transformationof cell wall deficient strains of C. reinhardtii by using theglass bead method, but also for stable transformation ofwild-type algae by using the particle gun.

Attempts to transform V. carteri using anaphVIII-based gene construct with Chlamydomonasflanking sequences

Encouraged by the results described earlier, we also in-tended to transform V. carteri using a similar aphVIII-basedgene construct, harboured within plasmid pSI103, whichwas constructed for transformation of C. reinhardtii earlier(Sizova et al. 2001). Plasmid pSI103 contains the aphVIIIgene with 5′ and 3′ UTRs from C. reinhardtii. More pre-cisely, in pSI103 the aphVIII gene is under control of aC. reinhardtii hsp70A-rbcS2 hybrid promoter and the con-struct also contains an 145 bp intron of the rbcS2 gene atposition − 42 bp upstream of the translation start codon(Fig. 1).

Before transformation, wild-type V. carteri algae wereexamined for their sensitivity towards paromomycin. Aconcentration of ≥ 3.0 µg/ml paromomycin killed allVolvox algae within 3–5 days of incubation.

Repeatedly, we tried to achieve nuclear transformation ofV. carteri by using plasmid pSI103. To that end exogenousDNA was introduced by the biolistic method (Schiedlmeier,Schmitt et al. 1994). Here we used a modified protocol anda PDS-1000/He biolistic device, gold microprojectiles with0.6, 1.0, or 1.6 µm in diameter (our standard size in Volvoxtransformation experiments is 1.0 µm), rupture disks with450, 650, 900, 1,100, 1,350 or 1,550 psi (our standard is900 psi), and varying target distances (our standard dis-tance is 16.5 cm) (see ‘Materials and methods’ section).This spectrum of conditions includes all the parameters thatnormally allow transformation of V. carteri, if Volvox spe-cific selectable markers are used (data not shown). Selectionfor paromomycin resistance was by adding 5.0 µg/ml paro-momycin to the medium at the fourth day and an additional5.0 µg/ml paromomycin at the eighth day.

All attempts to transform V. carteri with the DNA con-struct within plasmid pSI103 by particle gun and by usingthese and other conditions yielded not even a single se-lectable transformant (data not shown).

There are several possible reasons for this negative re-sult, like an inefficient DNA-transfer into the cells or nostable integration into the genome, incorrect or no splicingof the C. reinhardtii intron, a promoter that is inoperablein V. carteri, silencing of the gene after its illegitimate in-tegration into the Volvox genome, or a very low expressionrate and so on. We postulated that the aphVIII gene with5′ and 3′ UTRs from C. reinhardtii within plasmid pSI103was integrated into the genome of at least a few V. car-teri algae during the transformation procedure as intendedand speculated that it even was expressed correctly, how-ever, the expression rates might have been low, too lowfor identification of transgenic algae in a background ofuntransformed algae. To counter such a possible source offault, we wanted to enforce higher expression rates by anincreased gene dosage.

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Fig. 3 Cassette multiplication technique applied to construct plas-mids carrying repeated aphVIII cassettes in the same orientation. Theoriginal plasmid paphB contains the aphVIII gene, C. reinhardtii 5′and 3′ UTRs, and artificial cleavage sites of the restriction enzymesXbaI, SpeI, and EcoRI, which were inserted as indicated by recom-binant DNA technology at the ends of the aphVIII cassette. XbaI(T↓CTAGA) and SpeI (A↓CTAGT) produce compatible DNA-ends,but the sequence originating from ligation of these ends (TCTAGT)can no longer be cleaved by both XbaI or SpeI. The plasmids pa-phB, paphC, and paphD contain 1, 2, or 4, respectively, copies of theaphVIII cassette. X, XbaI; S, SpeI; E, EcoRI

Construction of plasmids carrying repeated aphVIIIcassettes with Chlamydomonas flanking sequences

Assuming that there is an at least low expression of theaphVIII gene when plasmid pSI103 with its 5′ and 3′ UTRsfrom C. reinhardtii is integrated into the Volvox genome,we intended to amplify this expression rate by cloningseveral copies of the selectable marker gene several timesin series by applying a ‘cassette multiplication technique’(Hallmann and Rappel 1999). During construction of plas-mid paphA (Fig. 1), cleavage sites of the restriction en-zymes XbaI, SpeI and EcoRI were inserted by recombinantDNA technology at the ends of what we call the ‘aphVIIIcassette’. The aphVIII cassette is ∼ 1.8 kb in size and con-sists of the aphVIII gene plus the 5′ and 3′ UTRs, obtainedfrom plasmid pSI103. Plasmid paphA has an XbaI-SpeI-XbaI constellation of cleavage sites on one side of theinsert. As unique restriction enzyme cleavage sites wereneeded for further construction, paphA was digested withXbaI and religated, resulting in plasmid paphB (Fig. 1).Plasmid paphB carries a unique XbaI site upstream of theaphVIII cassette and unique SpeI and EcoRI sites down-stream of the cassette. Plasmids pSI103, paphA and paphBwere tested upon functionality and compared with eachother by transformation of C. reinhardtii strain cw10; in

