| A gene is a region of DNA that controls a discrete hereditary characteristic, usually corresponding to a single mRNA carrying the information for constructing a protein. It contains one or more regulatory sequences that either increase or decrease the rate of its transcription. In the post-genome era, identifying the function and interactions of disease-associated proteins produced by individual genes and their roles in specific disease may provide a major opportunity to elucidate disease mechanisms, and to identify new diagnostic markers and therapeutic targets. Burn infection is an obstacle in therapying the severe burn patients, which is the most important lethal factor for burn patients till now. It is clear that excessive inflammatory reaction plays an important role in impairing endothelial cells (EC) process. Therefore, it is very meaningful to seek and identify correlated genes of inflammation in EC.The endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1) gene (GenBank Accession No. AY074889) was cloned by the effective technique of suppression subtraction hybridization (SSH) by comparing the differential gene expression between normal ECV304 and ECV304 treated by LPS. The full-length cDNA of the EOLA1 gene was generated with SMART RACE technique, which contains 1404 nucleotides, 474 nucleotides of the open reading frame that predicts 158 amino acids. The genomic DNA contains 5 exons, spans about 6294 bps, and is mapped to human chromosome Xq27.4. We found that EOLA1 secondary structure containsα-helix, β-Lamellosa and β-turn, and a helix-turn-helix (HTH) motif by bioinformatics analysis. We scanned the prosites of EOLA1 on line (http://genomic.sanger.ac.nk/prositepatterns), found that EOLA1 contains: (1) N-glycosylation site (36-39 NCTI), (2) Protein kinase C phosphorylation site (7-9 SFR, 33-35 SQR), (3) Casein kinase II phosphorylation site (100-103 TPDE). This information indicates that EOLA1 may play an important role in process of Human EC activation as a transcription factor. The extended expression profile of EOLA1 in some kinds of Human tissues and carcinoma cells was observed by Northern blotting. High expression of EOLA1 could be found in heart, skeletal muscle, liver and placenta, while low expression in the colon, small intestine, spleen and no expression in brain, thymus, lung and peripheral blood leukocyte. Besides, EOLA1 moderate expression also could be found in promyelocyte leukemia cell line HL-60, Hela cell line S3, chronic myelogenous leukemia cell line K-562, lung cancer cell line A54, and high expression in adult lymphocytic carcinoma MOLT-4, rectal glands carcinoma SW480, and no expression in melanoma G-361.The ORF of EOLA1 was amplified from the total RNA of ECV304 cells by RT-PCR technique, and directionally subcloned to the eukaryotic expressive vector (pEGFP-N2). The recombinant vector was transfected into ECV304 cells to express EGFP-EOLA1 fusion protein, and then the subcellular localization of EOLA1 was observed through the EGFP reporter molecule with fluorescent microscope. We found that EOLA1 localized in whole cell, and EOLA1 aggregation phenomenon was observed in nuclei of ECV304 cells. The ECV304 cell strain with stable expression of EGFP-EOLA1 fusion protein was obtained by culturing the ECV304 cells tansfected with pEGFP-N2/EOLA1 vector under the pressure of G418 (400ug/ml) in M199 medium for more than a month. This cell strain will facilitate the sequential study of this novel gene. The yeast two-hybrid system has been widely used for the identification of protein interactions. It is well established that interaction partners are an immediate lead into biological function and can potentially be exploited for therapeutic purposes. Therefore, in order to examine the biological functions of EOLA1, we searched for associated proteins and protein-protein interactions by a GAL4-based yeast two-hybrid system. Using the ORF of EOLA1 cDNA as bait to screen an adult human liver cDNA library, we found that eight positive clones could bind to th...
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