Isolation of cDNA clones for the light subunit of rat liver ferritin: evidence that the light subunit is encoded by a multigene family.

Abstract

The shell of the Fe-storage protein ferritin consists of 2 types of subunit (heavy, MW = 21,000 light, MW = 19,000). To study the structure and expression of the ferritin subunit genes, recombinant plasmids containing ferritin c[complementary] DNA were isolated. A cDNA library was constructed in the vector pBR322 from rat liver polysomal mRNA and screened by using 125I-labeled antibody to rat liver ferritin. Six positive clones were identified and were shown to contain cDNA inserts ranging in length from 800-950 base pairs. When these cDNA clones were used for hybrid selection of rat liver mRNA and the selected mRNA were translated in vitro, the products from each clone migrated on denaturing gels in a position similar to that of the light subunit of ferritin. No evidence of translation of the heavy subunit was obtained, indicating that the 2 subunits are encoded by separate mRNA. RNA blot analysis gave a length of 1100 nucleotides for the light-subunit mRNA. One of the cDNA inserts was fractionated into 4 fragments by using the restriction enzyme Sau3A. When the fragments were hybridized with Southern blots of rat spleen DNA, each fragment yielded similar patterns of hybridization to that obtained with the intact cDNA. All regions of the cDNA sequence contain homologous sequences to similar genomic restriction fragments. This is consistent with the existence of a family of genes that encode the light subunit of rat ferritin.