Mutation in a reporter gene depends on proximity to and transcription of immunoglobulin variable transgenes.
Mutation in a reporter gene depends on proximity to and transcription of immunoglobulin variable transgenes.
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报告基因的突变取决于免疫球蛋白可变转基因的接近度和转录。
DOI：
10.1073/pnas.88.11.4902
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发表时间：
1991
影响因子：
11.1
通讯作者：
Gearhart,PJ
中科院分区：
文献类型：
--
作者：
Umar,A;Schweitzer,PA;Levy,NS;Gearhart,JD;Gearhart,PJ
免疫球蛋白基因的体细胞突变定位于DNA周围2千碱基的区域，包括重排的可变区(V)、多样性和连接区(J)编码重链和轻链的基因片段。为了研究靶向突变的结构基础，我们开发了一种利用报告基因在质粒底物上进行突变评分的方法：将编码琥珀抑制tRNA分子的细菌基因放置在重排的kappa VJ基因的3‘端突变的边界内。报告基因非常适合于突变分析，因为它只有200个碱基对(BP)，不应该太大地破坏免疫球蛋白基因座的结构，而且基因的功能取决于二级结构，这意味着突变可以在许多不同的核苷酸位置得分。用该质粒制备转基因小鼠，然后免疫。穿梭载体通过质粒挽救被回收到指示菌株中，该菌株在其β-半乳糖苷酶基因中含有琥珀突变。TRNA分子的完整性通过菌落颜色进行监测，这使得许多转化子能够被直观地筛选出来。在体外培养的转基因B细胞系的DNA中没有发现突变，在转基因小鼠的非淋巴组织中也没有发现突变，这表明报告基因在细胞分裂和DNA操作中是稳定的。然而，当转基因小鼠免疫时，来自脾B细胞的DNA含有报告基因的点突变，每个转化子的频率为10(-3)。对17个突变的转基因进行了序列分析，发现这些突变是tRNA基因的1和2个碱基的缺失，其中1个质粒的V基因另外有2个碱基的缺失。相比之下，以前的研究表明，内源性VJ基因的突变主要是核苷酸替换，只有6%的缺失。在转基因株系中分析了另外两个质粒构造：当tRNA基因被放置在VJ基因的远端时没有发现突变，当免疫球蛋白启动子被删除时也没有发现突变。尽管我们缺乏直接证据表明tRNA基因的缺失是由作用于VJ基因的相同机制引起的，但我们已经证明，这种检测中的突变发生的方式与免疫球蛋白特异性突变一致，因为它们存在于脾B细胞而不是尾部组织中，取决于VJ基因旁边的位置，并且需要VJ基因的转录。
Somatic mutation in immunoglobulin genes is localized to a 2-kilobase region of DNA surrounding and including rearranged variable (V), diversity, and joining (J) gene segments encoding heavy and light chains. To examine the structural basis for targeted mutation, we developed an assay to score mutation on plasmid substrates by using a reporter gene: a bacterial gene encoding an amber-suppressor tRNA molecule was placed 3' of a rearranged kappa VJ gene within the boundaries of mutation. The reporter gene is exquisitely suited for mutational analysis because it is only 200 base pairs (bp), which should not greatly disrupt structure of the immunoglobulin locus, and gene function depends on secondary structure, which means mutation can be scored in many different nucleotide positions. The plasmid was used to make transgenic mice, which were then immunized. The shuttle vector was retrieved by plasmid rescue into an indicator strain of Escherichia coli that contained an amber mutation in its beta-galactosidase gene. Integrity of the tRNA molecule was monitored by colony color, which permitted many transformants to be screened visually. Mutations were not seen in DNA from a transfected B-cell line grown in vitro or in DNA from nonlymphoid tissue of transgenic mice, indicating that the reporter gene was stable during cell division and DNA manipulations. However, when the transgenic mice were immunized, DNA from splenic B cells contained point mutations in the reporter gene at a frequency of 10(-3) per transformant. Sequence analysis of 17 mutated transgenes revealed that the mutations were 1- and 2-bp deletions in the tRNA gene, and one plasmid had an additional 2-bp deletion in the V gene. In contrast, previous studies have shown that mutations in endogenous VJ genes are predominantly nucleotide substitutions and have only 6% deletions. Two other plasmid constructs were analyzed in transgenic lines: no mutations were found when the tRNA gene was placed distal to the VJ gene, and no mutations were seen when the immunoglobulin promoter was deleted. Although we lack direct evidence that the deletions in the tRNA gene are caused by the same mechanism that acts on VJ genes, we have shown that mutations in this assay occur in a manner consistent with immunoglobulin-specific mutation in that they are found in splenic B cells and not in tail tissue, depend on position next to the VJ gene, and require transcription of the VJ gene.
重排重链抗体可变区基因周围的突变分布
影响因子：
5.3
作者：
G. Both;L. Taylor;J. Pollard;E. Steele
通讯作者：
E. Steele