of August 7, 2018.This information is current as

Region of the Mouse Strain 129S1Heavy Chain Constant and Partial Variable Sequence and Characterization of the Ig

RibletAmerica Mauhar, Helmut Blöcker, Werner Müller and Roy Gabriele Nordsiek, Simone Severitt, Stephanie Thies,Conrad, Martin Hafner, Tschong-Hun Im, Monika Ludewig, Ida Retter, Christophe Chevillard, Maren Scharfe, Ansgar

http://www.jimmunol.org/content/179/4/2419doi: 10.4049/jimmunol.179.4.2419

2007; 179:2419-2427; ;J Immunol 

MaterialSupplementary http://www.jimmunol.org/content/suppl/2008/03/14/179.4.2419.DC1

Referenceshttp://www.jimmunol.org/content/179/4/2419.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2007 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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Sequence and Characterization of the Ig Heavy Chain Constantand Partial Variable Region of the Mouse Strain 129S11

Ida Retter,2* Christophe Chevillard,2,3‡ Maren Scharfe,† Ansgar Conrad,† Martin Hafner,*Tschong-Hun Im,† Monika Ludewig,† Gabriele Nordsiek,† Simone Severitt,† Stephanie Thies,†

America Mauhar,‡ Helmut Blocker,4† Werner Muller,4,5* and Roy Riblet4‡

Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particularcomplex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains.For example, the Ighb haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igha

haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3�

half of the Igha locus of 129S1/SvImJ, covering the CH region and approximately half of the VH region. This sequencecomprises 128 VH genes, of which 49 are judged to be functional. The comparison of the Igha sequence with the homologousIghb region from C57BL/6 revealed two major expansions in the germline repertoire of Igha. In addition, we found smallerhaplotype-specific differences like the duplication of five VH genes in the Igha locus. We generated a VH allele table bycomparing the individual VH genes of both haplotypes. Surprisingly, the number and position of DH genes in the 129S1 straindiffers not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguousgenomic sequence of the 3� part of the Igha locus allows a detailed view of the recent evolution of this highly dynamic locusin the mouse. The Journal of Immunology, 2007, 179: 2419 –2427.

T he Ig loci of humans and mouse have been heavily inves-tigated during the last three decades. One of the first mile-stones toward a molecular understanding of the mecha-

nisms generating Ab diversity was the work of Hozumi andTonegawa (1), who could show that the Ab coding sequence isgenerated in B cells through a DNA recombination event (1). Ittook only 7 more years to achieve a detailed description of thephenomena of Ig gene rearrangement and somatic hypermutation(2). Both in these early and many later studies, a main focus hasbeen the analysis of the notably complex H chain locus (Igh locus)of mouse. Three different types of gene segments, called VH (vari-able), DH (diversity) and JH (joining) genes, occur in the Igh locusin multiple copies. In early B cell development, one VH, DH, andJH gene is selected and recombined to form the variable part of thecoding sequence for the H peptide chain (IgH) of the Ab. In asimilar manner, the L chain VL region is subsequently formedfrom one VL and one JL gene in at least one of the L chain loci. Totarget these V(D)J rearrangements, the recombination machinery is

guided by conserved recombination signal sequences (RSS)6 to thesites of recombination. The successful rearrangement of one Hand one L chain locus results in the expression of a BCR on thecell surface, determining the further fate of the B cell. In themature B cell state, the Ab isotype can switch by another DNArecombination event in the constant region of the Igh locus(Igh-C region). This class switch recombination combines theVDJ coding sequence with another Igh-C exon group, resultingin the production of Abs with different effector functions. Themouse Igh-V locus is particularly complex concerning both thesize of the V region and the number of VH genes. Thus, specialeffort is required to elucidate the highly repetitive genomic se-quence of this locus.

The murine Igh locus is �3 million bases (Mb) in size and islocated close to the telomere on chromosome 12. The locus isknown to be highly polymorphic within the genus Mus (3). Basedon detailed Southern blot analyses, the Igh loci of inbred strainswere assigned to different haplotypes (4, 5). The strain 129/Svpossesses the Igha haplotype, as does BALB/c, the strain that per-haps has been most widely used in studies of Ab responses inmouse. The Ighb haplotype is present in C57BL/6 and in the con-text of the mouse genome project (6) the Ighb locus was entirelysequenced (7–9). In this study, we provide a 1.6-Mb sequence ofthe 129 substrain 129S1/SvImJ, abbreviated 129S1, spanning theCH � exons up to the beginning of the large VHJ558 family region.This Igha sequence expands the picture of the murine Igh locus: onthe one hand, the comparison of genomic sequence from the twohaplotypes allows a detailed view of recent evolutionary changesin copy number and sequence of VH genes and other features, e.g.,interspersed repeats. In contrast, the sequence is also of general use

