Programmed cell death-The molecular mechanism of tail regression during amphibian metamorphosis

为了研究尾巴吸收的分子机制，本研究尝试建立RNaseH特异性消减文库和聚合酶链式反应的文库消减方法，克隆退化尾巴中表达的变态特异基因，获得蝌蚪尾巴来源的细胞系，用于功能分析。在高潮之前，将少量退化的Tail基因与57期蝌蚪的大量Tail基因杂交，并用RNase H处理这种混合物以消化与cDNA杂交的mRNA。用聚合酶链式反应(PCR)扩增抗性基因片段，插入到载体中，用+/-法进行筛选。36个独立分离的克隆分为10个不同的基因，分为3个组，在不同组织中表现出不同的表达模式。第一组基因在所有组织中都有表达，包括大脑、眼睛、后肢和尾巴。第二个在后肢和尾巴被激活。第三组只在Tail中转录。蝌蚪尾部成肌细胞系维持了一年半以上，细胞在添加10 nM的甲状腺激素(T_3)时死亡，这是变态过程中血浆中的一个生理水平。在用甲状腺激素处理的这些细胞中，只有一种基因T6-5-12在一天内被激活。甲状腺激素受体βmRNA在T_3处理后4小时开始表达，而甲状腺激素受体α在T_3处理后持续表达。治疗8h后观察T6-5-12的表达。甲状腺激素受体α可能首先与T_3结合，该复合体激活甲状腺激素受体β基因，最终由这些受体与甲状腺激素的复合体诱导T6-5-12的表达，通过将退行性尾巴的特异性基因导入成肌细胞，将在不久的将来阐明两栖动物变态过程中尾巴吸收(细胞程序性死亡)的分子机制。

In order to study the molecular mechanism of tail resorption, I tried to establish the method of subtraction library using RNase H specificity and polymerase chain reaction, clone metamorphosis-specific genes expressed in regressing tail and obtain cell lines derived from tadpole tail for the functional assay. A small amount of regressing tail mRNA was hybridized to a large amount of tail cDNA from stage 57 tadpole before the climax and this mixture was treated by RNase H to digest the mRNA hybridized with cDNA. The cDNA synthesized from the resistant mRNA was amplified by PCR, inserted into plasmid and screened by the +/- method. 36 independently isolated clones fell into 10 different genes and were divided into 3 groups which show different expression patterns among tissues. The first group of genes are expressed in all tissues including brain, eye, hind limb and tail. The second one is activated in hind limb and tail. The third group are transcribed in only tail.A myoblastic cell line from tadpole tail is maintained more than one and half years and the cells die upon addition of 10 nM of thyroid hormone(T_3) which is a physiological level in plasma during metamorphosis. Only one gene, T6-5-12, is activated in these cells treated by thyroid hormone within one day. The expression of thyroid hormone receptor beta mRNA is induced 4 hours after T_3 treatment, whereas thyroid hormone receptor alpha is expressed constantly. The expression of T6-5-12 is observed 8 hours after the treatment. It is possible that thyroid hormone receptor alpha binds to T_3 at first, this complex activates thyroid hormone receptor beta gene, and finally the expression of T6-5-12 is induced by the compound of these receptors and thyroid hormone.By introducing specific genes of regressing tail into the myoblastic cells, the molecular mechanism of tail absorption during amphibian metamorphosis (programmed cell death) will be elucidated in the near future.