Nodulation of Legumes by Members of the Bêta-Subclass of Proteobacteria

t ,.i i i 3 letters to nature

Enos Lake. This work was supported by NSF-NATO and NSF-IntemationalResearch plants were very distant from known rhizobia. feloivships to J.W.B. and NSERC operating grants to D. Schluter. Strain STM678 was originally isolated from the South-African Correspondence and requests for materials should be addressed to J.W.B. legume Aspalathus carnosa, which was thought to be nodulated by (e-mail: boughman~zoology.ubcca). bacteria of the Bradyrhizobiutn genus6. However, we performed phylogenetic analysis of gene sequences of the small subunit of ribosomal RNA (16s rRNA), and found that strain STM678 does not belong to any ofthe four branches ofrhizobia described so far, nor even to the a-subclass ofProteobacteria, but instead belongs to ...... the ß-subclass of Proteobacteria (Fig. 1). From this analysis, we Nodulation d legumes by of found the most closely-related sequences to that of strain STM678 members (AJ302311) to be those ofBurkho1den.a kururiensis (96.9% identity), the ß-subclass of ProteObacteria B. brasilense (96.8% identity) and B. graminis (96.8% identity). Phylogenetic analyses of partial sequences of the 23s rRNA gene Lionel Moulin, Antonio Muniue, Bernardi Dreyfus (AJ302313) and the dnaK gene encoding the chaperon heat shock & Catherine oiuin-Masson protein (AJ302314) were consistedt with the 16s rRNA analysis, fr thus unambiguously positioning strain STM678 in the Burkholderia Laboratoire des Symbioses Tropicales et Méditerranéennes, IRD-INRA-CIRAD- genus within the ß-subdivision of ProteObacteria. ENSAM Baillarguet, PB 5035,34398 Montpellier Cedex 5, France To ensure that the Burkholderia strain STM678 was indeed a ...... rhizobium, we checked its ability to re-nodulate a leguminous plant. Members of the Leguminosae form the largest plant family on Because seeds of the original host plant, A. carnosa, were not Earth, with around 18,000 species. The success of legumes can available, we selected as test plant Macroptilium atropurpureum, a largely be attributed to their ability to form a nitrogen-fjxing tropical legume capable of establishing a symbiosis with diverse symbiosis with specific bacteria known as rhizobia, manifested by rhizobia. Over a three-week period, strain STM678 formed 5 to 20 the development of nodules on the plant roots in which the nodules per plant on the roots of M. atropurpureum (Fig. 2). The bacteria fix atmospheric nitrogen, a major contributor to the nodules displayed the classical determinate nodule structure, with a global nitrogen cycle. Rhizobia described so far belong exclusively central 'infected' tissue containing cells with intracellular bacteria to the or-subclass of Proteobacteria,where they are distributed in and a peripheral tissue with vascular bundles -(Fig. 2). Single four distinct phylogenetic branches'**. Although nitrogen-íking colonies re-isolated from surface-sterilized nodules exhibited the bacteria exist in other proteobacterid subclasses, for example characteristics ofstrain STM678, as assessed by 16s rDNA sequen- Herbaspirillum and Azoaras from the phylogeneticallydistant ß- cing and nod4 analysis using polymerase chain reaction-restriction subclass, none has been found to harbour the nod genes essential fragment length polymorphism (PCR-RFLP; see below). Hence, for establishing rhizobial symbiosis34Here we report the identi- Koch's postulates were verified. Td eliminate the possibility that fication of proteobaderia from the ß-subclass that nodulate strain STM678 is a mixture oftwo different bacteria, a Burkholderia legumes. This fìnding shows that the ability to establish a and a rhizobium, we isolated spontaneous mutants resistant to symbiosis with legumes is more widespread in bacteria than chloramphenicol, rifampicin and streptomycin, and showed by 16s anticipated to date. rDNA and nodA PCR-RFLP analyses that the individual mutants Recent taxonomic classifications portray rhizobia as belong- retained the characteristics of hi% Burkholderia and rhizobia. ing to three different branches of the or-subdass of Proteo- Nodules induced on M. atropurpureum by strain STM678 were bacteria: the Mesorhizobium-Sinorhizobium-Rhizobium branch, ineffective in terms of nitrogen kation, probably because the Bradyrhizobiunz branch and the Azorhizobium branch','. We M. atropurpureum is not the original symbiotic partner of strain recently described a fourth rhizobial branch within the a-subclass STM678. Supporting this conjecture, strain STM678 was indeed of Proteobacteria, containing methylotrophic rhizobial Methylo- found to contain the nipgene encoding dinitrogenase reductase, a bacterium'. To explore further the phylogenetic diversity ofnitro- key enzyme in nitrogen fixation (AJ302315). The highest identity gen-fixing legume symbionts, we characterized a collection of values with other nifi genes were 81.2% (the a-Proteobacterium rhizobia isolated from tropical legumes. We found here that two Azorhizobium caulinodans) and 81.1% (the ß-Proteobacterium bacteria isolated from nodules of Aspalathus and Machaerium Herbaspirillum seropedicae).

