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VH replacement is unlikely to contribute significantly to receptor editing due to an ineffectual embedded recombination signal sequence
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Molecular Immunology Molecular Immunology 24 "0887# 116Ð121
VH replacement is unlikely to contribute signi_cantly to receptor editing due to an ine}ectual embedded recombination signal sequence Bertrand Nadel\ Alan Tang\ Ann J[ Feeney The Scripps Research Institute\ Department of Immunology IMM!11\ La Jolla\ CA 81926\ U[S[A[ Received 3 February 0887^ received in revised form 12 March 0887^ accepted 13 March 0887
Abstract Receptor editing is a process consisting of replacement of pre!existing H or L chain rearrangements by secondary rearrangements[ This process could serve to remove autoreactive speci_cities\ or to rescue loci with non!functional rearrangements[ At the H chain locus\ functional replacement of a VHDJH rearrangement by an upstream VH requires the presence of an embedded RSS located in reverse orientation near the 2? end of the VH segment[ Although most VH genes contain a fairly consensus embedded heptamer\ the nonamer sequence bears little resemblance to the consensus RSS nonamer[ Therefore\ the physiologic rate of H chain editing by VH replacement is yet unknown[ In this study\ we used both conventional and sensitive competition recombination substrate assays to determine the recombination frequency of the VH70X embedded RSS relative to consensus and non!consensus RSS|s[ Results show no detectable recombination of the 70X embedded RSS in a recombination substrate\ and the competition substrate allows us to estimate that the 70X embedded RSS recombines at least 0299 fold less often than a consensus RSS[ This suggests that VH gene replacement is not responsible for the decrease in representation of the 70X gene during di}erentiation[ Furthermore\ since the sequence of the embedded RSS is very similar for many VH genes\ our results suggest that receptor editing of the H chain will be an infrequent event\ leaving L chain editing as the main mode of avoiding autoreactive speci_cities in vivo[ Þ 0887 Elsevier Science Ltd[ All rights reserved[ Keywords] Receptor editing^ Embedded heptamer^ VH70X^ V"D#J recombination^ Recombination Signal Sequences
0[ Introduction Receptor editing is the mechanism by which a pre! existing B cell receptor is replaced by secondary rearrangements "for review\ see Radic and Zouali\ 0885#[ Possible roles for receptor editing include "0# rescue of {dead!end| cells with a non!productive rearrangement at both alleles "Reth et al[\ 0875#\ and "1# providing a path! way to salvage autoreactive B!cells by elimination of self! reactive receptors "Gay et al[\ 0882^ Radic et al[\ 0882^ Tiegs et al[\ 0882#[ Receptor editing was initially described in the H chain locus "Klein_eld et al[\ 0875^ Reth et al[\ 0875#\ but ample data has been published since then documenting editing at the L chain loci "Chen et al[\ 0886^ Gay et al[\ 0882^ Han et al[\ 0886^ Luning Prak et al[\ 0883^ Luning Prak and Weigert\ 0884^ Melamed and Nemazee\ 0886^ Papavasiliou et al[\ 0886^ Tiegs et al[\ 0882#[ Editing of immunoglobulin light chain consists of Corresponding author[ Tel[] 990 508 673 1868^ fax] "508# 673!8089^ e!mail] feeneyÝscripps[edu Abbreviations] bp\ base pair^ RSS\ recombination signal sequence^ 70Xe\ VH70X embedded RSS[ S9050Ð4789:87 ,08[99 Þ 0887 Elsevier Science Ltd[ All rights reserved[ PII] S 9 0 5 0 Ð 4 7 8 9 " 8 7 # 9 9 9 1 8 Ð 6
replacement of an existing V!J exon by the secondary rearrangement of an upstream V segment to a down! stream J segment[ The L chain loci are suitably organized to easily permit such a mechanism[ For the IgH locus\ however\ receptor editing is not easily achieved[ After a VHDJH rearrangement\ all other D segments on that chromosome have been deleted and the 01Ð12 rule pre! cludes direct VH to JH recombination[ For this reason\ it has been proposed that receptor editing at the H locus would predominantly consist of recombination between the recombination signal sequence "RSS# ~anking an upstream VH segment and an embedded heptamer located near the 2? end of most VH segments\ resulting in VH replacement "Covey et al[\ 0889^ Klein_eld et al[\ 0875^ Reth et al[\ 0875#[ Prototypic RSS contain three sequence motifs] a very conserved heptamer\ a spacer of 01 or 12 bp and a con! served nonamer "see Fig[ 0#[ In the heptamer\ the [ [ [GTG motif "or CAC [ [ [\ depending upon the relative orien! tation of the RSS and coding end# adjacent to the coding end is the most conserved and the most crucial for e.cient recombination\ while changes at the four other positions have various e}ects on the frequency of recom!
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Fig[ 0[ Schematic representation of the murine heavy chain locus[ The location and sequence of the VH70X embedded RSS are shown[ Putative embedded heptamer and nonamer sequences are in bold[ Black triangles represent 12 bp RSS|s\ and white triangles represent 01 bp RSS|s[
bination"Akamatsu et al[\ 0883] Akira et al[\ 0876^ Hesse et al[\ 0876#[ In the nonamer\ the presence of at least 2 consecutive T|s is important for e.cient rearrangement "Akamatsu et al[\ 0883^ Hesse et al[\ 0878#[ The embedded heptamer is a 6 bp motif located 4Ð00 bp from the coding end of VH gene\ corresponding to the position of the invariant cysteine and highly conserved alanine in FR2 "Fig[ 0#[ Therefore\ in most VH segments\ the embedded heptamer contains 4Ð6 bp of the consensus heptamer positions\ including the critical GTG sequence[ Since VH genes are ~anked by RSS|s with 12 bp spacers\ one would predict that the nonamer should be separated from this heptamer by 01 bp to allow secondary rearrangements[ Although no appropriately spaced consensus nonamer was found\ Chen et al[ reported a fairly conserved 7Ð8 bp motif located 01 bp upstream of the embedded heptamer "Chen et al[\ 0884#[ They proposed that this novel non! amer!life motif\ in combination with the embedded hep! tamer\ could constitute a di}erent type of RSS allowing low rates of recombination resulting in VH replacement[ However\ this nonamer!like motif bears little resem! blance to the consensus RSS nonamer "Fig[ 0#\ and there! fore the recombination e.ciencies of such embedded RSS|s are unknown[ Also\ we have recently shown that the sequence of the spacer of the RSS can a}ect recom! bination frequency "Nadel et al[\ 0887#\ and thus the 70X embedded RSS {spacer| sequence could possibly com! pensate to some extent for the poor nonamer[ The fre! quencies of recombination of RSS|s lacking any conserved position in the nonamer motif have been shown to range from ³ 9[0Ð4) of that of a consensus RSS\ suggesting that the frequency of recombination of a cryptic heptamer is very dependent on the sequence present in lieu of the nonamer or upon the spacer sequence "Akamatsu et al[\ 0883^ Hesse et al[\ 0878^ Lewis et al[\ 0886#[ Therefore\ in this study we tried to estimate the fre! quency of recombination of the actual embedded RSS present in VH genes\ using a sensitive competition recom! bination substrate assay[ We chose to analyze the fre!