*Department of Experimental Immunology and †Department of Genome Analysis,Helmholtz Centre for Infection Research, Braunschweig, Germany; and ‡Torrey PinesInstitute for Molecular Studies, San Diego, CA 92121

Received for publication February 12, 2007. Accepted for publication May 29, 2007.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This research was supported by the German Bundesministerium fur Bildung undForschung through Grant PT DLR (FKZ 01KW0003) and the Bioinformatics Com-petence Centre “Intergenomics” Grant 031U110A/031U210A and National Institutesof Health Grant R01 AI23548.2 I.R. and C.C. are co-first authors.3 Current address: Institut National de la Sante et de la Recherche Medicale, Immunologyand Genetics of Parasitic Diseases, Marseille, France and Laboratory of Parasitology-Mycology, Faculte de Medecine, Universite de la Mediterranee, Marseille, France.4 H.B., W.M., and R.R. are co-last authors.5 Address correspondence and reprint requests to Prof. Werner Muller at his currentaddress: University of Manchester, Bill Ford Chair of Cellular Immunology, MichaelSmith Building, Oxford Road, Manchester, U.K.

6 Abbreviations used in this paper: RSS, recombination signal sequence; BAC, bac-terial artificial chromosome; STS, sequence-tagged site; PIP, percent identity plot;YAC, yeast artificial chromosome.

Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00

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and will support the elucidation of the special phenomena occurring inthe Igh locus: VDJ recombination, class switch recombination, andsomatic hypermutation, all of which are guided, regulated, or at leastinfluenced by different sequence motifs (10–12).

Materials and MethodsIdentification of Igh bacerial chromosome (BAC)

Igha locus BACs were isolated from the CitbCJ7, abbreviated CT7�, li-brary (see http://www.tree.caltech.edu/). This library was prepared from

the CJ7 ES cell line (13) derived from the 129S1/SvImJ mouse strain(ftp://ftp.informatics.jax.org/pub/reports/ES_CellLine.rpt). Library super-pools and high-density membranes for screening and individual BACclones were purchased from Research Genetics, now Invitrogen Life Tech-nologies, and are also available from OpenBiosystems. Superpools werescreened with a series of sequence-tagged site (STS) described in Ref. 14.These included sites in the 3� end of the CH region (IgA exon 3), JH region,DH region (DFL16.1), and various VH region sites (VHgroupIII-VH7183,VH11, VHS107, VHGAM3-8, and VHJ606), and a yeast artificial chromo-some (YAC) end (ADGC9-left arm) located in the VH10 region. Positive

FIGURE 1. Physical map of the 129S1 Igh locus, 3� part. The locus is shown in 5�33� orientation displayed in segments of 200 kb each. Positions of genesare depicted as vertical lines. Short vertical lines indicate pseudogenes and “possibly functional” genes. VH genes are colored corresponding to their sequencefamily. Positions of the underlying BACs are indicated as gray horizontal lines below the scaling line. The unfinished sequence of the most 5� BAC, 4K8, is markedwith a gray box; preliminary gene nomenclature in this 5� region is indicated by #. The sequence and annotation identifying each gene segment and its sequencecoordinates are available from EMBL/GenBank/DDBJ (accession no. AJ851868). VH gene sequences may also be downloaded from VBASE2(http://www.vbase2.org).

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mouse BAC clones were obtained from Research Genetics, plated on agarplates containing 12.5 �g/ml chloramphenicol, and confirmed by colonyPCR with the identifying primer sets. BAC ends were sequenced eitherdirectly from T7 and SP6 primers or following amplification by Vector-Hexamer PCR (15). BAC end sequences were deposited in EMBL/Gen-Bank/DDBJ accession nos. BH021141–BH021349. Contig assembly pro-ceeded by assessing STS content using the screening sites, D12Mit markersand others, and extensive Southern blot analysis using VH probes and BACand YAC ends, all as described in Ref. 14. Gaps were closed by developingnew screening PCR assays from BAC end sequences.

Sequencing and assembly

Sequencing of 23 BACs of the CT7 library was performed by a shotgunapproach as follows: sheared fragments of either 1 or 3 kb in length(GeneMachines) were subcloned separately into a pTZ18R vector. Atleast 800 clones were selected from each clone library, most of theplasmid DNA was prepared following a protocol supplied by Millipore.One-third of the selected clones were amplified by TempliPhi (384-wellformat) basically following the instructions of the supplier (AmershamPharmacia Biotech). Cycle sequencing was routinely performed using aDYEnamic ET terminator cycle sequencing premix kit (AmershamPharmacia Biotech) and UPO/RPO primer (MWG-BioTech). Most ofthe separations were run on Applied Biosystems 377 slab gel sequenc-ers and one-third of the samples on MegaBACE capillary sequencers.Data were assembled and edited using the GAP4 program (16).

