Proc. Natl. Acad. Sci. USAVol. 90, pp. 4912-4916, June 1993Cell Biology

Colocalization of centromeric and replicative functions onautonomously replicating sequences isolated from theyeast Yarrowia lipolytica

(chromosome/plasmid stability/DNA replication)

P. FOURNIER*t, A. ABBAS*, M. CHASLES*, B. KUDLA*, D. M. OGRYDZIAKt, D. YAVER*, J.-W. XUAN*,A. PEITO*, A.-M. RIBET*, C. FEYNEROL*, F. HE*, AND C. GAILLARDIN**Lab Genetique, Institut National de la Recherche Agronomique, 78850 Thiverval, Grignon, France; and tInstitute of Marine Resources, University ofCalifornia, Davis, CA 95616

Communicated by John A. Carbon, January 11, 1993

ABSTRACT Two sequences (ARS18 and ARS68) display-ing autonomous replication activity were previously cloned inthe yeast Yarrowia lipolytica. The smallest fragment (1-1.3 kb)required for extrachromosomal replication of a plasmid issignificantly larger in Y. lipolytica than in Saccharomycescerevisiae. Neither autonomously replicating sequence (ARS) ishomologous with known ARS or centromere (CEN) consensussequences. They share short regions of sequence similarity witheach other. These ARS fragments also contain Y. lipolyticacentromeres: (i) integration of marker genes at the ARS lociresults in a CEN-linked segregation of the markers, (it) anARSon a plasmid largely maintains sister chromatid attachment inmeiosis I, and (iii) integration of these sequences at the LEU2locus leads to chromosome breakage. Deletions performed onARS18 show that CEN and ARS functions can be physicallyseparated, but both are needed to establish a replicatingplasmid.

Yarrowia lipolytica sequences capable of supporting extra-chromosomal autonomous replication have been described(1). Plasmids containing ARS18 or ARS68 transform at highfrequency and are mitotically more stable than ARS plasmidsin Saccharomyces cerevisiae (2): loss per generation variesbetween 1% and 4% on nonselective medium. This value iscloser to the 1-3% observed in S. cerevisiae for artificialminichromosomes containing cloned centromeres (3) than tothe 5-50% found for ARS plasmids (4, 5).The copy number ofARS plasmids in Y. lipolytica has been

reported to be about 3 per plasmid-containing cell, which isalso very different from the 50-100 copies for S. cerevisiaeARS plasmids (6) but closer to the 1-2 copies per celldescribed for centromeric plasmids (3).We wondered whether ARS18 and ARS68 are ARS se-

quences associated with Y. lipolytica centromeres or withsome kind of stabilizing sequence like that described forSchizosaccharomyces pombe (7). The structure of S. cere-visiae centromeres has been extensively investigated (8):they contain two conserved sequences (CDEI and CDEIII)separated by an A+T-rich region (CDEII), and a functionalcentromere is contained within a 125-bp sequence (9). Incontrast, the three centromeres of the fission yeast Sch.pombe cover very large regions (35, 55, and 110 kb) display-ing a complex pattern of repetitive DNA sequences (10, 11).Thus the two types of centromeres from Sch. pombe and S.cerevisiae are very different (12). The Sch. pombe cen-tromeres have an organization similar to those of highereukaryotes, and the S. cerevisiae centromeres could be morerepresentative of those from other yeasts, such as Kluyver-

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omyces marxianus var. lactis (hereafter, K. lactis). IndeedDNA fragments probably corresponding to K. lactis cen-tromeres (13) are very similar in sequence to the S. cerevisiaecentromeres, except for a larger CDEII region (14). How-ever, they are not recognized as centromeres in S. cerevisiae.Our analysis of Y. lipolytica ARS18 and ARS68 shows thateach carries functional centromeres within a 1-kb DNAfragment and that they do not share any significant sequencesimilarity with known S. cerevisiae, K. lactis, or Sch. pombesequences.

MATERIALS AND METHODSStrains and Plasmids. The following Y. lipolytica strains

were used: INAG 33122 (MatB, leu2-35, xpr2, lys2-5), E122(MatA, leu2-270, ura3-302, lysll-23), 21805-9 (MatA, leu2-35, ura3-18), and 22301-3 (MatB, ura3-302, leu2-270, his-i).Mutations leu2-270 and ura3-302 are in vitro-generated de-letions ofLEU2 and URA3 (gift of E. Fabre, Institut Nationalde la Recherche Agronomique). The Y. lipolytica URA3 genewas obtained from L. Davidow (Pfizer). The ARS18-URA3plasmid pINA311 was given to us by E. Fabre.

