Recombination between kappa chain genetic markers in the mouse

In this study we report the first instance of recombination between kappa chain genetic markers in the mouse. The recombination frequency, 0.45% (95% limits, 0.12–1.61), is similar to that previously found for recombination between the kappa chain locus and the Lyt-2, 3 locus (0.3%, 95% limits, 0.05–1.6), but is relatively low in comparison with that found at the heavy chain locus (0.41–5.4%). Lyt-2, 3-typing of the recombinants permits a partial ordering of the kappa chain and Lyt-2, 3 loci as (Lyt-2, 3, Igk-Ef1) - Igk-Ef2. Light chains controlled by the two kappa markers include the V_k-(ser) subgroup (controlled by Igk-Ef1) and V_k–1 (controlled by Igk-Ef2). One of the recombinants has been recovered in a homozygous state (NAK) and should be suitable for V _k gene mapping studies.Abbreviations C complement - C_H constant region of the Ig heavy chain - CI cytotoxicity index - DNP dinitrophenyl - FMF flow microfluorimetry - IEF isoelectric focusing - IF immunofluorescence - Ig immunoglobulin - KLH keyhole limpet hemocyanin - V_H variable region of the Ig heavy chain - V_k variable region of the Ig kappa chain - V-region variable region     

Evolution of the immunoglobulin heavy chain variable region (Igh-V) locus in the genusMus

The evolution of the mouse immunoglobulin heavy chain variable region (Igh-V) locus was investigated by the comprehensive analysis of variable region (Vh) gene family content and restriction fragment polymorphism in the genusMus. The examination of naturalMus domesticus populations suggests an important role for recombination in the generation of the considerable restriction fragment polymorphism found at theIgh-V locus. Although the sizes of individualVh gene families vary widely both within and between differentMus species, evolutionary trends ofVh gene family copy number are revealed by the analysis of homologues of mouseVh gene families inRattus andPeromyscus. Processes of duplication, deletion, and sequence divergence all contribute to the evolution ofVh gene copy number. CertainVh gene families have expanded or contracted differently in the various muroid lineages examined. Collectively, these findings suggest that the evolution of individualVh family size is not driven by strong selective pressure but is relatively neutral, and that gene flow, rather than selection, serves to maintain the high level of restriction fragment polymorphism seen inM. domesticus.     

Deletion mapping of the mouse ornithine decarboxylase-related locus Odc-rs8 within Igh-V

The Odc-rs8 locus belongs to a family of mouse DNA sequences related to the gene encoding ornithine decarboxylase (ODC). Odc-rs8 was mapped by recombinant inbred (RI) strain analysis to the region of Chromosome (Chr) 12 occupied by the variable region genes of the immunoglobulin heavy chain (Igh) complex. In the present study, alleles at Odc-rs8 were shown to cosegregate with those for Igh variable region (Igh-V or V _H) genes among 37 inbred mouse strains that had been characterized previously for their haplotypes at Igh. For a more precise definition of the location of Odc-rs8 relative to Igh-V, DNAs from 17 Abelson murine leukemia virus (A-MuLV)-transformed pre-B cell lines cultured from mice heterozygous at Igh and Odc-rs8 were analyzed for the presence of DNA restriction fragments (RFs) derived from each parental Odc-rs8 allele. These cell lines, each of which has rearranged one or both Igh genes, previously were employed in mapping members of nine V _H gene families by deletion analysis (Brodeur et al. 1988). Comparing the deletion profiles of the cell lines for Odc-rs8 with those for the V _H gene families has located Odc-rs8 ^b within the V_HJ558/V_H3609 gene cluster and Odc-rs8 ^c either within or upstream of the 5-most 9% of V_HJ558, identifying Odc-rs8 as a potentially useful marker for the 5 end of the Igh complex.     