Sequence analysis

The VH genes were annotated using a procedure developed for the auto-matic generation of the database VBASE2 (17). In this procedure, VH

genes are detected by a BLAST search (18) of the BAC sequences withknown germline VH gene sequences. BLAST hits with a minimum identityof 80% and minimum alignment length of 200 bp are analyzed with theDNAPLOT program (W. Muller and H. H. Althaus, unpublished data;http://www.dnaplot.de) and matched to VDJ rearrangements from theEMBL/GenBank/DDBJ The DNAPLOT analysis is limited to exon 2 ofthe VH genes, but includes detection of RSS elements. Exon 1 of each VH

gene, DH gene, JH gene, and CH gene have been annotated manually bysequence comparisons with BLAST, PipMaker (19) and other alignmentprograms, referring to previously published annotations; the EMBL/Gen-Bank/DDBJ accession no. of the respective reference sequences are givenin the feature table of the EMBL/GenBank/DDBJ entry AJ851868.

Dot plots and percent identity plots (PIPs) have been generated with thePipMaker program, parameters: “search both strands,” “show all matches”sensitivity mode: “default,” “show all matches” for dot plots, and “chain-ing” for PIPs. Interspersed repeats have been detected by using the Re-peatmasker web server (A. F. A. Smit, R. Hubley, and P. Green, unpub-lished data; http://www.repeatmasker.org). The searches were performedagainst the Repbase mouse dataset with the “cross_match” search engineand slow sensitivity mode. Phylogenetic trees have been generated withIMGT alignments created with DNAPLOT (20) and visualized withMEGA version 3.1 (21). Further sequence analysis and formatting havebeen done with the emboss program package (22) and Perl scripts.

ResultsThe sequence of the 3� half of the Igha locus

To elucidate the genomic sequence of the murine Igha haplotype,23 BACs from the Citb, or CT7, library of the 129S1 mouse wereselected using a physical map of the locus assembled with YACand BAC end STS, D12Mit simple sequence length polymorphismsites, and Igh-C, J, D, and V gene segments (Refs. 14 and 15; C.Chevillard and R. Riblet, unpublished observations). The BACswere sequenced with a 10-fold coverage on average. The BACinserts represent an overall length of 2.5 Mb and were assembledto a sequence of 1.6 Mb. This sequence covers approximately one-half of the Igh locus, including the CH region, JH region, DH re-gion, and the JH-proximal part of the VH region (Fig. 1). Theassembly is contiguous between the BACs 407I12 and 34H6, ex-cept for two simple repeat stretches of unknown length, one in34H6 and one in the overlap of 459E6 and 436C3. The unfinished

Table I. Sequence comparison of VH pseudogenes and “possiblyfunctional” VH genes vs functional VH genes

Sequence Identityto the Most Similar

Functional Gene (%)No. of VH

Pseudogenes

No. of PossiblyFunctional VH

Genes

�70 44 070–79 8 180–89 5 390–99 6 12

Table II. Summary of the functional VH genes and VH pseudogenes in the JH-proximal part of the mouseIgha locus grouped by familya

129S1 C57BL/6

Family FunctionalPossibly

functional Pseudogene FunctionalPossibly

functional Pseudogene

VH7183 16 2 31 8 2 16 (11)VHQ52 14 3 10 4 (5) 4 3 (4)VHS107 2 1 1 3 0 2 (1)VHSM7 3 0 1 4 0 0VH36–60 7 1 2 3 3 2VH11 1 1 0 1 1 1 (0)VHX24 2 0 0 0 1 1VHGAM3–8 2 4 0 1 3 0VH3609N 0 1 2 0 2 (1) 2 (1)VH16 0 0 0 0 0 (1) 0VH12 0 1 4 1 0 2 (0)VHJ606 2b 2b 3b 1 4 1 (0)VH10 0b 0b 0b 2 0 4 (1)VH15 0b 0b 1b 0 1 1 (0)VHJ558 0b 0b 1b 29 (41) 9 (11) 36 (37)VH3609 0b 0b 0b 2 6 4 (8)Unclassified relics —c — 7 — — 0 (19)Total 49 16 63 59 (72) 36 (38) 75 (85)

128 170 (195)

a The number of VH genes in C57BL/6 published in Ref. 9 is given in parentheses when different from the number publishedin Ref. 7. Only VH genes with 100% sequence identity to a known VDJ rearrangement are listed as functional.

b The region of this family is not covered (VH10, VH3609) or is incompletely covered (VHJ606, VH15, VHJ558) by theavailable sequence.

c —, not applicable.

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sequence of BAC 4K8 overlaps 407I12 and extends the assemblytoward the JH distal part of the VH region with 12 single fragments.The order of these fragments is hypothetical, based on sequencecomparison with the homologous Ighb region in C57BL/6. Thecomplete assembled sequence and annotation are available fromEMBL/GenBank/DDBJ (accession no. AJ851868).