Y Rickettsia rickettsii Neisseria meningitidis Az&hus indigens Alcaligenes eutrophus w sr@& @$%%-%&e Rdstonia solanacearum7

BF !viuoxwiuno renua Azospiri//umbrasilense" Spiri//um vo/ufans

- 0.01 changes 6 E of Figure 1Unrooted 16s rDNA tree of Proteobacteria (purpie bacteria)..- -.- The figure shows using the neighbour-joiningmethod and adapted from ref. 5.16s rDNA sequences :he phy!ogenetic.relationshipsbetweenthediff~én€-ihizÖ~a~genera-a~represented by published bacteria are available in GenBank. 16s rDMfrom BU&ho/deria sp. STM 678 y+ species in bold-including the new rhizobial Burkholderhsp.strains (Y,ß, 6, y and and Burkhoderia sp. STM 815 are given in the text (A? 302311 and AJ 302312). F representthe different subdivisions of the Proteobacteria. The tree was constructed by - -. .- letters to nature

Nodulation of legumes by rhizobia is controlled by a set of la&-kanamycin-resistance cassette into the no& gene of strain bacterial nodulation (nod) genes involved in the production of STM678. The no& mutant did not form any nodules after lipo-chitooligosaccharides (Nod factors) that act as signalling mol- inoculation on M. atropurpureum, even after 30 days, indicating ecules for nodulating specificlegume hosts3j4.The nociABC genes are that the nod genes that we disrupted are required for nodulation of responsible for the synthesis of the core structure of.the Nod the Burkholderia sp. strain STM678. factor7+', and as such are present in all rhizobia. We thus looked By screening among bacteria isolated from root nodules collected for the presence of nodABC genes in the nodulating Burkholderia from various legumes in French Guiana, we found a second strain STM678 by PCR amplification (see Methods). Sequencingof Burkholderia rhizobium, strain STM815, isolated from the legume the amplified DNA revealed a genetic organisation of nodAB genes Machaerium li4natum. 16s rDNA sequencing (AJ302312) of this similar to that found in other rhizobia; that is, with nodAB in the strain revealed the following sequence identities with its closest same orientation and overlapping and preceded by a NodD-depen- phylogenetic neighbours: 96.9% (Burkholderia kururiensis), 96.8% dent regulatory sequence (nod box). A noK-like sequence was (B. brasilense), 96.6% (B. graminis) and 96.9% (strain STM678). found immediately downstream of nodB. However this sequence is These data clearly show that strain STM815 belongs to the unlikely to correspond to a functional nod% gene as it lacks the Burkholderia genus, and most probably to a different species to -600-base-pair 5' end of ho& nodC genes. We obtain evidence strain STM678. The nodulation ability of STM815 was confirmed, for the presence elsewhere in the STM678 genome of a longer nodC as described for strain STM678, by inoculation ofM.atropurpureum sequence that probably corresponds to the functional no& gene in axenic conditions and by re-isolation and characterizationof the (AJ306730). Such genetic unlinkage of nodABC genes in rhizobia is bacteria isolated from the induced nodules. not unprecedented9.The sequences of strain STM678 nodAB genes The ß-subdivision of Proteobacteria contains many bacteria that (AJ302321) revealed very high similarities with rhizobial Nod interact with eukaryotes, including human pathogens, such as protein sequences available in databases, with values ranging from Neisseria and Bordetella, and plant-associated bacteria. These 62.8% (Sinorhizobium meliloti) to 77.6% (Methylobacterium nodu- latter bacteria include pathogenic Raktonia solanacearum, rhizo- lans) for NodA and from 55.6% (Rhizobium galegae) to 70.9% spheric Burkholderia and endophytic Azoarcus. However the ß- (Mesorhizobium sp. N33) for NodB. To examinewhether these genes Proteobacteria had not been reported to include rhizobia, bacteria were functional, we constructed a nodA mutant by introducing a capable of nodulating leguminous plants. Here, we have identified two rhizobia belonging to the Burkholderia genus. These bacteria were isolated in different continents, from legumes belonging to different Papilionoideae tribes, and probably correspond to two distinct species. We have shown that the genetic control of nodulation by the Bmkholderia sp. strain STM678 involves nod genes. Moreover, this strain has been shown to produce Nod factors". Hence rhizobia from both the ci- and ß-proteobacteria (now termed CL- and ß-rhizobia) use the same strategy for establishing symbioses with legumes. Furthermore, we have performed phylogenetic analyses that iqdicate a much smaller phylogenetic distance between the nodAB genes of strain STM678 and other rhizobia (Fig. 3) than between the 16s rRNA genes of OL- and ß-ProteObacteria (Fig. 1). This suggests that the presence of nod genes in both OL- and ß-rhizobia probably occurred through horizontal gene transfer. This transfer may have occurred after the appearance oflegumes on Earth, about 70 million

