Sequence analysis of 0.5 Mb of the rabbit germline immunoglobulin heavy chain locus
Introduction
The immunoglobulin (Ig) locus encoding antibody heavy chains consists of several gene segments, including VH, DH, JH and various C elements which rearrange during B cell development to form functional antibody genes. In humans and mice, generation of a diverse antibody repertoire is accomplished through rearrangement of a large number of different V, D and J elements. This process of gene rearrangement appears to occur throughout life. In contrast, some other animals including rabbits and chicken rearrange one or a very limited number of VH elements early in life. Rearrangement of Ig genes stops in the chicken and diminishes in rabbits by maturity (for reviews see Knight and Crane, 1994, Weill and Reynaud, 1992). Subsequent to gene rearrangement, a diverse antibody repertoire in rabbits and chicken is generated by gene conversion of the rearranged V element using upstream V sequences as gene-conversion donors. Similar to somatic hypermutation, gene conversion appears to be an activation-induced deaminase-dependent-pathway of DNA repair. Consistent with the hypothesis that gene conversion requires high homology of V elements, rabbit and chicken Vs appear to belong to single families, although in the rabbit allelic forms of the frequently rearranged VH1 gene are found (Knight and Becker, 1990). In laboratory rabbits, there are three allelic allotypes a1, a2, a3 that reflect amino acids differences encoded in FR1 and FR3 (reviewed in Mage et al., 1984).
In an effort to understand the inheritance of VHa allotypes and antibody diversification by gene conversion, rabbit Ig loci have been characterized extensively and partial maps of the heavy chain locus have been established using plasmid, phage and cosmid clones. However, analysis of large complex genes requires libraries with large insert size. Here we describe construction of a rabbit bacterial artificial chromosome (BAC) library and subsequent isolation, characterization and sequencing of overlapping BACs representing a large part of the rabbit Ig heavy chain locus.
Section snippets
BAC libraries
High molecular weight DNA was isolated from an a2b5 (heavy chain haplotype F-I) male rabbit. Immediately after euthanization, spleen and kidneys were removed and rinsed in ice-cold phosphate-buffered saline (PBS). Fat and other associated tissues were removed and processed separately. The organs were cut into pieces and transferred to a pre-cooled Dounce homogenizer containing 2.5 ml PBS. After tissue grinding, supernatant was transferred into a cooled falcon tube and mixed with 50 ml PBS.
Library screening
Genomic DNA from a VHa2 (F-I) haplotype animal was partially digested with HindIII and cloned into pBeloBAC11. Digestion of BACs with NotI and subsequent gel electrophoresis revealed an average size of 124 kb. Therefore, our library of 105 clones represents 12,400 Mb, about four times the genome size of rabbits.
Screening of 105 BAC clones with probes for rabbit VH1, DH, and Cγ resulted in the identification of 56 positive clones. Southern Blot analysis revealed 51 BAC with fragments of the
Discussion
For the characterization of rabbit Ig loci we generated a genomic BAC library consisting of 105 clones with an average insert size of 124 kb, about four times the size of the rabbit genome. Screening with VH, DH, and Cγ specific probes resulted in the identification of several BAC representing large part of the rabbit Ig heavy chain locus, including 51 BACs (6.3 Mb) with VH elements. This suggests that the heavy chain locus spans about 1.6 Mb containing more than 200 VH elements separated by
Acknowledgements
We thank Dr. R. Mage for helpful comments about this manuscript, and Dominique Ostler for excellent technical assistance. We also thank the AGOWA (Berlin) for their excellent sequencing work.
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