Annotation of VH, DH, JH, and CH genes in the assembledsequence

The Igha sequence assembly was screened for VH genes using aBLAST search and a subsequent V gene analysis method based onthe program DNAPLOT (17). This method annotates the V genebeginning from the coding sequence of the mature peptide chainup to the noncoding RSS sites at the 3� end of each V gene. Theresults of this procedure were complemented by the annotation ofexon 1. VH genes with obvious defects like stop codons, defectivesplice sites, and abnormal RSS elements were marked as pseudo-genes. To further address the question of V gene functionality in adefined in silico approach, VH genes were matched against 6190 IgVDJ rearrangements extracted from EMBL/GenBank/DDBJ.Those VH genes with a 100% match to a rearranged sequence areclassified as functional. A physical map of the annotated regionis shown in Fig. 1.

In the 3� half of the Igha V region, 128 VH genes were anno-tated, of which 49 have been found in rearrangements. Of 79 VH

genes without clear evidence of functionality, 63 are obvious pseu-dogenes. The remaining 16 VH genes are designated as “possiblyfunctional.” To further analyze the potential functionality of theVH genes, we performed a pairwise sequence comparison of all 79possibly functional and pseudogenes with the 49 functional VH

genes. The result (summarized in Table I) shows that the majorityof pseudogenes have major changes in their sequence, whereassome of them are very similar to functional genes. Although 12possibly functional VH genes have at least 90% sequence identityto a functional gene, this is also the case for 6 pseudogenes. Thisindicates that a high sequence similarity to a functional gene can-

not be taken as evidence for functionality, and the functionality ofthe 16 possibly functional genes cannot be proven based on theavailable data. However, because somatic mutations in the VDJrearrangements prevent a 100% match to a germline sequence, it islikely that at least some of the possibly functional sequences areindeed functional.

One functional and four pseudogenes have an exact duplicatedcounterpart. The VH gene sequence family assignment revealedthat the currently elucidated part of the locus comprises membersfrom 12 of 15 known VH gene families, lacking representationfrom only the most 5� VH3609P, VH10, and VHJ558 families (Ta-ble II), with the exception of the pseudogene VHJ558.a1psi.119.Seven VH pseudogenes could not be assigned to a certain VH genefamily and were designated as VH relics, of which four are locatedin the VH7183 region and are closely related. Functional RSS el-ements were found at the 3� end of 72 VH genes. All RSS elementsfrom VH genes of the families VHGAM-8 and VHSM7, and 2 RSSelements of the VH36-60 family exhibit a 22-bp spacer. TwentyVH pseudogenes show abnormal RSS elements. A table listing allVH genes with their names, properties, and positions within theassembly is available as supplemental data.7

The VH genes have been named according to previously pub-lished nomenclature rules (4, 9). Each name consists of three partsseparated by dots: the first part gives the VH gene family. Thesecond part gives one letter for the Igh haplotype, in this case “a,”and the number of the VH gene within this particular family fol-lowed by the psi tag for pseudogenes. The third part gives a num-ber for the position of the VH genes within the locus, where boththe absolute and the family numbering run from the JH proximalto the distal side of the locus. Names for VH genes within the12 fragments of BAC 4K8 are given provisionally and thereforeare marked with # at the end. Because other nomenclature sche-mata for VH genes have been published (8, 23) and numerous

7 The online version of this article contains supplemental material.

FIGURE 2. Dot plots of the JH proximal VH and DH region. Positions of VH and DH genes from 129S1 are indicated as vertical lines, pseudogenesare marked as half-size vertical lines. A, 129S1 VH region, masked sequence vs C57BL/6 VH region, unmasked sequence. Two family regionsunderlying VH expansion in 129S1 are indicated as boxes. B, DH region of 129S1, masked (y-axis) vs unmasked (x-axis) sequence. Boxes indicatetwo homology blocks.

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trivial names are in use, we collected all names to our knowl-edge for each VH gene and make them available in the databaseVBASE2 (17).

The JH proximal VH region of the Igha and Ighb haplotype:sequence comparison and VH gene allele assignment

The distinct polymorphisms in the murine Ig receptor loci are dueto the strong evolutionary dynamics of these loci. The divergentevolution of VH genes has been described as resulting from diver-sifying selection and evolution by birth and death process (24).The inbred strains provide something like a snapshot of these loci,so that haplotype and even allele assignment is possible. We com-pared the Igha and Ighb haplotype by a dot plot of the homologousVH region sequences from 129S1 and C57BL/6 (Fig. 2A). Thisplot indicates that the overall structure, namely, the position of thediscrete VH gene family clusters, is very similar in both haplo-types, a finding that confirms previous experimental results basedon Southern blot analyses (25). Concerning individual VH genefamily clusters, the dot plot shows two regions of expansion in theIgha locus: the VH7183/VHQ52 region (previously described inRef. 26) and the interspersed VHGAM3-8/VH12 families. TheVH7183/VHQ52 cluster is well conserved between both strains atthe 3� end, followed by a sequence region which is much larger inIgha than in Ighb. This local expansion results in approximatelytwice as many functional VH 7183/VH Q52 genes in the Igha rep-ertoire compared with Ighb (Table II). Similarly, the VHGAM3-8/VH12 region is larger and comprises more VH genes in Igha com-pared with Ighb. A detailed view of the differences between Igha

and Ighb is provided by a percent identity plot of the homologousregions (supplemental data).7 The plot displays annotations andinterspersed repeats in the 129S1 sequence along with the percentidentity detected in the C57BL/6 sequence.