A linkage map of distal mouse chromosome 12

To refine the linkage map of distal mouse Chromosome 12, we have identified DNA restriction fragment variants associated with a creatine kinase gene (Ck-3), the Akt proto-oncogene, an Abelson proviral integration site (D12N1), and the immunoglobulin heavy chain V_H3609 variable region family (Igh-V36). The patterns of inheritance of these markers in backcross progeny and recombinant inbred mouse strains allowed their localization with respect to previously mapped genes to yield the linkage map: Aat-15.8 cM-Ck-3-0.9 cM-(Crip, Akt, Igh-C)-0.3 cM-(D12N1, Igh-V). This map confirms genetically the localization of the Igh-V gene complex distal to Igh-C on the chromosome. It differs from previous maps in placing D12N1 distal to Igh-C, and in suggesting that the Igh-V gene complex spans less than one centiMorgan (cM).Other DNA sequence variants detected with the creatine kinase probe allowed definition of four additional genetic loci: Ck-1 near Lmyc-1 on Chromosome 4; Ck-2 between Upg-1 and Hprt-ps1 (D17Rp10) on distal Chromosome 17; Ck-4 near Mpmv-17 and Mls-3 on Chromosome 16; and Ck-5 near Hba on Chromosome 11.     

Allelic polymorphism of mouse Igh-J locus,which encodes immunoglobulin heavy chain joining (JH) segments

The mouse genome contains four functional J _H genes, which encode immunoglobulin heavy chain joining segments. The J _H gene cluster is located a few kilobases 5 from the constant region genes (C genes) on chromosome 12. The polymerase chain reaction (PCR)-technique was used to amplify DNA stretches from mouse genome of approximately 1 340 nucleotides in length containing all four J _H genes (Igh-J locus). PCR products were directly used as templates in Sanger's dideoxy-sequencing, and sequences were determined. Twelve inbred mouse strains belonging to ten different Igh-C haplotypes were studied. The strains were: BALB/c, C58/J, RIII, DBA/2, CE, RF, CBA, NZB/J, AKR, C57BL/10, SJL, and A/J. Five allelic forms of the Igh-J locus were found among these strains. The A/J mouse has an allele (e) which differs from the BALB/c allele (a) by 15 nucleotides. C57BL and SJL have the allele (b) with eight differences from BALB/c. The CBA allele (j) has two differences, and the CE allele (f) has a single nucleotide difference compared with the BALB/c sequence. Based on the J _H , variable (V) and constant (C) region sequences we conclude that independent reshuffling of V _H ,J _H , and C _H gene clusters occurred during the evolution of Mus musculus.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X63146-X63175.     

The influence of Igh-1 genes on the class and subclass distribution of oxazolone-specific antibodies

Previous studies have demonstrated that the level of the oxazolone-specific antibody response induced by contact sensitization is under the control of H-2 and Igh-1-linked genes. The aim of the present study was to clarify the role of H-2 and Igh-1 genes in the regulation of antibody affinity and isotype composition of oxazolone-specific antibodies. Analysis of the antibody response to oxazolone has revealed different ratios of IgG2a and IgG2b antibodies in mice carrying the Igh-1 ^b allele and in strains carrying alleles a, c, and e. The characteristic ratio of IgG2a and IgG2b isotypes persisted during the whole period of the primary and secondary antibody response of CBA and CBA-Ig ^b Igh-C congenic mice. The Igh-1-linked genes influenced the isotype distribution and not the affinity of oxazolone-specific antibodies induced by contact sensitization.Abbreviations used in this paper c.Ig chicken immunoglobulin - Igh-C constant region of immunoglobulin heavy chain - DNCB 2,4-dinitrochlorobenzene - DTH delayed-type hypersensitivity - FITC fluorescein isothiocyanate - Igh-1 cluster of structural heavy chain allotype genes of IgG2a - KLH keyhole limpet hemocyanin - KSCN potassium-sulfocyanid - MHC major histocompatibility complex - Ox oxazolone (4-ethoxymethylene-2-phenyl-5-oxazolone - Ox-BSA oxazolone-bovine serum albumin - Ox-cap oxazolone-capronic acid - Ox-MSA oxazolone-mouse serum albumin - NP 4-hydroxy-3nitrophenyl acetyl - PVP polivinylpirrolidon - RIA radioimmunoassay - SRBC sheep red blood cell - T_H T helper - T_S T suppressor - Igh-V variable region of immunoglobulin heavy chain     

