Molecular characterization of the A/J J558 family of heavy chain variable region gene segments

The nucleotide sequences of the coding as well as the flanking regions of 11 A/J J558 heavy chain variable region (VH) gene segments are presented. Among these J558 VH segments was the unrearranged germline VH gene segment recruited in the predominant A strain-specific anti-arsonate response. Three other VH gene segments that are greater than 92% related to the p-azophenylarsenate (Ars) A VH gene segment were also isolated. Detailed analysis of the nucleotide sequences of these as well as the remaining seven J558 VH gene segments reveal that the J558 VH gene family is composed of distinct, but related, J558 VH subfamilies. Deletion mapping analyses were used to position the Ars A VH gene segment proximally with respect to the DH-JH clusters within the J558 VH gene family and distally with respect to its own J558 subfamily. The documentation of J558 VH subfamilies is discussed in the context of J558 VH family evolution and diversification.     

Variable region sequence analysis of anti-DNA antibodies: evidence for a family of closely related germ-line VH genes encoding lupus autoantibodies.

Cloning and sequencing of the V regions of the anti-DNA monoclonal antibodies (mAbs), H438 and H130, indicate that H438 is encoded by a J558 VH gene, a single D region nucleotide, and unmutated JH1, V kappa-1C and J kappa 1 genes, and the H130 L chain is encoded by a V kappa-21 subgroup gene J kappa 1 gene. Identification of VH438, which shared VH hybridization pattern with 6% of a panel of 352 MRL/lpr hybridomas, suggests that the frequency of J558 use among spontaneously activated B cells in MRL/lpr mice is greater than previously reported. The VHH438 J558 family gene is identical to VHPAR, which encodes the independently derived MRL/lpr autoantibody, MRP-2, and is highly homologous to the previously reported VHH130, which is identical to a BALB/c germ-line VH gene. Comparison of consensus sequences of homologous autoantibodies and previously reported restriction mapping suggest that a minimum of three highly related J558 germ-line genes encode lupus autoantibodies.     

Molecular analysis of heavy and light chains used by primary and secondary anti-(T,G)-A--L antibodies produced by normal and xid mice

The primary (1 degree) antibody response to (T,G)-A--L shows limited heterogeneity, consisting mostly of side chain-specific antibodies that bind GT and that express the TGB5 idiotype (Id). The secondary (2 degrees) response is very diverse: antibodies that bind the backbone A--L constitute a third of the response, and a high proportion of the side chain-specific antibodies do not bind GT and are TGB5 Id-. To provide a molecular basis for understanding this difference in repertoire expression, we analyzed the Ig genes used by heavy and light chains of 1 degree and 2 degrees side chain-specific anti-(T,G)-A--L hybridoma antibodies (HP). Southern blot restriction analysis and nucleotide sequence analysis of the expressed genes used by three TGB5 Id+ 2 degrees HP showed usage of three different VH genes in two VH gene families (36-60 and J558), different D segments, and two different Vk1 genes (the Vk1A and Vk1C subgroups). Thus, antibody heterogeneity in the 2 degrees response is contributed by combinatorial diversity of distinct germ-line genes. Nucleotide sequence analysis of the expressed genes used by TGB5 Id+ 1 degree HP showed use of highly homologous VH genes in the J558 VH gene family and highly homologous Vk1A genes. The majority of TGB5 Id+ 1 degree HP from different donors gave similar heavy and similar light chain gene rearrangements by Southern blot restriction analysis, after correction for known or potential J region differences. The combined nucleotide sequence and Southern blot restriction analysis data suggest that most 1 degree B cells use the same or very similar VH and Vk genes, i.e., the 1 degree response is paucigenic. Different D segments were used by the TGB5 Id+ 1 degree and 2 degrees HP that were sequenced, and there was no apparent correlation between TGB5 idiotypy and VH, D gene, or JH gene usage. However, all TGB5 Id+ HP sequenced used highly homologous genes from the Vk1 group. Expression of a Vk1 light chain correlates with, but is not sufficient for, TGB5 idiotypy, because one GT-binding, TGB5 Id- HP was found to use a Vk1C subgroup light chain. By Southern blot and nucleotide sequence analysis, the Vk genes used by two TGB5 Id+ 2 degrees HP from xid mice are highly homologous, if not identical to the Vk1A gene(s) used by 1 degree and 2 degrees Id+ HP from wild-type mice.     

