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.

我们通过分子技术研究兔免疫系统的基因 生物学加上免疫学测定。阿里西亚菌株的兔子 具有与免疫球蛋白重链分离的突变（ALI） （IGH）基因座，对重链的表达产生了CIS效应 编码A2异型的可变区域基因（VH）。我们一直在繁殖 这些动物在NIH。来自纯合突变体和野生型的基因组DNA 通过各种各样的分析，动物是无法区分的 限制酶消化和IGH探针。但是，在DNA的研究中 来自不经常切割酶的消化的碎片被分开 横向交流电泳，我们发现一个相对较小的 删除包含3'VH基因的段 功能，导致ALI表型的损失。我们对VH的研究 B细胞中的基因重排表明VH基因座的3'端 可能在调节兔子的VH基因表达中起关键作用 因为该区域中的VH基因是优先VDJ的目标 重排。这增加了替代机制的可能性 像基因转换一样正在起作用以生成抗体库 这个物种。 我们目前正在研究免疫球蛋白沉重之间的五种重组 链可变区（VH）和恒定区域（CH）基因。两个VH-CH 在我们的实验室中发现了重组，三只在巴塞尔 免疫学研究所。为了本地化 导致新单倍型的重组，我们一直在分析DNA 使用一组跨的探针从父母和重组单倍型 IGH基因座的VH，DH，JH，C（MU）和C（伽玛）区域。全部五个 重组位点是整个VH基因簇的下游。三 似乎发生在包含DH基因的区域内，一个 C（MU）的下游。 兔子卡帕轻链常数有两个基因的副本 区域，（Kappa）1和（Kappa）2。我们现在表明这些基因更多 相距1 MB，每个都具有V（Kappa）和J（Kappa）基因。 我们获得了表达的B细胞的分离和纯粹的种群 lambda或kappa 2轻链，表明（kappa）1基因已经 从表达Lambda光链的许多B细胞中删除。