The Function Research Of Human And Mouse Era Gene

era (Escherichia coli ras-like] was found as an open reading frame when sequencing the genome of E. coli in 1986. Later result showed that it was widely distributed in organisms. The N terminal of Era protein was a typical G domain and the C terminal of Era protein was a KH domain that could bind with RNA. Era was sorted as a novel G protein family because of its specific C terminal. There was great significance to study the new member of the G protein family.My research can be divided into two parts. The one is to fish the Era binding protein by using the yeast two-hybrid system. The other is to block the expression of era gene in mammalian cells by using RNAi.Part one: We acquired 15 candidate proteins from the fetal liver cDNA library by using hEra as the bait protein. One of them, A19, contained an NTR/C345C domain that could bind with metzincins. Metzincins is a Zn ion dependent metalloproteinase. Since YggG,which could bind with E. coli Era reported by Chen Na'n-chun in 1996, was a Zn ion dependent metalloproteinase, we determined to keep on studying the A19 fragment. The GST-pulldown results indicated that hEra could bind with the NTR/C345C domain only if the GTP was presented. These preliminary results make a good foundation for further research.Part two: We constructed an shRNA expression vector by ourselves, and we blocked the expression of hera and mera gene in the cells by resorting RNA interference. When the Era protein was decreased in the cells the "S" growth curve would move to the right. The electron microscopy results indicated that majority cells in which the Era protein was decreased existed just before the pre-division state, and there was almost no cell in the division phase. Flow cytometry results indicated that there was a cell cycle arrest in G2-M phase.The electron microscopy results also indicated that the cells had multi-nucleoli phenotype when era gene expression was blocked by RNA interference. The same phenotype was got when the mutant (mutated in KH domain) was transfected to the cells. Nucleolus is a compartment where the pre-rRNA is synthesized. These changes show that era gene was involved in rRNA gene transcription.