Structural characterization of mouse immunoglobulin allotypic determinants (allotopes) defined by monoclonal antibodies

We have generated a new series of monoclonal antibodies recognizing allotypic determinants on mouse IgG₁, IgG_2a, and IgG_2b. In this communication we describe their reactivity at the molecular level. A number of genetic specificities (as defined by reactivity with sera from inbred strains) were divided into subspecificities (allotopes) by these analyses. With the exception of one allotope located in the hinge region of Igh-1 ^b, all other 23 allotopes examined were preserved upon reduction and alkylation of immunoglobulin antigens. To further analyze the role of immunoglobulin conformation in presenting the allotopes, we assayed their presence on mixed Igh-1^a/Igh-4^a heavy chain molecules. The Igh-1^a determinants were maintained, but the Igh-4^a determinants were lost. Taken together, our results indicate that genetic polymorphisms at the Igh loci generate an enormous antigenic complexity, much of which relies on tertiary and quaternary protein structure for expression.     

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.     

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.     

Clonal analysis of B- and T-cell responses to Ia antigens

Thirty-five Ia^k-specific monoclonal alloantibodies, derived from hybridomas constructed by fusion between mouse myeloma and spleen cells from A.TH alloimmune mice (I ^S anti-I ^k ), have been used to estimate the allotypic polyporphism of the I^k-gene products. Cross-blocking studies using 17 mAb specific for the I-A molecule indicated that six determinants, which were associated with the conventional specificities Ia.2 and Ia.19, were organized in at least three distinct polymorphic areas of the I-A^k molecules. Similarly, another group of six determinants, which did not correspond to previously described conventional Ia specificities, were found to be topologically heterogeneous. By contrast, the five epitopes associated with the Ia. 1 specificity were clustered into a single region of this molecule. In addition the potentiation of binding observed between mAb specific for topologically distinct epitope regions of the I-A^k molecule, suggested that the latter may undergo conformational changes after binding of a given mAb. A similar analysis of 17 mAb specific for the I-E^k molecule indicated that specificity Ia. 7 of the E chain (as defined in this series by eight mAb) was composed of three topologically distinct polymorphic areas, one of which is also spatially related to a complex cluster of eight new determinants of the I-E^k molecule. Finally, one mAb identified a so far undescribed shared determinant of the I-A^k and I-E^k molecules. The present results, which provide a new estimate of the allotypic polymorphism of the Ia^k antigens, are discussed with regard to their functional, biochemical, and evolutionary implications.Abbreviations used in this paper mAb monoclonal antibodies - FCS Fetal calf serum - Con A concanavalin A - H-2 mouse major histocompatibility complex - NMS normal mouse serum - SaCI Staphylococcus aureus Cowan I strain - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Monoclonal antibodies to feline sarcoma virus gag and fes gene translational products

A series of hybridomas have been isolated which produce monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strains of FeSV. Within these are representatives of several immunoglobulin classes including IgG₁, IgG_2a, IgG_2b, IgG_2c, and IgM. Antibody produced by one hybridoma recognizes immunologic determinants localized within an FeLV gag gene structural component (p15) common to polyproteins encoded by all three FeSV isolates whereas antibody produced by a second is specific for p30 determinants unique to P170^gag-fms. Additional hybridomas secrete antibody directed against v-fes-encoded determinants common to the Gardner and Snyder-Theilen FeSV-encoded polyproteins. GA P110^gag-fes and ST P85^gag-fes immunoprecipitated by antibody directed against p15 exhibit tyrosine-specific protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components.     