To resolve the V region comparison to the level of single VH

genes, we performed a multiple sequence alignment of the com-bined VH gene repertoire of both strains. Whenever an Igha VH

gene had its best match within the Ighb repertoire and inversely thebest match of this Ighb VH gene was the referring Igha V gene, wedesignated this VH gene pair as alleles (Table III). In case of theduplicated Igha V genes, one Ighb allele refers to the pair of Igha

alleles. For all alleles a minimum sequence identity of 90% waspostulated. With this method, 53 pairs of alleles were assigned.The genes of the VHJ606 family were excluded from the alleleassignment, as both the order of these genes is hypothetical and itis unlikely that all VHJ606 genes are included in the current as-sembly. The allele assignment is in accordance with the dot plot ofthe genomic Igha and Ighb sequences (Fig. 2A); alleles occur inregions having high overall sequence similarity, whereas regionsof expansion have much less similarity and contain no alleles.

The Igh-D region of 129S1 consists of two separate homologyblocks

In the Igh-D region of 129S1, 15 DH genes are annotated andassigned to one of the sequence families DSP2, DFL16, DST4, or

FIGURE 3. Alignment of the DH gene families DST4 and DFL16. RSS elements are marked as gray boxes.

Table III. VH gene alleles in 129S1 and C57BL/6

129S1 C57BL6a C57BL/6b

VH7183.a1�.1 Vh7183.b1� 7183.1pg.1VHQ52.a1�.2 — Q52.1pg.2VH7183.a2.3 VH7183.b2 7183.2.3VHQ52.a2.4 VHQ52.b1 Q52.2.4VH7183.a3�.5 VH7183.b3� 7183.3pg.5VH7183.a4.6 VH7183.b4 7183.4.6VHQ52.a3.8 VHQ52.b2 Q52.3.8VH7183.a6�.9 VH7183.b5� 7183.6pg.9VH7183.a7.10 VH7183.b6 7183.7.10VH7183.a8�.11 VH7183.b7� PG.1.11VHQ52.a4�.12 VHQ52.b3� Q52.4pg.12VHQ52.a5.13 VHQ52.b4 Q52.5.13VH7183.a9�.14 VH7183.b8� 7183.8pg.14VH7183.a11�.16c VH7183.b10� —e

VHQ52.a6�.17c VHQ52.b5� Q52.6pg.17VHQ52.a7.18 — Q52.7.18VH7183.a12�.19c VH7183.b11� 7183.11pg.19VH7183.a13.20 VH7183.b12 7183.12.20VH7183.a14�.23 VH7183.b14� 7183.13pg.24VHQ52.a9.26 VHQ52.b6 Q52.8.22VHQ52.a13.37 VHQ52.b7 Q52.9.29VHQ52.a15.42 VHQ52.b6 Q52.8.22VH7183.a25.43 VH7183.b9 7183.9.15VH7183.a26�.44c VH7183.b10� —VHQ52.a16�.45c VHQ52.b5� Q52.6pg.17VH7183.a27�.47c VH7183.b11� 7183.11pg.19VH7183.a45�.73 VH7183.b19� —VHQ52.a25�.74 VHQ52.b9 Q52.11.34VH7183.a46.75 VH7183.b20 7183.18.35VH7183.a47.76 VH7183.b22 7183.20.37VH7183.a48�.77 VH7183.b23� 7183.21pg.38VHQ52.a26�.78 VHQ52.b10� Q52.12pg.39VHQ52.a27.79 VHQ52.b11 Q52.13.40VH7183.a49�.80 VH7183.b24� PG.7.41VHS107.a1.81 VHS107.b1 S107.1.42VHX24.a1.84 VHX24.b2 X24.2.50VHSM7.a2�.88 VHSm7.b2 SM7.2.49VH11.a2.92 VH11.b2 VH11.2.53VHSM7.a3.93 VHSm7.b3 SM7.3.54VHS107.a3.106 VHS107.b3 S107.3.62VH15.a1�.107 VH15.b1 —VHSM7.a4.108 VHSm7.b1 SM7.1.44VH36–60.a3�.109 VH36–60.b3 36–60.3.64VHS107.a4.110 VHS107.b4 S107.4.65VH36–60.a4.111 VH36–60.b4 36–60.4.66VH36–60.a5.112 VH36–60.b5 36–60.5.67VH3609N.a2�.113 VH3609N.b3psi 3609N.1pg.68VH36–60.a6.114 VH36–60.b6 36–60.6.70VHGAM3–8.a6.115 VHGam3–8.b4 VGAM3–8-4–71VH36–60.a8.117c VH36–60.b8 36–60.8.74VHJ558.a1�.119 VHJ558.b1psi PG.16.76VH3609N.a3.120 VH3609N.b4 3609N.2.77VH36–60.a9.121c,d VH36–60.b8 36–60.8.74

a Published in Ref. 7.b Published in Ref. 9.c Duplicated in 129S1.d Provisional nomenclature.e —, not annotated.

