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

兔同种异型——IG 基因的结构、组织和调控表达

• 批准号: 3809580
• 负责人: R G MAGE
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3809580
• 关键词: B lymphocyte; DNA; animal population genetics; electrophoresis; gene expression; gene mutation; gene rearrangement; genetic mapping; genetic markers; genetic recombination; genetic strain; immunoglobulin G; immunoglobulin genes; immunoglobulin structure; laboratory rabbit; linkage mapping; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; restriction mapping; tissue /cell culture

We study genes of the rabbit immune system by techniques of molecular biology coupled with immunological assays. Rabbits of the Alicia strain have a mutation (ali) that segregates with the immunoglobulin heavy chain (Igh ) locus and has a cis effect upon the expression of heavy chain variable region genes (VH) encoding the a2 allotype. We have been breeding these animals at the NIH. Genomic DNAs from homozygous mutant and wild type animals were indistinguishable by Southern analyses using a variety of restriction enzyme digests and Igh probes. However, in studies of DNA fragments from digests with infrequently cutting enzymes separated by transverse alternating field electrophoresis, 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. Our studies of VH gene rearrangement in B-cells suggest that 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. This raises the possibility that alternative mechanisms like gene conversion are at work to generate the antibody repertoire in this species. We are currently studying five recombinants between immunoglobulin heavy chain variable region (VH) and constant region (CH) genes. Two of the VH-CH recombinants were discovered in our laboratory and three at the Basel Institute for Immunology. In order to localize the sites of the recombinations that led to the new haplotypes, we have been analyzing DNAs from the parental and recombinant haplotypes using a set of probes spanning the VH, DH, JH, C(mu) and C(gamma) regions of the Igh locus. All five recombination sites were downstream of the entire VH gene cluster; three appear to have occurred within the region containing DH genes and one downstream of C(mu). There are two copies of the gene for the rabbit kappa light chain constant region, (kappa)1 and (kappa)2. We have now shown that these genes are more than 1 Mb apart and each has associated V(kappa) as well as J(kappa) genes. We have obtained separated and pure populations of B cells expressing lambda or kappa 2 light chains and shown that the (kappa)1 gene has been deleted from many of the B cells expressing lambda light chains.

我们用分子生物学技术研究兔免疫系统的基因 生物学与免疫学检测相结合。艾丽西亚品系的兔子 有一个与免疫球蛋白重链分离的突变(ALI) (IgH)基因座，对重链的表达有顺式效应 编码A2同种异型的可变区基因(VH)。我们一直在繁衍后代 美国国立卫生研究院的这些动物。纯合突变体和野生型的基因组DNA 根据南方的分析，动物是无法区分的，使用各种 限制性内切酶和免疫球蛋白探针。然而，在对DNA的研究中 用不常用的切割酶分离的消化液中的片段 横向交变磁场电泳法，我们发现了一个相对较小的 含有具有重要调控作用的3‘VH基因片段的缺失 功能缺失，导致ALI表型。我们对VH的研究 B细胞基因重排提示VH基因3‘端 可能在兔VH基因表达调控中起关键作用 因为该地区的VH基因(S)是VDJ的优先目标 重新安排。这增加了替代机制的可能性 像基因转换一样在工作中产生抗体库 这个物种。 我们目前正在研究免疫球蛋白重组体中的五个重组体 链可变区(VH)和恒定区(CH)基因。两个VH-CH 重组子是在我们的实验室发现的，在巴塞尔发现了三个 免疫学研究所。为了本地化的网站， 重组导致了新的单倍型，我们一直在分析DNA 从亲本和重组单倍型中 IgH基因的VH、DH、JH、C(MU)和C(伽马)区。所有五个人 重组位点位于整个VH基因簇的下游；三个 似乎发生在包含水解酶基因的区域和一个 C下游(亩)。 兔子kappa轻链常数的基因有两个拷贝 区域，(Kappa)1和(Kappa)2。我们现在已经表明，这些基因比 每一个都有相关的V(Kappa)和J(Kappa)基因。 我们已经获得了分离的和纯的B细胞群体，表达 Lambda或kappa 2轻链，表明(Kappa)1基因已经 从许多表达lambda轻链的B细胞中删除。