ENHANCED EXPRESSION OF HEPATIC GENES IN COPPER-DEFICIENT RATS DETECTED BY THE MESSENGER-RNA DIFFERENTIAL DISPLAY METHOD

The influence of copper (Cu) status on hepatic gene expression was exa mined by using the ''messenger RNA differential display'' technology. This method involves the distribution of mRNA in a two-dimensional arr ay for the rapid identification and cloning of differentially expresse d genes. Livers from male Sprague-Dawley rats that had been fed a Cu-d eficient (CD) diet (9.4 mu mol/kg) or a Cu-adequate (CA) diet (103.9 m u mol/kg) for 6 wk were used to supply cytosolic RNA. Cytosolic RNA we re reverse-transcribed in the presence of anchor primers and then ampl ified by polymerase chain reaction with anchor and arbitrary primer se ts. The amplified cDNA were then resolved by denaturing polyacrylamide gel electrophoresis. Differences in mRNA expression between the CD an d CA rats were identified. DNA fragments were cloned, sequenced and us ed as probes for Northern blot analysis to confirm that the identified genes were differentially expressed. The analysis of cDNA sequences b y computer searches against DNA and protein databases revealed that on e cDNA fragment, whose mRNA abundance was enhanced 1.2-fold by copper deficiency, is novel. Four other cDNA fragments were found to have sub stantial homology with rat ferritin mRNA; rat fetuin mRNA; rat mitocho ndrial 12S and 16S rRNA, phenylalanine-, valine- and leucine-tRNA gene s; rat mitochondrial genes for 16S rRNA, tRNA-leucine and tRNA-valine; and their mRNA abundance was 0.6- to 0.8-fold higher in Cu-deficient rats. Five additional cDNAs detected by this method appeared to repres ent novel genes because they exhibited no substantial homology to reco rded gene and protein sequences deposited in DNA and protein databases . These results demonstrate the usefulness of this technology in the d etection of genes which were differentially expressed as a result of t he deprivation of a single nutrient, dietary copper, in this research project.