A. caulinodans

S. meliloti R. tropici R. leguminosa" bv. viciae

Me. nodulans R. leguminosaium bv. trifolii \~." STM678 Burwlolderia sp. ' 94 78 100: 87 87 .I ..... R. gdegae 10.0 -.90 B. elkanji ' .99 . R. et/¡ Bradyrhizobium sp. NC92'. M. loti S. fedi Mesorhizobium sp. N33 M. - 0.05 changes huakuii

Figure 3 Unrooted NodA tree showing the close phylogenetic relationship between the NodA of strain STM678 and those of a-rhizobia.The tree is based on full-length sequences, and constructed by using geneighbour-joining method. Bootstrap values figure 2Nodules of MacropMumatropurpureum, three weeks after root inoculationwith (% from 1,000 replications) are indicated. NodA sequences of published rhizobia are Burwlolderiasp. strain STh4678. a, Root segments with nodules; b, longitudinalsection available in GenBank. NodA from 5ur~oMeriasp. STM 678 is given in the text zon? showing the typical structure of a determinate nodule with a central containing (AJ 302321). A, Azorhizobium, B., Bradyrhizobium.S, M, Mesarhizobium. infected cells oz), and a peripheral region with vascular bundles (vb). Me, Methylobacterium. ß, Rhizobium. Sinorhizobium.

949 letaers to nature

years ago”. Although rhizobia have been studied for more than 100 8. Perret, X, Staehelin, C. &Eroughton, W. I. Molecular basis ofsymbiotic promiscuity. MicrobioL MOL . E~OLRW. 64, 18o-zoi (moo). years, symbionts of less than 10% of the 750 legume genera have 9. Zhang, XX etal The commonnodulationgenes ofAsmgalussinim rhizobia are conserved despite been fully characterized. Our work suggests that characterization of chromosomal diversity. AppL Environ. MinobioL 66,2988-299s (2000). the symbionts of the yet unexplored legumes may reveal the 10. Boone, C., Olsthoom, M. M. A., Dakora, E D., Spaink, H. P. & Thomas-Oates,J. Structural rhizobial nature of additional members of the ß-ProteObacteria characterisation of lipo-chitinoligosaccharides isolated from Bradyrhizobium aspalati, microsym- bionts of commercially important South African legumes. Carbohydr..’Rer.317,155-163 (1999). and possibly other taxonomic classes. Such a study may contribute 11. Polhill,RM.,Raven,P.H.&Stirton,C.H.inA~~ancerinLrgumeSystem~ticcPart1 (edsPolhill,RM. significantly to the understanding of the origin, and the evolution &Raven, P. H.) 1-26 (Royal Botanic Gardens, Kew, 1981). of, the legume-rhizobia symbioses, and may open new perspectives 12. Thompson, J. D., Gibson, T. J., Plewniak, E, Jeanmougin,E & Higgins, D. G. The CLUSTALX for engineering beneficial associations. O windows interface:flexible strategies for multiple sequencealignment aided by quality analysis tools. Nudeic Acids Res. 25,4876-4882 (1997). 13. Swofford, D. L PAUP. PhylogeneticAnalysis Using Parsimony (and other Methods) Version 4 (Sinauer Associates, Sunderland, Massachusetts, 1998). J. Methods 14. Quandt, &Hynes,M.F.Versatilesuicidevectorswhich aUowdirectselectionforgenereplacementin Strains and culture conditions Gram-negative bacteria. Gene 127,15-21 (1993). 15. KokotekW.&Lotz,W. Constructionofalac-Z-kanamycin-resistancecassene,usefulfor site-directed STM678 was provided by H. P. Spainlc (UNv. Leiden). Cells were maintained and Strain mutagenesis and as a promoter probe. Gene 84,467-471 (1989). grown on yeast extract-mannitol medium2.

