Biochemical and Biophysical Research Communications
Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation☆
Section snippets
Materials and methods
3′ and 5′ rapid amplification of cDNA ends. For the amplification and cloning of full-length cDNAs from the subtracted cDNA fragments, the SMART RACE cDNA amplification kit (Clontech) was used. Total RNA from ECV304 cells stimulated with LPS was isolated as described elsewhere [9]. One microgram of RNA was reverse transcribed with Powerscript reverse transcriptase according to the manufacturer’s instructions. 3′-Rapid amplification of cDNA ends (RACE) was done using the supplied universal
EOLA1 cDNA cloning and sequence analysis
After SSH, one of the six clones without homology to known genes, designated ST55 (GenBank Accession No. BM121646), demonstrated an increased expression in HUVEC stimulated by LPS in Northern blot (figure not shown). RACE of ST55 clone resulted in a full-length cDNA and this gene was called EOLA1. The cDNA of this novel gene consists of 1404 nucleotides including a 542 nucleotide 5′ untranscription region (UTR), 474 nucleotides of an ORF predicting 158 amino acids with a molecular mass of 17.89
Acknowledgments
This work was supported by China National Natural Science Foundation of Grant (No. 30200101 and No. 30371468) and by an operating grant from China State Key Laboratory of Trauma, Burn and Combined Injury.
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Abbreviations: LPS, lipopolysaccharide; EOLA1, endothelial-overexpressed lipopolysaccharide-associated factor 1; MT2A, metallothionein 2A; HUVEC, human umbilical vein endothelial cells; SSH, suppression subtractive hybridization; LBP, LPS-binding protein; RACE, rapid amplification of cDNA ends.