Proc. Nadl. Acad. Sci. USAVol. 91, pp. 5080-5084, May 1994Genetics

Nuclear transformation of Volvox carted(cotrandormatlon/green alae/gold particles/heflum-flow particle gun/nonhomologous recombination)

BERNHARD SCHIEDLMEIER*, RCDIGER SCHMITT*, WALTRAUD MOLLER*, MARILYN M. KIRKt,HERIBERT GRUBER*, WOLFGANG MAGES*, AND DAVID L. KIRKt**fLehrstuhl fUr Genetik, Universitit Regensburg, 93040 Regensburg, Germany; and tDepartment of Biology, Washington University, St. Louis, MO 63130

Communicated by Richard C. Starr, January 6, 1994

ABSTRACT Stable nuclear transformation of Volvox car-ten was achieved using the cloned V. carteni itAr gene (whichencodes nitrate reductase) to complement a nitA mutation.Following bombardment of mutant cells with plasmid-coatedgold particles, putative transformants able to utilize nitrate asa nitrogen source were recovered with an efficiency of -2.5 x10-5. DNA analysis indicated that the plasmid integrated intothe genome, often in multiple copies, at sites other than the nitAlocus. Cotransformants were recovered with a frequency of40-80% when cells were cobombarded with a selected and anunselected marker. Thus, V. cartie becomes one ofthe simplestmulticellular organisms that Is accessible to detailed molecularstudies of genes regulating cellular differentiation and mor-phogenesis.

Volvox carteri is a multicellular organism with a completedivision of labor between somatic and reproductive cells (1,2). Genetic analysis (1-4) has led to the hypothesis that asmall number of loci act to cause differentiation of these twocell types (5), and patterns of cell-type-specific gene expres-sion in wild-type and mutant embryos are consistent with thathypothesis (6). However, detailed molecular analysis oftheseputative regulatory loci has awaited a method for transform-ing the organism with exogenous DNA.We repeatedly tried to transform V. carteri with various

bacterial or plant selectable markers that were introduced bymicroinjection, electroporation, particle bombardment (7),UV-laser microbeam irradiation (8), agitation with glassbeads (9), etc. As with the related unicellular alga, Chlamy-domonas reinhardtii, reproducible transformation with het-erologous selectable markers was not achieved, possiblybecause of an inability of these algae to express heterologousgenes. Again in parallel with C. reinhardtii (9-12), success intransforming V. carteri has come with the availability of ahomologous selectable marker: here we report use of therecently cloned nitrate reductase-encoding gene of V. carteri,nitA (13), to complement a nitA mutation.

MATERIALS AND METHODSRecipient Strains. Strains used as DNA recipients were F1

female progeny of HB11A, a previously described, multiplymarked strain of V. carteri f. nagariensis (4). All of thesestrains inherited from HB11A a stable mutant allele (rever-sion rate, <10-8) that confers resistance to chlorate, abol-ishes the ability to utilize nitrate as a nitrogen source, mapsto the nitA locus, and is therefore inferred to be a stableloss-of-function mutation of nitA, the gene encoding nitratereductase (13). Strain 153-81 was given the mnemonic Gls/Reg ("gonidialess/regenerator") because it inherited fromHB11A a regA mutation that causes somatic cells to redif-ferentiate as gonidia (asexual reproductive cells) and also has

a spontaneous mutation at the gis ("gonidialess") locus thatresults in an absence of any "true" gonidia (6). GIs/Reg wasused in initial studies because it has only one type of cells, allof which can reproduce; thus it provides a homogeneouspopulation of transformation targets. Strains 153-40, -45, and-48 are morphologically wild-type siblings of GIs/Reg.

Transformation Vectors. ANR106 is a A EMBL3 clonecontaining a 13.5-kb Volvox genomic insert that spans the5.8-kb coding region of the V. carteri nitA gene plus =1 kb ofdownstream and =6.7 kb ofupstream sequence (13). PlasmidpVcNR4 contains the same insert in the vector pJOE890 (14).Plasmid pVcNR1 contains the same coding and 3' region, butonly =1 kb of upstream DNA, in the vector pBS+ (13).Plasmids pLV13-6, pLV131-3, and pLV131-4, which wereused as unselected markers, were kindly provided by S. M.Miller (Washington University); each carries a different ADNA fragment plus a Volvox transposon (15) and will bedescribed in detail elsewhere (S. M. Miller, personal com-munication).

