Sakai T, Koga M, Ohshima Y
Department of Biology Faculty of Science, Kyushu University, Hakozaki Fukuoka, Japan.
J Mol Biol. 1996 Mar 1;256(3):548-55. doi: 10.1006/jmbi.1996.0107.
The let-23 gene in the nematode Caenorhabditis elegans encodes a receptor tyrosine kinase and is necessary for the induction of a vulva, survival past the L1 stage, hermaphrodite fertility and for male spicule development. We sequenced the entire let-23 genomic region of over 12 kb. The 5' end of the let-23 mRNA was mapped by sequencing polymerase chain reaction products, and two mRNAs were found which had alternative exons and were probably initiated at different sites. One transcript was trans-spliced to SL1. Expression of either cDNA rescued a let-23 vulvaless mutation in germline transformation. These results suggest that the let-23 gene encodes two proteins of 1323 or 1335 amino acid residues. We prepared various 5' deletion constructs of the let-23 gene, and examined their rescue activities for a let-23 lethal or vulvaless mutation. The results revealed that two regions were required for let-23 expression, one for larval survival and the other for vulva formation. We also cloned and analyzed a let-23 homologue from Caenorhabditis vulgaris. It can encode two proteins of 77% amino acid residue identity with the Let-23 proteins. The 12 kb fragment carrying the C. vulgaris gene rescued the let-23 vulvaless mutation in C. elegans. Seventeen sequences highly conserved between the two species were identified in the 5' upstream region or within an intron. Three of these sequences are contained in the two regions required for let-23 expression, suggesting that they are cis-acting elements for let-23 expression.
线虫秀丽隐杆线虫中的let-23基因编码一种受体酪氨酸激酶,对于阴门的诱导、L1期后的存活、雌雄同体的生育能力以及雄性交合刺的发育都是必需的。我们对超过12 kb的整个let-23基因区域进行了测序。通过对聚合酶链反应产物进行测序来定位let-23 mRNA的5'端,发现了两种具有可变外显子且可能起始于不同位点的mRNA。一种转录本进行了反式剪接至SL1。在种系转化中,任一cDNA的表达都挽救了let-23无阴门突变。这些结果表明let-23基因编码两种分别含有1323或1335个氨基酸残基的蛋白质。我们制备了let-23基因的各种5'缺失构建体,并检测了它们对let-23致死或无阴门突变的挽救活性。结果显示,let-23表达需要两个区域,一个用于幼虫存活,另一个用于阴门形成。我们还从普通秀丽隐杆线虫中克隆并分析了一个let-23同源物。它可以编码两种与Let-23蛋白氨基酸残基同一性为77%的蛋白质。携带普通秀丽隐杆线虫基因的12 kb片段挽救了秀丽隐杆线虫中的let-23无阴门突变。在两个物种之间的5'上游区域或一个内含子内鉴定出了17个高度保守的序列。其中三个序列包含在let-23表达所需的两个区域中,表明它们是let-23表达的顺式作用元件。