Lsolation and characterization of the Brain Forkhead gene

叉头基因家族的新成员脑叉头基因（在发现其表达谱后更名为间充质叉头-1 (Mesenchyme Fork Head-1, MFH-1)）有望在胚胎阶段发挥重要作用。Northern blot分析显示，MFH-1 mRNA主要存在于胚胎期，而不存在于成虫期。RNase的保护分析表明，其表达量在交配后第9.5天达到最大值，在胚胎后期逐渐降低。原位杂交分析显示，MFH-1 mRNA在体细胞中检测到9.5 d.p.c.，后来仅限于软骨组织和后肾。在新生小鼠中，MFH-1 mRNA主要存在于肾皮质。这些发现表明MFH-1基因可能在软骨和肾脏的发育中起重要作用。核苷酸序列分析表明，MFH-1蛋白在氨基端有叉头结构域，即dna结合结构域，在羧基端有富含脯氨酸和组氨酸的区域，即推测的反激活结构域。网状细胞裂解物中产生的重组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。进一步纯化大肠杆菌产生的重组MFH-1蛋白，获得抗MFH-1抗体。利用该抗体，我们发现MFH-1蛋白定位于细胞核内，其分子大小分别为60kda和58kda。

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.