The Expression Pattern And Function Of Human Era

Era (Escherihia coil ras-like) was originally named on the basis of limited similarity to the Ras protein. Further analysis of Era sequence suggested that the similarity was confined to the GTP-binding domain and that Era was not a number of Ras family, Era from different organisms is highly conserved and has been found in nearly every bacterial genome sequenced to date. Their unique C terminal make them form a new subfamily of GTPase. EAC0I1 Era is an essential membrane-associated GTPase. and has a regulatory role in cell cycle by coupling bacterial cell growth rate with cytokinesis. Cell division is signaled when a threshold of Era GTPase activity is reached. Artificially reducing the expression of ERA result in cell cycle arrest at a predivitional two-cell stage. The arrest lasts until Era activity accumulates to the threshold level. There are two domains in Era: The N-terminal GTP-binding domain is closely related to Ras p21, whereas the C-terminal domain is unique. Sequence study have shown that the C-terminal region of Era contains an RNA binding KH domain, consistent with the findings that Era bind RNA in vitro and in vivo. Recently, during professor CHEN Su-min worked in NIH, he and his college searched the dbEST database with the C-terminal of E.coli Era and found there are Era homologues in eukaryotes, such as Caenorhabditis elegans, mouse and human. Then they identified mouse and human Department of Biochemistry tzndMolecidar Biology, FMMU 2001 era-cDNA successfully. The aim of this study is to identify the expressive and contributive rule of human Era and the function of this protein on cell morphology and cell cycle. Here we report the analysis of the expression pattern and function of human Era. In the study, the expression pattern of human Era has been first analyzed. The coding region of human era-cDNA was labelled and used to probe human multiple-tissue Northen blot and human multiple-tissue Expression dot (CLONTECH) to detect the expression level of human era mRNA. The result demonstrated that human era mRNA is expressed in all tissues and cell lines analyzed but with different expression level, The probe identified an mRNA product of approximately 2.2kb. To analyze the expression level of human Era protein, the rabbit anti human Era polyclonal antibody prepared and purified in our former study has been used as the first antibody. The expression level of human Era in several fetal tissues was then identified by immunohistochemistry, This assay showed that human Era is expressed in almost all tissues detected. The order of positive signal strength is Pancreas >Lung>Heart>Spleen >Stomach >Small intestine> Liver>Kidney. To analyze the intercellular localization of human Era, we constructed two eukaryotic expressive vectors of EGFP-hEra fusion protein named pSMEGFP-hEra and pEGFP-C1-hEra, in which human Era localized in N or C terminal of EGFP separately. These vectors was transfected into mouse fibroblast cell line NIH3T3 by way of lipofectin. Fluorescence microscopy analysis revealed that human Era localized in cytoplasm around cell nucleus mainly. Then the intercellular localization of human Era with natural expression level was analyzed by indirect immunofluorescence assay. The results also demonstrated that human Era localized mainly in cytoplasm in several cultured cell lines such as osteosarcoma cell line MG63, stomach cancer cell line 7901 and rheumatoid arthritis cell line RA. This study suggested human Era ma...