Evaluation of the amino-acid polymorphism identified in the insulin gene transcription factor A1

两个碱性螺旋-环-螺旋转录因子E47和E12参与胰岛素基因的表达，它们是与顺式作用基序E-box相互作用的二聚体复合体的一部分。虽然两者都是通过选择性剪接的方式由单个基因(E2A)产生的，但这两个bHLH区的结构差异表明，两个bHLH蛋白可能在某些功能上存在差异。我们在亮氨酸拉链(LZ)基序的高度保守区发现了一个氨基酸(AA)多态。有趣的是，氨基酸缺失的位置和模式与先前描述的人类对应的氨基酸多态E47(E12)[Cell60(1990)547-555]相同。为了探索这种多态的可能的生理意义，我们调查了7个小鼠品系(C57BL/6ob/ob，C57BL/6J，C57BL/Ksj，ICR，BALB/c ByJ，DBA/2J和C3h/hej)的这种多态类型。…的LZ区进一步克隆了AI基因(nt31-401)，并对其进行了DNA测序。在我们检查的7个菌株中，有6个菌株具有谷氨酰胺(-)A1型，这表明就菌株数量而言，这种A1型相当普遍。只有C3H型菌株具有谷氨酰胺(+)-A1型。由于已知C3H株具有相对的高胰岛素血症，因此提出了可能的生理意义。然而，当这两种类型的A1在CHO细胞中表达时，显示出相同的DNA结合活性，这表明它们至少在CHO细胞中保持了相同的DNA结合活性。为了阐明E47和E12的个体意义，我们还研究了它们的同源物(分别为A1和KA1)在小鼠组织和细胞系中的mRNA表达比率。逆转录-聚合酶链式反应和特异的寡脱氧核苷酸杂交技术检测表明，A1和KA1mRNAs在所有被检测的组织中都普遍表达。然而，它们的比例是不同的：例如，骨骼肌，2.2；正负；0.3(平均值)，脾，2.0；正负0.2，胰岛细胞，1.2；正负0.2。研究的细胞系中的A1KA1比率与其原始组织中的A1KA1比率相似。在小鼠中观察到的E47和E12同源基因在mRNA表达中的普遍存在，为它们参与广泛的转录调控提供了支持。另一方面，A1/KA1表达比率的变化支持A1(E47)和KA1(E12)在这些E2A基因编码的bHLHp以下的功能中各自具有一些独特的作用的观点

Two basic helix-loop-helix (bHLH) transcription factors, E47 and E12, are involved in the insulin geneexpression as part of dimeric complexes which interact with the cis-acting motif, E-box. Although both generated from a single gene (E2A) by means of alternative splicing, the structural difference in these bHLH regions between the two suggests that two bHLH proteins may differ in some of their functions. We reccuntly indentified an amino acid (aa) polymorphism in the highly conserved region within the leucine zipper (LZ) motif. Interestingly, the location and pattern of aa delections are identical to those previously described for the aa polymorphism within the human counterpart, E47 (E12) [Cell60 (1990) 547-555], which was insertion or deletion of glutamine.To explore the possible physiological meaning of the polymorphism, we surveyed seven mouse strains (C57BL/6 ob/ob, C57BL/6J,C57BL/KsJ,ICR,BALB/c ByJ,DBA/2J and C3H/HeJ) in terms of the type of this polymorphism. The LZ regions of … More the AI cDNAs (nt31-401) were cloned and DNA-sequenced. Among the seven strains that we examined, six had the glutamine (-)-type A1, suggesting that this type of A1 is rather prevalent in terms of numbers of strains. Only the C3H strain had the glutamine (+)-type A1. Because the C3H strain is known to be relatively hyperinsulinemic, the possible physiological significance was suggested. However, the two types of A1, when expressed in CHO cells, revealed the same DNA-binding activity, suggesting that they retains the same DNA-binding activity at least in CHO cells.To elucidate the individual implications of E47 and E12, we also investigated the mRNA expression ratios of their homologues (A1 and KA1, respectively) in mouse tissues and cell lines. Measurement using reverse-transcription polymerase chain reaction and a specific oligodeoxyribonucleotide hybridization technique revealed that both the A1 and kA1 mRNAs were ubiquitously expressed in all tissues examined. However, their ratios varied : e.g.skeletal muscle, 2.2<plus-minus>0.3 (mean<plus-minus>SE), spleen, 2.0<plus-minus>0.2, pancreatic islet cells, 1.2<plus-minus>0.2. The A1 kA1 ratios in the cell lines investigated were similar to those of their original tissues. The ubiquity in mRNA expression observed for both the E47 and E12 homologues in mouse provides support for their involvement in a broad range of transcriptional regulation. The variation in the A1/kA1 expression ratios, on the other hand, supports the idea that A1 (E47) and KA1 (E12) each have some unique roles in the functions of these E2A geneencoded bHLH p Less