Genetic and Molecular Biology Techniques. ARS68 wascloned on a 2.3-kb BamHI-Bgl II fragment (see Fig. 2) intothe BamHI site of the pBluescript vector (Stratagene) in bothorientations. Both plasmids were sequentially digested withKpn I and HindIII and then treated with exonuclease III byusing the Erase-a-Base kit (Promega). The DNA was reli-gated, giving a series of plasmids with various deletions of theARS68 region. Several ARS18 restriction fragments werecloned in pBluescript. Dideoxy chain-termination sequenc-ing (15) was done with modified T7 polymerase (Sequenase,United States Biochemical) on double-stranded plasmids(16).

Y. lipolytica was transformed by lithium acetate treatmentas described (17) and by electroporation (18) using a Jouanelectroporator (Winchester, VA) at 2.2 kV/cm for 15 msec.Strain crosses, random spore analysis, and tetrad dissectionwere performed by published procedures (19-21). Mitoticstability of transformants was assessed as follows (1): 5-10transformants obtained with a given plasmid were grown onnonselective medium for about 15 generations, and 20 sub-clones of each culture were then checked for the loss of theplasmid-associated marker. Stable transformants showed nosegregation, whereas several plasmid free clones segregatedfrom unstable transformants. This test allowed detection ofplasmid loss per generation higher than 0.7%. Plasmids wereextracted from yeast transformants as from S. cerevisiae (22).Other molecular biology methods were performed by stan-dard procedures (23).

tTo whom reprint requests should be addressed.

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Proc. Natl. Acad. Sci. USA 90 (1993) 4913

Chromosome Separation. Cells grownwere harvested in exponential phase, wasi(10 mM Tris-HCl, pH 8.0/1 mM EDTA), ax 109 cells per ml in protoplasting buffer (U8.0/0.1 M EDTA/0.9 M sorbitol). Protop]monitored at 37°C for 20-30 min after addlOOT (Miles) at 0.3 mg/ml. An equal vcmelting agarose in 0.25 M EDTA (pH 8.0) Nmixture was poured into appropriate m(incubated for 48 hr at 50°C in 0.5M EDTA (sodium N-lauroylsarcosine (1%, wt/vol) amg/ml) (Boehringer). The blocks were stoEDTA. After extensive dialysis in TE buffiwas conducted in the Beckman TAFE syst(per liter: 1.21 g of Tris, 0.145 g of EDTAacetic acid) for 168 hr at 18 mA in a 0.8% 2with a pulse time of 70 min.

RESULTSDeletion Mapping of ARS Function. Cli

5.4-kb insert in plasmid pINA119 and Ainsert in plasmid pINA123 do not cross-hiem blots (1) and their restriction maps are1 and 2).ARS18 was subcloned and deletion rr

structed by use of restriction sites (Fig.deletion plasmids carrying the Y. lipolytwere used to transform strain INAG 3312efficiencies and stabilities of transformantrvarious deletions are reported in Fig. 1. Itransformation and plasmid instability weARS function. We checked by hybridizatiothat unstable clones actually contained (plasmids and that stable ones resulted eiintegration into the chromosome or fromversion. Results with the first seven delethe ARS activity was contained within thcarried by pINA176 or pINA237. Deleti0.6-kb BstBI fragment (pINA292 and pINi10-fold decrease in the transformation fr4the transformants remained unstable.(pINA173, pINA382, pINA384, and pINAform at high frequency and gave rise only

H Ba H Bga BgpINA119 1 I I 1115.*pINA232 B BpINA121 Bg a Ba 69pINA120 H 6BpINA172 H HpINA122 BHpINAl 71 ,pINA1 47 i2Ba s Ba 2.2

x !BSBE,\-splNA237,pINA176 6Ba: 1.3IkbplNA292,pINA175 X BseB B SpiNA173 x Ba BYplNA382M htspINA384 x B SpINA385x L.PplNA725 _Ba E_s