Acknowledgements DNA amplification, sequencing and analysis We thank H. P. Spaink for providing strain STM678, M. Neyra for providing the 23s rRNA Nearly full-length 16s rDNAwas amplified and sequenced previously described*. 16s as primers, Prin for help in microscopy studies and C. Hutte1 for sending plant material. rDNA PCR-RFLP analysis was performed as described’ except that Cfrl31, Hinfl and MspI Y. We also thank J. Cullimore and J. Batut for comments and suggestions. were used. A 870-bp paof dnaKwas amplified and sequenced using the primers 5’- GAMGTCAARCGBATCATCAA-3’ and 5’-TGTCnTTVGCBSMNACRTGCAG-3’.A 370- Correspondence and requests for materials should be addressed to C.B.-M. bp fragment of 23s rRNA gene was amplified and sequenced using the universal primers (e-mail: [email protected]).EMBL accession numbers: A7302311 to AJ302315, 5’-AGAGGCGATGAAGGACGT-3’and S’-ACCTITCCCTCACGGTACT-3’.A 1,509-bp AJ302321, AJ306730. fragment containing partial nifH and nipgenes were amplified using the primers 5‘- GCCWTCTWGGNAARGGNGG-3’and S’-ATCAGGCCGATCGGGC3’ and further sequenced. A 2.6-kb fragment containing the nodAB genes of strain STM678 was amplified using the primers S’-CAGATCNAGDCCBTTGAARCGCA-3’(located at the end of nodD in rhizobia) and 5’-CTNCGNGCCCARCGNAGTTG-3’(located within nodC in rhizobia). This fragment was further sequenced using pairs of degenerateprimers ...... defined from conserved motifs of no&, no&, nodC, and nod box. Two 1-kb overlapping fragments containing part of the nodC and nod1 genes of strain STM678 were amplified Spatial awareness is a function using the primer pairs 5’-TAYRTGGTYGAYGACGGWTC-3’/5’-CCATACGCACCGTGG TGCTCTTGC-3’ and 5’-GGTTATCGGACCGAGTACG/S‘-TCTl’CCAWAWRTGVGT temporal NGTCA-3’ (forward primer located at the beginning of nodC in rhizobia, reverse primer of the not the located within nodIin rhizobia) and further sequenced. nodA PCR-RFLP analysis was performed on a 455-bp PCR product obtained with the primer pair 5’-TCACARCTC posterior parietal lobe KGGCCCGTTCCG-3’/5‘-TGGGaGGNGCNAGRCCBGA-3’and digested with Cfil31, Hinfl and HaeIII. Multiple alignments were performed with CLUSTALX’’. Phylogenetic Hans-Otto Kamath, Susanne Ferber & Marc Himmelbach analyses used the neighbour-joining method and the programmes in PAUP version 4.0b5”. Department of CognitiveNeurolon, University of Tubingen,Hoppe-Sqler-Strasse 3, 72076 Tubingen, Germany Constructionof a nod4 mutant ...... A 2.5-kbXhoI-XhoI fragment containing the aodtLB genes of strain STM678 was obtained Our current understandihg of ’spatial behaviour AdParietal lobe by PCR amplification using modifiedprimers containing additional’Xho1sites and doned in the SalIsiteofpJQ200mp18 suicideve~tor’~.The4.7-kb SanlacZ-kanamycin-resistance function is largely based on the belief that spatial neglect in cassette ofpKOK51Swas inserted at the SaII site of the nodA gene cloned in pJQ200mp18. humans (a lack of awareness of space on the side of the body The pJQ200 derivatives obtained, which encoded a counterselectablesaCB marker, were contralateral to a brain injury) is typically associated with lesions transformed into Escherkhia coli XLII, and fuher introduced by conjugation into a of the posterior parietal lobe. However, in monkeys, this disorder spontaneous chloramphenicol-resistantderivative of strain STM678. Transconjugant ml-’ is observed after lesions of the superior temporal a colonies grown on YM medium containing 50 pg kanamycin and 100 wgml-’ cortex’, chloramphenicol were plated onto YM medium containing 7% sucrose and kanamycin. puzzling discrepancy between the species. Here we show that, Sucrose-resistant colonies were screened by PCR to ensure replacement of the wild-rype contrary to the widely accepted view, the superior temporal cortex nodA gene by the n0dA::lacZmutated gene. The Biirkholderia status of the mutated strain is the neural substrate of spatial neglect in humans, as it is in was assessed by 16s rDNA Pa-RFLP. monkeys. Unlike the monkey brain, spatial awareness in humans a function largely confined to the right superior temporal Plant tests is min. cortex, a location topographicallyreminiscent of that for language Seeds were surface-sterilizedwith concentmted sulphuric add for 5 Plant cultivation on the leP. Hence, the decisive phylogenetic transition from and nodulation tests were canied out as described’. Effectivenesswas estimated by visual monkey to human brain seems to be a restriction of a formerly observation of plant vigour and foliage colour of 30-day-old plants. Sections were made using a LeicaVTlOOoSVibratome, and examined after staining with 0.01% methyleneblue. bilateral function to the right side, rather than a shift from the temporal to the parietal lobe. One may speculate that this Received 22 Januaq;accepted 2 May 2001. lateralization of spatial awareness parallels the emergence of an 1. Young, J. P. W. & Haukka, K. E. Diversity and phylogeny of rhizobia. New PhytoL 133, 87-94 elaborate representation for language on the left side. (1996). Spatial neglect is a characteristic failure to explore the side of 2. A. et al. Methylotrophic Methylobacteriumnodulate and fix nitrogen in symbiosis with legumes. J.Sy, space contralateral to a brain lesion. Patients with this disorder Banm’oL 183,214-220 (2001). 3. Lemuge, P. et al. Symbiotic host-specificity ofRhizobium meliloti is determined by a sulphated and behave as if one side of the surrounding space had ceased to exist. acylated glucosamine oligosaccharide signal. Nahm 344,781-784 (1990). Since the early post-mortem studies, we have believed that, in 4. Spaink, H. P. et al. A novel highly unsaturated fatty acid moiety of lipo-oligosaccharidesignals humans, lesions located predominantly in the posterior parietal determines host specificity of Rhizobium. Nafure 354,125-130 (1991). 5. vanBerkum,P.&Eartly,B.D.inTheRhuobiacae(edsSpainlc,H.P.,Kondorosi,A.&Hooykaas,P.J.l.) lobe are critical for this disorder. Analyses of computerized tomog- 1-24 (Kluwer Academic, Dordrecht, 1998). raphy scans of right-hemispheric stroke patients with neglect found 6. Deschodt, C. C. & Snijdom, E. W. Effective nodulation ofAspalaBuc linearir spp. linaris by rhizobia that superimposed lateral projections of these lesions centred on the from other Malathus species. Phytophylucfiui 8, 103-104 (1976). J., J. inferior parietal lobule (IPL)334and the temporo-parieto-occipital 7. D’marié, DebeU4 E &Prom&, C. Rhizobium lipo-chitooligosaccharide nodulation factors: sjgnaling molecules mediating recognition and morphogenesis.Annu. Rev. Biochem. 65,503-535 (TPO)junction4. More recent studies have confirmed the validity of (1996). this conclusion although evidence for additional pathology leading

