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

我们的目的是克隆在两栖类变态过程中由甲状腺激素(TH)诱导的蝌蚪退化尾巴中的凋亡诱导基因。我们已经分离了非洲爪哇Caspase家族的所有基因作为凋亡执行基因，并将它们导入了尾部来源的成肌细胞系。具有较长原结构域的Caspase过表达具有较强的细胞杀伤活性。研究结果表明：1.非洲爪蛙Caspase家族基因在变性蝌蚪退化尾部表达上调，但在TH处理的死亡尾部来源的细胞系中表达上调，这表明Caspase的新RNA合成对细胞程序性死亡是必不可少的。许多报道支持谋杀模型，即TH诱导的分泌细胞外基质降解酶的增加会导致肌腱连接的破坏，从而使肌肉细胞从细胞外基质中分离出来，导致细胞死亡。然而，当TH信号被Dominan…的强制表达所阻断时更多的T阴性甲状腺激素受体(DNTR)，TH处理的成肌细胞系的死亡被抑制，提示细胞的程序性死亡是通过自杀机制发生的。为了证实这一点，将DNTR表达载体注射到活的蝌蚪尾部肌肉细胞中，并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与DNTR表达载体共注射的尾巴中观察到。这一结果表明，肌肉细胞的死亡是对退化尾巴中TH的一种反应。利用体内电穿孔技术将标记基因和cDNA文库导入蝌蚪尾巴中，几天后取出进行器官培养，检测标记基因的表达情况。没有一个克隆被分离出来作为抑制基因表达的标记。到目前为止，通过退化尾巴中表达增加而确定的几个基因的过度表达也没有表现出任何抑制活性。我们正在分析一组在尾部来源的巨噬细胞系的筛选中对标记基因的表达具有显着抑制活性的cDNA克隆。较少

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