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

为了研究蝌蚪尾部再吸收的分子机制，本研究尝试利用RNase H特异性和聚合酶链反应建立消减文库的方法，克隆蝌蚪尾部再吸收的变态特异性表达基因，并获得蝌蚪尾部再吸收的细胞系用于功能检测。将少量退化的尾mRNA与大量的57期蝌蚪的尾cDNA杂交，用RNase H消化与cDNA杂交的mRNA。通过PCR扩增抗性mRNA合成的cDNA，插入到质粒中，并通过+/-方法筛选。36个独立分离的克隆分属于10个不同的基因，并分为3组，这些基因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。第二个是在后肢和尾部激活。第三组仅在尾部转录，一个来自蝌蚪尾部的成肌细胞系维持了一年半以上，细胞在加入10 nM的甲状腺激素（T_3）后死亡，这是变态期间血浆中的生理水平。只有一个基因，T6-5-12，在这些细胞中被甲状腺激素处理一天内激活。T_3处理后4 h，甲状腺激素受体β mRNA表达被诱导，而甲状腺激素受体α mRNA表达持续。在处理后8h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，这种复合物激活甲状腺激素受体β基因，最后这些受体与甲状腺激素复合物诱导T_6 -5-12的表达。两栖类变态过程中尾部吸收的分子机制（程序性细胞死亡）将在不久的将来得到阐明。

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.