Investigation Of The Relationship Between Structure And Function Of The JWA-A Retinoic Acid Responsible And Cytoskeleton Associated Gene

la1laliOX1cOk)gy I lxmtoy, Schx)l oliublic I I lth,Nanjing Nlxlical Univcity. Nuing2l(X)2). P.R. Chii .1 WA gene was primarily cloned ironi human trachearonchial cpithel ial cells by using mRNA di Hential ci isplapolymerase chain reaction (11)1)?PC?R) technique by zhou ci. al. (1 998). As a novel gene. it has been submitted to the NC13I and got the offered olficiai accession number at AF070523 in ( iei I aiik. ihe lull length cl)NA sequence ol the gene contains 2114 nucleotidcs and a 567 base pairs (include stop coden TAA) of open reading frame(ORF) has been identi fled which encodi ig 1 88 aim no acids. the computer predicted IHOlCCLilii weight of JWA protein is 21.5 kDa. Our previous data showed that JWA may functioning as a signal molecular joins cellular cli fferenti ation and niorphologicaily as a cytoskeleton similar location pattern in investigated cells. Here, we further investigated the genic structure characteristics and the potential relationship betwcen the structure and the function of the gene. Firstly. we used both PCR and recombinant DNA techniques to identi fv the potential introns and the promoter secjuence of JWA gene. Both genomic DNA and lii] I length cJ)NA Fragnicnls were used as the templates of the PC R. and the initial comparable results showed that there was no introns identified in this gene. Within the identified 300() base pairs of promoter sequence of the gene. we found several cis-ction elements. such as pliorbol ester response eicment( RI ). stress response element(SRE), aiid the retinoic acid response element Semi-ite ci al.. we supposed that JWA might be regulated by these factors under the controled cellular signal. In another hand, for understanding J WA homo logue acne in other species. we cloned a consensus homologue fragment lion rat tissues, which contains 567. nucleotides and four distinct base pairs existed within the coding region sequence. so its to four distinct amino acids were identified in the translated product. For figuring out the relationship between the structure and the location of JWA protein in cells, we constructed site deleted recombinant express clones for JWA protein(with or without P1CC phosphorylation sites) with live fluorescent vector pEGFP. After confirmation, stable transfection and screening, the distributions and locations of the recombinant JWA proteins were identified under the laser confeal microscopy. The results showed that both full length and shortest fragment (without two PKC phosphorylation sites ) clones revealed a similar protein locations in cytoplasm. However, the clones with one of PKC phosphorylation sites showed a diffemtnt protein distribution pattern. that vtts. the protein focused and gathered around the nuclear envelop of the cell. Secondly, for confirming the structure-function relationship of die gene, we carried out the cell culture models to see if ATRA and TPA regulate the transcription of JWA and how differences of the role of the drugs among cell lines(NIH3T3, MCF-7, K562, FIL-60). The results indicated that both ATRA and WA did regulate the expression of the gene at transcription level mid lied a distinct pattern among cell lines. It was suggest that during both ATRA and WA or anyone itself induced cellular differentiation and/or apoptosis. it matv rel distinct signal pathways in different cell lines. JWA might be a potential marker for the pathways. At the same time, several other cell differentiation induccrs (41IPR, DM50) also were investigated in these cell culture models, and w...