B

FIG. 1. Deletion mapping ofARS18. The frauplasmids are shown. Plasmids marked with an YpBluescript, others in pBR322. All contained thgene as a marker. Transformation frequencytransformants per jig ofDNA. Mitotic stability i!U, unstable; ND, not determined). B, BstBI; BaEv, EcoRV; H, HindlIl; S, Sau3Al.

in YPD mediumhed with TE buffermnd suspended at 5.1 M Tris HCl, pHlast formation wasition of zymolyase)lume of 1% low-was added, and theDlds. Blocks were(pH 8.0) containingLnd proteinase K (1tred at 4°C in 0.5 Mer, electrophoresisem in TAFE buffer, 0.25 ml of glacialagarose gel at 10°C

oned ARS18 on aLRS68 on a 6.6-kbybridize on South-: not similar (Figs.

pINA123plNA242plNA243plNA248plNA386

pINA734pINA751plNA752plNA736PINA737pINA753pINA750plNA731

TransformationBg Ba Bg E B9 Frequency Stability

I 'a I 16.6 kb 10 000 U

10 S10 S

10 000 U10 000 U

kb6:31 1 S N p8pI I B1

10 00010 00010 000

101 0

10 0001 01 0

uuussuss

location of ARS68

FIG. 2. Deletion mapping ofARS68. The fragments carried on theplasmids are shown. Plasmids were constructed in pBR322. Markergene, transformation frequency, and stability are as described forFig. 1. B, BstBI; Ba, BamHI; Bg, Bgl II; E, EcoRI; H, Hpa I; N, NheI; P, Pst I; Sc, Sca I; Ss, Sst I; St, Sty I.

not shown). We conclude that efficient ARS function needs)utaNatiwerARSon8 the central BstBI fragment and some of the flanking se-1catLEU2 arkSJ quences: ARS18 maps to a region of 0.6 kb to 1.3 kb.tica LEU2marker In the case ofARS68, the 6.6-kb insert in pINA123 (Fig. 2)s. trans ormation was first reduced to a 2.3-kb BamHI-Bgl II fragment byobtained with the subcloning (pINA386). This DNA was further shortened by

High frequency of exonuclease III treatment on either side. The left boundaryre associated with of the functional ARS68 mapped to the right of the Nhe I site,n (data not shown) and the right boundary to the right of the Hpa I site.extrachromosomal Comparison ofthe fragments contained in plasmids with ARSither from plasmid function (pINA752 and pINA753) with those in plasmids thati LEU2 gene con- had lost ARS function (pINA736 and pINA750) indicates thattions indicate that ARS68 maps to a region of 0.7 kb to 1 kb.ie 1.3-kb fragment Nucleotide Sequence. The 1305-bp BamHI-Sau3Al ARS18ion of the central fragment, which is the smallest ARS18 insert displaying fullA175) resulted in a ARS activity in vivo, and the 2309-bp BamHI-Bgl II frag-equency, although ment, which includes ARS68, were sequenced. The overallAll other clones G+C content is 32.5% for ARS18 and 36% for the smallest385) failed to trans- functional (1049-bp) ARS68 insert, much lower than 49o forto integrants (data total DNA of Y. lipolytica (24), and in accordance with the

A+T-richness of several ARS sequences in S. cerevisiae (2).Transformation Comparison of the sequences revealed two distinct regions

F10ec000 U of similarity. The left-hand end of each ARS contains a 15-bp10 000 U (A/C)Y(A/T)(A/T)NTACAAGTAY(A/C) consensus (se-

1 0 U quence 1 in Fig. 3 A and C) present twice in opposite1 0 S orientations. Each consensus lies in 50-bp stretches ofDNA1 0 S that are 60% identical. The distance between the repeats is1 0 S 194 bp in ARS68 and 130 bp in ARS18. Another 60-bp region

kb 10 000 U displaying 60% identity between both ARS68 and ARS18contains the 19-bp consensus TA(A/C)AYATGGAT-10 000 U AGYATA(A/C)A (sequence 2 in Fig. 3 B and C). It is located

1 000 U at the right-hand end of each ARS in opposite orientations100 S and near a 75% A+T stretch of differing length in the two10 ND ARS sequences (120 bp in ARS18 and 180 bp in ARS68).1 0 S Other 20- to 40-bp direct repeats are also present in each ARS10 S (data not shown). No long open reading frame is present in