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DQ52. In addition, three copies of the truncated DH gene DMB1(27), DMB1–DMB3, were found. The DH gene DSP2.2 is dupli-cated and is designated as DSP2.2a and DSP2.2b. Two new DH

genes are seen, DFL16.3 and DST4.3, which are probably notfunctional (see below). The dot plot of the DH region (Fig. 2B)shows that the DH genes are arranged in two separate homologyblocks: The main block, including all DSP2 genes, starts beforeDFL16.1 and ends after DST4. Upstream of this major DH clusterthere is an additional small block consisting of the genes DST4.2,DMB1, and DFL16.3. The DH gene DQ52 constitutes the 3� end ofthe DH region and is separate from the main DH gene block. Boththe main and the small blocks are composed of multiple relatedrepeats of a 3-kb sequence containing one DH gene.

The sequences of the DH genes DFL16.3, DST4.2, and DST4.3(Fig. 3) were checked in silico for potential functionality. ABLAST search of the DH gene sequences was performed againsta database comprised of the junctional regions of 6190 Ig VDJrearrangements extracted from EMBL/GenBank/DDBJ. ForDFL16.3 and DST4.3, no specifically matching rearrangementswere found, so that both DH genes are classified as nonfunctional.For DST4.2, six rearrangements were found that shared theDST4.2-specific insertion of a cytidine nucleotide in the secondposition. Interestingly, three rearrangements stem from a mouseline where the main block of DH genes has been deleted (27).Thus, it seems that the lack of DH genes from the main blockenforces the usage of the otherwise seldom or nonused DH genesof the small block.

Comparison of the DH genes of 129S1, BALB/c, and C57BL/6

Although the Igh-V regions of different inbred strains of mice al-low a clear assignment to a relatively small number of haplotypes,the Igh-D regions show even more polymorphism (28). AlthoughDH gene usage and the role of the DH region within the process ofVDJ rearrangement have been intensively studied (29–33), a com-plete nucleotide sequence of the DH region of BALB/c has neverbeen published. The existing map is based on Southern blot hy-bridization experiments (34, 35). The Mouse Genome SequencingConsortium provided a complete sequence of the DH region ofC57BL/6, which has recently been annotated (7, 36). Fig. 4 com-pares the DH regions of 129S1, BALB/c, and C57BL/6. The threemaps have a common feature, namely, the main DH gene block isseparate from the downstream DH gene DQ52. The small DH geneblock is present in 129S1 and C57BL/6, but it is unknown whetherit exists in BALB/c. Compared with 129S1 and BALB/c, the mainDH gene block is smaller in C57BL/6 and contains fewer genes.The main blocks of 129S1 and BALB/c contain different DSP2genes. Differences in the size of the main DH blocks may be dueto the fact that the BALB/c map is of limited resolution. However,the available data show notable differences in the DH region ofboth Igha haplotype strains 129S1 and BALB/c.

The JH and CH regions of 129S1

Close to the most downstream DH gene DQ52, four JH genes arelocated within a short stretch of 1.5 kb. The adjacent CH regioncontains eight genes coding for eight different isotypes of the Hchain of the Ab. Each CH gene consists of a group of three to fiveexons coding for different domains in the Ab molecule. B cells canswitch the isotype of the produced Ab by class switch recombi-nation. This is mediated by repetitive sequences upstream of theparticular isotype exon group, the switch (S) regions. JH genes, CH

region exons, and S regions were annotated referring to previouslypublished annotation of these sequences from BALB/c mice (forreferences, see EMBL/GenBank/DDBJ AJ851868). In addition tothe previously known CH� pseudogenes (37), one new CH� pseu-dogene group was found, consisting of CH2, CH3, and a truncatedM1 exon. The new pseudogene group was designated as CHpsi�0and is in an inverted orientation located upstream of the CH�3group, thereby heading the CH� cluster.

To depict the genomic structure of the CH region, comprisinginternal sequence homologies as well as location and size of re-petitive sequences, we performed a dot plot of the genomic se-quence (Fig. 5). In the dot plot, S regions appear as black boxesadjacent to the I exon. The S region of CH�1 is noticeably en-larged. Four sequence blocks comprising the CH� exon groupsshow strong conservation, starting upstream of the I exons and

FIGURE 4. Map of DH genes in 129S1, BALB/c, and C57BL/6. The single maps are scaled equally. The DH gene DQ52 is used as a bench-mark forpositioning. A, 129S1; B, BALB/c (adapted from Ref. 35); and C, C57BL/6 (adapted from Ref. 36).

