Isolation and Characterization of a Novel Gene , Jpk , from Murine Embryonic cDNA Library

During the study on the regulation of Hox gene expression, one novel cDNA (C171, Jopock, Jpk) has been isolated, and the total nucleotide sequence (1148 bp) of cDNA has been determined. It encodes an 187 aa-long ORF (open reading frame) containing one trans-memberane domain (AA22-51) in it. Computer analysis revealed that a human homologue of C171 (Jpk) is a hypothetical uni-gene, AD-015 located on the locus, LOC113213 of human chromosome 15q26. When EGFP (enhanced green fluorescent protein) fusion construct, EGFP-C171 (JPK) was generated and transfected into the F9 teratocarcinoma cells in an attempt to trace the localization of the JPK protein inside of the cell, most of the fluorescent cells died except small portion: viable cells expressed the fusion protein in the cytoplasm, particularly in the granular structure. The viability of the transfected cell was reduced dramatically in a time dependent manner; less than 50% of the cells survived after 72 hours transfection. The tansfection of Jpk induced DNA fragmentation, generating nucleosomal ladder and the sub-G1 fraction of DNA in the nucleus, and increased the exposure rate of phosphatidyl serine on the cell surface. In order to identify the sequence necessary for trans-activation of JPK, several reporter constructs containing different regions of PSRE (position specific regulatory element) were generated. Cotransfection analysis revealed that the construct NS218 containing two GAGA binding sites was the most responsible fragment. The results altogether indicate that the expression of JPK should be regulated precisely during development since a trace amount of the protein is very toxic, leading the host cell to death.ope