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

我们开发了一种新的方法（表位标记的转录因子的靶基因），在线虫线虫体内分离的转录因子的结合位点。一般来说，这类实验到目前为止是非常困难的。我们克隆了unc-86基因的完整DNA片段。我们将GFP（绿色荧光蛋白）cDNA插入转录因子的编码区，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细胞核，将核蛋白与DNA交联，然后将细胞核裂解成核小体。用抗GFP单克隆抗体对GFP-86/GFP-靶DNA序列复合物进行免疫纯化。将以这种方式分离的DNA片段克隆并测序。迄今为止，利用线虫基因组序列联盟的数据库，已鉴定出29个与线虫染色体上的某些基因组1 oci相对应的独特序列。其中24个出现了某些基因的调控序列。24个中的4个对应于已知基因，其中3个是unc-86基因本身，一个已知为在HSN神经元分化的遗传途径中作用于unc-86下游的基因。其他基因分为两类。第一种是与已知基因有某种同源性的基因，这些基因可以在神经系统中起作用。其他基因与已知基因没有任何同源性，未来将分析其在神经系统中的可能功能。

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.