Genetic 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 reactivities with immunoglobulins of the inbred mouse strains. Comparison with serology charts indicates that many of these monoclonal antibodies detect allotypic specificities previously defined by conventional antisera; others define previously undescribed specificities. Strain and isotype distribution allows us to assign five new allotypic specificities to Igh-1 and three new specificities to Igh-3. In addition, on the basis of reactivity with the monoclonal antibodies, we have defined a new Igh haplotype in SWR/J mice, Igh ^p.Abbreviations used in this paper Igh immunoglobulin heavy chain - SDS sodium dodecyl sulfate     

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.     

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.     

Evidence for structural homology between murine and human Ia antigens

The serological cross-reactivity and the structural homology of murine and human Ia alloantigens were analyzed. Both normal human peripheral blood B lymphocytes and chronic lymphocytic leukemia (CLL) cells were shown to be lysed in the presence of complement by both murine anti-Ia and human anti-HLA-DR alloantisera. A mouse A.TH anti-A.TL (anti-I ^k ) alloantiserum reacted with determinants expressed on all of the 20 normal human B cell populations tested. Only 3 of these 20 B cell populations were lysed with an A.TL anti-A.TH anti-I ^s alloantiserum. The frequency of cytotoxic cross-reactivity concordant with anti-I ^k appears to be greater for anti-I-EC ^k than for anti-I-A ^k alloreactivity. An immunochemical analysis demonstrated that Iaα-chain andβ-chain polypeptides may be immunoprecipitated from CLL cell lysates by either a mouse anti-I ^k alloantiserum or various human anti-HLA-DR alloantisera. The Ia molecules detected with the mouse and human antisera are coprecipitable as revealed by one-dimensional gel electrophoresis. Two-dimensional gel electrophoresis studies indicated that the human CLL cell Ia antigens analyzed possess considerable molecular heterogeneity. They are structurally more similar, with respect to molecular size and charge, to mouse Ia antigens determined by the murineH-2-linkedI-EC subregion rather than theI-A subregion. The structural, genetic and functional implications of these findings are discussed.     

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.     

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     

Biochemical characterization of murine Ia antigens with xenoantisera

Rabbit anti-Ia sera was produced by immunization with detergentsolubilized extracts from splenic, lymph-node and thymus cells. The antisera contained activity against H-2 as well as Ia molecules. By a sequential immunoprecipitation assay it was shown that the rabbit anti-mouseH-2 ^s serum precipitated a second Ia molecule in theH-2 ^s haplotype. Previous studies with alloantisera have shown only one Ia molecule associated with this haplotype. Sequential precipitations with alloantiserum against the wholeI region were used to show that this second Ia molecule is coded by genes within theI region. Since only I-A- and I-E-region coded molecules are immunoprecipitable in most haplotypes, we presume that the rabbit antiserum could be identifying the I-E-subregion coded molecule in theH-2 ^s haplotype. The rabbit antiserum reacts with an isotypic specificity on the molecule. The studies suggest that theI-E subregion does exist in theH-2 ^s haplotype even though alloantiserum cannot be produced to identify allotypic variants associated with this subregion.     

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.     

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     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An (H-2k/H-2d)F1 sarcoma cell line was subjected to immunoselection using ascites fluid from a mouse growing a hybridoma secreting an anti H-2Kk antibody.One hundred random clones were picked from the surviving population and screened by direct cytolysis using the hybridoma antibody or alloantisera against H-2Kk and H-2Dk. Fifty-nine clones were resistant to all three antisera, indicating that they no longer expressed the entire H-2k haplotype. Thirty-two were resistant to the ascites and to the anti H-2Kk alloantiserum, but sensitive to the anti H-2Dk serum, indicating that they had lost H-2Kk antigen, but retained H-2Dk. Nine clones were sensitive to the alloantisera, but resistant to the hybridoma, indicating that, though they retained the product(s) recognized by the alloantiserum against H-2Kk, they had lost the site(s) that bound the hybridoma antibody. Quantitative absorption assays using lymph-node cells from young BALB.K (H-2Kk) mice as targets show that one representative clone from the last group absorbs the anti H-2Kk activity in the alloantiserum. This implies that the sensitivity of the variant clone to the alloantiserum is not due to contaminating anti C-type virus antibodies in the serum. The possible implications of these data are discussed.     