The mouse Igh-1 a and Igh-1 b H chain constant regions are derived from two distinct isotypic genes

Genetic and structural analyses of the mouse genes encoding constant region of immunoglobulin subclasses (Igh-C) have shown that recombination is rare within this cluster which is inherited as a set designated the Igh haplotype. Recent molecular analyses have demonstrated that either DNA exchanges or gene duplications have probably occurred during the evolution of this set of genes. In order to assess the generality of the duplication processes, the presence and expression of two allelic forms of the Igh-1 (2a) gene (Igh-1 ^a and Igh-1 ^b) were examined in a large panel of wild mice belonging to Mus musculus domesticus and Mus musculus musculus species. Our data indicate that certain M. m. domesticus animals and most animals in the M. m. musculus group coexpress the two allelic forms of Igh-1. Moreover, genetic studies show that these two immunoglobulin types are encoded by tandemly arranged genes. We propose that wild mice, from which laboratory mice are derived, carry three isotypic 2 genes (Igh-1 ^a, Igh-1^b, Igh-3), and these have given rise to the two isotypes seen in laboratory strains by a deletion/insertion mechanism.     

Crystallization of intact monoclonal antibodies

Attempts were made to crystallize four monoclonal antibodies, one IgG_2ak and three IgG_1k. Using a PEG 3350 screen combined with detergents, and developed from our experiments with an IgG_2ak antibody specific for canine lymphoma cells,^1,2 crystals have now been obtained of two of these four immunoglobulins, an antiphenytoin and an antiphenobarbital antibody. A complex between the antiphenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG_1k antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic, with unit cell dimensions a = 67 Å, b = 193 Å, c = 74 Å, and β = 110°. These crystals have an entire IgG_1k molecule as the asymmetric unit and they diffract to at least 3.2 Å resolution. © 1995 Wiley-Liss, Inc.     

Monoclonal antibodies to a nitroxide lipid hapten

The isolation and characterization of a hybridoma cell line producing a monoclonal IgG₁ antibody against a spin-label nitroxide group is described. The antibody recognizes a synthetic hapten containing linked dinitrophenyl and 2,2,6,6-tetramethylpiperidinyl 1-oxy groups, having an affinity of 3.6±1.0·10⁶ M^?1 for the soluble hapten at 25°C. The antibody binds to phospholipid vesicles containing 2 mol% of spin label-derivitized lipid (lipid hapten) with an affinity of 1.5±0.2·10⁸ M^?1. This monoclonal IgG₁ mediates the binding of hapten-bearing lipid vesicles to mouse macrophage RAW264 cells bearing Fc receptors. The cellular responses to this binding are similar to those observed previously using polyclonal rabbit anti-hapten IgG. As with the heterogeneous antibodies, the monoclonal IgG₁ is more efficient in mediating cellular uptake when the vesicles are in the ‘fluid’ physical state (dimyristoylphosphatidylcholine at 37°C) compared to ‘solid’ (dipalmitoylphosphatidylcholine at 37°C). Despite the enhanced binding of ‘fluid’ phospholipid vesicles to cells, only the ‘solid’ vesicles triggered a significant respiratory burst in RAW264 macrophages.     

The IgG2a antibody response to thyroglobulin is linked to the Igh locus in mouse

The IgG-subclass usage by several strains of mice in the response to immunization with mouse thyroglobulin (mTg) was examined in the experimental autoimmune thyroiditis model. While the subclass usage by most mouse strains was similar, the Igh^b allotype-bearing mice consistently produced lower IgG2a levels to mTg. Using CBA-Igh ^b congenic and recombinant inbred strains of mice, the lower level of IgG2a in the Igh^b mouse was mapped to the Igh locus. The regulation of IgG2a appeared to be cis controlled, as the CBA x C57BL/6F₁ mouse also produced reduced IgG2a of the Igh^b (B6) allotype but not Igh^j (CBA) allotype.     

