Background: Recent advances in molecular biology, such as polymerase chain reaction (PCR) differential display, have enabled the screening of mRNAs transcriptionally regulated by chronic ethanol treatment. Screening of gene expression after ethanol exposure will be the most needed for new biological insights into alcoholism.
Methods: We used PCR differential display to detect differentially expressed RNAs in N18TG2 cells treated for 4 days with physiologic concentrations of ethanol (25 mM).
Results: We succeeded in identifying two differentially expressed RNAs in the ethanol-treated cells. The increase in the expression of the two RNAs was verified by Northern hybridization analysis. Sequence analyses and searches 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 product of a novel gene. The increase in the level of HSC73 mRNA after ethanol administration 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 channel L-type alpha I subunit.
Conclusions: The function of this new gene product is presently unknown, but our results indicate that an investigation of the pathophysiological significance of the gene in alcoholism would be worthwhile. Identification of genes that are influenced by chronic ethanol will certainly increase of the molecular mechanisms underlying physiologic dependence.