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I. Yamag~chi-Iwa~,E d aL Homologous recombhation, but not DNA repair, is reduced in vertebrate ...... *...... *. cells defident in -52. MOL CelL Bid. 18,6430-6435 (1998). 1. Liu, N. et d. XRCC? and XRCC3, new human Rad51-family members, promote chromosome . mtum stability and protedagakm DNAcross-linksand other damages. MOL Cell 1,783-793 (1998). IO. Johnson,R D., Liu, N. & Jasin,M. Mammalian XRU2 promotes the repair of DNA double-strand . breaks by homologousrecombination Nahire401,397-399 (1999). Il. Pierce,A. J.,,Johnson,RD.,Thompson,LH.&Jasin,M.XRCOpromoteshomologydirectedrepair of DNA damage in mammalian cells. Gmes Dew 13,2633-2638 (1999). Nodulation legumes by members of J. J. of 12. Brenneman,M. A., Weiss, A. E., Nickoloff, A. & Chen, D. XRCC3 is required for efficientrepair of chromosome breakr by homo!ogous recombination. Mufur Ra. 459,89-97 (2000). the ß-subclass of Pmteobacteria 13. Cui, X et al. The XRCC2 and XRCC3 repair genes we required for chromosome stahilityin . mammkan cells. Mufut Res. 434,75-88 (1999). C. J. Lionel Moulin, Antonio Munive, Bernard 14. Deans, B., Griffin, S., Mamnochie, M. & Thacker, Xrq2 is requkd for genetic stability, Owyfus . embryonic neurogenesis and viability in mice. EMBO I.19,6675-6685 (2000). & Catherine Bohrin-Masson J., J. 15. Grit%, C. S., Simpson, P. Wbon, C R &"hacker, Mammalian recombination-repair genes XRCCZ and,XRCC3 promote correct chromosome segreptioo. Nature Cell Biol. 2 757-761 Nature 948-950 (2001). (2000). 411, J. 16. Sale, E &Neuberge[, M. S. TdT-accessible breaks arescatterd over the immunogIobulinVdomdu In Figs 1and 3 the tree branches were very faint. .The corrected in a constitutivelyhypermutating B cell line. Immunity9,859-869 (1998). figures are shown below. CI 17. Rada, C, Ehrenstein, M. R, Neubergu, M. S. & Milstein, C Hot spot fomking of somatic two hypermutation in MS€E-ddcient mice suggests stages of mutational targeting. Immuniry 9, 135-141 (1998). Y 18. Diaz, M., Flajnik, M. F. & Klinman, N. Evolution and~emolecularbasiofsomatichypermutation of antigen receptor genes. Phi¿ Trans. R Soc Lond B 35667-72 (2041). 19. Muramatsu, M. et al. Class-switchricombination and hypermutation require activation-induced ' cytidine deaminase (AID), a potential RNA editing enyme. Cd 102 553-563 (2000). 10. Maizels, N. Somaticbypermutation: bow many mechanisms diversify V region sequences? Ce11 83, 9-12 (1995). J. C. i' 11. Weill, &Reynaud, C. A. Rearrangement/hypermn~tio~gmeconversion:when, where and why? Immunol Toduy 17,92-97 (1996). U.Xu, B. & Seking, E. Analysis of sequence transfers resembling gene conversion in a mouse antibody transgene. Science 265,1590-1593 (1994). 13. Papamsiliou, F. N. & Schatz, D. G. Cell-cyde-regulated DNA double-sixandedbreaksjn somatic bypermutation of immunoglobulin genes. Nufure 408,216-221 (2000). 14. Bros. L ef al. DNA double-strand breaks in immunoglobulin genes undergoing somatic hypermu- ' tation Immunity 13,589-597 (2000). 25. T&ta,M. etuLTheRad51 pmlogRad51Bpromotes homologousrecombinationalrepair.MoLcell Figure 1