Genetic controls of T cell-independent Thy-1 alloantibody responses

Early and late primary IgM antibody responses of mice to Thy-1.1 antigens showed different antigenic and cellular requirements. We studied genetic controls of the early primary responses, which could be induced by subcellular thymocyte antigens independently of host T-cell activity. All Thy-1.2 mouse strains of Igh ^a(BALB/c and BC8), Igh-V ^aC^b(BAB14), Igh ^d(AKR/Cum), Igh ^j(CBA/J, C3H/HeN, C3H.SW, and C3H.JK), and Igh ⁿ(NZB) definitely responded early to Thy-1.1 antigens from AKR/J (Igh ^d), A.Thy-1.1 (Igh ^e), or B10.Thy-1.1 (Igh ^b) mice or SD rats, whereas all strains of Igh ^b(C57BL/6, C57BL/10, B10.D2, B10.BR, B10.A, CB20 and CWB), Igh ^c(DBA/2), Igh ^e(A/J), and Igh ^o(C.AL20) responded poorly to the same antigens. This contrasts with the observation that both strains of Igh ^j(C3H/HeN) and Igh ^b(B10.BR) responded well at later times. As was the case for late responses, the matching of H-2 between donor and recipient resulted in early responses of exceptional quality in high-responder strains. It was concluded that under the influence of H-2, whose incompatibility between donor and recipient partially interferes with responses, early but not late primary Thy-1.1-specific antibody responses are selectively controlled by Igh-V or closely linked Ir gene(s) as a new V _Hmarker.Abbreviations used in this paper Tl T cell-independent - TD T cell-dependent - PFC plaque-forming cell(s) - Igh immunoglobulin heavy chain - V _H variable region of heavy chain - C _H constant region of heavy chain     

The immunoglobulin κ locus: polymorphism and haplotypes of Caucasoid and non-Caucasoid individuals

The immunoglobuline locus has previously been characterized by comparing the restriction patterns of the DNA of 23 Caucasoid individuals and defining various polymorphisms and haplotypes. This study has now been extended to a group of 28 Blacks and another group of 13 individuals of different ethnic origins. The predominant haplotype of the Caucasoid group, called haplotype N, was also found frequently in the other groups. Some of the restriction fragment length polymorphism markers typical of haplotype G, on the other hand, were seen 2–3 times more frequently in the black than in the Caucasoid group. Haplotype 11, which is characterized by the absence of about half of the variable gene segments (V) and which had been observed in 3 out of 46 Caucasoid alleles, has been found twice in the 82 alleles of the two new groups. A number of new polymorphisms was detected and new haplotypes were defined, although the structure of the immunoglobulin locus seems generally to be well conserved among different populations.     

Allelic polymorphism of murine IgE controlled by the seventh immunoglobulin heavy chain allotype locus

Two alloantisera against hybridoma-derived IgE detected allotypic determinants expressed on the murine s chain. An antiserum raised in BALB/c mice against monoclonal IgE of C57BL/6 origin reacted exclusively with IgE of strains having Igh-1^b (IgG_2a) allotype. The second antiserum, C57BL/6 anti-BALB/c monoclonal IgE, reacted with IgE of strains having Igh-1^a, Igh-1^d, Igh-1^e and Igh-1^j allotypes. The genetic studies of (BALB/c x C57BL/6)F₁ and backcross F₂ animals indicated that the locus controlling the IgE allotype is linked to the Igh-1 locus. This was further confirmed by the possession of respective IgE allotypes by Igh-C congenic mice, BALB/c and BAB-14, C3H.SW/Hz and CWB/Hz. Thus, the allotype detected on the chain is controlled by the seventh murine immunoglobulin allotype locus, and should be designated as the Igh-7 allotype.Abbreviations used in this paper PCA passive cutaneous anaphylaxis - RID radioimmunodiffusion - i.p. intraperitoneally - EA egg albumin - Igh-C immunoglobulin heavy chain constant region locus - DNP 2,4-dinitrophenyl - PBS phosphate-buffered saline - NMS normal mouse serum - KLH keyhole limpet hemocyanin Visiting investigator supported by the Scientific and Humanistic Development Council from the Central University of Venezuela, currently at the following address: Consejo de Desarrollo Cientifico y Universidad Central de Venezuela, Av. Principal Urb. La Floresta Ota., Silenia Caracas, Venezuela.     