quency of rearrangement of the VH70X embedded RSS for several reasons[ First\ the VH70X gene segment is the most D!proximal VH segment in the mouse[ VH70X!D!J rearrangements are therefore the most likely joints to be replaced by the receptor editing process in vivo[ In addition\ VH70X has been observed to rearrange at high frequencies in bone marrow pre!B cells\ but becomes under represented in bone marrow B cells as well as in the peripheral repertoire "Lawler et al[\ 0876^ Marshall et al[\ 0885^ Yancopoulos et al[\ 0873#[ The reduction of VH70X usage in the periphery is likely to be due at least in part to its inability to bind to surrogate light chain e}ectively "Keyna et al[\ 0884^ ten Boekel et al[\ 0886#\ and this compromised binding could favor rescue of these VH70X!D!JH rearrangements by receptor editing\ as pre! viously suggested by Radic and Zouali "Radic and Zouali\ 0885#[ Furthermore\ the initial decline in the fre! quency of VH70X in the bone marrow also occurs in mutant mice which lack the membrane exon of Cm "Mar! shall et al[\ 0885#[ Replacement of VH70X!D!JH rearrangements by editing is therefore a candidate mech! anism to explain this reduction of 70X rearrangements\ as proposed by Marshall et al[\ "Marshall et al[\ 0885#[ Finally\ the 70X embedded heptamer contains 5 out of 6 consensus positions in the heptamer\ as do most VH6072 genes\ and thus is likely to contain any recombinogenic potential which may be present in this novel RSS[ Impor! tantly\ most of the VH6072 genes contain the identical {nonamer|!spacer!heptamer sequence of the VH70X embedded RSS "Carlsson et al[\ 0881^ Chukwuocha et al[\ 0883#[ Thus\ the data presented here are generalizable for the entire VH6072 family[ The 70X embedded RSS "70Xe# was therefore inserted in competition and conventional recombination sub! strates and assayed for recombination[ Results show no recombination of the 70X embedded RSS[ By comparing relative frequencies of various RSS|s in competition sub! strates\ we estimate that the 70X embedded RSS rearranges at a minimum 0299 times less frequently than a prototypic consensus RSS[ Hence\ receptor editing at
B[ Nadel et al[:Molecular Immunology 24 "0887# 116Ð121
the IgH locus is unlikely to play a signi_cant role in repertoire development or elimination of autoreactive receptors[
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by removing the SalI:NotI cassette containing VkA1b and its RSS\ blunting and religating the plasmid[ The RSS and surrounding regions of all constructs were con! _rmed by sequencing[
1[ Materials and Methods 1[1[ Recombination assay 1[0[ Plasmid constructs The Comp12 competition substrate is shown in Fig[ 1A\ and it was derived from the {Comp| plasmid series "Nadel and Feeney\ 0886^ Nadel et al[\ 0887\ and B[N[\ A[T[\ G[L[\ V[Love\ G[E[\ A[J[F[\ manuscript in prep! aration#[ The full VkA1b and Jk0 coding ends ~anked by their RSS|s were obtained by PCR "½199 bp segments#\ with primers containing the appropriate restriction sites to enable insertion into the Comp01 backbone[ 70Xe RSS was inserted as a double stranded oligonucleotide cassette ~anked by NotI and MluI restriction sites "Fig[ 1A#[ The 70Xe recombination substrate was derived from Comp12
07[7 A!MuLV transformed pre!B cells "19x095 are tran! siently transfected with 19 mg of Qiagen!puri_ed plasmid by electroporation "859 mF\ 9[2Kv# as previously described "Nadel and Feeney\ 0886# and resuspended in 09 ml of RPMI 0539 supplemented with 09) FCS\ 1 mM glutamine\ 4×09−4 M 1!mercaptoethanol\ and 0 mM ca}eine[ After 37 h\ plasmids are recovered from trans! fected cells by alkaline lysis\ followed by DpnI:SpeI diges! tion[ Digested plasmids are then electroporated into E[ coli and plated on plates containing ampicillin "099 mg:ml# and chloramphenicol "4 mg:ml#[ PCR are per! formed from the colonies by resuspending bacterial cells
Fig[ 1[ "A# Comp12 competition substrate map[ Sequences of part of the Jk0 coding end ~anked by its RSS\ 70X embedded RSS "70Xe# and the A1b coding end ~anked by its RSS are shown[ Coding end sequences are in bold[ Jk0 is separated from the competing 70Xe and A1b RSS by a transcriptional terminator\ called {stop| in the _gure[ B\ BamHI^ M\ MluI^ N\ NotI^ Sc\ SacII\ S\ SalI^ Sp\ SpeI[ "B# PCR screen used to analyze rearrangement status[ Locations of the PCR primers "AF63\ P3# and size of the PCR products are shown[ The total number of recombined colonies obtained is indicated[
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containing plasmid directly in the PCR mixture\ and amplifying with P3 and AF63 primers "Fig[ 1B# for 29 cycles at the following conditions] 0 min at 83>C\ 0 min at 44>C\ and 1 min at 61>C[ Random clones were sim! ultaneously miniprepped and sequenced[
Table 0 Comparison of VH70Xe and VkA07 frequencies of rearrangement in recombination substrates
VH70Xe VkA07
è Recombinant colonies
è Transfections
9 209
3 1