Eleven MRL-lpr/lpr anti-DNA autoantibodies are encoded by genes from four VH gene families: a potentially biased usage of VH genes

The genes encoding 11 independently derived anti-DNA autoantibodies from the lupus-prone mouse strain, MRL-lpr/lpr, were examined with VH, D, and JH gene probes. These autoantibodies do not define new VH gene families, since all of the autoantibodies were encoded by VH genes from four of the nine known gene families. A minimum of nine different VH genes encoded this panel of 11 anti-DNA autoantibodies. These results are consistent with the stochastic use of the VH gene repertoire and the expression of multiple VH genes. However, the data is also consistent with a biased usage of the VH gene repertoire. First, two pairs of autoantibodies, one from the J558 family and one from the 7183 family, appear to express identical or closely related VH genes as determined by the position of two restriction enzyme sites 5' of the expressed VH genes. In addition, three autoantibodies that appear to be sister clones might define a third VH gene that is used repeatedly. Secondly, about 45% of the panel is encoded by the Q52 and 7183 families, which are the 3' most families. These families have been shown to be preferentially rearranged early in B cell ontogeny. This suggests that some anti-DNA autoantibodies might originate from a population of B cells that predominate early in ontogeny. An alternative hypothesis is that the potential bias in VH gene and gene family usage could be due to antigen selection. All four JH genes are expressed, although the JH1 gene appears to be underutilized in both expressed and unexpressed rearrangements. Two members of the panel that bind double-stranded DNA were encoded by two different VH gene families, the S107 family and the J558 family.     

Variable region cDNA sequences and antigen binding specificity of mouse monoclonal antibodies to isomaltosyl oligosaccharides coupled to proteins. T-dependent analogues of alpha(1----6)dextran

Four mouse hybridomas specific for alpha(1----6)dextran, 16.4.12E (IgA kappa, C57BL/6), 28.4.10A (IgM kappa, BALB/c), 35.8.2H (IgG1 kappa, BALB/c), and 36.1.2D (IgM kappa, BALB/c) were obtained by immunization with the T-dependent Ag isomaltohexaose or isomaltotriose coupled to keyhole limpet hemocyanin or to BSA. Immunochemical characterization of the hybridoma antibodies showed that 16.4.12E and 36.1.2D had cavity-type combining sites, recognizing the terminal non-reducing end of alpha(1----6)dextran, whereas 28.4.10A and 35.8.2H had groove-type sites, recognizing internal linear segments of the dextran. The V region cDNA of the H and L chains of the antibodies were cloned and sequenced. VH of 16.4.12E and VH of 36.1.2D belonged to the X24 and Q52 germ-line gene families, respectively. The VH and V kappa sequences of 16.4.12E and V kappa sequence of 36.1.2D were highly homologous to those of W3129, the only anti-alpha(1----6)dextran mAb with a cavity-type site thus far sequenced; 16.4.12E differed from W3129 in the D, JH, and J kappa. VH genes of 28.4.10A and 35.8.2H were homologous to those of several anti-alpha(1----6)dextrans with groove-type sites, but belonged to the J558 germ-line gene family, differed from the other J558 anti-alpha(1----6)dextrans, probably representing a different germ-line subfamily. The L chain sequence of 28.4.10A encoded by V kappa-Ars and J kappa 2 was almost identical to other groove-type anti-alpha(1----6)dextrans obtained by immunizing with the T-independent glycolipid Ag, stearyl-isomaltotetraose. Use of T-dependent Ag such as isomaltosyl oligosaccharide-protein conjugates provides an additional parameter for probing the fine structure of antibody combining sites and evaluating the V-gene repertoire of anti-alpha(1----6)dextrans.     

Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri.