Higher cytolytic efficiency of an IgG2 alpha than of an IgG1 monoclonal antibody reacting with the same (or spatially close) determinant on a human high-molecular-weight melanoma-associated antigen

Serological and immunochemical assays showed that the monoclonal antibody (MoAb) 225.28S, an IgG_2α, and the MoAb 653.40S, an IgG₁, react with the same (or spatially close) antigenic determinant expressed on a set of molecules carrying a high-molecular-weight human melanoma-associated antigen. Neither monoclonal antibody mediates complement-dependent lysis of cultured melanoma cells, but both of them specifically mediate lysis of target cells in an antiglobulin cytotoxic assay and in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. In the latter two assays the IgG_2α displays a higher lytic activity than the IgG₁. The differential lytic activity of the IgG_2α and IgG₁ monoclonal antibodies was detected also when the sensitivity of the ADCC assay was increased either by boosting the cytolytic activity of the effector cells or by enhancing the susceptibility to lysis of target cells.     

T cell allotypic determinants encoded by genes linked to the immunoglobulin heavy chain locus. I. Establishment of monoclonal antibodies against allotypic determinants

Monoclonal alloantibodies for T cell allotypic determinants were obtained by hybridizing SP-2 tumor cells with BALB/c (H-2d, Igh-1a) spleen cells, which had been repeatedly immunized with Con A-stimulated CB-20 (H-2d, Igh-1b) spleen cells. It was found that these monoclonal anti-CB-20 antibodies detect the new allotypic determinants (distinct from the B cell Igh-C region determinant) expressed only on the augmenting or suppressor T cells. Genetic analysis of these antigenic determinants revealed these antibodies react with the gene products located on the telomeric side chromosome of the Igh variable region gene (Igh-V) cluster. These antibodies when given in vivo caused a modification of antibody production. The antibody activity was absorbed by Con A-stimulated B10.BR (H-2b, Igh-1b), C57BL/6 (H-2b, Igh-1b), CWB (H-2b, Igh-1b), CB-20 (H-2d, Igh-1b), and BAB-14 (H-2d, Igh-1b) spleen cells, but not by Con A-stimulated C3H.SW (H-2b, Igh-1j), BALB/c (H-2d, Igh-1a), A/Sn (H-2a, Igh-1e), and C.AL-20 (H-2d, Igh-1d) spleen cells. In addition, in vivo these monoclonal antibodies modified anti-SRBC antibody production only in Igh-1b allotype-bearing mice. One monoclonal antibody reacted with 4-hydroxy-3-nitrophenyl acetyl- (NP) hapten-specific augmenting T cells, and the other two batches of monoclonal antibodies reacted with NP-specific suppressor T cells of NP-mediated cutaneous responses. A mapping study with these recombinants limits the gene coding for the T cell-specific determinants to a gene within the variable region to the telomeric side of NP-VH and to the centromeric side of prealbumin. This segment is inclusive of all immunoglobulin genes, the region Owen named IgT-C, and a histocompatibility gene (H-Ig).     

Immunoglobulin isoantigens (allotypes) in the mouse

Murine antisera raised against allogeneic lymphoid cells often contain antibodies to IgM allotypes. Rarely, allotypic antibodies to IgM have been found after immunization withB. pertussis anti-B. pertussis conjugates. Using both types of antibodies, we have defined a new constant-region locus for both secreted and membrane-bound chains. This locus,Ig-6, is closely linked to the previously described H-chain constant-region loci (Ig-1 throughIg-5) and is subject to allelic exclusion. We have identified three alleles and four antigenic specificities ofIg-6.Authors listed alphabetically     

Localization of murine Igh-1a allotypic determinants by using a panel of mouse myeloma variant immunoglobulins

A number of monoclonal antibodies are available that are reactive with distinct mouse immunoglobulin allotypic determinants. By determining which ones are present on a panel of hybrid IgG2b-IgG2a immunoglobulins, we have localized some of the allotypic determinants present on the IgG2a heavy chain of the "a" allotype (Igh-1a proteins). In particular, one group of determinants--Ig(1a)9.8 (20.6B8), 17.2 (20.19.2), and 14.4 (21.74.4)--has been placed in the CH2 domain. A second group--Ig(1a)8.3 (20.8.3), 21.2 (20.11.2), and 15.3 (21.66.3)--is located in a segment spanning the C terminal 8 residues of the CH2 domain and the complete CH3 domain.     