10 000 u these sequences, and the conserved features described forcentromeric DNA in S. cerevisiae (4, 25, 26) are not found ineither ARS. There is no perfect match with the S. cerevisiaegments carried on the ARS consensus (2), but there are several 10- or 9-bp matches

were constructed in in ARS18 and ARS68. Sequences partially matching the

e is given as no. of consensus can behave as a bona fide ARS in vivo in S.sindicated (S, stable; cerevisiae (5, 27). pINA119 (ARS18-LEU2) and pINA123, BamHI; Bg, Bgl II; (ARS68-LEU2) yielded about 5000 transformants per ,ug in

S. cerevisiae. Their replicative ability was recognized in S.

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AARS68 818 Gc CTTAtTACAArTAtA IttcAaGcAagt ATAtCcCTA l--- i laTtGTaGcACt- IgGTACTTGTACaaag 1015ARSIB 104 Gg ICTTAcTACAAGTAcA agtAgGtAtac IATAgCgrA - | -TaGTtGtACga |tTACTTCTACttgt 237

®KYIWTACAArTAYh KRTACTTGTANWRK (BARS68 1583 TTTtcGTAGAtaAtggaaTAcAAATGgaTATcc-AgAgta TAcAcATGGATAGtATAcA ctGAcA 1646ARS18 958 TTTatGTAGAatAaatgtTAtAAATGcgTATgggAaAtct TAaAtATGGATAGcATAaA t-GAtA 895

() TAKAYATGGATAGYATAMA

ARS68 820

7'40

1014

PstI1084

1622AT rich -_S*region i)

PstI Sc&I1414 1434

ARSIO 106 236

a6Bin'I

0BatBI283

ZcoRV SpaI 17831611 1671

918AT rich

W region

BatBI EcoRV876 895

I

ARtS

I IpINA385IpINA173

CEN

FIG. 3. Sequence comparison ofARS18 and ARS68. The GenBank accession numbers M91600 and M91601 were given to the 1.3-kb ARS18and the 2.3-kb ARS68 sequence, respectively. The numbers indicated along the sequences refer to the position in the GenBank record. The mainstretches of similarity are boxed. W stands for A/T, M for A/C, Y for pyrimidine (C or T), R for purine (A or G), and N for A/C/G/T. (A)Similarities at the left-hand end of the sequences in direct orientation. (B) Similarities at the right-hand end of the sequences on opposite strands.(C) Position of the similar regions in ARS18 and ARS68; the numbers within circles refer to the conserved stretches shown inA and B. Deletionplasmids obtained from ARS18 are shown at the bottom. Locations of ARS and CEN in ARS18 are deduced from the last section of Results.

cerevisiae but their mitotic stability on complete medium (1%prototrophs after 10 generations) was much lower than in Y.lipolytica and similar to that of S. cerevisiae ARS plasmids.

Genetic Segregation Data. Segregation patterns of markergenes integrated

Proc. Natl. Acad. Sci. USA 90 (1993) 4915

Transf. No of No of ConclusionTarget Plasmid Freasf. clones stable fromq. analysed clones Southern

ARS18 plNA232/BamHl 19400 50 20 1 LEU2 gene conversion19 plasmid integrations

at ARSI8

LEU2 pINAl 76/Xhol 840 21 1 1 pasnmid integrationat LEU2

LEU2 pINAl 1 9/Apal 4000 20 3 2 LEU2 gene conversions1 plasrmid integration

at LEU2

FIG. 4. Integration ofARS18 in the Y. lipolytica genome. (Upper)Maps of the plasmids used for the integration ofARS18. The ARS18inserts are described in Fig. 1. The sites used for integration in yeastare underlined. (Lower) Results of yeast transfonnations obtainedwith different centromeric plasmids. Transformation frequency isexpressed as no. of transformants per jig of plasmid DNA.

