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.

我们通过分子技术研究兔免疫系统的基因 生物学和免疫学。 Alicia菌株的兔子有突变 （ALI）与免疫球蛋白重链（IGH）基因座分离 并对重链变量区域的表达产生顺式影响 编码A2异型的基因（VH）。 一个相对较小的删除 影响包含3'VH基因的段，其损失导致 阿里表型。 VH基因座的3端可能在 VH1基因是兔子中VH基因表达的调节 优先VDJ重排的目标。这增加了可能性 诸如基因转化和超名之类的躯体机制使得 产生兔子抗体库的主要贡献。 来自纯合VH-CH重组兔的DNA和适当的DNA 使用南部的非重组父母单倍型表征 与来自克隆区域的探针杂种的印迹 兔免疫球蛋白重链基因复合物。 在这三个 重组者，该站点是整个VH群集的下游， JH簇的上游约50 kb区域内 重复序列DNA和DH基因的膨胀。 兔子具有两种同种型形式的免疫球蛋白喀巴灯光 链，K1和K2可能是通过重复而产生的。 在正常情况下 兔子，仅产生K2轻链的痕迹。 我们使用了脉冲 场和横向交流电泳以获得 巨像图，发现两个Ckappa基因大约是1兆巴 除外。 K2表达不佳的一种解释可能很棒 与Vkappa基因的物理距离。 但是，我们发现有 vkappa，jkappa和ckappa2基因在约105 kb的片段中。 因此 vkappa与ckappa2的物理距离可能不是穷人的基础 K2表达。 我们有兴趣寻找第二个增强剂3' Ckappa基因是因为在K2基因座中没有3'增强子 可以解释K1同种型的优先利用。 强壮 发现增强剂活性的区域大约是下游的7 kb CKAPPA2基因。 没有3'增强器的TBU可能不是 K2基因低表达的解释。