Chu X, Thompson D, Yee L J, Sung L A
Department of Bioengineering and Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0412, USA.
Gene. 2000 Oct 3;256(1-2):271-81. doi: 10.1016/s0378-1119(00)00327-9.
Erythrocyte tropomodulin (E-Tmod), a globular protein of 359 residues, is highly expressed in the erythrocyte, heart and skeletal muscle. By binding to the N-terminus of tropomyosin (TM) and actin, E-Tmod blocks the elongation and depolymerization of the actin filaments at the pointed end. In erythrocytes, the E-Tmod/TM complex contributes to the formation of the short actin protofilament, which in turn defines the geometry of the membrane skeleton. In juvenile mice, over-expression of E-Tmod is associated with dilated cardiomyopathy. We have previously cloned the human E-Tmod cDNA, identified its TM-binding region, and mapped its gene to chromosome 9q22. Through genomic library screening and PCR-based genomic walking we have now cloned the mouse E-Tmod gene, whose coding region spans approximately 60kb containing nine exons and eight introns. The human E-Tmod gene obtained by PCR has an identical exon-intron organization. In sanpodo, a Tmod homologue in Drosophila, the exon boundaries are also conserved except that exons 2-5 and 6-7 are 'fused' and alternative splicing of two additional 5' exons and the 3' exons may give rise to several sanpodo isoforms. In a Tmod-like gene of C. elegans, exons 2-3 are 'fused', boundaries of exons 1, 7, 8, and 9 are conserved and exon/intron junctions of exons 4, 5 and 6 are shifted by a few residues. Analyses of 15 Tmod members from six species show no insertions or deletions of residues in the region of exons 6 and 7. A 5' rapid amplification of cDNA ends reveals that mouse E-Tmod transcripts obtained from embryonic stem cells, skeletal muscle and heart, but not smooth muscle, contain an additional 86bp untranslated cDNA sequence further upstream from exon 1. Thus, alternative promoters may provide a possible mechanism for tissue-specific expression and regulation of E-Tmod. This study is the first to report the exon organization of E-Tmod genes, which allows their regulation, manipulation, and disease relevance to be further investigated.
红细胞原肌球蛋白调节蛋白（E-Tmod）是一种由359个氨基酸残基组成的球状蛋白，在红细胞、心脏和骨骼肌中高度表达。通过与原肌球蛋白（TM）和肌动蛋白的N端结合，E-Tmod可阻止肌动蛋白丝在其尖端的延长和解聚。在红细胞中，E-Tmod/TM复合物有助于短肌动蛋白原丝的形成，进而决定膜骨架的几何形状。在幼年小鼠中，E-Tmod的过度表达与扩张型心肌病有关。我们之前已克隆了人类E-Tmod cDNA，确定了其TM结合区域，并将其基因定位到9号染色体的q22区域。通过基因组文库筛选和基于PCR的基因组步移，我们现已克隆出小鼠E-Tmod基因，其编码区跨度约为60kb，包含9个外显子和8个内含子。通过PCR获得的人类E-Tmod基因具有相同的外显子-内含子结构。在果蝇的Tmod同源物sanpodo中，外显子边界也保守，只是外显子2-5和6-7是“融合”的，另外两个5'外显子和3'外显子的可变剪接可能产生几种sanpodo异构体。在秀丽隐杆线虫的一个类Tmod基因中，外显子2-3是“融合”的，外显子1、7、8和9的边界保守，外显子4、5和6的外显子/内含子连接点有几个残基的移位。对来自6个物种的15个Tmod成员的分析表明，在外显子6和7区域没有氨基酸残基的插入或缺失。5' cDNA末端快速扩增显示，从小鼠胚胎干细胞、骨骼肌和心脏而非平滑肌中获得的E-Tmod转录本，在1号外显子上游还有一个额外的86bp非翻译cDNA序列。因此，可变启动子可能为E-Tmod基因的组织特异性表达和调控提供一种可能机制。本研究首次报道了E-Tmod基因的外显子结构，这使得对其调控、操作及与疾病的相关性研究得以进一步开展。