2[ Results 2[0[ No recombination of the 70X embedded RSS when in competition with the weak VkA1b RSS To test the frequency of recombination of the 70X embedded RSS\ we used a {competition| plasmid in which two segments are in competition for rearrangement with a unique joining partner "Fig[ 1A#[ This strategy allowed us to compare directly the frequency of rearrangement of one RSS relative to another one[ We have shown in other studies that the distal or proximal position of the various gene segments in the plasmid has no e}ect on the relative frequencies of recombination "B[N[\ A[T[\ G[ Lugo\ V[ Love\ G[ Escuro and A[J[F[\ manuscript in preparation\ and Nadel et al[\ 0887#[ Since we anticipated that the 70X embedded RSS would not be an e.cient RSS due to the many changes from the consensus RSS\ we placed it in competition with a RSS that rearranges at a very low frequency[ The human VkA1b gene is a defective allele of the VkA1a segment "Feeney et al[\ 0885#[ Compared to the consensus RSS\ the VkA1b RSS contains a change in the sixth nucleotide of the heptamer and another change in the 3th position of the nonamer "Fig[ 1A#[ We found a 02!fold decrease in the frequency of rearrange! ment of VkA1b compared to the consensus RSS in similar competition substrate experiments "B[N[\ A[T[\ G[L[\ V[ Love\ G[E[ and A[J[F[\ manuscript in preparation#[ We therefore modi_ed a competition recombination sub! strate containing VkA1b to create Comp12\ a plasmid containing on one side the Jk0 coding end ~anked by its RSS\ and on the other side both the 70X embedded RSS\ and the VkA1b coding end ~anked by its RSS "Fig[ 1A#[ These two segments are therefore in competition for rearrangement to the Jk0 segment[ We performed four independent transfections of 07[7 pre!B cells with Comp12 and screened colonies by PCR[ Out of 88 recom! bined colonies\ none gave rise to the 149 bp band cor! responding to the Jk0!70Xe and 88 gave rise to the 279 bp band expected for Jk0!VkA1b rearrangement "Fig[ 1B#[ In addition\ a few colonies gave rise to a 0089 bp PCR band corresponding to the background unre! combined plasmids[ These results suggest that rearrangement of the 70X embedded RSS is at least 099 times less e.cient than the VkA1b RSS[ Since we have shown using similar com! petition substrates that the VkA1B RSS\ with its non! consensus heptamer and nonamer\ is about 02!fold less e.cient than the corresponding consensus RSS "B[N[\
A[T[\ G[L[\ V[ Love\ G[E[ and A[J[F[\ manuscript in preparation#\ we can estimate at least a 0299 fold decrease in the frequency of rearrangement of the 70X embedded RSS compared to a consensus RSS[ We have\ however\ recently observed that the spacer sequence also plays a role in the e.ciency of the recombination process "Nadel et al[\ 0887#[ Since the A1 spacer is one of the best spacers that we tested so far\ this estimate may be slightly high[ 2[1[ No recombination is observed for the 70X embedded RSS To test the ability of the 70X embedded RSS to recom! bine in absence of other competing RSSs\ we generated the Jk0!70Xe plasmid\ a classical recombination sub! strate containing the germline sequence of the Jk0 coding end ~anked by its RSS on one side of the termination signal\ and on the other side\ the germline sequence of the 70X embedded RSS without any other segment in competition[ 07[7 cells were transfected in four inde! pendent transfections with the Jk0!70Xe plasmid in pres! ence of 1 mM ca}eine to increase to a maximum the level of recombination activity of the cell line "Menetski and Gellert\ 0889#[ Only rare colonies were obtained from ampicillin:chloramphenicol plates\ and those were mini! prepped and sequenced[ None of the clones were identi! _ed as Jk0!70Xe recombination\ all clones consisting of background unrearranged plasmids "Table 0#[ To rule out the possibility that the absence of rearrangement could be due to a putative defect in the transfection or harvesting processes\ a positive control was included in the same experiments[ 07[7 cells were transfected with a similar plasmid "Jk0!VkA07# containing Jk0 and the VkA07 coding end ~anked by its RSS\ instead of the 70X embedded RSS[ Although VkA07RSS has a nucleotide change in the nonamer resulting in a 1[6!fold reduction of the recombination frequency compared to consensus "B[N[\ A[T[\ G[ Lugo\ V[ Love\ G[ Escuro and A[J[F[\ manuscript in preparation#\ 209 colonies were obtained on ampicillin:chloramphenicol plates from two trans! fections\ and all sequenced clones showed proper rearrangement of the Jk0!VkA07 plasmid[ This result indicates that the 70X embedded RSS is the element responsible for the lack of rearrangement of the Jk0!70Xe plasmid[ These results con_rm that the 70X embedded
B[ Nadel et al[:Molecular Immunology 24 "0887# 116Ð121
RSS is a very ine.cient RSS and demonstrate that it will promote rearrangement at extremely low frequencies[
3[ Discussion Two main questions have emerged from the initial VH replacement studies] "0# from a mechanistic point of view\ can the embedded RSS promote speci_c V"D#J recom! bination< and "1# from a functional point of view\ does receptor editing of the heavy chain locus play a signi_cant role in vivo< Use of the embedded RSS for editing VHDJH rearrangements was initially shown in transformed B and pre!B cell lines "Brokaw et al[\ 0881^ Covey et al[\ 0889^ Deane and Norton\ 0889^ Klein_eld et al[\ 0875^ Klein! _eld and Weigert\ 0878^ Reth et al[\ 0875#[ Subsequently\ two transgenic mice carrying a site!directed transgene in which the unrearranged DQ41!JH region has been replaced by a complete VHDJH rearrangement have been generated "Chen et al[\ 0884^ Taki et al[\ 0884#[ In both reports\ replacement of the transgenic VHDJH joint occurred\ but in one mouse two di}erent heptamer!like sequences were used instead of the embedded heptamer\ and in the other mouse\ the use of the embedded hep! tamer was observed in cells pre!selected for the expression of surface Ig[ Similarly\ the more frequent use of alternate GTG sites in lieu of the embedded