BioL.ZO,6476-6482 (2000). . // 16. Thta, M. et al. Homologousrecombination and non-homologousend-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in A cauiinodans ' vertebrate cells. EMBOJ 17,5497-5508 (1998). J. 17. Bonfield, K, Smith, K & Staden, RAnew DNAsequence assembly pro& NudeicAcidr Res. U, 4992-4999 (1995). J. 18. Reynaud, CA,Anquer, V., GrimaI, H. &Weill, C. A byperconversionmeçhanism generate5 the S. meliloti ' I chicken light chain prehune repextoire. Cell 48,379-388 (1987). leguminosa" bv. viciae 19. McCnrmack, W. T., Hurley, E A. &Thompson, C B. Germ line maintenance of the pseudogene donor pool for somatic immunoglobulingene conversion in chidcens. MOL GIL BioL 13,821-830 R. leguminos" bv. trifolii (1993). @u&hoide6a sp. STMG $0. Reynaud, C. A., Dahan,h,Anqner,V. &Weill, I.C Somatichyperconversion divers%es thesinglevu gene of the chickenwith a high incidence in the D region. Cell 59,171-183 (1989). .

SradyMzobium sp. NC92 Ncknowledgements orhkobim sp. N33 We thank A. Jobnson for cell sorting and C. 'Milstein for helpful discussions.

&riespondence should be addressed to J.E.S. (e-mail: [email protected]) or M.S.N. (e-mail: [email protected]. Figure 3

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