Study On The Function Of Mammalian Era In Cell Cycle

The gene of era was firstly discovered in E. coli, and the protein it codes has limited similarity to the Ras protein. Thus, the protein was named the Escherichia coli (E. coli) Ras-like protein (Era). Further analysis of Era sequence suggested that it has similarity to the Ras protein only in the GTP-binding domain. And it wasn't a member of Ras family. The crystal structure of E. coli Era has showed that the N terminal of Era was a typical G domain and the C terminal was a KH domain, which could bind RNA.The gene of era is necessary to bacteria survival, and the deficiency or mutant of Era in bacteria is lethal. Analysis of temperature-sensitive mutants indicates that era is related to cell cycle of bacterium, and it plays a role in the phase after DNA replication and chromosome segregation but before cell division.Many studies have implicated E. coli era in diverse biological processes after era cloned from bacterium, but the function of era in eukaryotic cells is still unclear. Since the mechanism of controlling the cell cycle in eukaryotes is different from in prokaryotes, and bacterium era plays an important role incell cycle, one aim of this study is to investigate the function of mammalian era in cell cycle.Inducible Mammalian Expression System can produce high expression level of target gene and turn on/off gene expression rapidly, it is a strong means to study gene function. This system has vector pEGSH and receptor expression vector pERV3 in the experiment. After induced by PonA, target gene can express.Wild type human era gene was cloned into inducible expression vector pEGSH and co-transferred into cell line HEK293 with receptor expression vector pERV3. After induced by PonA, the effect of over-expressed Era on cells was observed. The result showed that over-expression of wild type human Era could stimulate cell growth and proliferation, and the number of G2/M phase decreased dramatically. Wild type human Era plays an important role in cell cycle in that it can promote mitosis and accelerate cell proliferation.To study the functions of two domains, 4 mutants were constructed: 2 point mutants heraS36N, heraI310N(the mutant points were located in Era G domain and KH domain respectively), and 2 truncated mutants heraN, heraC(KH domain in C terminal and G domain in N terminal were deleted). They were cloned into inducible expression vectors, and then transfected these expression vectors into HEK293 cell lines in which pERV3 was stably transfected. After induced, over-expressed mutant type human Era could depress cell proliferation, and increase the number of G2/M phase dramatically. The two domains of human Era are important to the function as promoting mitosis. The changes in two domains are dominant negative, and they can depress the function of wild type human Era, inhibit cell proliferation,and arrest cells in G2/M phase.To understand more about the function of human era in cell cycle control, RNAi vector which target was human era gene was constructed. The results showed it could effectively decrease the expression of wild type human Era, inhibit cell proliferation, arrest cells in G2/M phase. So wild type human Era plays an important role in cell cycle, can promote mitosis and accelerate cell proliferation. Decreased Era expression can inhibit cell proliferation, arrest cells in G2/M phase.To investigate the role of human era in cell cycle regulator net, cDNA micro-array was conducted for screening up- and down-regulated genes after the expression of human era gene was blocked by using RNAi. We found that 10 genes up-regulated, and 7 genes down-regulated. These genes play important roles in cell cycle or apoptosis system, and give us some clues in studying the biological functions of human era gene.To study the function of wild type mouse Era, inducible mera expression vector was cloned. After induced by PonA, the over-expression of wild type mouse Era could stimulate cell proliferation, and decrease the percentage of G2/M cells dramatically. So wild type mouse Era plays an important role in cell cycle, it can promote mitosis and accelerate cell proliferation.To study the functions of two domains of mouse Era, 4 mutants were constructed: 2 point mutants meraS21N, meraI295N(the mutant points were located in Era G domain and KH domain respectively), and 2 truncated mutants meraN, meraC(KH domain in C terminal and G domain in N terminal were deleted respectively).When they were over-expressed, cells proliferation was depressed, and the number of cells in G2/M phase increased dramatically. So these mutants are dominant negative mutants, they candepress the function of promoting mitosis, inhibit cell proliferation, arrest cells in G2/M phase.When our lab cloned mouse era cDNA, two splicing mouse era-cDNAs from mouse brain and blastula tissues were obtained. The two kinds of mouse era were analyzed by using Bioinformatics. The results showed that one kind of mouse era (meraS) was shorter 249bp than another (meraW); correspondingly, mEraS protein is shorter 83aa than mEraW protein. All of two splicing Era proteins have two domains (GTP-binding domain and KH domain). They have highly homology with the human and E.coli Era protein. MBP-mEraW and MBP-mEraS fusion proteins were expressed in E.coli, and purified by amylose affinity chromatography. The purity came to approximately 67% and 61% of the total bacterial protein respectively. The rabbit anti-human Era antibody has high specificity to two splicing mouse Era proteins in Western-blot, the antibody can be used in studying the function of these two kinds of splicing mouse Era proteins.To study the intercellular localization of mouse Era, we constructed Era-EGFP fusion expressive vectors: pSMEGFP-meraW and pSMEGFP-meraS, and then transfected them into mouse fibroblast cell line NIH3T3. Fluorescence microscopy analysis revealed that two splicing mouse Era-EGFP proteins localized mainly in cytoplasm around cell nucleus. The intercellular localization of mouse Era in nature was analyzed by Immunocytochemistry techniques and the results showed that mouse Era localized in all subcellular parts. The reason of different results may be that Era-EGFP fusion protein is difficult into cell nucleus.To study the effects of two alternative splicing mouse era genes on cells growth, the two alternative splicing mouse Era were over-expressed in cell...