Lsolation and characterization of the Brain Forkhead gene

脑叉头基因的分离和表征

基本信息

批准号：
05680592
负责人：
MIURA Naoyuki
金额：
$ 1.28万
依托单位：
Akita University (1994)Osaka University (1993)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05680592/
关键词：
Forkhead domain HNF3 Transcription factor Kidney cortex Cartilage Gene family DNA binding Development フォークヘッド MFH-1 ノックアウトマウス胎児腎臓 fork head 形態形成間充織

项目摘要

A new member of the forkhead gene family, the Brain Forkhead gene (which is renamed Mesenchyme Fork Head-1 (MFH-1) after the finding of its expression profile) was expected to play an important role in the embryonal stage. Northern blot analysis showed that the MFH-1 mRNA is detected mainly in the embryonal stage, but not in the adult stage. RNase protection analysis indicated that its expression is maximum on day 9.5 postcoitum and decreasing in the later embryonal stage. In situ hybridization analysis revealed that the MFH-1 mRNA is detected in the somite at 9.5 d.p.c. and later restricted to the cartilaginous tissues and metanephros. In the neonatal mice, the MFH-1 mRNA is mainly detected in the kidney cortex. These findings suggest that the MFH-1 gene might play an important role in the development of cartilages and kidneys.Nucleotide sequence analysis indicated that the MFH-1 protein has the forkhead domain, the DNA-binding domain, in the amino-terminal half and the proline-and histidine-rich region, the putative transactivating domain, in the carboxy-terminal half. The recombinant MFH-1 protein produced in a reticulocyte lysate, could bind to the HNF3-binding site.Next we cloned and analyzed the chromosomal MFH-1 gene. We found that the MFH-1 gene is an intronless gene and its major transcriptional initiation site is located 584 bp upstream from the translation initiation site.Furthermore we purified the recombinant MFH-1 protein produced in E.coli and obtained anti-MFH-1 antibody. Using this antibody, we found that the MFH-1 protein is localized in the nucleus andits molecular sizes are 60 kDa and 58 KDa.

作为叉头基因家族的新成员，Brain Forkhead 基因（在发现其表达谱后更名为 Mesenchyme Fork Head-1 (MFH-1)）预计将在胚胎阶段发挥重要作用。 Northern印迹分析显示MFH-1 mRNA主要在胚胎期检测到，但在成体阶段不检测到。 RNase保护分析表明，其表达在交配后第9.5天达到最大，并在胚胎后期逐渐减少。原位杂交分析表明，在 9.5 d.p.c. 的体节中检测到 MFH-1 mRNA。后来仅限于软骨组织和后肾。在新生小鼠中，MFH-1 mRNA 主要在肾皮质中检测到。这些发现表明MFH-1基因可能在软骨和肾脏的发育中发挥重要作用。核苷酸序列分析表明MFH-1蛋白在氨基末端具有叉头结构域，即DNA结合结构域，富含脯氨酸和组氨酸的区域，即推定的反式激活结构域，位于羧基末端的一半。网织红细胞裂解液中产生的重组MFH-1蛋白可以与HNF3结合位点结合。接下来我们克隆并分析了染色体MFH-1基因。我们发现MFH-1基因是一个无内含子基因，其主要转录起始位点位于翻译起始位点上游584 bp处。进一步纯化了大肠杆菌中产生的重组MFH-1蛋白，获得了抗MFH-1蛋白。抗体。使用该抗体，我们发现MFH-1蛋白定位于细胞核，其分子大小为60 kDa和58 KDa。