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，我试图使用RNase H特异性和聚合酶链反应建立减法文库的方法，克隆变质特异性基因在回归尾巴中表达并获得tadpole尾巴的细胞系进行功能分析。在高潮之前，将少量的回归尾mRNA杂交与大量的尾cDNA杂交，并通过RNase H处理该混合物，以消化与cDNA杂交的mRNA。通过PCR扩增从抗性mRNA合成的cDNA，插入质粒中，并通过+/-方法进行筛选。 36独立分离的克隆分为10种不同的基因，分为3组，这些基因在组织之间显示出不同的表达模式。第一组基因在包括大脑，眼睛，后肢和尾巴在内的所有组织中表达。第二个是在后肢和尾部激活的。第三组仅在尾部转录。t尾的肌细胞细胞系保持超过一年半，并且在添加10 nm的甲状腺激素（T_3）后死亡，这是变质过程中血浆中的生理水平。在一天之内通过甲状腺激素治疗的这些细胞中，只有一个基因T6-5-12被激活。 T_3处理后4小时诱导甲状腺激素受体βMRNA的表达，而甲状腺激素受体α不断表达。治疗后8小时观察到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T6-5-12的表达是由这些受体和甲状腺激素的化合物诱导的。通过引入肌细胞造成的细胞的特定基因，在肌母细胞中的特定基因，促进了肌细胞的特定基因，该基因是肌细胞的质量造成的。细胞死亡）将在不久的将来阐明。