Localization of the rat immunoglobulin lambda light chain locus to chromosome 11

Previous experiments using rat/mouse somatic cell hybrids have localized the rat c-myc gene to chromosome 7 (Sümegi et al. 1983), the rat immunoglobulin kappa locus to chromosome 4 (Perlmann et al. 1985), and the rat immunoglobulin heavy chain locus to chromosome 6 (Pear et al. 1986). Using a similar approach, we now report the localization of the rat immunoglobulin lambda light chain locus to chromosome 11.     

Duplication of J κ genes within genus Rattus

We have isolated a region containing the immunoglobulin kappa chain joining segments from a liver DNA library of the Australian rat Rattus villosissimus, and determined its nucleotide sequence. While the laboratory rat (Rattus norvegicus) had previously been shown to contain three recently duplicated copies of J 2, R. villosissimus has only two. Furthermore, all three copies of J 2 in R. norvegicus share an 11 by deletion in their 5 flanking regions which is not evident in either copy of J 2 in R. villosissimus. This suggests that the initial duplication events occurred separately in the two lineages, and were followed by a second duplication in R. norvegicus, all three duplications having occurred within the last 6–12 million years (although more complicated schemes involving gene conversion events cannot be excluded). These results indicate that there is a high degree of plasticity in this region of the genome, and that selective forces must exist which have maintained the number of expressible J segments in humans (5) and rodents (4–6) within their narrow range.     

Familial clustering of IGHC deletions and duplications: functional and molecular analysis

The human immunoglobulin heavy chain constant region locus (IGHC) comprises nine genes and two pseudogenes clustered in a 350 kilobase (kb) region on chromosome 14q32. Several IGHC haplotypes with single or multiple gene deletions and duplications have been characterized. The most likely mechanism accounting for these unusual haplotypes is the unequal crossing-over between homologous regions within the locus. Here we report the analysis of an unusual case of familial clustering of deletions/duplications. In the two branches of the BON family, three duplicated and two deleted haplotypes, all probably independent in origin, have been characterized. The structure of the haplotypes, one of which is described here for the first time, supports the hypothesis of homologous unequal crossing-over as the origin of recombinant haplotypes. The analysis of serological markers in a subject carrying one deleted and one duplicated haplotype allowed us the first direct inferences concerning the functions of the duplicated IGHC haplotypes.     

Sites that direct nuclear compartmentalization are near the 5' end of the mouse immunoglobulin heavy-chain locus

VDJ rearrangement in the mouse immunoglobulin heavy chain (Igh) locus involves a combination of events, including a large change in its nuclear compartmentalization. Prior to rearrangement, Igh moves from its default peripheral location near the nuclear envelope to an interior compartment, and after rearrangement it returns to the periphery. To identify any sites in Igh responsible for its association with the periphery, we systematically analyzed the nuclear positions of the Igh locus in mouse non-B- and B-cell lines and, importantly, in primary splenic lipopolysaccharide-stimulated B cells and plasmablasts. We found that a broad approximately 1-Mb region in the 5' half of the variable-gene region heavy-chain (Vh) locus regularly colocalizes with the nuclear lamina. The 3' half of the Vh gene region is less frequently colocalized with the periphery, while sequences flanking the Vh gene region are infrequently so. Importantly, in plasmacytomas, VDJ rearrangements that delete most of the Vh locus, including part of the 5' half of the Vh gene region, result in loss of peripheral compartmentalization, while deletion of only the proximal half of the Vh gene region does not. In addition, when Igh-Myc translocations move the Vh genes to a new chromosome, the distal Vh gene region is still associated with the nuclear periphery. Thus, the Igh region that interacts with the nuclear periphery is localized but is likely comprised of multiple sites that are distributed over approximately 1 Mb in the 5' half of the Vh gene region. This 5' Vh gene region that produces peripheral compartmentalization is the same region that is distinguished by requirements for interleukin-7, Pax5, and Ezh2 for rearrangement of the Vh genes.     