Genetic chasing of T helper cell idiotype and allotype genes

The present experiments were performed to study whether the genes responsible for the expression of T-cell idiotypes and allotypes could be mapped in relation to immunoglobulin (Ig) heavy chain V- and C-genes. Use was made of our antiserum 5936, which detects idiotypes in B6 anti-B10.BR sera and on Lyt-1⁺, 2.3^–B6 anti-B10.BR T-cell populations, and antiserum 6036, which detects allotypes on Lyt-1⁺, 2.3^–B6 T cells, but which does not react against Ig. The reactivity of these antisera with T cells from (B6 x C3H.OH) x C3H.OH backcross mice and CBA-allotype congenic B6 mice was investigated because 5936 idiotypes and 6036 allotypes appeared to be associated with Igh-1 ^b genes (B6) and not with Igh-1 ^b genes (C3H.OH, CBA). Our results will show, first, that 5936 idiotypes on Lyt-1⁺, 2.3^–B6 anti-B10.BR T cells are synthesized by genes linked to Igh-1 ^b allotype genes and they are situated either within Ig heavy chain V-genes or centromeric to them. Second, our results will show that 6036 allotypes on Lyt-1⁺, 2.3^–B6 T cells are produced by genes also linked to Igh-1 ^b -allotype genes, and the 6036 allotype genes are situated between Ig-VH and prealbumin genes.Abbreviations used in this paper BCGF B cell growth factor - B6 C57B1/6 - C_H constant region of immunoglobulin heavy chain - Con A concanavalin A - FCS fetal calf serum - Id idiotype - Ig immunoglobulin - LPS lipopolysaccharide - M mouse - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MRBC mouse red blood cells - NMS normal mouse serum - NP nitrophenacetyl - NRS normal rabbit serum - PFC plaque forming cell - R rabbit - Tcf T cell factor - Tcr T cell receptor - TNP Trinitrophenol - V_H variable region of Ig heavy chain Definitions of terms used in this paper: T-cell idiotypes, structures on T-cell membranes or released T-cell molecules detected by an anti-idiotypic antiserum (5936) produced against specific immunoglobulin idiotypes. The 5936 T-cell idiotypes are related to the specific binding of IA^k gene products by certain Igh-1^b T cells. T cell allotypes, structures on T-cell membranes or released T-cell molecules detected by an antiserum (6036) produced against 5936 idiotype-bearing T-cell molecules. The 6036 T-cell allotypes are related to the binding by Igh-1^b T cells of all Ia gene products tested, and they are non-cross-reactive with immunoglobulin allotypes.     

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     

Tumor localization of Lewis lung carcinoma with radiolabeled monoclonal antibodies

Summary Mouse 6D6 IgG_2a and 5B5 IgM monoclonal antibodies which specifically bind murine lung carcinoma cells (3LL cells) were injected to healthy and tumor-bearing mice. In vivo localization was analyzed by counting the tissue radioactivity and by external gamma ray scintigraphy at various times after IV injection of ¹²⁵I- or ¹³¹I-labeled antibodies. The clearance of the two monoclonal antibodies was not modified by the presence of the tumor, and the 6D6 IgG_2a was cleared at a rate slower than the 5B5 IgM. Both antibodies gave a high specific uptake at the tumor level; the tumor-to-healthy tissue ratios were higher with the 6D6 IgG_2a than the 5B5 IgM; unspecific mouse immunoglobulins (IgG₂) did not localize in the tumor. The amount of 6D6 IgG_2a antibody still associated with the tumor after 2 days following IV injection was 10 times higher than that of 5B5 IgM, and was still high enough to localize the tumor after 5 days.Imaging experiments confirmed the ability of 6D6 IgG_2a to detect the presence of tumor cells. The targeting kinetics determined by computer analysis of camera images indicated a rapid targeting of antibodies in tumor with a maximal concentration after 4–6 h; after 48 h the background was quite low and the 6D6 IgG_2a appeared to be specifically localized in the tumor.     