A lambda gt11 expression library of Tn5-tagged invasion plasmid pWR110 (from Shigella flexneri serotype 5, strain M90T-W) contained a set of recombinants encoding a 60-kilodalton protein (designated IpaH) recognized by rabbit antisera raised against S. flexneri invasion plasmid antigens (J. M. Buysse, C. K. Stover, E. V. Oaks, M. M. Venkatesan, and D. J. Kopecko, J. Bacteriol. 169:2561-2569, 1987). Southern blot analysis of wild-type S. flexneri serotype 5 invasion plasmid DNA (pWR100) digested with various combinations of five restriction enzymes and hybridized with defined ipaH probes showed complex hybridization patterns resulting from multiple copies of the ipaH gene on pWR100. DNA sequence analysis of a 2.9-kilobase (kb) EcoRI fragment directing IpaH antigen synthesis in plasmid recombinant pWR390 revealed an open reading frame coding for a 532-amino-acid protein (60.8 kilodaltons); this size matched well with the estimated size of IpaH determined by Western blot analysis of M90T-W cells and maxicell analysis of Escherichia coli HB101(pWR390) transformants. Examination of the amino acid sequence of IpaH revealed a hydrophilic protein with six evenly spaced 14-residue (L-X2-L-P-X-L-P-X2-L-X2-L) repeat motifs in the amino-terminal end of the molecule. Southern blot analysis of HindIII-digested pWR100 DNA probed with defined segments of the pWR390 2.9-kb insert demonstrated that the multiple band hybridization pattern resulted from repeats of a significant portion of the ipaH structural gene in five distinct HindIII fragments (9.8, 7.8, 4.5, 2.5, and 1.4 kb). Affinity-purified IpaH antibody, used to monitor the expression of the antigen in M90T-W cells grown at 30 and 37 degrees C, showed that IpaH synthesis was not regulated by growth temperature.     

One heavy chain variable region gene segment subfamily in the BALB/c mouse contains 500-1000 or more members

Nucleic acid hybridization studies suggest that 500-1000 or more heavy chain variable (VH) gene segments related to one VH gene segment (J558) are present in the genome of the BALB/c mouse. The implications of this result regarding the overall size, sequence organization, and evolution of the mouse family of VH gene segments are discussed.     

A V region determinant (idiotope) expressed at high frequency in B lymphocytes is encoded by a large set of antibody structural genes.

Idiotope Ac38, a V region determinant of the lambda 1 chain-bearing, germ line encoded antibody B1-8, is expressed at high frequency (approximately 1/40) in lambda 1 chain-bearing B cells. Here, we describe the isolation of lambda-positive hybridomas from C57BL/6 mice which had been immunized with antibody Ac38, the antibody recognizing idiotope Ac38. In Northern blot analysis, mRNA isolated from 10 such hybridomas hybridizes with a cDNA probe from the VH gene expressed in the cell line B1-8. Amino acid sequence analysis of the VH regions of four of the hybridoma proteins reveals that they are all derived from related, though distinct, germ line VH genes. In one case the sequence data suggest that extensive somatic mutation has taken place. Only one of the four sequences derives from the same VH gene that is expressed in the cell line B1-8. Together with earlier evidence, the present data demonstrate that the Ac38 idiotope is a marker for at least five VH and three D region genes in the C57BL/6 germ line. This explains the high frequency at which this idiotope is expressed in the B cell population. In addition, our sequence determinations identify two VH genes in the C57BL/6 strain which are closely related (and possibly allelic) to two known BALB/c VH genes. One of these genes is the gene expressed in the BALB/c myeloma MOPC 104E.     

VH gene analysis of spontaneously activated B cells in adult MRL-lpr/lpr mice. J558 bias is not limited to classic lupus autoantibodies

To determine the genetic origins of lupus auto-antibodies, we analyzed the relationship between VH gene usage and auto-Ag-binding properties of 352 B cell hybridomas derived from MRL-lpr/lpr mice. The hybridomas were derived from neonatal, 1-month-old, 3-month-old, and 6-month-old mice. The experimental strategy provided that the hybridomas were monoclonal at initial evaluation, so the Ag binding and V gene frequencies of the entire population could be determined. Initially, 1032 Ig-producing hybridomas were evaluated for binding to six Ag; VH gene family use was determined in 119 anti-DNA and anti-rabbit thymus extract (RTE) antibodies (autoantibodies) and in 233 age-matched Ig that did not bind to any of the six Ag (nonbinders). Neonatal B cells, including cross-reactive IgM autoantibodies and nonbinder IgM, used relatively 3' VH genes. The majority of B cells in adult mice used VH genes of the J558 family. Although J558 use was significantly higher among the autoantibodies (anti-DNA and anti-RTE) than among the nonbinder Ig, this difference was due to a higher frequency of J558 use by 1-month-old mice. At 3 months, J558 use by the nonbinder Ig increased to the same frequency of J558 use as in the autoantibody population. J558 use in both groups of antibodies exceeded a previously reported estimation of J558 expression in the functional B cell repertoire of young adult MRL-lpr/lpr mice. Several subgroups of antibodies that share properties with pathogenic Ig, including IgG, cross-reactive Ig, and anti-dsDNA autoantibodies, demonstrated a marked preferential expression of the J558 family. These results suggest that there is an age-related bias in the activation of B cells using J558 VH genes in MRL-lpr/lpr mice that is under the influence of a selective force distinct from, or in addition to, an ssDNA or RTE auto-Ag-driven response.     