Cultivation Conditions. Recipient strains were grown inaerated flasks, as described (16). Standard Volvox medium(SVM) contained 0.5 mM urea plus 0.5 mM calcium nitrate(16), but selective medium (NSVM) contained only nitrate asa nitrogen source. Synchronous Gls/Reg cultures were ob-tained by inoculating 300 ml of SVM with 50 spheroids(selected when embryos were in early cleavage) and incu-bating for 3 days; this yielded =5 x 106 gonidia or embryos.Strains 153-40, -45, and -48 were cultured to a density of t10spheroids per ml in flasks containing -1.5 liters of SVM; -2x 105 gonidia or embryos could be recovered from such aculture.

Transformation Protocols. In initial successful experi-ments, mechanical disruption of cytoplasmic bridges linkingembryonic cells (17) was used to provide cells access toexogenous DNA, as follows: Gls/Reg embryos (16- to 64-cellstage) were suspended in SVM containing nitA DNA and 50mM sorbitol, broken into single cells in a Dounce homoge-nizer with a tight-fitting pestle (clearance, =2.5 Pm), andcultured in NSVM solidified with 0.375% SeaPlaque agarose(FMC). Plates were screened at intervals, and green, growingcolonies were transferred to tubes containing NSVM.

In all recent experiments, flowing helium (He) was used tobombard cells with gold particles (1-3 gam in diameter;Johnson Matthey GmbH, Karlsruhe, Germany, or Aldrich)that had been coated with nitA plasmid DNA by ethanolprecipitation. Recipient spheroids were harvested by filtra-tion and broken mechanically to release gonidia or embryos,which were then collected by mild centrifugation. For mor-phologically wild-type strains, 7% Percoll (Pharmacia) inSVM was used for the centrifugation step, so that the(floating) somatic cells could be removed from the (pelleted)gonidia. Target gonidia or embryos were resuspended inNSVM at a density of -4 x 105 per ml, cultured in the lightfor 15-120 min, and then bombarded by minor modifications

tTo whom reprint requests should be addressed.

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of the methods of Takeuchi et al. (18): 10 y4 of DNA-coatedgold particles in EtOH was placed on the screen ofa Swinnex(Millipore) filter holder that was connected by tubing to a Hetank; recipient cells were spotted on sterile filter paper in thebottom of a Petri plate and placed below the filter holder; asolenoid valve was then opened for -0. 1 sec to permit a rapidflow of He, which propelled the particles toward the cells.Parameter ranges tested extensively included: gun-to-targetdistance, 6-9 cm; He exit pressure, 4-6 bars (1 bar = 100kPa); particle suspension, 125-250 ,g ofgold and 125-750 ngof DNA; target, 104-2.5 x 105 mature gonidia or early-cleavage embryos. Controls included use of uncoated parti-cles. Two selection protocols were tested: either the platecontaining the bombarded filter was flooded with 30 ml ofNSVM, covered, and returned to the culture chamber or cellswere washed from the filter and cultured 2 days in 25 ml ofNSVM and then aliquoted to wells of a multiwell plate. Inboth cases cultures were observed regularly, and each green,growing organism was transferred to a tube of NSVM.Putative transformants were tested for chlorate sensitivity byexposure to 8 mM potassium chlorate.

Preparation and Analysis of DNA. Plasmid DNAs wereprepared by alkaline extraction (19). Genomic DNA wasCsCl purified as described (4) or prepared by a newerminiprep method (15). Endonuclease digestion and electro-phoresis of DNA (1-4 pg per lane on 0.8% agarose gels),preparation, hybridization, and autoradiography of Southernblots were all by previously described, conventional methods(4, 19), except that some Southern blots were probed withdigoxigenin-labeled DNAs, according to directions providedby the vendor (Boehringer Mannheim). In all cases, finalmembrane washes were at 650C in 45 mM NaCl/4.5 mMsodium citrate/0.2% SDS/5 mM EDTA.

RESULTSThe nitA locus of V. carteri encodes nitrate reductase (13);nitA mutants require reduced nitrogen (nitrite, ammonium, orurea) for growth and are resistant to chlorate. Successfultransformation of a nitA- mutant with the cloned nitA+ geneshould generate Nit+ cells that can utilize nitrate but arekilled by chlorate.