Monoclonal Antibodies to Spirosin of Yersinia enterocolitica and Analysis of the Localization of Spirosome by Use of Them

Two hybridomas producing monoclonal antibodies (MAbs) were prepared by fusing myeloma cells (Sp2/0-Ag14) with mouse spleen cells immunized with purified spirosin from Yersinia enterocolitica SYT-11–72 (YE72). The antibodies produced by them were designated MAbs-S5 and S27. They were IgG_2a and IgG₁, respectively, both with χ light chains. MAbs-S5 and S27 reacted specifically with spirosin from YE72. On Western blotting after limited proteolysis with Staphylococcus aureus V8 protease, YE72 spirosin revealed peptide fragments of 35 and 37 kDa reacting markedly with MAb-S5, which suggested the presence of an antigenic determinant on these fragments. By cellular fractionation of YE72 and subsequent EIA and Western blot analysis, spirosome was shown to be present in the cytoplasm of YE72.     

In vitro inhibition of the helper activity of GAT-specific T-cell lines by a syngeneic anti-idiotypic serum: preferential effect on the IgG response

We described in this paper the characteristics of a syngeneic anti-idiotypic serum made in BALB/c against BALB/c anti-poly (Glu⁶⁰ Ala³⁰ Tyr¹⁰) (GAT) antibodies. This serum recognizes idiotypic determinants present in all anti-GAT sera whatever the allotypic markers of the mice used to prepare the sera. The functional effect of this serum on two helper cell lines is also described. Cell line BDF₁/52 was obtained from GAT immunized lymph node cells (LNC). Cell line BDF₁/E₃ was selected from splenic T-cells educated in vitro on GAT-pulsed adherent cells. Both lines were propagated in presence of filler cells, antigen, and medium containing T-cell growth factor(s) from splenic cells activated with concanavalin A. Both cell lines exhibit a helper activity as measured by the plaque-forming cell (PFC) response they induce in vitro in the presence of DNP-GAT and DNP sensitized B cells. Their helper activity is specific and they require a hapten-carrier bridge to activate B cells. These lines are able to induce IgG₁, IgG_2a and IgG_2b anti-TNP PFC. Syngeneic anti-idiotypic serum B 658 inhibits specifically the function of these two lines but does not affect the helper activity of an OVA-specific T-cell line. The blocking activity of the serum can be adsorbed on a hybridoma protein with anti-GAT activity. This inhibition affects more dramatically the IgG₁ response than the IgG_2a and IgG_2b responses.     

Selective toxicity of neocarzinostatin-monoclonal antibody conjugates to the antigen-bearing human melanoma cell line in vitro

Summary Monoclonal antibodies (IgG₁) against high molecular weight antigen A-1-43 on human melanoma cell line A-375 were successfully linked to the anti-tumour protein neocarzinostatin (NCS) using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The conjugate retained both the reactivity of the antibody and the toxicity of the drug. The antigen-bearing cell line A-375, antigen-lacking cell line MeWo and normal skin fibroblasts were exposed to NCS-monoclonal antibody conjugates. As negative control, cells were also treated with free NCS and NCS coupled to normal mouse IgG₁ antibodies. Inhibition of ³H-thymidine uptake after treatment was used to measure the biological activity of the cytotoxic drug complex or substance, respectively.Comparing the inhibition dose for 50% uptake (ID₅₀) it was found that the monoclonal antibody-drug complex is about 100 times more toxic for the antigen-bearing cell line than free NCS or normal mouse IgG₁-NCS. This high toxicity is due to a local increase of drug concentration on these cells. With the two cell lines lacking the appropriate antigen no significant differences in the ID₅₀ values were observed. A selectivity factor of 40–50 was obtained by comparing the cytotoxic effect of the monoclonal antibody-NCS conjugate upon the antigen-bearing as opposed to the antigen-lacking cell type. These data demonstrate, that the toxicity of NCS can be directed by monoclonal antibodies to human tumour cells carrying the corresponding surface antigen.     

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.