an integration at LEU2. Their electrophoretic karyotypeswere analyzed. The recipient strain INAG 33122 probablyharbors 5 chromosomes (Fig. 5A, lane 6): a doublet at 2.5 Mb,a band at 3.4 Mb, and two close bands at 4.0-4.5 Mb. Thesignal obtained by hybridization with LEU2 in the recipientstrain INAG 33122 (Fig. 5B, lane 6) corresponded to achromosome of 4.5 Mb that was not present in the pINA176transformant (lane 5). The LEU2 signal was found on a newchromosome band of 0.8 Mb, which also hybridized toARS18 (Fig. 5C). A band of 3.7 Mb was also visible (Fig. SA,lane 5), which corresponded to the other part of the brokenchromosome. Similar chromosomal breakage was observedin the transformant obtained with pINA119 (data not shown).The stability of the new small chromosome was investi-

gated by crossing the transformant with the leu2 strain21805-9. The resulting diploid was cultivated for 50 genera-tions in liquid YPD medium and the percentage of Leu-colonies was scored. The frequency of loss per generation ofthe LEU2-containing chromosome was 490 times higher inthis strain than in a control diploid (3.8 x 10-3 vs. 7.8 x 10-6).Either the cloned ARS18 centromere lacks some elementsneeded for correct chromosome segregation in mitosis, orsmaller chromosomes are poorly propagated in this yeast.

Integration experiments with ARS68 also resulted in chro-mosome breakage when ARS68 was integrated at the LEU2

locus. ARS68 and ARS18 hybridized to different chromo-somes, as would be expected for different centromeres (datanot shown).

Fine Dissection of ARS18. The previous experiments sug-gested the presence ofa functional centromere (CEN) in eachARS18 and ARS68 sequence. We investigated whether or notthe ARS and CEN functions of ARS18 could be separated.We used some of the deletions shown at the bottom of Fig.3, and the strategy of forced integration described in thepreceding section: Xho I digestion in the LEU2 gene, trans-formation, screening for stable clones, Southern hybridiza-tion to show that they were integrants and not the result ofLEU2 gene conversion, and analysis of their chromosomeson pulsed-field gels. We expected that deletions retaining anentire CEN should transform at a lower frequency due to thegeneration of dicentrics, which upon breakage should resultin lethal transformation events. Indeed, linearized pINA384and pINA382 yielded 50-100 transformants per ,ug, whereaspINA173 and pINA385 yielded 500-2000 transformants per,ug. The chromosome separations showed that integrantsobtained with pINA173 (Fig. 5A, lane 1) and pINA385 (Fig.5A, lane 3) had patterns identical to that of the parent (Fig.5A, lane 6), whereas those obtained with pINA384 (Fig. SA,lane 2) and pINA382 (Fig. 5D) clearly showed the appearanceof two new bands, one of 3.2 Mb and one of about 1.3 Mb.The LEU2 hybridization confimed the disappearance of a4.5-Mb band (Fig. 5 B and D). Hybridization with ARS18(Fig. 5C) showed that this sequence was present on the2.5-Mb band, as in the recipient strain, and also on the 1.3-Mbbroken chromosome. These results are similar to thoseobtained when the entire ARS18 sequence was used (Fig. SA,lane 5). The CEN function was therefore contained in the410-bp sequence in pINA382. Because plasmids pINA292and pINA175 (Fig. 1) containing this 410-bp sequence and a283-bp BamHI-BstBI sequence are replicative, the replica-tion origin is probably located in the BamHI-BstBI fragment.Plasmid pINA725 (Fig. 1), which contains these two frag-ments separated by pBR322 sequence, transformed at highfrequency.The CEN plasmid pINA384 was used to clone ARS se-

quences from the genome. Ifassociation ofany chromosomalARS with this CEN generated a replicative plasmid and ifARS sequences were randomly distributed on the genomeevery 20-40 kb, then about 1 in every 10 clones in a bank withinserts of about 4 kb should contain an ARS. We constructedsuch a bank, and it transformed Y. lipolytica 10 times moreefficiently than an integrative pBR322-LEU2 plasmid.Eighty-seven percent of the yeast transformants were unsta-ble and were shown to contain autonomously replicatingplasmids. The presence of CEN on the vector, therefore,

A1 2 3 4 5 6 7

5.74.63.5

Bt 2 3 4 5 6

A lw

*D 0*0.~~~~~~~~~~..

