We analyzed VDJ and VJ rearrangements in IgM‐secreting B lymphocytes from a cow infected with bovine leukemia virus (BLV). BLV causes expansion of CD5+ and IgM+ B lymphocytes regardless of antigen specificity. The data showed that single point mutations contribute to the diversification of IgM antibodies. The most striking observation, however, is that approximately 9 % of theVDJ rearrangement in IgM‐secreting B cells encode an exceptionally long third complementarity‐determining region of the heavy chain (CDR3H; 56 to 61 amino acids) with multiple cysteine residues. Such an exceptionally long CDR3H is the first ever to be documented for an antibody in a species. These VDJ rearrangements encode functional IgM antibodies as some of these show polyspecific reactivity. The presence of even‐numbered cysteine residues in the CDR3H may provide hitherto unknown configurational ability to the antigen combining site via intra‐CDR3H disulfide bridging. In addition, the VDJ rearrangements encoding exceptionally long CDR3H paired with either novel Vλ1 or Vx1x genes, earlier noted not to be expressed. Overall, these experiments provide evidence that somatic hypermutations and generation of an exceptionally long CDR3H contribute to the diversification of IgM antibodies in cattle.

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Naturally occurring antibody repertoires of cattle (Bos taurus) include a group of IgMlambda antibodies with exceptionally long complementarity-determining region 3 of the heavy chain (CDR3H) segments, containing multiple Cys residues. These massive CDR3H segments will greatly influence the tertiary and quaternary structures of the bovine IgM combining sites. As an antibody's combining site is formed by both heavy and light chains, we have analyzed the nucleotide sequences and structural properties of the lambda-light chains that pair with micro -heavy chains containing exceptionally long CDR3H. There appears to be an exquisite selective pressure for the use of three V(lambda)1 genes (V(lambda)1x and two new V(lambda)1d and V(lambda)1e genes) in IgM with unusually long CDR3H. The V(lambda)1d and V(lambda)1e genes are similar to each other, but diverge from the other V(lambda)1 genes into two closely related subfamilies. The available bovine V(lambda) genes are classified into three V(lambda) gene families: V(lambda)1, V(lambda)2 and V(lambda)3 based on nucleotide similarity >/=80%. Further, analysis of total Ser content and positions of Ser residues in the sequences was found to be sufficient to classify the cattle V(lambda)1 subfamilies. Patterns of Ser residues differ for V(lambda) domains from ruminant species (e.g. cattle, sheep and goats) and other mammals (e.g. humans and mice). These 'Ser signatures' can be used to track divergent evolution in lambda-light chains. Interestingly, Ser90L in complementarity-determining region 3 of the light chain (CDR3L) occurred in all V(lambda) domains that pair with V(H) regions containing exceptionally long CDR3H. A structural role for Ser90L was revealed in homology models of V(lambda) domains, i.e. to hold the ascending polypeptide of CDR3L in a relatively tight space between the N-terminal segment and residues from CDR1L. The CDR3L of V(lambda) domains also occupied smaller volumes if paired to V(H) domains with extremely long CDR3H (>/=48 residues), and were more variable in their conformation and filled larger volumes if CDR3Hs were

A cDNA clone encoding the variable region of the heavy chain of a BALB/c antibromelinized mouse red blood cell (BrMRBC) monoclonal antibody has been characterized. The nucleic acid sequence indicates that the variable region of the heavy chain is likely encoded by variable-VCP12, diversity-DSP 2 (5, 7 or 8) and joining-JH1 germ-line genes. An identical combination of V genes was observed for six other CBA/J and NZB anti-BrMRBC hybridomas. The BALB/c VCP12 nucleotide sequence is less than 80% homologous to members of the 10 known VH families. Moreover, the genomic restriction fragments detected under moderate stringency conditions with the radiolabeled cDNA probe did not correspond to those obtained with probes of the most homologous VH families (7183 and X24). The results indicate that the VCP12 gene defines a new VH family which we propose to designate VH11. The observation of 1, 2 or 3 genomic restriction fragments at the most, with mice bearing the Igh-Vd or j, Igh-Va or b or Igh-Vc haplotypes, suggests the existence of a few VCP12-related genes.