Preliminary Studies. Transient Nit+ revertants, but nostable Nit+ lines, were recovered when attempts were madeto transform V. carteri nitA mutants by gently agitating acell-wall-less strain in the presence of nitA DNA and glassbeads (9) or by using a UV microbeam (8) to introduce nitADNA into plasmolyzed gonidia. The first stable Nit+ trans-formant of V. carteri was recovered when embryos, andhence the cytoplasmic bridges that link embryonic cells (17),were disrupted in the presence of ANR106 DNA. DNAanalysis indicated that this transformant possessed integratedA DNA (data not shown); this indicated that the ANR106insert carried a functional nitA+ gene and that Volvox cellswere capable of integrating and expressing exogenous DNA.However, efforts to establish an efficient, reproducible trans-formation system using this method ofDNA introduction didnot succeed.Recovery of Nit+ Transformants After Bombardment with

njL4+ DNA. Stable Nit+ transformants were repeatedly re-covered when a device (18) that employs a rapid flow of Hegas (rather than the explosive force used in other devices; ref.7) was used to propel gold particles that had been coated withnitA DNA toward V. carteri cells carrying a stable nitA-mutation. The first recipient used was the morphologicaldouble mutant, Gls/Reg (6), in which all cells reproduce andare therefore potential targets for transformation rescue.Subsequently, parallel experiments were successfully per-formed with gonidia derived from three morphologically

wild-type siblings ofGls/Reg, strains 153-40, -45, and -48 thatcarry the same nitA allele as Gls/Reg.

In 17 experiments (10 with Gls/Reg and 7 with 153-45 or-48) in which variables were held within the ranges describedin Materials and Methods, and in which the survival ofaliquots of the bombarded cells on nonselective plates wasmonitored, the mean frequency of stable transformantsamong the survivors was 2.5 x 10-5 (SD, ± 2.4 x 10-5). Nostatistically significant differences were detected that couldbe attributed to any of the variables, including the genotypeor developmental stage of the recipient cells. Under theseconditions, as many as 40 stable transformants were recov-ered in a single series of shots that took <2 hr. No stabletransformants were recovered from experiments in whichplasmid DNA was omitted.The first plasmid used for transformation was pVcNR4,

which contains =6 kb of genomic DNA upstream of the nitAcoding region (14); subsequently, pVcNR1, which containsonly =1 kb of upstream DNA, was found to be as effective.Transformation efficiency was not improved when linearizedplasmids were used. No transformants were recovered inexperiments in which an aqueous solution of plasmid DNAwas merely mixed with either the gold particles or the targetcells prior to bombardment.

Stability of Transformation and Integrated Plamid Se-quences. A small and variable fraction of the putative trans-formants that initially grew in NSVM (indicating that theypossessed a functional nitA gene) stopped growing, bleached,and died after a few days or weeks, indicating that the nitAfunction had not been stably transmitted. Most transform-ants, however, have been stably maintained-in many casesfor more than a year, or >1500 mitotic divisions-suggestingthat plasmid DNA had been stably integrated.Genomic DNA from recipient strains and many indepen-

dent Nit+ transformants was analyzed on Southern blots forthe presence of vector sequences. Sequences hybridizing tothe vector were not detected in recipients but were in thetransformants; moreover, different transformants often ex-hibited different numbers, sizes, and relative intensities offragments hybridizing to the vector (Fig. 1A; also see Fig. 3A;other data not shown). When similar blots were hybridizedwith a probe detecting part of the nitA coding region, eachtransformant was found to possess a hybridizing fragmentthat comigrated with the one present in the recipient strain,plus one or more novel fragments (Fig. 1B). These datasuggested that in each case one or more copies of thetransforming plasmid had integrated into the genome vianonhomologous recombination at sites outside the nitA lo-cus.Transformant cultures grown for >100 mitotic cycles un-

der nonselective conditions retained most of the vectorsequences that were present in cultures maintained undercontinuous selection (Fig. 2) and also retained their Nit+phenotype when again tested. on selective medium (data notshown). This reinforced the inference that transformantscontained plasmid sequences that had been stably integratedinto the nuclear genome. Occasionally, however, strains thathad integrated multiple copies of the plasmid were observedto have lost or gained one or two bands following prolongedcultivation (Fig. 2); we have not yet determined whether suchevents occur more frequently under selective or nonselectiveconditions.