F

C D7 1 2 3 4 5 6 7 a b a b

irn i... -V* :4t

.,,0UW**#

FIG. 5. Chromosome separations by pulsed-field gel electrophoresis. (A) Chromosomes of Y. lipolytica integrative transformants obtainedwith pINA173 (lane 1), pINA384 (lane 2), pINA385 (lane 3), pINA232 (lane 4), or pINA176 (lane 5) and with the recipient strain INAG 33122(lane 6). Sch. pombe chromosomes are shown as markers (lane 7) and their size is expressed in megabases (Mb). The lowest bands correspondto the front of migration. (B) Hybridization with a LEU2 probe. (C) Hybridization with an ARS18 probe. (D) (Left) Chromosomes of thetransformant obtained with the recipient strain INAG 33122 (lane a) and with pINA382 (lane b). (Right) Hybridization with a LEU2 probe.

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permitted the successful cloning of ARS sequences at highfrequency in Y. lipolytica, as is the case in S. cerevisiae.

DISCUSSIONWe have shown that ARS18 and ARS68 contain Y. lipolyticacentromeres. Integration of marker genes near ARS18 orARS68 led to centromere linkage on different chromosomes.The absence of tetratypes in 38 tetrads indicates that ARS18and ARS68 are less than 1.3 centimorgans from the cen-tromeres. The ARS18 sequence on a plasmid provided cen-tromere-like meiotic functions. Nonrandom assortment ofthe plasmids was shown by the excess of the 2+:2- segre-gation ratio over the 3+ :1 - and 1+ :3- ratios and, moreclearly, by maintenance of sister chromatid attachment dur-ing meiosis I, as evidenced by the lack of tetratypes withrespect to the centromere-linked marker. Dicentric chromo-somes have been reported to be very unstable in S. cerevi-siae, giving rise to breakage, telomere healing, centromeredeletion, or chromosome loss (31-33). When a plasmid con-taining the whole ARSI$ sequence was integrated at theARS18 locus in Y. lipolytica, the two copies of ARS18 were11 kb apart; the construct was nevertheless stable, suggestingthat the two copies could not be recognized as two separatecentromeres. But when ARS68 or ARS18 was integrated atthe LEU2 locus, chromosome breakage was observed, show-ing that the cloned sequences contained a functional cen-tromere.ARS18 contains two separate domains. One harbors the

replication function. It is probably included in the left, 283-bpBamHI-BstBI fragment (Figs. 1 and 3). This is slightly largerthan in S. cerevisiae, where about 100 bp are sufficient forreplication function (34, 35). The second domain correspondsto the CEN function and is carried on the 410-bp EcoRV-Sau3Al fragment (Figs. 1 and 3) in pINA382. Interestingly,these two domains correspond to the two regions showingsequence similarities with ARS68 (Fig. 3, sequences 1 and 2).Therefore, we expect that ARS68 also consists of two func-tional domains and that the regions of similarity indeedcorrespond to a biological consensus.The size of the Y. lipolytica CEN is similar to that of S.

cerevisiae but its structure is different. Further, the instabil-ity of the Y. lipolytica CEN in S. cerevisiae suggests that itis not recognized as a centromere.

Colocalization of ARS and CEN has been found in S.cerevisiae in the case of CEN3, but this ARS has been shownnot to be functional in vivo (36). The results of fine dissectionof ARS18 by chromosomal integration suggest that in Y.lipolytica, both a centromere and an ARS are required toobtain a replicative extrachromosomal plasmid. Plasmidscarrying only an ARS would not segregate properly in theprogeny of a transformed cell, due to an unexplained verystrong mother segregation bias in this yeast. Only two ARSsequences were cloned in Y. lipolytica (1), so ARS and CENmay be closely linked on two of its chromosomes. By usingthe cloned fragment bearing the ARS function as a recipientvector, it should now be possible to clone the other Y.lipolytica centromeres and thereby better define their con-sensus structure. The identification of a consensus will be ofgreat interest, as no sequence similarity was found withcentromeric DNA from either S. cerevisiae or Sch. pombe.

We thank E. Fabre and L. Davidow for gifts of strains andplasmids. This study was conducted with the financial help of the

Mission des Biotechnologies, ofthe Institut National de la RechercheAgronomique, of the Centre National de la Recherche Scientifique,and of Pfizer, Inc. (Groton, CT). D.M.O. and D.Y. were supportedby the National Science Foundation. A.A., J.-W.X., and F.H. wererecipients of grants from the Algerian, French, and Chinese govern-ments, respectively.

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