Serological expression after sequential double transfection with purified HLA-11 gene of mouse fibroblasts carrying human beta-2 microglobulin

A genomic cosmid library constructed from DNA from a genotyped individual (JF = HLA-A11, Cw–, B38/A26, Cw7, B51) was screened for clones containing class I histocompatibility genes. Among these clones, one was found to carry a 4.8 kb Hind III fragment which is highly correlated with HLA-A11. This clone was used to transfect LMTK⁺ cultured mouse fibroblast transformants expressing human beta-2 microglobulin. The human beta-2 microglobulin heavy chain-associated determinant was positively detected by the M18 monoclonal antibody. HLA-A11 expression on these doubly transformed cells was specifically demonstrated by complement-dependent cytotoxicity with HLA-A11 + A3-specific but not with HLA-A3-specific monoclonal antibodies. Absorption studies with human alloantisera confirmed the presence on these cells of HLA-A11 determinants and of cross-reacting determinants which absorbed anti-HLA-A1 and –A3 alloantisera. The JF5-J27 transfected cell expressed both heavy and light chains of human class I histocompatibility genes.Abbreviations used in this paper ₂m beta-2 microglobulin - CTL cytolytic T lymphocytes - FCS fetal calf serum - HAT hypoxanthine-azaguanine-thymidine - kb kilobase pair - MHC major histocompatibility complex - MoAb monoclonal antibodies - PBL peripheral blood lymphocytes - PEG polyethylene glycol - r correlation coefficient This study is dedicated to the memory of Jean-Jacques Metzger.     

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.     

Structural and evolutionary comparisons of four alleles of the mouse Igk-J locus which encodes immunoglobulin kappa light chain joining (J K ) segments

The Igk-J locus of the mouse encodes the immunoglobulin light chain joining (J) segments. Four Igk-J alleles have been described on the basis of restriction enzyme length polymorphisms. The nucleotide sequences of the Igk-J ^a allele (type strain, C.C58), Igk-J ^c allele (type strain, SJL/J), and Igk-J ^d allele (type strain, SK/CamRk) have been determined and are compared with the previously reported Igk-J ^b allele sequence (type strain, BALB/c). The mouse sequences are also compared with published sequences for rat and human J _k sequences. Far more differences were found between the Igk-J ^a allele and the other mouse alleles than between any two of the latter. These result in two amino acid substitutions which distinguish the J2 and J3 ¹ segments of the Igk-J ^a allele from the other three alleles. Use of the Phylogenetic Analysis Using Parsimony program to generate a phylogenetic tree strongly indicates that after divergence from the rat ancestor, there appears to have been an early split between the Igk-J ^a allele and the evolutionary precursor of the other mouse alleles. There also appears to have been far less divergence from the ancestral condition in the Igk-J ^a allele than in the other alleles. Also, the presence of only one convergent mutation among the four mouse alleles provides strong evidence against any crossing over within the Igk-J locus during the history of these alleles. Finally, the differences in rates of evolution of the Igk-J alleles are in marked contrast to the relatively uniform rates of divergence of four alleles of a mouse V _k gene, Igk-VSer.     

Evolution of pseudogenes in the immunoglobulin V H-gene family of the mouse

A quantitative analysis of the complexity of the J558 V _H -gene family in the mouse immunoglobulin heavy chain (Igh) gene locus has been performed. Considerable variations in the degree of complexity are observed in various Igh haplotypes derived from laboratory mice and wild mice. The BALB/c strain shows the highest degree of complexity of the J558 V _H -gene family when all mice are compared. Multiple gene duplications seem to have occurred in the BALB/c-derived J558 V_H-gene family less than 1–2 million years ago. This dating is supported by the divergence in coding and flanking regions of three strongly homologous V _H -region genes. Two of these genes were generated by the duplication of a pseudogene about 1.5 × 10⁵ years ago. A recent expansion of the J558 V _H -gene family and therefore little time for evolutionary drift may explain why most of the pseudogenes in this family exhibit a largely intact structure. We also describe two V _H -region genes which represent older pseudogenes in states of progressive disintegration.     