heptamer was observed in in vitro experiments "Usuda et al[\ 0881#[ Thus\ although it is clear that secondary rearrangement using the embedded heptamer does occur in vivo\ it is less obvious if replacement occurs at that site at a higher frequency than at any other random GTG sites "Lewis et al[\ 0886#[ Recent studies have shown the critical role of the non! amer in the recognition and binding of RAG!0 on the synapse "Di_lippantonio et al[\ 0885^ Spanopoulou et al[\ 0885^ Nagawa et al[\ 0887#[ The absence of an appropriate nonamer associated with the embedded heptamer is therefore probably very detrimental to the frequency of recombination[ In the nonamer\ the presence of at least 2 consecutive T|s is important for e.cient rearrangement "Akamatsu et al[\ 0883#\ and essentially none of the non! amer!like elements found in association with embedded heptamers contain this motif "Chen et al[\ 0884#[ It\ in physiological situations\ receptor editing of the IgH chain would play any signi_cant role in generation of diversity and:or elimination of self!reactive speci_cities\ the embedded RSS must function at some reasonable frequency[ However\ we estimate in this study that the 70X embedded RSS recombines at least 0299!fold lower than a consensus RSS[ This suggests that VH gene replace! ment is not responsible for the great decrease in rep! resentation of the 70X gene during di}erentiation[ Moreover\ since the 70X embedded heptamer contains essentially all of the consensus heptamer sequence\ and both embedded heptamer\ spacer and {nonamer| motifs
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are well conserved among all members of the 6072 family\ our results suggest that receptor editing of the heavy chain is unlikely to participate to any signi_cant extent in the establishment of the peripheral B cell repertoire[ Thus\ rescue of pro!B cells with two non!productive H chain rearrangements by VH gene replacement is likely to be a very rare event\ and L chain editing\ rather than H chain editing\ would be the predominant mechanism to avoid autoreactive speci_cities in vivo[ Acknowledgements This work was supported by National Institute of Health grant AI18561[ B[N[ was supported by an Amer! ican Cancer Society!California Division fellowship 0!22! 85B[ This is manuscript 00244!IMM from The Scripps Research Institute[
References Akamatsu\ Y[\ Tsurushita\ N[\ Nagawa\ F[\ Matsuoka\ M[\ Okazaki\ K[\ Imai\ M[\ Sakano\ H[\ 0883[ Essential residues in V"D#J recom! bination signals[ J[ Immunol[ 042\ 3419Ð3418[ Akira\ S[\ Okazaki\ K[\ Sakano\ H[\ 0876[ Two pairs of recombination signals are su.cient to cause immunoglobulin V!"D#!J joining[ Sci! ence 127\ 0023Ð0027[ Brokaw\ J[L[\ Wetzel\ S[M[\ Pollok\ B[A[\ 0881[ Conserved patterns of somatic mutation and secondary VH gene rearrangement create aberrant Ig!encoding genes in Epstein!Barr virus!transformed and normal human B lymphocytes[ Int[ Immunol[ 3\ 086Ð195[ Carlsson\ L[\ O Ý vermo\ C[\ Holmberg\ D[\ 0881[ Developmentally con! trolled selection of antibody genes] Characterization of individual VH6072 genes and evidence for stage!speci_c somatic diversi_cation[ Eur[ J[ Immunol[ 11\ 60Ð67[ Chen\ C[\ Luning Prak\ E[\ Weigert\ M[\ 0886[ Editing disease!associ! ated autoantibodies[ Immunity 5\ 86Ð094[ Chen\ C[\ Nagy\ Z[\ Luning Prak\ E[\ Weigert\ M[\ 0884[ Immu! noglobulin heavy chain gene replacement] A mechanism of receptor editing[ Immunity 2\ 636Ð644[ Chukwuocha\ R[U[\ Hartman\ A[B[\ Feeney\ A[J[\ 0883[ Sequences of four new members of the VH6072 gene family in BALB:c mice[ Immunogenetics 39\ 65Ð67[ Covey\ L[R[\ Ferrier\ P[\ Alt\ F[W[\ 0889[ VH to VHDJH rearrangement is mediated by the internal VH heptamer[ Int[ Immunol[ 1\ 479Ð471[ Deane\ M[\ Norton\ J[D[\ 0889[ Immunoglobulin heavy chain gene rearrangement involving V!V region recombination[ Nucl[ Acids Res[ 07\ 0541Ð0542[ Di_lippantonio\ M[J[\ McMahan\ C[J[\ Eastman\ Q[M[\ Spanopoulou\ E[\ Schatz\ D[G[\ 0885[ RAG0 mediates signal sequence recognition and recruitment of RAG1 in V"D#J recombination[ Cell 76\ 142Ð 151[ Feeney\ A[J[\ Atkinson\ M[J[\ Cowan\ M[J[\ Escuro\ G[\ Lugo\ G[\ 0885[ A defective VkA1 allele in Navajos which may play a role in increased susceptibility to Haemophilus in~uenzae type b disease[ J[ Clin[ Invest[ 86\ 1166Ð1171[ Gay\ D[\ Saunders\ T[\ Camper\ S[\ Weigert\ M[\ 0882[ Receptor editing] An approach by autoreactive B cells to escape tolerance[ J[ Exp[ Med[ 066\ 888Ð0997[ Han\ S[\ Dillon\ S[R[\ Zheng\ B[\ Shimoda\ M[\ Schlissel\ M[S[\ Kelsoe\ G[\ 0886[ V"D#J recombinase activity in a subset center B lympho! cytes[ Science 167\ 290Ð294[
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Hesse\ J[E[\ Lieber\ M[R[\ Gellert\ M[\ Mizuuchi\ K[\ 0876[ Extra! chromosomal DNA substrates in pre!B cells undergo inversion or deletion at immunoglobulin V!"D#!J joining signals[ Cell 38\ 664Ð 672[ Hesse\ J[E[\ Lieber\ M[R[\ Mizuuchi\ K[\ Gellert\ M[\ 0878[ V"D#J recombination] A functional de_nition of the joining signals[ Genes Dev[ 2\ 0942Ð0950[ Keyna\ U[\ Beck!Engeser\ G[B[\ Jongstra\ J[\ Applequist\ S[E[\ Jack\ H[!M[\ 0884[ Surrogate light chain!dependent selection of Ig heavy chain V regions[ J[ Immunol[ 044\ 4425Ð4431[ Klein_eld\ R[\ Hardy\ R[R[\ Tarlinton\ D[\ Dangl\ J[\ Herzenberg\ L[A[\ Weigert\ M[\ 0875[ Recombination between an expressed immu! noglobulin heavy!chain gene and a germline variable gene segment in a Ly 0¦ B!cell lymphoma[ Nature 211\ 732Ð735[ Klein_eld\ R[W[\ Weigert\ M[G[\ 0878[ Interspersion of the VhQ41 and Vh6072 gene families in the NFS:N mouse[ J[ Immunol[ 031\ 3372Ð 3381[ Lawler\ A[M[\ Lin\ P[S[\ Gearhart\ P[J[\ 0876[ Adult B!cell repertoire is biased toward two heavy!chain variable!region genes that rearrange frequently in fetal pre!B cells[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 73\ 1343Ð1347[ Lewis\ S[M[\ Agard\ E[\ Suh\ S[\ Czyzyk\ L[\ 0886[ Cryptic signals and the _delity of V"D#J joining[ Mol[ Cell[ Biol[ 06\ 2014Ð2025[ Luning Prak\ E[\ Trounstine\ M[\ Huszar\ D[\ Weigert\ M[\ 0883[ Light chain editing in k!de_cient animals] A potential mechanism of B cell tolerance[ J[ Exp[ Med[ 079\ 0794Ð0704[ Luning\ Prak E[\ Weigert\ M[\ 0884[ Light chain replacement] a new model for antibody gene rearrangement[ J[ Exp[ Med[ 071\ 430Ð 437[ Marshall\ A[J[\ Wu\ G[E[\ Paige\ C[J[\ 0885[ Frequency of VH70X usage during B cell development[ J[ Immunol[ 045\ 1966Ð1973[ Melamed\ D[\ Nemazee\ D[\ 0886[ Self!antigen does not accelerate immature B cell apoptosis\ but stimulates receptor editing as a consequence of developmental arrest[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 83\ 8156Ð8161[ Menetski\ J[P[\ Gellert\ M[\ 0889[ V"D#J recombination activity in lymphoid cell lines is increased by agents that elevate cAMP[ Proc[ Natl[ Acad[ U[S[A[ 76\ 8213Ð8217[ Nadel\ B[\ Feeney\ A[J[\ 0886[ Nucleotide deletion and P addition in V"D#J recombination] a determinant role of the coding!end sequence[ Mol[ Cell[ Biol[ 06\ 2657Ð2667[ Nadel\ B[\ Tang\ A[\ Escuro\ G[\ Lugo\ G[\ Feeney\ A[J[\ 0887[ Sequence
of the spacer in the RSS a}ects V"D#J rearrangement frequency and correlates with non!random Vk usage in vivo[ J[ Exp[ Med[ in press[ Nagawa\ F[\ Ishiguro\ K[!I[\ Tsuboi\ A[\ Yoshida\ T[\ Ishikawa\ A[\ Takemori\ T[\ Otsuka\ A[J[\ Sakano\ H[\ 0887[ Footprint analysis of the RAG protein recombination signal sequence complex for V"D#J recombination[ Mol[ Cell[ Biol[ 07\ 544Ð552[ Papavasiliou\ F[\ Casellas\ R[\ Suh\ H[\ Qin\ X[!F[\ Besmer\ E[\ Pelanda\ R[\ Nemazee\ D[\ Rajewsky\ K[\ Nussenzweig\ M[C[\ 0886[ V"D#J recombination in mature B cells] A mechanism for altering antibody responses[ Science 167\ 187Ð290[ Radic\ M[Z[\ Erikson\ J[\ Litwin\ S[\ Weigert\ M[\ 0882[ B lymphocytes may escape tolerance by revising their antigen receptors[ J[ Exp[ Med[ 066\ 0054Ð0062[ Radic\ M[Z[\ Zouali\ M[\ 0885[ Receptor editing\ immune diver! si_cation\ and self!tolerance[ Immunity 4\ 494Ð400[ Reth\ M[\ Gehrmann\ P[\ Petrac\ E[\ Wiese\ P[\ 0875a[ A novel VH to VHDJH joining mechanism in heavy!chain!negative "null# pre!B cells results in heavy!chain production[ Nature 211\ 739Ð731[ Reth\ M[G[\ Jackson\ S[\ Alt\ F[W[\ 0875b[ VHDJH formation and DJH replacement during pore!B di}erentiation] Non!random usage of gene segments[ EMBO J[ 4\ 1020Ð1027[ Spanopoulou\ E[\ Zaitseva\ F[\ Wang\ F[!H[\ Santagata\ S[\ Baltimore\ D[\ Panayotou\ G[\ 0885[ The homeodomain region of Rag!0 reveals the parallel mechanisms of bacterial and V"D#J recombination[ Cell 76\ 152Ð165[ Taki\ S[\ Schwenk\ F[\ Rajewsky\ K[\ 0884[ Rearrangement of upstream DH and VH genes to a rearranged immunoglobulin variable region gene inserted into the DQ41!JH region of the immunoglobulin heavy chain locus[ Eur[ J[ Immunol[ 14\ 0777Ð0785[ ten Boekel\ E[\ Melchers\ F[\ Rolink\ A[G[\ 0886[ Changes in the VH gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre!B cell receptor[ Immunity 6\ 246Ð 257[ Tiegs\ S[L[\ Russell\ D[M[\ Nemazee\ D[\ 0882[ Receptor editing in self! reactive bone marrow B cells[ J[ Exp[ Med[ 066\ 0998Ð0919[ Usuda\ S[\ Takemori\ T[\ Matsuoka\ M[\ Shirasawa\ T[\ Yoshida\ K[\ Mori\ A[\ Ishizaka\ K[\ Sakano\ H[\ 0881[ Immunoglobulin V gene replacement is caused by the intramolecular DNA deletion mech! anism[ EMBO J[ 00\ 500Ð507[ Yancopoulos\ G[D[\ Desiderio\ S[V[\ Paskind\ M[\ Kearney\ J[F[\ Bal! timore\ D[\ Alt\ F[W[\ 0873[ Preferential utilization of the most JH! proximal VH gene segments in pre!B!cell lines[ Nature 200\ 616Ð622[
Molecular Immunology Molecular Immunology 24 "0887# 116Ð121
VH replacement is unlikely to contribute signi_cantly to receptor editing due to an ine}ectual embedded recombination signal sequence Bertrand Nadel\ Alan Tang\ Ann J[ Feeney The Scripps Research Institute\ Department of Immunology IMM!11\ La Jolla\ CA 81926\ U[S[A[ Received 3 February 0887^ received in revised form 12 March 0887^ accepted 13 March 0887
Abstract Receptor editing is a process consisting of replacement of pre!existing H or L chain rearrangements by secondary rearrangements[ This process could serve to remove autoreactive speci_cities\ or to rescue loci with non!functional rearrangements[ At the H chain locus\ functional replacement of a VHDJH rearrangement by an upstream VH requires the presence of an embedded RSS located in reverse orientation near the 2? end of the VH segment[ Although most VH genes contain a fairly consensus embedded heptamer\ the nonamer sequence bears little resemblance to the consensus RSS nonamer[ Therefore\ the physiologic rate of H chain editing by VH replacement is yet unknown[ In this study\ we used both conventional and sensitive competition recombination substrate assays to determine the recombination frequency of the VH70X embedded RSS relative to consensus and non!consensus RSS|s[ Results show no detectable recombination of the 70X embedded RSS in a recombination substrate\ and the competition substrate allows us to estimate that the 70X embedded RSS recombines at least 0299 fold less often than a consensus RSS[ This suggests that VH gene replacement is not responsible for the decrease in representation of the 70X gene during di}erentiation[ Furthermore\ since the sequence of the embedded RSS is very similar for many VH genes\ our results suggest that receptor editing of the H chain will be an infrequent event\ leaving L chain editing as the main mode of avoiding autoreactive speci_cities in vivo[ Þ 0887 Elsevier Science Ltd[ All rights reserved[ Keywords] Receptor editing^ Embedded heptamer^ VH70X^ V"D#J recombination^ Recombination Signal Sequences