Molecular analysis of recombination sites within the immunoglobulin heavy chain locus of the rabbit

Previously, recombinations involving genes of the rabbit immunoglobulin heavy chain locus have been documented serologically. These data indicated that the sites at which the causative recombination events occurred could have been anywhere from within the V _H gene cluster up to, or 3 of, C. Since these sites could not be localized further by serological methods, we attempted to do this using techniques of molecular biology. DNAs from homozygous recombinant rabbits and from the appropriate non-recombinant parental haplotypes were characterized using Southern blots hybridized with a panel of probes derived from cloned regions of the rabbit immunoglobulin heavy chain gene complex. In all three recombinants, the site was downstream of the entireV _H cluster and upstream of the J _Hcluster within an 50 kilobase (kb) egion containing expanses of repetitive-sequence DNA as well as D _H genes. D _H-specific probes further showed that in two of the recombinants, the recombination appears to have occurred within or 5 of D _H1 and 5 of D _H2 genes; in the third it occurred 3 of the D _H2 genes but at least 5 kb 5 of the J _H region. Address for correspondence and offprint request to: R. G. Mage.     

New mouse immunoglobulin a heavy chain allotype specificities detected using the hybridoma-derived IgA of I/St Mice

Immunizations of C57BL/6 and A mice with IgA derived from the I/St mouse strain yield alloantisera which detect two allotypic determinants of immunoglobulin A. The two determinants display discrete strain distributions. The first, identified by the alloantiserum C57BL/6 anti-IgA of I/St strain hybridoma ID150, follows the Igh ^c haplotype, and the second, identified by the alloantiserum A anti-IgA of I/St strain hybridoma ID150, correlates with Igh ^c and Igh ^c haplotypes. Absorption with monoclonal IgM, which has the same idiotype as the ID150 IgA clone, removed idiotype-specific antibodies from both alloantisera. The remaining antibodies are directed against determinants associated with the chain constant region, as shown by absorption with monoclonal IgA. By use of recombinant inbred strains of mice and mice congenic at the Igh locus, the loci controlling both C allotypic determinants have been mapped to the Igh region on chromosome 12.Abbreviations used in this paper Ig immunoglobulin - NMS normal mouse serum (sera) The genetic nomenclature of Green (1979) for mouse immunoglobulin loci was used in this report.     

Two closely related κ variable region pseudogenes pose an evolutionary paradox

Two pseudogenes belonging to the Igk-V1 variable region group have been isolated from BALB/c mice. The genes share >96.5% identity of nucleotide sequence in a 1800 base pair (bp) region surrounding the coding region, but deletions of 221 bp and 84 bp have removed essential sequences from the two genes. As the deletions are different in the two pseudogenes, they must have occurred independently in each gene during or subsequent to the duplication event which gave rise to the genes from a common ancestral gene. Polymerase chain reaction analysis was used to identify the pseudogenes in inbred strains of mice. BALB/c (Igk ^c) and AKR (Igk ^a), prototype strains representative of the predominant haplotypes, possess both pseudogenes but no intact copy. Only one of the pseudogenes was present in SJL (Igk ^a). Strains C58, c.C58 (Igk ^d) and NZB (Igk ^b) possessed an intact version of the gene. This distribution of haplotypes is consistent with a close linkage of the pseudogenes with other Igk-V1 genes on chromosome 6. The translated amino acid sequence of the pseudogenes indicates that prior to their acquiring deletions they encoded typical Igk-V1 variable regions except for an unusual FR2 region, in which the conserved proline at position 44 is replaced by leucine and the normally hydrophobic position 36 was occupied by histidine. Possible mechanisms to explain the occurrence of deletions in both of the pseudogenes in the recent evolution of BALB/c are discussed. One explanation would be that the two genes were already nonfunctional at the time of the duplication so that the subsequent deletions represent neutral events which became fixed in the inbred strains by a process of genetic drift. Alternatively, if the genes were functional at the time of duplication, their rapid loss due to deletion events suggests that negative selection may have acted to eliminate the genes from the V-region repertoire. Address correspondence and offprint requests to: D. M. Gibson.     

