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: 6098890
• 负责人: Rose 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/6098890
• 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 study genes of the rabbit immune system by techniques of molecular biology and immunology. The rabbit has a limited number of VH genes that rearrange. As in the chicken, the 3?-most VH1 gene is rearranged in most rabbit B lymphocytes. Rabbit appendix and chicken bursa of Fabricius are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. By six weeks after birth, diversification of rearranged VH genes occurs at least in part, by gene conversion-like events in the appendix suggesting that this organ is a homologue of the avian bursa of Fabricius. Rad51 contributes to repair of double strand breaks in DNA during somatic and meiotic recombination. The gene was first identified in lower eukaryotes and later in vertebrates including chicken, as encoding an E. coli RecA-like protein. We cloned and sequenced RAD51 cDNA from the rabbit. Because the chicken bursa was shown to express high levels of RAD51 message, we investigated the expression of RAD51 in the rabbit appendix and other tissues. Using a quantitative PCR mimic assay and conventional Northern analyses, we found high RAD51 expression in young rabbit appendix comparable to levels in testis where there is an abundance of meiotic recombination. RAD51 levels were three times higher in appendix B lymphocytes compared to T lymphocytes and were lower in adult appendix, as well as in spleen and Peyer?s patches of young rabbits. We measured the levels of message in several appendix cell subpopulations obtained by fluorescence activated cell sorting (FACS) and found that subpopulations of B lymphocytes corresponding to different stages of B-cell development as well as B cells undergoing isotype switch did not have significantly different mRNA levels. We conclude that although Rad51 may have roles in gene conversion and isotype switching in young rabbit appendix, they are played against a background of high steady state RAD51 mRNA levels in developing B cells (Schiaffella, et al., 1998). We investigated the molecular basis for the appearance of VHa2 allotype-bearing B cells in mutant Alicia rabbits. The mutation arose in an a2 rabbit; mutants exhibit altered expression of VH genes because of a small deletion encompassing VH1a2, the 3? most gene in the VH locus. The VH1 gene is the major source of VHa allotype because this gene is preferentially rearranged in normal rabbits. In young homozygous ali/ali animals, the levels of a2 molecules found in the serum increase with age. In adult ali/ali rabbits, 20 to 50% of serum immunoglobulins and B cells bear a2 allotypic determinants. Previous studies suggested that positive selection results in expansion of a2 allotype-bearing B cells in the appendix of young mutant ali/ali rabbits (reviewed in Pospisil and Mage 1998a). We separated appendix cells from a 6- wk-old Alicia rabbit by FACS based on the expression of surface IgM and a2 allotype. The VDJ portion of the expressed immunoglobulin mRNA was amplified from the IgM+ a2+ and IgM+ a2- populations by RT-PCR. The cDNAs from both populations were cloned and sequenced. Analysis of these sequences suggested that in a2+ B cells, the first D proximal functional gene in Alicia rabbits, VH4a2, rearranged and was altered further by a gene conversion-like mechanism. Upstream VH genes were identified as potential gene sequence donors; VH9 was found to be the most frequently used gene donor. Among the a2- B cells y33 was the most frequently rearranged gene (Sehgal et al., 1998).

我们通过研究兔子免疫系统的基因 分子生物学和免疫学技术。这只兔子有一只 重排的VH基因数量有限。就像鸡肉一样， 3？-大多数兔B淋巴细胞VH1基因重排。 兔阑尾和鸡法氏囊是主要的肉鸡 B细胞抗体库形成的淋巴器官 生发中心主要通过类似基因转换的过程进行。在六点之前 出生几周后，重排的VH基因的多样化发生在 至少部分是由附录中类似基因转换的事件引起的 表明这个器官是鸡的法氏囊的同源物。 法布里修斯。RAD51有助于修复双链断裂 体细胞和减数分裂重组过程中的DNA。基因是第一个 在低等真核生物中发现，后来在脊椎动物中发现，包括 鸡肉，编码一种类似E.ColiRecA的蛋白。我们克隆并 对兔RAD51基因进行了序列测定。因为鸡肉 Bursa被证明表达高水平的RAD51信息，我们 探讨RAD51在兔盲肠和盲肠组织中的表达 其他纸巾。使用定量的PCR模拟分析和 常规Northern分析，我们发现RAD51高表达 在幼兔的阑尾中，与睾丸中的水平相当 是大量的减数分裂重组。RAD51水平为三级 与T淋巴细胞相比，附录B淋巴细胞的数量增加了一倍 淋巴细胞在成人阑尾和脾中含量较低。 而佩尔呢？S则是一块块的小兔子。我们测量了它们的水平 在几个附录细胞亚群中获得的消息 荧光激活细胞分选(FACS)发现 不同阶段的B淋巴细胞亚群 B细胞发育与B细胞同型转换的关系 没有显著差异的mRNA水平。我们的结论是 尽管RAD51可能在基因转换和同工型中发挥作用 切换到幼兔的阑尾，他们与一只 高稳定状态RAD51基因表达的背景 发展中的B细胞(Schiaffella等人，1998年)。我们调查了 VHA2异型B细胞产生的分子基础 在突变的Alicia兔身上。突变发生在一只a2兔身上；突变体 由于一个微小的缺失而表现出VH基因表达的变化 包括VH1a2、3？VH基因座上的大多数基因。VH1 基因是VHA同种异型的主要来源，因为该基因 在正常兔中优先重排。在年轻的纯合子 ALI/ALI动物血清中A2分子水平的变化 随着年龄增长。在成年ALI/ALI兔中，20%至50%的血清 免疫球蛋白和B细胞携带a2异型决定簇。 以前的研究表明，积极的选择会导致 青少年阑尾a2同种异型B细胞的扩增 突变型ALI/ALI兔(见Pospisil和MAGE 1998a综述)我们 用流式细胞仪分离6周龄艾丽西亚兔的阑尾细胞 根据表面免疫球蛋白M和a2同种异型的表达。VDJ 扩增了部分免疫球蛋白mRNAs 通过RT-PCR从IgM+a2+和IgM+a2-群体中分离出。这个 对两个种群的cDNA进行克隆和测序。 对这些序列的分析表明，在A2+B细胞中，第一个 艾丽西亚兔D近端功能基因VH4a2重排 并被一种类似基因转换的机制进一步改变。 上游VH基因被确定为潜在的基因序列 供体；VH9被发现是最常用的基因供体。 在a2-B细胞中，Y33基因重排频率最高 (Sehga等人，1998年)。