Preliminary Study On The Functions Of TMEM66 Gene

The immune system is the essential part of the body to protect itself to counter detrimental agents from outside. The malfunction of the system sometimes caused lethal problems of normal life. The immune cells play a central role in the system; they derived from hematopoietic progenitor cell in bone marrow. So, the development and differentiation of hematopoietic progenitor cell is vital importance for the normal functioning of immune activities. TMEM66 (transmembrane protein 66) is a protein involved in the differentiation of the immune cells. Present studies focus in the gene function both in vitro and in vivo. The main results are as follows:1 The predicted function of TMEM66 geneUntil now, there is no report in the scientific literature regarding the function of TEME66 gene. At first, we colleted some related information about this gene employ some bioinformatics methods to deduce the possible biological function. This will be better for future study. The TEME66 protein is a tri-transmembrane protein which contains a DUF1183 domain. In human, the gene expression of TEME66 is high in differentiated immune cells such as B lymphocytes, T lymphocytes, monocytes and dendritic cells. It also showed high expression in brain, spinal cord, lymph note and blood. However, it showed low expression level in leukemic cell (HL60, Molt4 and K562), malignant lymphoma cells (Raji, Daudi) and other undifferentiated immune cells. It is down-regulated in carcinomatous tissues too. These results indicated that TEME66 is involved in the differentiation of immune cells and its aberrant low expression is associated with cancer.2 Molecular characterization of TEME66 geneTEME66 is a new gene which in relate to cell differentiation in our bioinformatics predictions. Present study cloned, mapped the porcine TEME66 gene, then identified several polymorphism sites in pig populations and detected its expression pattern in both human and pig, provided basic information for further research of this gene. Our PCR results showed the pig TMEM66 gene contains 1886bp including an integrity ORF and encoding a protein with 337 animo acids which shared 84% homologous with its human counterpart. The pig TMEM66 gene located in the q arm of porcine chromosome 15 and closely linked with marker SW1673. An A/C SNP is identified in the pig TMEM66 locus. The genotyping results showed the A allele is only presented in Bama pigs and it is the dominant allele in this population. However, there is only C allele in other pigs investigated. Pig TEME66 gene expression is abundant in lung, spleen, lymph note and brain, but in low level in skeletal muscle, heart and fat. The expression of human TEME66 gene is down-regulated in carcinomatous tissues or cells compared with normal tissues. The TMEM66 gene is up-regulated through the embryo development. After ATRA induction in U937 cells, the expression of TEME66 gene is increased, reached the plateau around 48 hours and then decreased. These results indicated that TEME66 gene involved in cell differentiation. In this study, there were two other transcriptions of human TMEM66 gene founded. They may participate to the regulation of the function of TMEM66 gene.3 TEME66 function in cellsTEME66 protein is a membrane protein; the subcellular localization of TEME66 protein could implicate its function. This study detected the subcellular localization of human TEME66 protein. We also studyed the overexpression of TMEM66 gene using lentivirus vector. The results indicated that TEME66 protein located in the nuclear membrane. The third transcript distributed across the cytoplasm and also nuclear membrane. The IFIT5 which was interacted with TMEM66 localized to cytoplasm. After overexpression of TEME66 by lentivirus infection in U937 cells, the apoptosis intensified. Then, ATRA induction was introduced in and the G2 period of U937 cells with overexpression of TEME66 is higher than that of control cells without TEME66 overexpression. The apoptosis percent is also higher in the overexression cell lines. These results indicated that TEME66 protein mainly localized to the nuclear membrane. During differentiation, TEME66 could cause the G2 period prolongation and induce the apoptosis of targeted G2 period cells.4 The TEME66 mutation transgenic mouseTo investigate the TEME66 function in vivo, we produced the TEME66 mutation transgenic mice by conventional micro injection method. After PCR and southern blot verification, we found 6 positive individuals among the 55 neonatal pups. Three positive mice could stably transfer the transgene to next generation through passage. The transgene is incorporated into the host genome in a serial multi-copy manner. RT-PCR analysis indicated that the transgene was expressed in many tissues. The results showed at least 3 mice embraced the TEME66 mutation transgene which could be stably passaged; provide a valuable model for the further study of TEME66 function.