FIGURE 5. Dot plot of the 129S1 CH region. Masked (y-axis) vs un-masked (x-axis) sequence. To display S regions, simple repeats were notremoved from the masked sequence. Positions of CH genes and CH pseu-dogenes are indicated as vertical lines and half-size lines, respectively.

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extending beyond the membrane exons of each group. Parts of thisconserved block are inversely inserted upstream of CH�3, CH�2b,and CH�2a. These findings about the overall CH region structureare in accordance with data published on the CH region of BALB/c(37, 38). A refined view of the relationship between the JH and CH

loci of both strains is gained by comparison of the coding se-quences. The sequences of the four JH genes of 129S1 and BALB/care identical, except for a silent point mutation in the third valinecodon of JH1 (C vs T). Remarkably, the CH region coding se-quences from 129S1 and BALB/c show several amino acid ex-changes concerning the isotypes IgD, IgG1, IgG2b, IgE, and IgA(Table IV). However, the CDS of IgM, IgG3, and IgG2a areidentical.

DiscussionNomenclature of the mouse Igh-V genes

For a sensible gene nomenclature, the gene name should provide areasonable amount of information about the gene. In particular forthe Igh locus of mouse, numerous nomenclature suggestions havebeen made, each representing different aspects of the genes. TheIMGT (http://imgt.cines.fr/) established a systematic nomenclatureby assigning a number to each V gene family and numbering Vgenes within each family (39). This nomenclature regards genesindependent of their chromosomal position and haplotype, but itaccounts for V gene alleles. de Bono (8) applied another rule set tothe Ighb-V genes, introducing position-dependent numbering.The nomenclature rules that Johnston et al. (9) and we appliedto the genomic sequence of the Ighb and Igha loci, respectively,include both haplotype and positional information, thereby in-creasing the information content of the name. Concerning thefamily nomenclature, we used the well-established familynames that trace back to the original myelomas and other celllines from which the first family members were derived. It isobvious that there is no benefit in maintaining different nomen-clatures in parallel. However, until the Mouse Genomic No-menclature Committee establishes a definite standard, we haveto accept diverse opinions and the resulting parallel nomencla-tures. To generate transparency, we have made available a listof corresponding names for each V gene in the germline V genedatabase VBASE2 (http://www.vbase2.org).

Heterogeneity within the Igha haplotype

Serological studies using polyclonal antiallotype sera classified129/Sv and BALB/c together as Igha (40), and later studies with

mAbs to BALB/c (i.e., IgM, IgD, and IgG1) bound 129/Sv Ig aswell (41, 42). RFLP analyses of the VH region showed a strongcorrespondence between the CH region and VH region haplotypes(4, 43), meaning that strains with identical CH region patterns oftenalso exhibit the same VH region patterns. For 129/Sv, comprehen-sive Southern blot analyses revealed restriction patterns identicalto the VH region of BALB/c, with the exception of the pattern forthe VH3909P family at the distal part of the locus (5).

Based on this, it was unexpected to find the remarkable differ-ences between the CH coding sequences of 129S1 and BALB/cthat are listed in Table IV. The DH region sequence of 129S1shows a mixed haplotype, represented by a DST4-Igha allele anda DQ52-Ighb allele. A detailed comparison with the DH region ofBALB/c is limited by the lack of BALB/c genomic sequence.However, based on the available sequence information, there areobvious differences between the DH regions of BALB/c and129S1. Taken together, our results point to a distinct heterogeneitywithin the Igha haplotype that had not been detected in previousexperiments and which might affect, to a minor degree, also the VH

region of 129S1.

Interspersed repeats in the murine Igh locus

An unusually high content of interspersed repeats has been repeat-edly noted for the murine Igh and Igk loci (14, 15, 44). In theelucidated VH region of 129S1, Repeatmasker analysis shows thatinterspersed repeats occupy 54% of the sequence: the content ofLINE-1 (L1) elements is 36%, whereas the SINE content is �2%.This considerably deviates from the average for the mouse ge-nome, where 19% L1 and 8% SINE content have been reported(6). The distribution of repetitive elements in the VH region of129S1 is very similar to the distribution that was reported for theVH region of C57BL/6 (9). This unusually high density of LINEelements has several interesting parallels; a high density has beennoted around other monoallelically expressed genes (45), and theX chromosome has a high LINE density. Lyon (46) has proposedthat this L1 density is a factor in the heterochromatization of theinactive X. To visualize interspersed repeats in relation to the V, D,J, and C gene positions, we performed a percent identity plot (PIP)of the Igha vs Ighb sequence with the PipMaker program (supple-mental data).7 The PIP shows not only differences between thehaplotypes, but also the position and class of interspersed repeatsin the 129S1 sequence and can therefore be taken as a high-reso-lution map of the region. It graphically displays the relatively uni-form distribution of L1 elements throughout the VH region andrarity in the CH region; also apparent are insertions of L1 elementsin 129S1 that are absent in C57BL/6, consistent with the continu-ing evolution of this complex locus.

