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: 3803117
• 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/3803117
• 关键词: gene deletion mutation; gene expression; gene rearrangement; genetic enhancer element; genetic mapping; genetic recombination; genetic strain; immunoglobulin genes; immunoglobulin isotypes; immunoglobulin structure; laboratory rabbit; nucleic acid probes; nucleic acid sequence; phenotype; pulsed field gel electrophoresis; southern blotting

We study genes of the rabbit immune system by techniques of molecular biology and immunology. 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. A relatively small deletion affects a segment containing 3'VH genes, 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 the VH1 gene is the target of preferential VDJ rearrangement. This raises the possibility that somatic mechanisms such as gene conversion and hypermutation make major contributions to generating the rabbit's antibody repertoire. DNAs from homozygous VH-CH recombinant rabbits and from the appropriate non-recombinant parental haplotypes were characterized using Southern blots hybridized with a panel of probes derived from cloned regions of the rabbit immunoglobulin heavy chain gene complex. In all three recombinants, the site was downstream of the entire VH cluster and upstream of the JH cluster within an about 50 kb region containing expanses of repetitive-sequence DNA as well as DH genes. The rabbit has two isotypic forms of the immunoglobulin kappa light chain, K1 and K2 which probably arose by duplication. In the normal rabbit, only traces of K2 light chains are produced. We used pulsed field and transverse alternating field electrophoresis to obtain megabase maps and found that the two Ckappa genes are about 1 megabase apart. One explanation for the poor expression of K2, could be great physical distance from Vkappa genes. However, we found that there are Vkappa, Jkappa and Ckappa2 genes within a about 105 kb fragment. Thus physical distance of Vkappa from Ckappa2 may not be the basis for poor K2 expression. We were interested in looking for second enhancers 3' of the Ckappa genes because the absence of a 3' enhancer in the K2 locus could explain the preferential utilization of the K1 isotype. A strong region of enhancer activity is found about 7 kb downstream of the Ckappa2 gene. Tbus absence of the 3' enhancer is probably not the explanation for low expression of the K2 gene.

我们用分子生物学技术研究了兔免疫系统的基因， 生物学和免疫学。 艾丽西娅品系的兔子有一种突变 (ali)与免疫球蛋白重链（Igh）基因座分离 并且对重链可变区的表达具有顺式效应 编码α 2同种异型的基因（VH）。 一个相对较小的删除 影响含有3 'VH基因的片段，其缺失导致 ali表型。 VH基因座的3 '端可能在以下方面起关键作用： 由于VH 1基因是兔中VH基因表达的调控基因， 优先VDJ重排的目标。这就提出了一种可能性， 基因转换和超突变等体细胞机制 对产生兔子的抗体库做出了重大贡献。 来自纯合VH-CH重组兔和来自适当的 非重组亲本单倍型的特征在于使用Southern 与来自克隆区域的一组探针杂交的印迹 兔免疫球蛋白重链基因复合物。 在所有三 在重组体中，该位点在整个VH簇的下游， 在JH簇的上游约50 kb区域内， 重复序列DNA和DH基因的扩增。 兔有两种同种型形式的免疫球蛋白κ轻 链，K1和K2可能是由重复产生的。 正常 兔，仅产生痕量的K2轻链。 我们用脉冲 场和横向交变场电泳， 发现两个Ckappa基因约为1兆碱基， apart. K2表达不佳的一种解释可能是很好的 与Vkappa基因的物理距离 然而，我们发现， Vkappa、Jkappa和Ckappa 2基因在约105 kb片段内。 因此 Vkappa与Ckappa 2的物理距离可能不是差的基础。 K2表达。 我们有兴趣寻找第二个增强子3'的 Ckappa基因，因为在K2基因座中缺少3'增强子 可以解释K1同种型的优先利用。 一个强大 增强子活性区域位于该区域下游约7 kb处。 Ckappa 2基因。 Tbus 3'增强子的缺失可能不是 K2基因低表达的原因。