0[ Introduction Receptor editing is the mechanism by which a pre! existing B cell receptor is replaced by secondary rearrangements "for review\ see Radic and Zouali\ 0885#[ Possible roles for receptor editing include "0# rescue of {dead!end| cells with a non!productive rearrangement at both alleles "Reth et al[\ 0875#\ and "1# providing a path! way to salvage autoreactive B!cells by elimination of self! reactive receptors "Gay et al[\ 0882^ Radic et al[\ 0882^ Tiegs et al[\ 0882#[ Receptor editing was initially described in the H chain locus "Klein_eld et al[\ 0875^ Reth et al[\ 0875#\ but ample data has been published since then documenting editing at the L chain loci "Chen et al[\ 0886^ Gay et al[\ 0882^ Han et al[\ 0886^ Luning Prak et al[\ 0883^ Luning Prak and Weigert\ 0884^ Melamed and Nemazee\ 0886^ Papavasiliou et al[\ 0886^ Tiegs et al[\ 0882#[ Editing of immunoglobulin light chain consists of Corresponding author[ Tel[] 990 508 673 1868^ fax] "508# 673!8089^ e!mail] feeneyÝscripps[edu Abbreviations] bp\ base pair^ RSS\ recombination signal sequence^ 70Xe\ VH70X embedded RSS[ S9050Ð4789:87 ,08[99 Þ 0887 Elsevier Science Ltd[ All rights reserved[ PII] S 9 0 5 0 Ð 4 7 8 9 " 8 7 # 9 9 9 1 8 Ð 6
replacement of an existing V!J exon by the secondary rearrangement of an upstream V segment to a down! stream J segment[ The L chain loci are suitably organized to easily permit such a mechanism[ For the IgH locus\ however\ receptor editing is not easily achieved[ After a VHDJH rearrangement\ all other D segments on that chromosome have been deleted and the 01Ð12 rule pre! cludes direct VH to JH recombination[ For this reason\ it has been proposed that receptor editing at the H locus would predominantly consist of recombination between the recombination signal sequence "RSS# ~anking an upstream VH segment and an embedded heptamer located near the 2? end of most VH segments\ resulting in VH replacement "Covey et al[\ 0889^ Klein_eld et al[\ 0875^ Reth et al[\ 0875#[ Prototypic RSS contain three sequence motifs] a very conserved heptamer\ a spacer of 01 or 12 bp and a con! served nonamer "see Fig[ 0#[ In the heptamer\ the [ [ [GTG motif "or CAC [ [ [\ depending upon the relative orien! tation of the RSS and coding end# adjacent to the coding end is the most conserved and the most crucial for e.cient recombination\ while changes at the four other positions have various e}ects on the frequency of recom!
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Fig[ 0[ Schematic representation of the murine heavy chain locus[ The location and sequence of the VH70X embedded RSS are shown[ Putative embedded heptamer and nonamer sequences are in bold[ Black triangles represent 12 bp RSS|s\ and white triangles represent 01 bp RSS|s[
bination"Akamatsu et al[\ 0883] Akira et al[\ 0876^ Hesse et al[\ 0876#[ In the nonamer\ the presence of at least 2 consecutive T|s is important for e.cient rearrangement "Akamatsu et al[\ 0883^ Hesse et al[\ 0878#[ The embedded heptamer is a 6 bp motif located 4Ð00 bp from the coding end of VH gene\ corresponding to the position of the invariant cysteine and highly conserved alanine in FR2 "Fig[ 0#[ Therefore\ in most VH segments\ the embedded heptamer contains 4Ð6 bp of the consensus heptamer positions\ including the critical GTG sequence[ Since VH genes are ~anked by RSS|s with 12 bp spacers\ one would predict that the nonamer should be separated from this heptamer by 01 bp to allow secondary rearrangements[ Although no appropriately spaced consensus nonamer was found\ Chen et al[ reported a fairly conserved 7Ð8 bp motif located 01 bp upstream of the embedded heptamer "Chen et al[\ 0884#[ They proposed that this novel non! amer!life motif\ in combination with the embedded hep! tamer\ could constitute a di}erent type of RSS allowing low rates of recombination resulting in VH replacement[ However\ this nonamer!like motif bears little resem! blance to the consensus RSS nonamer "Fig[ 0#\ and there! fore the recombination e.ciencies of such embedded RSS|s are unknown[ Also\ we have recently shown that the sequence of the spacer of the RSS can a}ect recom! bination frequency "Nadel et al[\ 0887#\ and thus the 70X embedded RSS {spacer| sequence could possibly com! pensate to some extent for the poor nonamer[ The fre! quencies of recombination of RSS|s lacking any conserved position in the nonamer motif have been shown to range from ³ 9[0Ð4) of that of a consensus RSS\ suggesting that the frequency of recombination of a cryptic heptamer is very dependent on the sequence present in lieu of the nonamer or upon the spacer sequence "Akamatsu et al[\ 0883^ Hesse et al[\ 0878^ Lewis et al[\ 0886#[ Therefore\ in this study we tried to estimate the fre! quency of recombination of the actual embedded RSS present in VH genes\ using a sensitive competition recom! bination substrate assay[ We chose to analyze the fre!
