Cloning of rat intestinal mRNAs affected by zinc deficiency

Male rats were fed a purified diet containing 1 mg Zn/kg to induce zinc deficiency (-Zn). A second set of rats were fed a 30 mg Zn/kg diet, which supplied adequate levels of zinc (+Zn). The zinc-adequate rats were pair-fed to the rats fed the 1 mg Zn/kg diet to eliminate differences in diet intake. After 16 d, the two conditions of dietary zinc status were confirmed by assaying serum zinc concentrations and by Northern analysis for metallothionein mRNA abundance in the kidney. Messenger RNA was purified from small intestine by oligo(dT) chromatography and pooled within conditions. A cDNA plasmid library was constructed from the mRNA derived from the intestines of zinc-deficient rats. The library was then screened by the method of differential hybridization using 32P-labeled first strand cDNA probes derived from the +Zn and -Zn mRNA. After screening 10,000 independent cDNA clones, nine cDNAs were isolated and studied further, corresponding to mRNAs that are down-regulated by zinc deficiency. The ratio of the abundance between the two conditions for the nine mRNAs ranged from 1.5- to sevenfold as determined by Northern analysis of the +Zn and -Zn intestinal mRNAs. Two of these clones seemed to be specific to the intestine, and four others were abundant in the intestine and one or two other tissues. We are using these cDNAs as models to study gene regulation under various conditions of dietary zinc intake and to explore the genes most sensitive to zinc deficiency.