Sites of Recombination Within the Transforming Plasmid.To substantiate the conclusion that stable transformantscontained integrated plasmid DNA, and to determine wherewithin each plasmid recombination had occurred duringintegration, a more detailed Southern blot analysis wasperformed. First, blots containing transformant DNAs di-gested by BamHI, EcoRI, or BamHI plus EcoRI werehybridized with a probe that detects the vector region of the

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A ,

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FiG. 1. Southern blots of DNA from the transforming plasmid(pVcNR4), the recipient strain (Gls/Reg), and eight transformants(identified by strain number). Sizes of A DNA standards (plain lines)and selected hybridizing bands (arrows) are given at the left. (A)DNA digested with Sca I and blot probed with plasmid pJOE890 (14),which shares withpVcNR4 only the vector region. (B)DNA digestedwith HindI and blot probed with a subcloned 4.3-kb HindIfragment from the 3' end of the nitA gene (13) that detects a 6.5-kbgenomic HindIII fragment; note that all transformants possess sucha 6.5-kb fragment.

transforming plasmid (Fig. 3A and C). Free pVcNR1 plasmidyields a 9.6-kb EcoRJ, a 4.8-kb BamHI, and a 3.2-kb EcoRI-BamHI fragment that hybridize to this probe (Fig. 3A and C).In a plasmid that underwent recombination near the 5' end of

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FiG. 2. Southern blots ofDNA from transformants (identified bystrain number) that had been either maintained under continuousselective pressure in NSVM (+) or grown >100 mitotic cycles in theabsence of selection (-). Size markers as in Fig. 1. DNA wasdigested with BamHI and the blot probed with a 3.2-kb BamHI-EcoRI fiagment representing the vector (pBS+) region of pVcNR1(see Fig. 3C).

the nitA insert (region I, Fig. 3C), the 9.6-kb EcoRI fragmentshould be replaced by a novel EcoRI fragment containinggenomicDNA flanking the integration site; in copies in whichrecombination occurred within the 3' region of the insert(region III, Fig. 3C), a novel BamHI fragment should bepresent; and in copies that had recombined within the vector(region II, Fig. 3C), six novel fragments should be present inplace ofthe three fragments characteristic offree plasmid. Inthe second part of this analysis, Southern blots of HindEI-digestedDNA from the same transformants were probed witha fragment representing the 3' end of the nitA gene (Fig. 3 Band C). This permitted us to define a fourth region (region IV,Fig. 3C) and thereby subdivide region I into two sections,since only plasmids that had recombined within region IVshould retain the 8.3-kb HindIII fragment of pVcNR1 butlack the 9.6-kb EcoPJ fragment.

In each transformant analyzed in the above manner, hy-bridizing fragments different from those offreepVcNR1 wereobserved, indicating that every transformant contained atleast one recombinant plasmid. But many hybridization pat-terns were more complex than could be accounted for by asingle plasmid integrant. It was often possible to account forall of the bands in a transformant as a reflection of a small,integral number of plasmids that had integrated by somecombination of the types of recombination events describedabove. For example, one may infer from the data in Fig. 3Athat transformant Hill 143 arose via integration oftwo copiesof the plasmid: one that recombined in region III (leaving its9.6-kb EcoRI fragment and 3.2-kb EcoRI-BamHI fragments

A a.V.XA:a;Ri:1i..........4 zh |i!R H4A H.~

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FIG. 3. Southern blots of DNA from the transforming plasmid (pVcNR1), the recipient strain (Gls/Reg), and two transformants (Hill 142and 143). Size markers as in Fig. 1. (A) DNA digested with eitherBamHI (B), EcoRI (E), or both (B/E) and probed with a 3.2-kb BamHI-EcoRIfragment ofpVcNR1 that represents the vector (pBS+) region of the plasmid (C). (B) DNA digested with HindM and probed with a subcloned4-kb HindM-BamHI fragment of pVcNR1 representing the 3' region of the nitA insert (cross-hatched box in C) that hybridizes to a 6.5-kbgenomic fragment. (C) Partial restriction map ofpVcNR1, shown for clarity in a linear form with a partial repeat (broken lines) at the right end.Stippled box, vector (pBS+) region; open box, coding region ofthe nitA insert (pointed end is 3'); plain lines, flanking regions ofthe insert. Arrowsat bottom of diagram: four regions in which recombination of the plasmid would have different effects on fragments detected in Southern blotssuch as those in A and B.

