Cloning And Verification Of The Promoter Of Endothelial-overexpressed Lipopolysaccharide-associated Factor 1(EOLA1)

After finishing the human genomic plan,most genes have been located.Identification and characteristicing the human genes has been the primary work in back genomic era. Now, the function and transcription mechanism of most genes have been found, but some novel genes also maintain unkown,including EOLA1(endothelial-overexpressed lipopolysa -ccharide-associated factor 1, EOLA1).Endothelial cell (EC) is one of the main target cells that are directly activated by LPS, and many researches have been done in signal transmitting, specific receptor and the route of action in EC by LPS stimulation. The EOLA1 gene (GenBank Accession No. AY074889) was cloned by our laboratory by the effective method of mRNA differential display (differential gene expression between normal ECV304 and thosed treated with LPS). Using the SMART RACE technique, the full-length cDNA of the EOLA1 gene was cloned. This gene is 1404 base pair (bp) long and the open reading frame is 474bp, encoding a 158aa product. This gene locates at the human chromosome Xq27.4, containing 5 exons spans about 6294bp.α-helix,β-Lamellosa,β-turn and a helix-turn-helix (HTH) motif were found by bioinformatics analysis. This information indicated that EOLA1 may be as a transcription factor, which plays an important role in the process of activating Human EC.Northern blot analysis between EOLA1 and some human tissues and carcinoma cells revealed an extended expression profile of EOLA1. It was highly expressed in skeletal muscle, heart ,liver, kidney, placenta and expression in the spleen, colon and small intestine, and expression in brain, thymus, lung and peripheral blood leukocyte was not observed. EOLA1 was found to be expressed in different human cancer cells, such as promyelocytic leukemia HL-60, HeLa cell S3, chronic myelogenous leukemia K-562, lymphoblastic leukemia MOLT-4 and colorectal adenocarcinoma SW-480. Expression in lymphoblastic leukemia MOLT-4 and colorectal adenocarcinoma SW-480 was especially highly. But it was not found in lung carcinoma A-549 and melanoma G-361.The enhanced green fluorescent protein-EOLA1 was found in the whole ECV-304 after the pressure bolting of G418.The yeast two-hybrid experiment has showed the interaction of EOLA1 and MT2A, which may play some roles in the apoptosis, growth and anti-inflammation course in human EC. But the biological function of EOAL1 and its regulation is unknown.It is important to make the transcriptional control known before understanding the differential expression of identical gene. The transcriptional control system consists of promoter, enhancer, silencer, cis-acting element and the related trans-acting element, but it is the promoter that determines the basic transcriptional control. Most of the gene promoters have the TATA box, or there are many transcriptional start sites. There are two types of mRNA in EOLA1 due to the different transcriptional start site. Moreover, the transcriptional factor combining with the cis-acting element can boost the transcriptional activity of the promoter, or hold up its transcription. This is the reason why genes display different transcriptional activity in different cells or at the different stage in the same cell.The aim of this research was to seek the relatively accurate position of the promoter region of EOAL1 by Polymerase chain reaction (PCR), nested deletion, recombination , dual-luciferase reporter assay and Bioinformatics analysis, and this would be the foundation of subsequent research.First the genomic DNA was extracted from the ECV-304 cells, and this genomic DNA was used to clone the upstream regions of EOAL1 with different length. Then these DNA fragments were cloned into the luciferase reporter vector, pGL3-Basic, and three recombinant vectors with different length of upstream fragment of EOAL1 were obtained, naming pGL3-Basic-1076bp,pGL3-Basic-1723bp,pGL3-Basic-2426bp.These recombinant vectors were identified by specific endonuclease digestion and gene sequencing analysis to warrant the correctness. Then these vectors were contransefected into the ECV-304, with a interior control pRL-TK. Compared with pGL3-Basic,the relative luciferase activities of pGL3-Basic-1076bp, pGL3-Basic-1723bp and pGL3-Basic-2426bp are remarkably enhanced, and the improvement of pGL3-Basic-1723bp was especially significant. Bioinformatics analysis by promoter prediction program shows the region between -671 bp and -721 bp was the most probable promoter location region, with the score of 1.00. This result was consistent with that obtained in the reporter gene system.In conclusion, this research specified the promoter of EOAL1, which located between the region of -1672bp～+51bp. This work is of important means for the following research on the regulation and expression of EOAL1.