Expansion of functional and nonfunctional VH genes by blockduplications

Duplications of both large and small sequence blocks are a com-mon phenomenon in the Ig loci (9, 47–49) and are assumed to bean essential force in the generation of multiple gene copies in theseloci. Ota and Nei (24) explain the maintenance of diversity withinthe large VH gene family by selective forces favoring diversifica-tion of the duplicated genes. To explain the high number of pseu-dogenes within the Ig loci, Kawasaki et al. (48) suggested thecoamplification and fixation of pseudogenes along with adjacentfunctional V genes. We tested this hypothesis on the VH7183/VHQ52 region of the murine Igh-V locus, which is expanded in the129S1 strain compared with C57BL/6 (Fig. 2A). We generateda phylogenetic tree of the VH7183 Igha family (Fig. 6A) andnoticed that there is a clear separation of nonfunctional andfunctional VH7183 sequences, indicating that the nonfunctional

Table IV. Differences in CDS of CH region genes between 129S1 andBALB/c

C RegionGene Exon/Domain

NucleotidePosition

within ExonAmino Acid Difference

129S1 vs BALB/c

C� CH3 125..127 S vs G �V00788�C�1 CH1 219..224 TW vs PR �J00453�C�1 CH3 174..176 D vs N �J00453�C�1 CH3 180..182 D vs N �J00453�C�2b CH1 72..74 L v. S �V00763�C�2b CH1 105..107 S vs. P �V00763�C�2b CH3 27..29 I vs T �V00763�C� CH1 93..95 G vs N �X01857�C� CH1 45..47 A vs V �J00475�C� CH1 51..53 S vs C �J00475�C� Hinge-CH2 195..197 V vs A �J00475�

EMBL/GenBank/DDBJ accession no. of BALB/c reference sequences are givenin brackets.

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sequences have evolved independently from the functional se-quences and there are, with the exception of VH7183.a1psi.1and VH7183.a43psi.70, no pseudogenes related to the func-tional sequences. Looking at the physical map of the VH7183region, one can see repeated patterns where a certain functionalgene is close to a pseudogene in a conserved distance and order(Fig. 6B). The first, most obvious example of such a block con-sists of a VH relic sequence next to a functional VHQ52 gene.This block, indicated by a noncolored box in Fig. 6B, appearsfour times within the VH7183/VHQ52 region. In addition, wefind other blocks involving pseudogenes of the VH7183 familynext to functional genes of the VHQ52 or the VH7183 family,indicated as colored boxes in Fig. 6B. When we superimposethese blocks on the phylogenetic tree shown in Fig. 6A, we cannicely see clustering of the pseudogenes within the phyloge-netic tree. From our analysis, we conclude that functional VH

genes are duplicated as large stretches of DNA containing moreflanking sequences than necessary to encode for a functional Vgene and by this “blockwise” duplication pseudogenes are ex-panded as well. In Fig. 6A, we further show the relationship tocorresponding alleles of the C57BL/6 locus, indicating that thephylogenetic distribution between the functional and nonfunc-tional VH genes remains valid also in case of the C57BL/6strain. When we analyzed the order of functional and nonfunc-tional sequences within the entire Igha region, we could see anunderrepresentation of two functional VH genes next to eachother (data not shown). In case of a random distribution, wewould have expected a higher fraction of neighboring func-tional VH genes. This finding supports the idea of blockwiseexpansion of large segments of DNA containing at least onefunctional VH gene. We cannot rule out that the adjacent pseu-dogenes comprise functional regulatory elements that are used,for example, for the opening of the locus during B lymphocytedevelopment or later by the functional VH genes and are therebypositively selected. However, the latter possibility seems un-

likely since there are examples of pseudogenes located bothupstream and downstream of a functional gene within the indi-cated homology blocks. In addition, the fact that we find onlyone orientation of VH genes, irrespective if these are functional,nonfunctional, or relics, points to a directed mechanism in theevolution of this locus. Currently, we have no conclusive ex-planation for the driving forces underlying the evolution of VH

genes. Given the complexity of the genealogical trees of mouseinbred strains (50), we cannot state when and how modificationsof the VH genes happened. Either they occurred during the gen-eration of inbred strains within the last 100 years or these dif-ferences represent allelic variants of wild mice selected over atime span of 100,000 years or more that have been fixed duringthe generation of inbred lines. As BALB/c and 129S1 mice,although distantly related in the genealogical tree of mouse in-bred strains, have similar Igh haplotypes, we favor the latterpossibility.

AcknowledgmentsWe thank Klaus Rajewsky and Blair Prochnow for critical review of thismanuscript.

DisclosuresThe authors have no financial conflict of interest.

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