Differential display of ethanol-induced gene in N18TG2 cells

Background: Recent advances in molecular biology, such as polymerase chain reaction (PCR) differential display, have enabled the screening of mRNAs tr anscriptionally regulated by chronic ethanol treatment. Screening of gene e xpression after ethanol exposure will bet the most needed for new biologica l insights into alcoholism. Methods: We used PCR differential display to detect differentially expresse d RNAs in N18TG2 cells treated for 4 days with physiologic concentrations o f ethanol (25 mM). Results: We succeeded in identifying two differentially expressed RNAs in t he ethanol-treated cells. The increase in the expression of the two RNAs wa s verified by Northern hybridization analysis. Sequence analyses and search es of the sequence databases revealed that one of the RNAs was that of the heat shock cognate protein 73 (HSC73) gene and that the other was the produ ct of a novel gene. The increase in the level of HSC73 mRNA after ethanol a dministration was consistent with similar reports from other laboratories, and indicated that our assay system would be applicable to the screening of up-regulated gene expression during ethanol treatment. Rapid amplification of cDNA 5'-ends (5'-RACE) allowed us to determine the upstream sequence of the uncharacterized mRNA that would code for a protein of 113 amino acids. A homology search by MPsrch indicated very low homology to the calcium cha nnel L-type alpha 1 subunit. Conclusions: The function of this new gene product is presently unknown, bu t our results indicate that an investigation of the pathophysiological sign ificance of the gene in alcoholism would be worthwhile. Identification of g enes that are influenced by chronic ethanol will certainly increase of the molecular mechanisms underlying physiologic dependence.