RABBIT ALLOTYPES--STRUCTURE, ORGANIZATION AND REGULATED EXPRESSION OF IG GENES

利用分子技术研究兔免疫系统的基因

We study genes of the rabbit immune system by techniques of molecular biology and immunology. Alicia rabbits have a mutation in the immuno- globulin heavy chain locus that affects the expression of heavy chain variable region genes (VH) encoding the a2 allotype. Earlier, we found a relatively small deletion of a segment containing 3' VH genes with important regulatory functions, the loss of which leads to the ali phenotype. The 3' end of the VH locus probably plays a key role in regulation of VH gene expression in rabbits because VH gene(s) in this region are the targets of preferential VDJ rearrangements. Alternative mechanisms such as gene conversion may contribute to the diversification of the antibody repertoire in this species. We used the PCR technique to amplify the expressed VH genes of mutant Alicia and normal a2 rabbits of different ages, in order to determine the sequences of VHa genes coding for a2 molecules as well as VHa- genes coding for the a- molecules whose expression is elevated in mutant Alicia rabbit. We found that the products of the first functional VH gene (VH4a2) or VH4a2-like gene(s) were expressed in two to eight-week-old mutant Alicia; expression of both the VHx and VHy types of a-negative genes was also elevated but the relative proportions of VHx and VHy, especially VHx, decreased with age; meanwhile, the relative levels of expression of the VH4a2 or VH4a2-like gene(s) increased. The appearance of sequences resembling that of the VH1a2 gene which is deleted in the mutants may be due to gene conversions that altered the sequence of the rearranged VH4a2; rearrangement of upstream VH1a2-like genes may also occur later in development. We used reverse transcription and PCR amplification to isolate the complete coding region of the rabbit RAG-2 gene. Two cDNA clones from thymic mRNA were sequenced and compared to the sequence of a genomic DNA clone from Dr. K Roux. Genetic polymorphism of the rabbit RAG-2 gene was observed by sequencing and Southern anal-yses. Northern analysis of thymic mRNA showed that the rabbit RAG-2 transcript was almost twice the size of the homologous mouse transcript. Southern analyses using a probe from 5' of the RAG-2 coding region indicate that the rabbit has several exons contributing to the mRNA sequence. The rabbit mRNA for RAG-1 is of comparable size to those of mouse and man. A fragment of RAG-1 coding sequence has also been cloned by similar PCR techniques and the sequence compared with genomic RAG-1.