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: 6160565
• 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/6160565
• 关键词: B lymphocyte; animal genetic material tag; flow cytometry; gene conversion; gene expression; gene mutation; gene rearrangement; genetic polymorphism; gut associated lymphoid tissue; immunoglobulin genes; immunoglobulin structure; laboratory rabbit; leukopoiesis; monoclonal antibody; northern blottings; nucleic acid sequence; polymerase chain reaction; thymus

We studied genes of the rabbit immune system by techniques of molecular biology and immunology. We used anti-RAG-1 and 2 antibodies in studies of developing rabbit appendix tissues. The surface markers CD43 and IgM distinguished appendix cell populations from 6 to 9-week-old rabbits that contained RAG-1 and RAG-2 proteins. The appearance of CD43 during different stages of B-cell maturation may be related to the function of the appendix as a site of both B-cell development and diversification. In the ali mutant rabbit, a small deletion at the 3' end of the V/H gene cluster led to loss of V/H1 and one V/H pseudogene. Although homozygous mutant ali/ali rabbits lack the V/H1a2 gene, B cells with a2-like surface Ig develop and expand in numbers. We sequenced immunoglobulin heavy chain variable regions (V/H) from expressed mRNA of sorted a2-positive and negative appendix B cell populations from young ali rabbits. The a2 positive cells appear to have rearranged V/H4, the first functional gene in the mutants' V/H cluster. Sequence alterations in FR1 and FR3 can be accounted for by gene-conversion-like changes that utilized candidate donor sequences upstream V/H4. Our studies of the appearance of cells bearing a2-like epitopes in the appendix of mutant rabbits suggested that there was positive selection and expansion based on framework region structures (V/Ha allotypes). We suggested that the preferential expansion and survival of B cells based on FR1 and FR3 expression may involve "superantigen"-like interactions with endogenous and exogenous ligands. One endogenous ligand appears to be CD5. In man and mouse, the 3'-most D/H gene, DQ52, is preferentially rearranged early in B-cell development. To test whether this preference for rearranging a D/H gene segment based on 3' end proximity exists in rabbit, we cloned and sequenced the rabbit DQ52 gene. The coding region sequence is identical to a mouse DQ52 but the 3' recombination signal sequence has an atypical nonamer. The DQ52 gene was utilized very infrequently; one VDJ sequence from 28 day fetal liver had 8 bp that matched the germline DQ52 sequence. In contrast to man and mouse, instead of rearranging DQ52 early in B cell ontogeny, rabbits preferentially express another D/H gene (Df) located in the middle of the D/H region about 32 kb upstream of the JH genes. This may correlate with more frequent initial rearrangement of V/H to D/H in rabbit B cells.

本研究利用分子生物学技术对兔免疫系统基因进行了研究， 生物学和免疫学。 我们在研究中使用了抗RAG-1和2抗体 发育中的兔子阑尾组织。 表面标志物CD 43和IgM 从6 - 9周龄的兔中分离出不同的阑尾细胞群 含有RAG-1和RAG-2蛋白。 CD 43的出现， B细胞成熟的不同阶段可能与 阑尾是B细胞发育和多样化的场所。 在ali突变兔中，V/H基因3'端有一个小缺失 簇导致V/H1和一个V/H假基因的丢失。 虽然纯合子 突变型ali/ali兔缺乏V/H1 a2基因，B细胞具有a2样 表面免疫球蛋白IG在数量上发展和扩大。 我们对免疫球蛋白 来自分选的细胞的表达mRNA的重链可变区（V/H） 来自年轻阿里的a2-阳性和阴性阑尾B细胞群 家兔 α 2阳性细胞似乎重排了V/H4， 突变体V/H簇中的第一个功能基因。 序列改变 在FR 1和FR 3中，可以通过基因转换样变化来解释， 利用V/H4上游的候选供体序列。 我们的研究 在突变体的阑尾中出现携带A2样表位的细胞 家兔的实验结果表明， 框架区结构（V/Ha同种异型）。 我们建议 基于FR 1和FR 3的B细胞的优先扩增和存活 表达可能涉及“超抗原”样与内源性 和外源配体。 一种内源性配体似乎是CD 5。 在人 而小鼠的3 ′端D/H基因DQ 52优先重排 在B细胞发育的早期 要测试此首选项是否 基于3'端邻近的D/H基因片段重排存在于 克隆了兔DQ 52基因并进行了序列测定。 编码区 序列与小鼠DQ 52相同，但3'重组信号 序列具有非典型九聚体。 DQ 52基因被广泛应用于 很少;来自28天胎肝的一个VDJ序列具有8bp， 与生殖系DQ 52序列相匹配 与人和老鼠相比， 在B细胞个体发育早期，兔没有重排DQ 52， D/H基因（Df）位于细胞的中部， JH基因上游约32 kb处的D/H区。 这可能与 在兔B中，V/H至D/H的初始重排更频繁 细胞