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

细胞程序性死亡——两栖动物变态过程中尾部退化的分子机制

基本信息

批准号：
03670134
负责人：
YAOITA Yoshio
金额：
$ 1.28万
依托单位：
TOKYO METROPOLITAN INSTITUTE FOR NEUROSCIENCES
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670134/
关键词：
Programmed Cell Death Metamorphosis Xenopus Developmental Biology Genetic Engineering

项目摘要

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.

为了研究尾部吸收的分子机制，我尝试利用RNase H特异性和聚合酶链式反应建立消减文库的方法，克隆回归尾部表达的变态特异性基因，并获得蝌蚪尾部来源的细胞系进行功能测定。。将少量的退化尾部mRNA与来自高潮前的57期蝌蚪的大量尾部cDNA杂交，并用RNase H处理该混合物以消化与cDNA杂交的mRNA。将抗性mRNA合成的cDNA通过PCR扩增，插入质粒中并用+/-法筛选。 36个独立分离的克隆分为10个不同的基因，并分为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，包括大脑、眼睛、后肢和尾巴。第二个在后肢和尾部被激活。第三组仅在尾部转录。来自蝌蚪尾部的成肌细胞系维持超过一年半，在添加10 nM甲状腺激素（T_3）（变态过程中血浆中的生理水平）后细胞死亡。在经过甲状腺激素处理的这些细胞中，只有一个基因 T6-5-12 在一天内被激活。 T_3治疗后4小时诱导甲状腺激素受体β mRNA的表达，而甲状腺激素受体α持续表达。治疗8小时后观察T6-5-12的表达。可能是甲状腺激素受体α首先与T_3结合，该复合物激活甲状腺激素受体β基因，最后这些受体与甲状腺激素的复合物诱导T6-5-12的表达。将尾巴回归到成肌细胞中，两栖动物变态过程中尾巴吸收（程序性细胞死亡）的分子机制将在不久的将来得到阐明。