quency of rearrangement of the VH70X embedded RSS for several reasons[ First\ the VH70X gene segment is the most D!proximal VH segment in the mouse[ VH70X!D!J rearrangements are therefore the most likely joints to be replaced by the receptor editing process in vivo[ In addition\ VH70X has been observed to rearrange at high frequencies in bone marrow pre!B cells\ but becomes under represented in bone marrow B cells as well as in the peripheral repertoire "Lawler et al[\ 0876^ Marshall et al[\ 0885^ Yancopoulos et al[\ 0873#[ The reduction of VH70X usage in the periphery is likely to be due at least in part to its inability to bind to surrogate light chain e}ectively "Keyna et al[\ 0884^ ten Boekel et al[\ 0886#\ and this compromised binding could favor rescue of these VH70X!D!JH rearrangements by receptor editing\ as pre! viously suggested by Radic and Zouali "Radic and Zouali\ 0885#[ Furthermore\ the initial decline in the fre! quency of VH70X in the bone marrow also occurs in mutant mice which lack the membrane exon of Cm "Mar! shall et al[\ 0885#[ Replacement of VH70X!D!JH rearrangements by editing is therefore a candidate mech! anism to explain this reduction of 70X rearrangements\ as proposed by Marshall et al[\ "Marshall et al[\ 0885#[ Finally\ the 70X embedded heptamer contains 5 out of 6 consensus positions in the heptamer\ as do most VH6072 genes\ and thus is likely to contain any recombinogenic potential which may be present in this novel RSS[ Impor! tantly\ most of the VH6072 genes contain the identical {nonamer|!spacer!heptamer sequence of the VH70X embedded RSS "Carlsson et al[\ 0881^ Chukwuocha et al[\ 0883#[ Thus\ the data presented here are generalizable for the entire VH6072 family[ The 70X embedded RSS "70Xe# was therefore inserted in competition and conventional recombination sub! strates and assayed for recombination[ Results show no recombination of the 70X embedded RSS[ By comparing relative frequencies of various RSS|s in competition sub! strates\ we estimate that the 70X embedded RSS rearranges at a minimum 0299 times less frequently than a prototypic consensus RSS[ Hence\ receptor editing at
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the IgH locus is unlikely to play a signi_cant role in repertoire development or elimination of autoreactive receptors[
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by removing the SalI:NotI cassette containing VkA1b and its RSS\ blunting and religating the plasmid[ The RSS and surrounding regions of all constructs were con! _rmed by sequencing[
1[ Materials and Methods 1[1[ Recombination assay 1[0[ Plasmid constructs The Comp12 competition substrate is shown in Fig[ 1A\ and it was derived from the {Comp| plasmid series "Nadel and Feeney\ 0886^ Nadel et al[\ 0887\ and B[N[\ A[T[\ G[L[\ V[Love\ G[E[\ A[J[F[\ manuscript in prep! aration#[ The full VkA1b and Jk0 coding ends ~anked by their RSS|s were obtained by PCR "½199 bp segments#\ with primers containing the appropriate restriction sites to enable insertion into the Comp01 backbone[ 70Xe RSS was inserted as a double stranded oligonucleotide cassette ~anked by NotI and MluI restriction sites "Fig[ 1A#[ The 70Xe recombination substrate was derived from Comp12
07[7 A!MuLV transformed pre!B cells "19x095 are tran! siently transfected with 19 mg of Qiagen!puri_ed plasmid by electroporation "859 mF\ 9[2Kv# as previously described "Nadel and Feeney\ 0886# and resuspended in 09 ml of RPMI 0539 supplemented with 09) FCS\ 1 mM glutamine\ 4×09−4 M 1!mercaptoethanol\ and 0 mM ca}eine[ After 37 h\ plasmids are recovered from trans! fected cells by alkaline lysis\ followed by DpnI:SpeI diges! tion[ Digested plasmids are then electroporated into E[ coli and plated on plates containing ampicillin "099 mg:ml# and chloramphenicol "4 mg:ml#[ PCR are per! formed from the colonies by resuspending bacterial cells
Fig[ 1[ "A# Comp12 competition substrate map[ Sequences of part of the Jk0 coding end ~anked by its RSS\ 70X embedded RSS "70Xe# and the A1b coding end ~anked by its RSS are shown[ Coding end sequences are in bold[ Jk0 is separated from the competing 70Xe and A1b RSS by a transcriptional terminator\ called {stop| in the _gure[ B\ BamHI^ M\ MluI^ N\ NotI^ Sc\ SacII\ S\ SalI^ Sp\ SpeI[ "B# PCR screen used to analyze rearrangement status[ Locations of the PCR primers "AF63\ P3# and size of the PCR products are shown[ The total number of recombined colonies obtained is indicated[
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containing plasmid directly in the PCR mixture\ and amplifying with P3 and AF63 primers "Fig[ 1B# for 29 cycles at the following conditions] 0 min at 83>C\ 0 min at 44>C\ and 1 min at 61>C[ Random clones were sim! ultaneously miniprepped and sequenced[
Table 0 Comparison of VH70Xe and VkA07 frequencies of rearrangement in recombination substrates
VH70Xe VkA07
è Recombinant colonies
è Transfections
9 209
3 1
2[ Results 2[0[ No recombination of the 70X embedded RSS when in competition with the weak VkA1b RSS To test the frequency of recombination of the 70X embedded RSS\ we used a {competition| plasmid in which two segments are in competition for rearrangement with a unique joining partner "Fig[ 1A#[ This strategy allowed us to compare directly the frequency of rearrangement of one RSS relative to another one[ We have shown in other studies that the distal or proximal position of the various gene segments in the plasmid has no e}ect on the relative frequencies of recombination "B[N[\ A[T[\ G[ Lugo\ V[ Love\ G[ Escuro and A[J[F[\ manuscript in preparation\ and Nadel et al[\ 0887#[ Since we anticipated that the 70X embedded RSS would not be an e.cient RSS due to the many changes from the consensus RSS\ we placed it in competition with a RSS that rearranges at a very low frequency[ The human VkA1b gene is a defective allele of the VkA1a segment "Feeney et al[\ 0885#[ Compared to the consensus RSS\ the VkA1b RSS contains a change in the sixth nucleotide of the heptamer and another change in the 3th position of the nonamer "Fig[ 1A#[ We found a 02!fold decrease in the frequency of rearrange! ment of VkA1b compared to the consensus RSS in similar competition substrate experiments "B[N[\ A[T[\ G[L[\ V[ Love\ G[E[ and A[J[F[\ manuscript in preparation#[ We therefore modi_ed a competition recombination sub! strate containing VkA1b to create Comp12\ a plasmid containing on one side the Jk0 coding end ~anked by its RSS\ and on the other side both the 70X embedded RSS\ and the VkA1b coding end ~anked by its RSS "Fig[ 1A#[ These two segments are therefore in competition for rearrangement to the Jk0 segment[ We performed four independent transfections of 07[7 pre!B cells with Comp12 and screened colonies by PCR[ Out of 88 recom! bined colonies\ none gave rise to the 149 bp band cor! responding to the Jk0!70Xe and 88 gave rise to the 279 bp band expected for Jk0!VkA1b rearrangement "Fig[ 1B#[ In addition\ a few colonies gave rise to a 0089 bp PCR band corresponding to the background unre! combined plasmids[ These results suggest that rearrangement of the 70X embedded RSS is at least 099 times less e.cient than the VkA1b RSS[ Since we have shown using similar com! petition substrates that the VkA1B RSS\ with its non! consensus heptamer and nonamer\ is about 02!fold less e.cient than the corresponding consensus RSS "B[N[\
