Human Liver-specific Expression Cloning And Functional Studies Of Gene Tcp10l

Liver is an important multifunctional organ performing detoxification, substance synthesis and metabolic balance of nutrients. However some diseases such as hepatoma, hepatitis B, cirrhosis may distort it's function. The satisfactory treatment of these diseases is yet to come and depend largely on the further study of genes involved in the functions of liver. Grounded on a differentially displayed cDNA fragment down-regulated in hepatoma tissues, we cloned a novel cDNA of 957bp, TCP10L (T-complex protein 10 like), from human liver cDNA library. The expression pattern of this novel gene in 16 adult human tissues was examined. The result of Northern hybridization revealed that TCP10L expressed specifically in human liver and testis. By bioinformation analysis TCP10L is located to chromosome 21q22.11, and composed of 5 exons and 4 introns; its transcript contains a 645-bp open reading frame encoding a deduced polypeptide of 215 amino acids with a typical leucine zipper motif in N-terminal. As leucine zipper is a basic DNA binding motif of many known transcription factors, we examined the transcriptional function of the TCP10L using dual luciferase assay system. In three different cell strains:HEK293, SK-HEP-1 and CHO, TCP10L significantly inhibited the expression of reporter plasmid pGAL45tkLUC compared with that of the negative control (pM). Recombinant gene of TCP10L was constructed to pET32a vector and was expressed in E coli(BL21DE3)system. Purified recombinant protein is 42KD as expected and was used as antigen to make polyclonal antibody in New Zealand rabbits. After purifying and desalting, the polyclonal antibody was applied to immunohistochemical research of human tissues. Robust signals were seen specifically in the tissues of liver and testis, coincident with the Northern blot result. In the tissue of liver, the cells in hepatoma beside hepatoma were preferentially stained while in connective tissues were undetectable. In testis tissues, hybrid signals were showing in seminiferous tubles and confined specifically in primary spermatocyte.Two genes that may interact with TCP10L were obtained using yeast two hybrid technique. One gene is PMF1,another MAD4,both genes are transcription factors. Considering the false positive results of yeast two hybrid results, subcellular colocalization of TCP10L with PMF1 and with MAD4 is performed. Protein of TCP10L(in red) with that of PMF1(in green) and with MAD4(in green) coexpressed and perfectly overlapped in nuclear of HEK293 cells. However, the diffuse distribution of the three proteins made us hesitate to certify their colocalization. Further exploration is needed to make a persuasive photo that proving their colocalization. Coimmunoprecipitation that can offer the evidence of their interaction on a different aspect is undertaking.