each case ∼ 100 paromomycin-resistant colonies in glassbead transformations could be obtained and, consequently,no significant difference in efficiency could be detected.Plasmid paphB, carrying one aphVIII cassette, was cut withSpeI and EcoRI on the one hand or with XbaI and EcoRIon the other hand (Fig. 3). The products, each containingone aphVIII cassette, were ligated. The restriction enzymesXbaI (T↓CTAGA) or SpeI (A↓CTAGT) produce compati-ble DNA-ends, but the sequence originating from ligationof these ends can no longer be cleaved by both XbaI andSpeI. Thus, in the product, plasmid paphC (Figs. 1 and 3)with its two aphVIII cassettes, the cleavage sites of XbaI,SpeI and EcoRI are arranged just as in the original plas-mid paphB. Then the product with two cassettes, paphC,was used in the same way to generate a construct with fourcopies (paphD) and so on (Fig. 3). The cassette multipli-cation technique was continued until the aphVIII cassettewas repeated 16 times (plasmid paphG, Fig. 1). As any ofthis 1-, 2-, 4-, 8,- and 16-mers can be combined with eachother by this strategy, plasmids with any number of aphVIIIcassettes might be constructed, only limited by the capacityof the vector or the host organism. The constructed plas-mids, paphB (1 × aphVIII cassette), paphC, (2 × ) paphD(4 × ), paphE (8 × ), and paphG (16 × ) were confirmed bysequencing and restriction analysis (Fig. 4). Plasmid pa-phG has a total size of 31.4 kb, which is quite extensivefor a pBluescript II derivative, making more than 16 copiesof the 1.8 kb aphVIII cassette almost impracticable withinthis E. coli vector.

Fig. 4 Restriction analysis of plasmids paphB, paphC, paphD, pa-phE, and paphG. Plasmid DNAs were analyzed by double restrictionusing the unique restriction enzyme cleavage sites XbaI and SpeI,which allow excision of the complete insert from the pBluescript IIvector in all given plasmids. The total sizes of plasmids paphB, pa-phC, paphD, paphE, and paphG are 4.7, 6.5, 10.1, 17.2, and 31.4 kb,respectively. The number of repeats of the aphVIII cassettes and thesizes of the excised inserts of paphB, paphC, paphD, paphE, andpaphG are 1.8 kb (1 × aphVIII cassette), 3.6 kb (2 × ), 7.1 kb (4 × ),14.2 kb (8 × ), and 28.4 kb (16 × ). The size of the pBluescript IIvector is 3.0 kb

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Stable transformation of C. reinhardtii using 16 ×repeated aphVIII cassettes with Chlamydomonasflanking sequences

First of all we intended to prove the functionality of plas-mid paphG (16 × repeated aphVIII cassettes) by trans-forming both C. reinhardtii strains cw10 (cell wall assem-bly defective) and CC-125 (wild-type) using the modifiedglass bead protocol or the particle gun protocol, respec-tively. After 18 h under non-selective conditions, cellswere transferred onto selective TAP-agar plates containing10.0 µg/ml paromomycin. Like plasmid pPmr3, plasmidpaphG resulted in ∼ 100 paromomycin-resistant coloniesin glass bead transformations of cw10 or ∼ 40% less inparticle gun transformations. Again, re-analysis of paphG-transformants was in liquid medium with increasing con-centrations of paromomycin. The integration of plasmidpaphG into the genome of transformants was verified by ge-nomic PCR analysis using aphVIII-specific primers. PCRfragments of 776 bp were predicted in transformants. Allparomomycin-resistant transformants were positive in ge-nomic PCR analysis. The PCR result with a representa-tive C. reinhardtii paphG-transformant of the parent cellwall assembly defective strain cw10, a transformant ofthe parent wild-type strain CC-125, and results of bothcorresponding parent strains are shown in Fig. 5a and b.After a 3-month propagation period without selective pres-sure through paromomycin the paromomycin resistance as-say and the aphVIII-specific genomic PCR were repeated;again both came to the same result.

Transgenic algae, both from glass bead- and particlegun transformations, clearly were resistant towards paro-momycin, but did not tolerate more than 200 µg/mlparomomycin, just as pPmr3-transformants. So it wasnot possible to raise the high tolerance of pPmr3-transformants towards paromomycin any further by in-creasing the gene dosage in paphG-transformants of C.reinhardtii. As one possible cause we took into con-sideration that the concentration of aminoglycoside 3′-phosphotransferase could have been already such high inpPmr3-transformants (1 × aphVIII/plasmid) that a furtherincrease of this protein concentration could be impossi-ble in paphG-transformants (16 × aphVIII/plasmid) dueto physiological reasons or that a further increase of theaminoglycoside 3′-phosphotransferase concentration didnot yield a further increase of antibiotic tolerance due toother unknown reasons. But if this problem would existonly at high aminoglycoside 3′-phosphotransferase- andparomomycin concentrations, it would not influence theintended transformation experiments in Volvox.