Localization of the rat immunoglobulin heavy chain locus to chromosome 6

We have previously used rat/mouse somatic cell hybrids to localize the rat c-myc gene to chromosome 7 (Sümegi et al. 1983) and the rat immunoglobulin kappa locus to chromosome 4 (Perlmann et al. 1985). We now report that by utilizing rat/mouse somatic cell hybrids, we have localized the rat immunoglobulin heavy chain locus to chromosome 6.     

Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins.

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre-) B cell differentiation, we find that most Abelson murine leukemia virus (A-MuLV)-transformed pre-B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A-MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre-B cell lines from normal mice, attempted V kappa-to-J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back-to-back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site-specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.     

A linkage map of the mouse immunoglobulin lambda light chain locus

The mouse immunoglobulin lambda light chain locus has been linked using field inversion gel electrophoresis. The lambda light chain locus classically contains two V and four J-C gene segments in inbred mouse strains, and was physically mapped in the BALB/c cell line Wehi-3 which contains unrearranged lambda light chain gene segments. The locus is relatively small and spans 300 kb, as defined by a variety of single and double digests using methylation-sensitive restriction enzymes. The order of the lambda gene segments is V2-J2C2J4C4-V1-J3C3J1C1, as was originally proposed. No evidence for nonmethylated CpG rich areas (HTF islands) within the region was found. Fine mapping using the 1, 3 rearranged cell line J558 mapped the gap between the V and J-C gene segments in the lambda 1 gene cluster (VI-J3C3JIC1) to approximately 70 kb. The similar distance (60–100 kb) found in the lambda 2 gene cluster (V2-J2C2J4C4) is further evidence that duplication of an ancestral locus occurred.     

Structural and evolutionary comparisons of four alleles of the mouse immunoglobulin kappa chain gene,Igk-VSer

The mouseIgK-VSer gene encodes an immunoglobulin light chain variable region which gives rise to two phenotypic polymorphisms of mouse chains. The nucleotide sequences of coding and flanking regions of theIgk-VSer ^c andIgk-VSer ^d alleles found in recently inbred strains of wild mice are compared with those of theIgk-VSer ^a andIgk-VSer ^b alleles described previously. Results suggest that the gene is evolving randomly and that framework 2 and complentarity determining region 2 are preserved, presumably for overall light chain structure. Results indicate that all four allels have an octamer motif upstream of the gene which should be functional and allow prediction of whether or not the product of the germ line gene will be detectable as either the I_B-peptide or Ef1^a phenotypic polymorphism. Southern hybridization of genomic DNA using as probe a 1-kbXba I-Xba I fragment located approximately 4 kb upstream of the BALB/cIgk-VSer ^b coding region demonstrated the presence of homologous DNA in mice bearing theIgk-VSer ^a allele and absence from mice bearing theIgk-VSer ^c andIgk-VSer ^d alleles. Nucleotide sequence comparison of BALB/c and SK/CamRk (Igk-VSer ^d ) DNA in this region demonstrated that BALB/c contained an insertion 2.4 kb in length which was absent from SK/CamRk. Both strains contain DNA homologous to the reverse complement of the mouse Bam5 repetitive element at the point of the insertion, with BALB/c containing approximately 70 nucleotides more of the element than SK/CamRk. Surprisingly, the strains containing DNA related to theXba I-Xba I probe are not those determined to be the most similar by nucleotide sequence comparisons and by the Phylogenetic Analysis Using Parsimony program. The evolutionary relationship of the alleles and a possible basis for the inconsistency presented by theXba I-Xba I fragment-related DNA are discussed.