Enhanced Expression of Hepatic Genes in Copper-Deficient Rats Detected by the Messenger RNA Differential Display Method

Abstract

The influence of copper (Cu) status on hepatic gene expression was examined by using the “messenger RNA differential display” technology. This method involves the distribution of mRNA in a two-dimensional array for the rapid identification and cloning of differentially expressed genes. Livers from male Sprague-Dawley rats that had been fed a Cu-deficient (CD) diet (9.4 µmol/kg) or a Cu-adequate (CA) diet (103.9 µmol/kg) for 6 wk were used to supply cytosolic RNA. Cytosolic RNA were reverse-transcribed in the presence of anchor primers and then amplified by polymerase chain reaction with anchor and arbitrary primer sets. The amplified cDNA were then resolved by denaturing polyacrylamide gel electrophoresis. Differences in mRNA expression between the CD and CA rats were identified. DNA fragments were cloned, sequenced and used as probes for Northern blot analysis to confirm that the identified genes were differentially expressed. The analysis of cDNA sequences by computer searches against DNA and protein databases revealed that one cDNA fragment, whose mRNA abundance was enhanced 1.2-fold by copper deficiency, is novel. Four other cDNA fragments were found to have substantial homology with rat ferritin mRNA; rat fetuin mRNA; rat mitochondrial 12S and 16S rRNA, phenylalanine-, valine- and leucine-tRNA genes; 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 represent novel genes because they exhibited no substantial homology to recorded gene and protein sequences deposited in DNA and protein data-bases. These results demonstrate the usefulness of this technology in the detection of genes which were differentially expressed as a result of the deprivation of a single nutrient, dietary copper, in this research project.