A[T[\ G[L[\ V[ Love\ G[E[ and A[J[F[\ manuscript in preparation#\ we can estimate at least a 0299 fold decrease in the frequency of rearrangement of the 70X embedded RSS compared to a consensus RSS[ We have\ however\ recently observed that the spacer sequence also plays a role in the e.ciency of the recombination process "Nadel et al[\ 0887#[ Since the A1 spacer is one of the best spacers that we tested so far\ this estimate may be slightly high[ 2[1[ No recombination is observed for the 70X embedded RSS To test the ability of the 70X embedded RSS to recom! bine in absence of other competing RSSs\ we generated the Jk0!70Xe plasmid\ a classical recombination sub! strate containing the germline sequence of the Jk0 coding end ~anked by its RSS on one side of the termination signal\ and on the other side\ the germline sequence of the 70X embedded RSS without any other segment in competition[ 07[7 cells were transfected in four inde! pendent transfections with the Jk0!70Xe plasmid in pres! ence of 1 mM ca}eine to increase to a maximum the level of recombination activity of the cell line "Menetski and Gellert\ 0889#[ Only rare colonies were obtained from ampicillin:chloramphenicol plates\ and those were mini! prepped and sequenced[ None of the clones were identi! _ed as Jk0!70Xe recombination\ all clones consisting of background unrearranged plasmids "Table 0#[ To rule out the possibility that the absence of rearrangement could be due to a putative defect in the transfection or harvesting processes\ a positive control was included in the same experiments[ 07[7 cells were transfected with a similar plasmid "Jk0!VkA07# containing Jk0 and the VkA07 coding end ~anked by its RSS\ instead of the 70X embedded RSS[ Although VkA07RSS has a nucleotide change in the nonamer resulting in a 1[6!fold reduction of the recombination frequency compared to consensus "B[N[\ A[T[\ G[ Lugo\ V[ Love\ G[ Escuro and A[J[F[\ manuscript in preparation#\ 209 colonies were obtained on ampicillin:chloramphenicol plates from two trans! fections\ and all sequenced clones showed proper rearrangement of the Jk0!VkA07 plasmid[ This result indicates that the 70X embedded RSS is the element responsible for the lack of rearrangement of the Jk0!70Xe plasmid[ These results con_rm that the 70X embedded
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RSS is a very ine.cient RSS and demonstrate that it will promote rearrangement at extremely low frequencies[
3[ Discussion Two main questions have emerged from the initial VH replacement studies] "0# from a mechanistic point of view\ can the embedded RSS promote speci_c V"D#J recom! bination< and "1# from a functional point of view\ does receptor editing of the heavy chain locus play a signi_cant role in vivo< Use of the embedded RSS for editing VHDJH rearrangements was initially shown in transformed B and pre!B cell lines "Brokaw et al[\ 0881^ Covey et al[\ 0889^ Deane and Norton\ 0889^ Klein_eld et al[\ 0875^ Klein! _eld and Weigert\ 0878^ Reth et al[\ 0875#[ Subsequently\ two transgenic mice carrying a site!directed transgene in which the unrearranged DQ41!JH region has been replaced by a complete VHDJH rearrangement have been generated "Chen et al[\ 0884^ Taki et al[\ 0884#[ In both reports\ replacement of the transgenic VHDJH joint occurred\ but in one mouse two di}erent heptamer!like sequences were used instead of the embedded heptamer\ and in the other mouse\ the use of the embedded hep! tamer was observed in cells pre!selected for the expression of surface Ig[ Similarly\ the more frequent use of alternate GTG sites in lieu of the embedded heptamer was observed in in vitro experiments "Usuda et al[\ 0881#[ Thus\ although it is clear that secondary rearrangement using the embedded heptamer does occur in vivo\ it is less obvious if replacement occurs at that site at a higher frequency than at any other random GTG sites "Lewis et al[\ 0886#[ Recent studies have shown the critical role of the non! amer in the recognition and binding of RAG!0 on the synapse "Di_lippantonio et al[\ 0885^ Spanopoulou et al[\ 0885^ Nagawa et al[\ 0887#[ The absence of an appropriate nonamer associated with the embedded heptamer is therefore probably very detrimental to the frequency of recombination[ In the nonamer\ the presence of at least 2 consecutive T|s is important for e.cient rearrangement "Akamatsu et al[\ 0883#\ and essentially none of the non! amer!like elements found in association with embedded heptamers contain this motif "Chen et al[\ 0884#[ It\ in physiological situations\ receptor editing of the IgH chain would play any signi_cant role in generation of diversity and:or elimination of self!reactive speci_cities\ the embedded RSS must function at some reasonable frequency[ However\ we estimate in this study that the 70X embedded RSS recombines at least 0299!fold lower than a consensus RSS[ This suggests that VH gene replace! ment is not responsible for the great decrease in rep! resentation of the 70X gene during di}erentiation[ Moreover\ since the 70X embedded heptamer contains essentially all of the consensus heptamer sequence\ and both embedded heptamer\ spacer and {nonamer| motifs
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are well conserved among all members of the 6072 family\ our results suggest that receptor editing of the heavy chain is unlikely to participate to any signi_cant extent in the establishment of the peripheral B cell repertoire[ Thus\ rescue of pro!B cells with two non!productive H chain rearrangements by VH gene replacement is likely to be a very rare event\ and L chain editing\ rather than H chain editing\ would be the predominant mechanism to avoid autoreactive speci_cities in vivo[ Acknowledgements This work was supported by National Institute of Health grant AI18561[ B[N[ was supported by an Amer! ican Cancer Society!California Division fellowship 0!22! 85B[ This is manuscript 00244!IMM from The Scripps Research Institute[
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