Organization and rearrangement of immunoglobulin M genes in the amphibian Xenopus.

Sequences of immunoglobulin (Ig) cDNA clones of Xenopus laevis show that at least three different VH families are expressed in association with different JH elements and different isotypes of Ig constant regions. In genomic Southern blot analysis, the VH probes for each family hybridize to a distinct set of multiple DNA fragments. In contrast, the genomic JH elements and the IgM constant region gene are localized in a single DNA fragment of approximately 15 kb. Genomic VH elements contain regulatory sequences similar to those in VH genes of shark, fish and mammals and have a leader peptide sequence that contains an intron; they encode the VH region until residue 95 and have heptamer--23-bp--nonamer motifs similar to the rearrangement signal sequences (RSS) in all other vertebrate VH elements. The six genomic JH elements so far sequenced have a nonamer--23-bp--heptamer motif at their 5' end. These RSS motifs imply the existence of DH elements. The comparison of cDNA clones that contain similar constant regions but different VH regions or JH elements suggest rearrangement events. This is shown by Southern blot analysis of erythrocyte and B cell DNA with a JH probe. Thus, the overall organization of the Xenopus Ig gene locus is similar to that of mammals but strikingly different from shark.     

Direct quantitative in situ hybridization studies of Ig VH utilization. A comparison between unstimulated B cells from autoimmune and normal mice

This study examines Ig VH utilization in murine lupus with emphasis on the relative contribution of 3' and 5' gene families. We used in situ hybridization with 35S-labeled ssRNA probes to detect VH expression in individual spleen cells. Cells were taken from unmanipulated animals, and were not stimulated in vitro. This approach allows analysis of VH usage among only those B cells which have undergone activation in vivo, while minimizing the potential for skewing in vitro. We compared usage of the 3' 7183 and Q52 families with the more 5' J558 family in adult NZB, MRL-lpr/lpr, and nonautoimmune NIH Swiss mice. VH utilization in the autoimmune strains was proportionate to VH family size, and was not biased toward the 3' families when compared with the Swiss repertoire. Moreover, 3' skewing did not develop in NZB mice with increasing age. Thus, systemic autoimmunity is not associated with impaired normalization of the adult repertoire away from the 3' bias of early ontogeny. Instead, our data support a stochastic model for VH gene usage in the activated B cells and plasma cells of adult lupus mice.     

Germ Line Variable Regions That Match Hypermutated Sequences in Genes Encoding Murine anti-Hapten Antibodies

We asked whether there are germ line immunoglobulin variable (V) segments that match sites of hypermutation in V regions encoding murine antibodies. Murine germ line DNA was probed with a panel of short deoxyoligonucleotides identical in sequence to segments of hypermutated V regions from hybridomas generated in the BALB/c response to the hapten 2-phenyloxazolone (Ox). Germ line sequences that match mutations in both heavy and kappa light chain V regions were identified, and clones of some of these germ line V segments were obtained. Comparison of these clones with hypermutated V regions revealed regions of identity ranging in size from 7 to over 50 nucleotides. In an effort to separate the effects of antigen selection from the mutagenic process, we also searched for matches to a panel of silent mutations in VH regions from germinal center B cells. Fourteen silent mutations occur among a collection of 36 hypermutated VH regions from two separate germinal centers of C57BL/6 mice stimulated with the hapten 4-hydroxy-3-nitrophenyl. Matches to nine of these silent mutations can be found among published sequences of C57BL/6 VH regions of the J558 family. Taken together, these data are consistent with the possibility that a template-dependent mutational process, like gene conversion, may contribute to somatic hypermutation.     

