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Analysis of the mechanisms of neuronal cell-type diversification by the transcription factor UNC-86

转录因子UNC-86对神经元细胞类型多样化的机制分析

基本信息

批准号：
08680847
负责人：
MITANI Shohei
金额：
$ 1.6万
依托单位：
Tokyo Women's Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08680847/
关键词：
nematode neurodevelopment transcription factors epitope-tagging GFP C.elegans ニューロン種

项目摘要

We developed a novel method (epitope-tagging of target genes of transcription factors) to isolate in vivo binding sites of transcription factors in the nematode C.elegans. Generally, this kind of experiments are very difficult so far. We cloned the whole DNA fragment for unc-86 gene. We inserted GFP (green fluorescent protein) cDNA in frame to the coding region of the transcription factor, and used the DNA construct to rescue unc-86 mutant phenotypes. We isolated nuclei from such a strain, cross-linked the nuclear protein to DNA.Then, the nuclei were fragmented to nucleosomes. The UNC-86/GFP-target DNA sequence complexes were immuno-purified with an anti-GFP monoclonal antibody. The DNA fragments isolated in this way were cloned and sequenced. So far, 29 unique sequences corresponding to certain genome 1oci on the C.elegans chromosomes were identified using the database by C.elegans genome sequence consortium. Twenty-four of which appeared regulatory sequences of certain genes. Four of 24 corresponding to known genes, three of which are unc-86 gene itself and one known as a gene which works downstream of unc-86 in a genetic pathway for HSN neuron differentiation. Other genes are categorized into two. First are genes sharing somehow homologies to known genes which can work in nervous systems. Others didn't share any homology to known genes and are to be analyzed in the future on the possible functions in the nervous systems.

我们开发了一种新方法（转录因子靶基因的表位标记）来分离线虫体内转录因子的结合位点。一般来说，迄今为止这种实验是非常困难的。我们克隆了unc-86基因的整个DNA片段。我们将 GFP（绿色荧光蛋白）cDNA 插入到转录因子的编码区中，并使用 DNA 构建体来拯救 unc-86 突变表型。我们从这样的菌株中分离出细胞核，将核蛋白与DNA交联。然后，将细胞核片段化为核小体。使用抗 GFP 单克隆抗体对 UNC-86/GFP 靶 DNA 序列复合物进行免疫纯化。对以此方式分离的DNA片段进行克隆和测序。到目前为止，线虫基因组序列联盟利用该数据库鉴定了与线虫染色体上某些基因组1oci相对应的29个独特序列。其中二十四个出现了某些基因的调控序列。 24 个基因中的 4 个对应于已知基因，其中 3 个是 unc-86 基因本身，还有一个已知为在 HSN 神经元分化的遗传途径中在 unc-86 下游起作用的基因。其他基因分为两类。首先是与已知基因具有某种同源性的基因，这些基因可以在神经系统中发挥作用。其他基因与已知基因没有任何同源性，未来将对其在神经系统中可能的功能进行分析。