Generation of cytotoxic T lymphocytes by the H-2-encoded E molecules

Primary CML was generated in strain combinations 4R anti-2R, R107 anti-3R, 7R anti-9R, and GD anti-R101 — combinations differing only in the chromosomal interval between the I-A subregion and the Ss locus. No CML could be obtained in any of the reciprocal combinations of these strains. This unidirectionality of the CML reaction correlates with the expression or nonexpression of the E molecules encoded by this interval: the reaction occurred in combinations in which the responder strain lacked and the stimulator strain expressed the E molecules in the cell membrane. The CML reaction was positive when tested on LPS-stimulated blast cells but weak on Con A-stimulated blasts and negative on la-negative tumor cells. The reaction could partially be inhibited by monoclonal antibodies to the Ia.m7 determinant presumably carried by the E chain; it was not inhibited by monoclonal antibodies specific for Ia determinants carried by the A molecule. Cytotoxic lymphocytes specific for a particular combination of E and E chains reacted with all cells expressing the particular E chain, no matter what the origin of the E chain associated with the E chain was. Attempts to generate cytotoxic lymphocytes specifically reactive with allotypic determinants on E chains failed. In F₁ hybrids expressing one type of E chain and two types of E chain, the single E chain was found to associate with both chains, producing two types of E molecule. We conclude from these experiments that the CML determinants detected in the strain combinations used are encoded by the same loci as those coding for the serologically detectable la determinants. The CML determinants are carried by the E chains; the E chain does not contribute in any way to the specificity of determinant recognition by the cytotoxic lymphocytes. No evidence for allotypic variation of the E chain as detected by the CML assay could be found in this study.     

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.     

Immunochemistry of the HLA class II molecules isolated from a mouse cell transfected with DQ alpha and beta genes from a DR4 haplotype

Cells from a mouse B lymphoma were transfected by DQ alpha and DQ beta genes derived from a DR4 haplotype. Quantitatively, the resulting expression of human class II molecules was similar to that of human B lymphoblastoid cell lines. Qualitatively, the transformant class II molecules differed from normal class II molecules in their carbohydrate moiety. As for their antigenic specificity, they were shown to carry two determinants previously identified on DQ molecules controlled by DR4 haplotypes, i. e., DQw3 and DCHON. The transformant molecules did not carry a third DR4-associated specificity, DC5 (equivalent to TA10), and must possess a structure allelic to DC5. However, no corresponding alloantigenic specificity was detected by a screening of relevant alloantisera.     

Genetic analyses of alloreactions between recently wild and classical inbred strains of syrian hamsters: Evidence in favor of a major histocompatibility complex

Cytotoxic alloantisera were raised between recently wild and classical inbred strains of Syrian hamsters. Antisera produced by immunizing the classical inbred strains with tissue from the partially inbred, recently wild hamsters detect several specificities shared between the classical and recently wild strains. Reciprocal mixed lymphocyte reactions between the two different groups of hamsters suggest that the new source of hamsters possesses several unique MLR phenotypes which may represent new Hm-1 haplotypes. Moreover, several recently wild strains express MLR phenotypes quite similar if not identical to the Hm-1 ^a haplotype of the inbred strain, MHA. Genetic analyses of alloreactions between domestic inbred and recently wild strains suggest that a single locus or chromosomal region encodes the allodeterminants that induce strong MLR reactivity. Six unique MLR phenotypes have been defined which most likely represent haplotypes of the hamster MHC equivalent, Hm-1. Genetic linkage studies indicate that some alloantisera detect determinants encoded by loci closely linked to the MLR locus, and therefore define Hm-1 determinants. Moreover, other alloantisera recognize determinants encoded by a locus that is unlinked to Hm-1. These studies suggest that Syrian hamsters express a polymorphic MHC equivalent, Hm-1, which encodes determinants that induce both cell-mediated and humoral alloreactivity.     

Antigenic properties of T cell antigen-specific receptors isolated from the surface of rabbit and mouse spleen and lymph node cells

The presence ofa allotypic determinants was tested in fractions obtained by gel filtration of antigen-specific receptors isolated by immunoadsorption from lymphoid cells of antigen-stimulateda3-3 rabbits. This technique, as well as the inhibition of the reaction of isolated receptors with anti-T cell receptor antisera (anti R) by anti-a3 antibodies failed to demonstrate the presence of a allotypie determinants. The inhibitory effect of antigen-specific receptors isolated from the lymphoid cells of stimulated A/J mice on the cytotoxic effect of anti-Ia antibodies on mouse spleen cells in the presence of rabbit complement was tested. All preparations inhibited the cytotoxic reaction with the average effectivity of 60%. In order to confirm the presence of Ia determinants on the rabbit and mouse T cell receptor molecules it was shown that the reactions of three anti-R antisera with 12 different receptor preparations were inhibited by anti-la antibodies. SDS-PAGE analyses of¹²⁵I-labelled mouse specific receptors and the precipitate obtained by anti-R antisera showed that T cell receptors were present in fractions with molar mass 100 and 85 kg/mol. The molar mass of the former fraction after reduction and alkylation was 45 kg/mol.     

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.