Complete amino acid sequence of the heavy-chain variable region from an A/J mouse antigen-nonbinding monoclonal antibody bearing the predominant arsonate idiotype

The 1F6 hybridoma protein, exhibiting the predominant cross-reactive idiotype (CRI) associated with the immune response to p-azophenylarsonate in A/J mice but failing to bind the hapten arsonate, was elicited following immunization with rat anti-CRI [Wysocki, L.J., & Sato, V. (1981) Eur. J. Immunol. 11, 832-839]. The dissociation of idiotype and antigen binding in this hybridoma provides an opportunity to determine structural features involved in antigen binding and idiotypic sites. The complete heavy-chain variable region (VH) amino acid sequence was obtained by automated Edman degradation of the intact chain and fragments due to CNBr cleavage, trypsin digestion, mild acid hydrolysis, and carboxypeptidase A digestion of a CNBr fragment. Comparison of the CRI+ arsonate-nonbinding 1F6 sequence with the CRI+ germ-line VH gene sequence reveals that the 1F6 heavy chain differs from the germ-line-encoded amino acid sequence at seven positions within VH [Siekevitz, M., Gefter, M. L., Brodeur, P., Riblet, R., & Marshak-Rothstein, A. (1982) Eur. J. Immunol. 12, 1023-1032]. The 1F6 VH appears to arise from the CRI+ germ-line VH by somatic mutation at at least seven amino acid residues, each of which could be due to a single nucleotide base change. The diversity (D) gene-encoded segment of 1F6 is similar to that of the CRI+ antigen-binding hybridoma 36-65 except for two amino acid substitutions. Further, the idiotype (CRI) is preserved despite use of a JH4 gene segment in 1F6 as compared to JH2 in all CRI+ arsonate-binding hybridomas examined to date.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequences of the VH and VL regions of murine monoclonal antibodies against 3-fucosyllactosamine

Many mAb that bind the carbohydrate antigenic determinant 3-fucosyl-lactosamine (3-FL), Gal beta 1-4[Fuc alpha-3]GlcNAc-R have been raised in BALB/c mice, and we are studying the structure and regulation of these antibodies. In this report, we present the first information about their amino acid sequences and the Ig gene segments used to encode them. V regions of the H and L chains of three anti-3-FL antibodies, PMN6, PMN29, and PM81, were sequenced by a combination of mRNA and amino acid sequencing. The L chain sequences of PMN6 and PM81 antibodies indicate that their VK and JK regions are encoded by VK24B and JK1 germ-line genes, respectively. The nucleotide and amino acid sequences of the H chains suggest that the three anti-3-FL antibodies are encoded by the VH441 gene segment of the X24 VH family, and this conclusion was supported by Southern filter hybridization with VH441 and JH3-JH4 probes. PMN29 has at least 11 amino acid substitutions, which is an unusually large amount of somatic mutation for an IgM antibody. Previous analyses of BALB/c genomic libraries with VHX24 and VH441 probes make it unlikely that this VH family contains additional germ-line genes, but this possibility cannot be excluded. All three antibodies use the DQ52 and JH4 gene segments. The single VH and VL gene segments used to encode the anti-3-FL antibodies is in contrast to the multiple VH and VL segments used by antibodies against other carbohydrate Ag such as alpha 1-6 dextran and group A streptococcal carbohydrate. VH441 also encodes the VH regions of antibodies against galactan and levan (beta 2-6 fructosan). The similarities among VH segments of antibodies against 3-FL, levan, and galactan, and the striking differences in their CDR3 sequences, suggest that CDR3 plays an important role in the formation of the Ag binding site. The use of a single VH segment from the smallest VH gene family by antibodies against at least three different carbohydrate determinants is noteworthy. It raises the possibility that the amino acid sequence encoded by VH441 has some general structural features that make it particularly well adapted for binding to carbohydrate sequences.     

Cloning, nucleotide sequence, and expression of the flavodoxin gene from Desulfovibrio vulgaris (Hildenborough)

The gene coding for the flavodoxin protein from Desulfovibrio vulgaris (Hildenborough) has been identified, cloned, and sequenced. DNA fragments containing the flavodoxin gene were identified by hybridization of a mixed synthetic heptadecanucleotide probe to Southern blots of SalI-digested genomic DNA. The nucleotide sequences of the probe were derived from the published protein primary structure (Dubourdieu, M., LeGall, J., and Fox, J. L. (1973) Biochem. Biophys. Res. Commun. 52, 1418-1425). The same oligonucleotide probe was used to screen libraries (in pUC19) containing size-selected SalI fragments. One recombinant, carrying a 1.6-kilobase (kb) insert which strongly hybridizes to the probe, was found to contain a nucleotide sequence which codes for the first 104 residues of the amino-terminal portion of the flavodoxin protein sequence but lacked the remainder of the gene. Therefore, a PstI restriction fragment from this clone was used as a probe to isolate the entire gene from a partial Sau3AI library in Charon 35. Of the plaques which continued to hybridize strongly to this probe through repeated screenings, one recombinant, containing a 16-kb insert, was further characterized. The entire flavodoxin gene was localized within a 1.4-kb XhoI-SacI fragment of this clone. The complete nucleotide sequence of the structural gene for the flavodoxin protein from Desulfovibrio vulgaris and flanking sequences which may include promoter and regulatory sequences are reported here. The cloned flavodoxin gene was placed behind the hybrid tac promoter for overexpression of the protein in Escherichia coli. Modification to the 5'-end of the gene, including substitutions at the second codon, were required to obtain high levels of expression. The expressed recombinant flavodoxin protein is isolated from E. coli cells as the holoprotein with physical and spectral properties similar to the protein isolated from D. vulgaris. To our knowledge, this is the first example of the expression of a foreign flavodoxin gene in E. coli using recombinant DNA methods.     

