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，我尝试利用RNase H特异性和聚合酶链反应建立减文库的方法，克隆退化尾巴中表达的变态特异性基因，获得蝌蚪尾巴衍生细胞系进行功能分析。将少量回归的尾部mRNA与57期蝌蚪高潮前的大量尾部cDNA杂交，并用RNase H对杂交后的mRNA进行消化处理。用PCR扩增抗性mRNA合成的cDNA，插入质粒，用+/-法筛选。36个独立分离的克隆分为10个不同的基因，分为3组，在组织中表现出不同的表达模式。第一组基因在所有组织中表达，包括脑、眼、后肢和尾巴。第二个在后肢和尾部被激活。第三组只转录在尾部。蝌蚪尾成肌细胞系维持1年半以上，细胞在加入10 nM的甲状腺激素（T_3）后死亡，T_3是变态过程中血浆中的生理水平。在甲状腺激素治疗的这些细胞中，只有一个基因T6-5-12在一天内被激活。甲状腺激素受体β mRNA在T_3处理后4小时被诱导表达，而甲状腺激素受体α则持续表达。治疗后8小时观察T6-5-12的表达情况。可能甲状腺激素受体α首先与T_3结合，该复合物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复合诱导T6-5-12的表达。通过在成肌细胞中引入尾巴退化的特定基因，将在不久的将来阐明两栖动物变态（程序性细胞死亡）过程中尾巴吸收的分子机制。

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.