Stable transformation of V. carteri using 16 ×repeated aphVIII cassettes with Chlamydomonasflanking sequences

Nuclear transformation of wild-type V. carteri by particlegun was attempted again, but this time instead of pSI103 the

plasmid paphG with its 16 × repeated aphVIII cassettes,each with 5′ and 3′ UTRs from C. reinhardtii, was used. Forthis purpose the PDS-1000/He biolistic device was loadedwith paphG-coated gold microprojectiles (diameter 1 µm),the distance to reproductive cells was adjusted to 16.5 cm,and acceleration of particles was by flowing helium at900 psi within the evacuated bombardment chamber (see‘Materials and methods’ section). Reproductive cells weretransferred to fresh medium and 5.0 µg/ml paromomycinwas added to the medium at the fourth day and the sameamount was added again at the eighth day. This timeseveral green and viable Volvox algae were detectedstereomicroscopically after 7–14 days. Re-analysis ofidentified clones was in liquid medium with increasingconcentrations of paromomycin. Transformants toleratedbetween 12 and 23 µg/ml paromomycin. Genomic PCRwas used to verify the integration of plasmid paphGinto the genome of V. carteri transformants. Again PCRfragments of 776 bp were predicted in transformants. Allparomomycin-resistant transformants were positive in ge-nomic PCR analysis, demonstrating that the 16 × repeatedaphVIII cassette with C. reinhardtii flanking sequencesworks as a dominant selectable marker in Volvox. Theresult with a representative V. carteri paphG-transformantand of the parent wild-type strain as a control is shown inFig. 5c. These experiments were repeated after a 3-monthpropagation period without selective pressure throughparomomycin. After this period, which corresponds to∼ 45 generations, the transformed strains still were posi-tive in the genomic PCR that verifies the presence of theaphVIII cassette and they still retained their paromomycinresistance.

Conclusions

Heterologous expression of a dominant resistance markernormally takes the existence of appropriate 5′ and 3′ se-quences from the target organism for granted. If no such ge-nomic sequences are available, use of 5′ and 3′ UTRs from arelated species might be an alternative that can be realizedquickly. In Volvox transformation experiments with 16 ×repeated aphVIII cassettes and Chlamydomonas flankingsequences (plasmid paphG) roughly the same number oftransformants could be obtained as in Volvox transforma-tion experiments using selectable marker genes with Volvoxpromoters. Likewise, in Chlamydomonas transformationexperiments with the aphVIII gene and Volvox flanking se-quences (plasmid pPmr3), approximately the same numberof transformants could be achieved as in Chlamydomonastransformation experiments using Chlamydomonas flank-ing sequences (plasmid paphA). Thus, promoters froma related species can be used in transformation experi-ments with Volvocales at least if a putative low expressionrate is compensated by an enforced high gene dosage asshown.

Our next aim will be to apply this strategy to genera suchas Gonium, Pandorina, Eudorina, and Pleodorina.

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Fig. 5 Detection of the aphVIII gene originating from plasmid pa-phG (containing C. reinhardtii 5′ and 3′ UTRs) in transformants ofC. reinhardtii cell wall assembly defective or wild-type strains trans-formed by glass-beads or by particle gun, or in V. carteri wild-typestrain transformed by particle gun. Genomic DNA was extracted fromuntransformed C. reinhardtii (cw10 or wild-type strain CC-125) or V.carteri wild-type strain (EVE), or from the respective transformantswhich integrated aphVIII genes originating from plasmid paphG,which contains C. reinhardtii 5′ and 3′ UTRs within each of its16 copies of the aphVIII cassette. Only transformants were expected

to yield a 776 bp fragment in PCR. a The cell wall assembly de-fective C. reinhardtii strain cw10 was transformed by glass-beads.(Left side): parent strain; (right side): transformant. b The wild-type C. reinhardtii strain CC-125 (WT) with its intact cell wall wastransformed by particle gun. (Left side): parent strain; (right side):transformant. c The wild-type V. carteri strain EVE (WT) with itsintact extracellular matrix was transformed by particle gun. (Leftside): parent strain; (right side): transformant. Products of PCR werecloned and sequenced

Acknowledgements We wish to thank Irina Sizova and PeterHegemann for providing plasmid pSI103, Thomas Jakobiak andRudiger Schmitt for providing plasmid pPmr3, and Kordula Pulsfor expert technical assistance. This work was supported by theDeutsche Forschungsgemeinschaft (SFB 521).

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