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intact, but replacing its 4.8-kb BamHI fragment with a novelone) and one that recombined in region III (replacing all threeof the pVcNR1 BamHI/EcoRI fragments with novel ones).The pattern obtained with a HindIII digest of Hill 143 (Fig.3B) is consistent with this interpretation: in addition to the6.5-kb fragment derived from the mutant nitA gene of therecipient strain (which is retained by all transformants yetstudied), Hill 143 has two novel HindIII fragments, asexpected for a strain possessing two copies of the plasmidthat recombined outside region IV. Similarly, one can inferfrom the more complex pattern of fragments generated fromHill 142 DNA (Fig. 3A) that this strain resulted from fourseparate plasmid-integration events, two in region I, one inregion II, and one in region III. Data from the HindIII digest(Fig. 3B) confirm and extend this interpretation: Hill 142 hasfour HindIII fragments (in addition to the 6.5-kb mutant-genefragment), but the fact that one of these is of the same size asthat present in pVcNR1 indicates that of the two plasmidsthat recombined within region I, one recombined withinregion IV, and the other recombined outside that region.

In some transformants the numbers and intensities ofBamHI, EcoRI, BamHI-EcoRI, and HindIII fragments in-dicated that as many as 10 copies of the plasmid had inte-grated, and in some cases fragments were present that couldonly be accounted for by assuming that certain plasmids hadrearranged before or during integration (data not shown).Cot formatlon ofan Unselected Marker. Ultimately, the

utility of a nitA-based transformation system for Volvoxinvestigators will depend on the ease with which cotransfor-mation can be achieved with an unselected marker, such asone of the genes believed to regulate cellular differentiationin Volvox (2). In the related unicellular organism, C. rein-hardtii, cotransformation frequencies of -50% wereachieved when the selected and unselected markers weredelivered on separate plasmids (20). To determine whetherthe same might be true of V. carteri, transformation wasperformed with a mixture of DNAs from pVcNR1 orpVcNR4 and another plasmid (pLV13-6, pLV131-3, orpLV1314) that contained a A DNA marker that does notcross-hybridize to Volvox DNA or to either of the nitAplasmids. All Nit+ transformants recovered were analyzed todetermine whether they contained the A DNA marker. In thefirst experiment in which Gls/Reg cells were bombarded witha mixture of pVcNR1 and pLV1314, 16 of 20 Nit+ trans-formants that were examined were found to contain the ADNA marker and the nitA plasmid (representative data inFig. 4). Other experiments involving different recipientstrains (153-40, -45, and -48), a different transforming plasmid(pVcNR4), and/or different A-marked plasmids have allyielded cotransformants with frequencies of -40-80%.

DISCUSSIONWe report here a method for achieving stable nuclear trans-formation of the multicellular alga, V. carteri. As with therelated unicellular alga, C. reinhardtii (10, 11), success indeveloping a useful transformation system required a homol-ogous selectable marker, since prolonged, intensive efforts toachieve such an outcome with heterologous selectable mark-ers met with failure.The selectable marker used here, the V. carteri nitA gene

(13), was cloned on the basis of its homology to the C.reinhardtii nit) gene, one of two genes now widely used fortransformation of C. reinhardtii (10, 11). However, methodsused successfully to transform C. reinhardtii with nit) havenot worked with V. carteri, undoubtedly because of differ-ences between the target cells ofthe two organisms. Every C.reinhardtii cell has reproductive potential and thus is apotential transformation target. Somatic cells of Volvox re-semble C. reinhardtii cells cytologically but are not suitable

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FIG. 4. Southern blots used to test for cotransformation in sixNit+ transformants that had been cobombarded with a selectablemarker (nitA in pVcNRl) and an unselected marker (A in pLV131-4).DNA samples were digested with HindIR. (A) Blot probed with the4-kb Hindfll-BamHI fragment of pVcNR1. (B) Blot probed with a5.6-kb BamHI fragment of A DNA homologous to the A insertcontained in pLV131-4. Note that four of six strains carry theunselected as well as the selected marker.

