Lsolation and characterization of the Brain Forkhead gene

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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.