Cloning of apoptosis-inducing genes by introducing genes into living tadpole

我们的目的是克隆两栖动物蜕变过程中蝌蚪退化尾中甲状腺激素诱导的凋亡诱导基因。我们分离出所有爪蟾Caspase家族基因作为凋亡执行基因，并将其导入尾源性成肌细胞系。长前体Caspases的过表达显示出较强的细胞杀伤活性。爪蟾Caspase家族基因在蜕变蝌蚪的退化尾中表达上调，而在TH处理的濒死尾源细胞系中表达上调，说明Caspase的新RNA合成对于程序性细胞死亡是必不可少的。许多报道支持谋杀模型，即TH诱导分泌的细胞外基质降解酶的增加导致肌腱连接的破坏，从而使肌肉细胞与细胞外基质分离并导致细胞死亡。然而，当th信号被显性…More t阴性甲状腺激素受体（DNTR）的强制表达阻断时，th处理的成肌细胞系的死亡受到抑制，这表明细胞程序性死亡是通过自杀机制发生的。为了证实这一点，我们将带有标记基因的DNTR表达构建体注射到活蝌蚪尾肌细胞中，在尾巴完全吸收之前检测标记蛋白的活性。标记基因在肌肉细胞中的表达仅在共注射DNTR表达构建体的尾部观察到。这一结果表明，肌肉细胞自杀死亡是对TH的一种反应。将标记基因和cDNA文库引入蝌蚪尾，在体内电穿孔的表达载体中，几天后切除进行器官培养，检测maker基因的表达。未分离到任何一个克隆作为一个抑制标记基因表达。目前在回归尾中发现的几个基因的过表达也没有表现出任何抑制活性。我们通过转染尾源性动母细胞系进行筛选，分析了一组对标记基因表达有显著抑制活性的cDNA克隆。少

Our purpose is to clone apoptosis-inducing genes that are induced by thyroid hormone (TH) in the regressing tail of tadpole during amphibian metamorphosis.1. We have isolated all Xenopus Caspase family genes as apoptosis-excuting genes, and introduced them into tail-derived myoblast cell line. The overexpression of Caspases with a long prodomain showed a strong cell-killing activity. The expression of Xenopus Caspase family genes was upregulated in the regressing tail of metamorphosing tadpole, but was not in dying tail-derived cell line treated with TH, which demonstrates that new RNA synthesis of caspases is dispensable to programmed cell death.2. Many reports supported the murder model that the increase of secreted extracellular matrixdegrading enzymes induced by TH results in the disruption of the myotendinous junctions, which detaches muscle cells from the extracellular matrix and causes their cell death. However, when TH-signaling was blocked by the enforced expression of dominan … More t-negative thyroid hormone receptor (DNTR), the death of TH-treated myoblast cell line was repressed, suggesting that the programmed cell death takes place through a suicide mechanism. To confirm it, DNTR expression construct was injected into the tail muscle cells of living tadpole with a marker gene, and the activity of marker protein was detected just before tail resorbed completely. The marker gene expression in muscle cells was observed only in tails co-injected with DNTR expression construct. This result indicates that muscle cell dies by suicide as a response to TH in the regressing tail.3. Tadpole tail was introduced with a marker gene and cDNA library in a expression vector using in vivo electroporation, cut out for the organ culture several days later, and examined on the maker gene expression. Any clone was not isolated as one inhibiting marker gene expression. The overexpression of several genes identified so far by the increased expression in the regressing tail did not show any suppressive activity, either. We are analyzing a group of cDNA clones which has the significant suppressive activity to the marker gene expression in the screening by transfection of tail-derived mvoblastic cell line. Less