as transformation targets because they lack reproductivepotential, whereas the reproductive cells of Volvox (gonidia)are very different from Chlamydomonas cells cytologically:they are much more highly vacuolated and more sensitive tomechanical stresses. One method widely used for transfor-mation of C. reinhardtii involves vortexing protoplasts in thepresence of exogenous DNA and glass beads (9). Whennaked V. carteri gonidia derived from "cell-wall-less" strainswere subjected to this treatment, >99.99%o were killed in thefirst few seconds. When the protocol was modified byomitting glass beads, or using gentle inversion to mix nakedcells, beads, and DNA, or by using wild-type gonidia (en-closed in extracellular matrix) as targets, cell survival wasmarkedly improved, but no stable transformants were recov-ered. Similarly, Volvox gonidia were incapable of withstand-ing the extreme pressure transients experienced in the typesofparticle guns that have been used successfully with Chlam-ydomonas (10-12), even when metal particles and DNA wereomitted from the protocol. Thus, it became clear that othermethods of DNA introduction, more compatible with theproperties of the organism, were required.The first way in which nitA was successfully used to

transform V. carteri exploited an unusual feature of itsbiology: the existence of numerous cytoplasmic bridgeslinking all cells during embryogenesis (17). When embryoswere mechanically disrupted in the presence of nitA DNA,

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stable transformation occurred at a very low frequency,presumably because broken cytoplasmic bridges transientlyprovided channels for uptake of DNA. Although initial suc-cess with this method was important in establishing that afunctional nitA gene was present in the cloned element, andthat V. carteri was capable of integrating and expressingexogenous DNAs, attempts to achieve reproducible andefficient transformation with this method ofintroducing DNAdid not succeed.The more efficient method reported here for DNA intro-

duction uses a He-flow particle gun (18), which differs fromthe earlier ballistic devices (7, 10, 12) in that it is operated atambient pressure (rather than in a vacuum chamber) and thatit uses a rapid flow of He (rather than an explosive force) topropel the particles toward the target cells. The use of He asthe propellant is thought to be important, since He has a muchhigher diffusivity than other gases and is inert and thusappears to have fewer toxic side effects (18). In our hands,survival of bombarded Volvox gonidia is several orders ofmagnitude higher with the He gun than with other particleguns we have tested (=25% vs. <0.01%).Among other things, the present results demonstrate that

the cloned insert in plasmid pVcNR1, which includes only 1kb of upstream DNA (13), carries all of the informationnecessary for expression of the nitA gene. It remains to bedetermined, however, whether it also carries the informationrequired for normal regulation of nitA transcription by am-monium and nitrate ions (13).

Southern blot analysis indicated that in many cases thestable transformants produced here possess multiple inte-grated copies ofthe transforming plasmid but also retain whatappears to be an unmodified version of the (defective) nitAlocus present in the recipient strain (Figs. 1B and 3B). Thusit appears that integration of plasmid DNA occurs by non-homologous Campbell-type (21) recombination at sites otherthan the nitA locus. This parallels observations made withChlamydomonas (9, 10). In many cases the restriction pat-terns of Volvox transformants can be accounted for byassuming that multiple plasmids integrated independently,with one recombination event per plasmid (Fig. 3). In othercases, fragments are present that cannot be accounted for thisway, suggesting that sometimes recombination may occuramong copies of the plasmid before or during integration.Even in cases in which restriction analysis indicates thatseveral plasmids integrated independently, however, we can-not rule out the possibility that they are tightly clustered inthe genome, as has been observed in Chlamydomonas trans-formants (12).Although the efficiency of transformation achieved here

(r2.5 x 10-5) is modest, this is partially compensated for bythe high efficiency (40-80%6) with which cotransformationwas observed for unselected markers.

Volvox is significantly more complex developmentally thanSaccharomyces or Chlamydomonas, and yet much simpler

than Arabidopsis, Caenorhabditis, or Drosophila. Hence, ithas often been said to have unique promise as a develop-mental-genetic model system (1), particularly for analyzingthe genetic basis for germ-soma differentiation (2). Theavailability of a cotransformation system-together with theVolvox transposons that have recently been identified (15, 22)and that may permit tagging and recovery ofdevelopmentallyimportant genes-should help bring that promise closer torealization.

This paper is dedicated to Prof. Lothar Jaenicke in honor of his70th birthday. We are grateful to Stephen M. Miller for the plasmidsused in cotransformation studies and for helpful advice. Support wasprovided by the Deutsche Forschungsgemeinschaft (SFB 43), theNational Institutes of Health (GM 27215), the National ScienceFoundation (DMB-9005233), and the North Atlantic Treaty Organi-zation (870065).

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