Detection and characterization of idiotype-specific enhancing cells generated in mice immunized with idiotype

Cellular interaction between MOPC-104E (M104E) cross-reactive idiotypic (CRI) antibody-producing B lymphocytes and lymphocytes generated by immunization with the relevant idiotype, M104E, was investigated. Adoptive transfer of M104E idiotype-primed and normal spleen cells into 600R x-irradiated syngeneic recipient mice resulted in striking enhancement of the M104E-CRI positive antibody response upon simultaneous immunization of recipients with dextran B1355S. The enhancement was not attributable to a simple additive effect but was due to synergistic cooperation between the two lymphocyte populations. This synergistic enhancement of the anti-idiotype immune cells producing CRI antibody was specific for MOPC-104E CRI, and was reproducible in an in vitro culture system. Because of the cellular characteristics of the enhancing cells, they were assumed to be B lymphocytes specific for the corresponding idiotype, since the activity was not abrogated by treatment with anti-Thy-1, anti-Lyt-1, anti-Lyt-2, or anti-brain-associated theta antisera plus complement, but was eliminated by means of a planning method using a rabbit-anti-mouse immunoglobulin-coated or idiotype-coated dish. The mechanisms of interaction between the CRI-positive B cells and anti-idiotypic B cells in response to the thymus-independent antigen dextran B1355S are discussed.     

Strain-dependent expression of VH gene families

The tremendous diversity of the antibody specificity repertoire stems from the ability of each developing B cell to select one out of many possible variable, diversity, and joining gene segments by specific rearrangement of the DNA. The mechanism by which V region gene segments is selected is not known. Moreover, evidence for both random and nonrandom expression of VH genes in mature B cells has been presented previously. In this report, the technique of in situ hybridization is used to accurately measure at the single cell level VH gene family expression in LPS-induced cells from several strains. In this way, at least one-third of the B cells are stimulated and a large sampling of activated splenocytes from each strain analyzed. The use of in situ hybridization eliminates any potential biases resulting from transformation protocols. In addition, because all populations of cells are analyzed by both in situ hybridization and immunocytochemical staining with anti-IgM, the proportion of cells detected by in situ hybridization could be compared with the proportion of B cells, blasts, and plasma cells in the population. It was concluded from these comparisons that the cells being detected by in situ hybridization under the conditions described are plasmablasts and plasma cells. Therefore, an accurate measure of the functional and expressed VH gene repertoire could be made. The results clearly demonstrate strain-dependent variation in VH gene family expression, particularly VH 7183 and VH J558 with up to three-fold differences observed. Thus, either there is considerable strain variation in the number of functional VH gene family segments or the expression of VH genes is not entirely random.     

Organization of the murine immunoglobulin VH complex in the inbred strains.

Deletion mapping analyses have been employed to order the heavy chain variable region (VH) gene families in three inbred murine strains. These nine VH gene families have been positioned with respect to the J558 and 3660 VH families in A/J (Ighe) as follows: 3609-J558-(J606,VGAM3-8,S107)-3660-(X24,Q52,7183 )-DH. Maps generated with respect to J558 in the BALB/c (Igha) and C57BL/6 (Ighb) strains are consistent with these results. The organization of the VH complex produced by deletion mapping is quite different from the accepted map generated by other methods, particularly in that J558 is more DH distal and 3660 is more DH proximal than previously thought. The order presented here is compatible with VH rearrangement frequencies suggesting preferential utilization of DH-proximal VH gene segments. Our data also indicate that interspersion of some VH family members may be a common feature of the murine VH complex since the 3609 VH family is interdigitated in the three strains and a Q52 VH gene segment is interspersed in C57BL/6.