Abstract
Exceptionally long third complementarity determining regions of the heavy chain (CDR3H) were previously described as a specificity of bovine IgG and IgM immunoglobulins. In addition, the genomic organization of the immunoglobulin heavy chain locus remains to be elucidated with a special focus on the number of variable segments (IGHV). By analyzing the variable regions according to the isotype-specific PCR using cDNA-PCR, we were able to prove the existence of exceptional long CDR3H in all bovine isotypes. The corresponding sequences of three distinct amplicons were grouped according to the length of the CDR3H. Sequences of CDR3H possessed 5 to 10, 12 to 31 or at least 48 amino acid residues. Long and mid-length CDR3H were composed of mainly hydrophilic amino acid residues, while short CDR3H also contained hydrophobic amino acid residues. All sequences with long CDR3H were related to the germline variable segment 10. Using the current genome assembly, Bos taurus NCBI build 6.1, the genomic organization of the bovine immunoglobulin heavy-chain locus was analyzed. A main locus was investigated on BTA21. Exons coding for variable, diversity, and joining segments, as well as for the constant regions of different isotypes, were also localized on BTA7, BTA8, and BTA20. Together with the information from unplaced contigs, 36 IGHV were detected of which 13 are putatively functional. Phylogenetic analysis revealed two bovine IGHV families (boVH1, boVH2). Thus, the existence of the two bovine families suggested was demonstrated, where boVH1 comprises all functional segments. This study substantially improves the understanding of the generation of immunoglobulin diversity in cattle.
Highlights
The generation of antibody diversity in vertebrates is subjected to a sequence of steps such as the recombination of separated germline gene segments for both heavy (V, D, and J) and light (V and J) chains
The functional locus was mapped to BTA21q23-q24 [21,22,23], where we detected ten variable and two diversity segments together with the exons coding for IgM(F), IgE, and IgA(F)
The human 5pter part is inversely syntenic to BTA20qter [54]
Summary
The generation of antibody diversity in vertebrates is subjected to a sequence of steps such as the recombination of separated germline gene segments for both heavy (V, D, and J) and light (V and J) chains. Somatic hypermutations contribute to antibody diversity – dependent or independent of antigen contact [5,6,7] While these general processes of diversification are very similar in all vertebrate species, considerable differences were found in the available pool of the germline V, D, and J segments. In chicken gene conversion, the use of pseudogene sequences is a frequent post-recombinatorial strategy for the generation of the preimmune antibody repertoire [5,14]. This mechanism was confirmed for l-light chains in cattle [15] and is discussed in horses [16]
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