Ross D A, Magor B G, Middleton D L, Wilson M R, Miller N W, Clem L W, Warr G W
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425, USA.
J Immunol. 1998 Apr 15;160(8):3874-82.
The Ig heavy chain enhancer of the channel catfish (Ictalurus punctatus) has an unusual position and structure, being found in the 3' region of the mu gene and containing eight functional octamer motifs of consensus (ATGCAAAT) and variant sequences. The presence of multiple octamer motifs suggests that an Oct2 homologue may play an important role in driving expression of the Ig heavy chain locus in a teleost fish. To test this hypothesis, two catfish Oct2 cDNAs (alpha and beta) were cloned by screening a catfish B cell cDNA library. Catfish Oct2 alpha and beta isoforms are derived by alternative RNA splicing; as determined by Southern analysis, Oct2 is a single copy gene. In comparisons with mammalian Oct2, the catfish Oct2 isoforms show high sequence conservation in their N-terminal regions and POU domains, but extensive divergence in their C-terminal regions. Catfish Oct2 a and beta are tissue restricted, bind both consensus and variant octamer motifs, and activate transcription in both catfish and murine cells. In contrast, mouse Oct2 activated transcription in mouse but not catfish cells. Catfish Oct2 beta is a more potent transcriptional activator than Oct2 alpha. In transient expression assays, catfish Oct2 beta showed a marked preference for the octamer variant, ATGtAAAT, which occurs twice in the catfish enhancer. Mouse Oct2 also showed increased activity with the variant octamer when tested in mouse B cells. Gel-shift analysis competition assays indicated that catfish Oct2 binds the consensus octamer motif with an apparently higher affinity than it does the variant motif.
斑点叉尾鮰（Ictalurus punctatus）的免疫球蛋白重链增强子具有不同寻常的位置和结构，它位于μ基因的3'区域，包含八个具有共有序列（ATGCAAAT）和变体序列的功能性八聚体基序。多个八聚体基序的存在表明Oct2同源物可能在硬骨鱼免疫球蛋白重链基因座的表达驱动中发挥重要作用。为了验证这一假设，通过筛选斑点叉尾鮰B细胞cDNA文库克隆了两个斑点叉尾鮰Oct2 cDNA（α和β）。斑点叉尾鮰Oct2α和β异构体是通过选择性RNA剪接产生的；通过Southern分析确定，Oct2是单拷贝基因。与哺乳动物Oct2相比，斑点叉尾鮰Oct2异构体在其N端区域和POU结构域显示出高度的序列保守性，但在其C端区域存在广泛的差异。斑点叉尾鮰Oct2α和β具有组织限制性，能结合共有和变体八聚体基序，并在斑点叉尾鮰和小鼠细胞中激活转录。相比之下，小鼠Oct2在小鼠细胞而非斑点叉尾鮰细胞中激活转录。斑点叉尾鮰Oct2β是比Oct2α更强的转录激活剂。在瞬时表达试验中，斑点叉尾鮰Oct2β对八聚体变体ATGtAAAT表现出明显的偏好，该变体在斑点叉尾鮰增强子中出现两次。在小鼠B细胞中测试时，小鼠Oct2对变体八聚体的活性也有所增加。凝胶迁移分析竞争试验表明，斑点叉尾鮰Oct2与共有八聚体基序的结合亲和力明显高于变体基序。
