ISOLATION, SEQUENCING AND EXPRESSION OF CDNA SEQUENCES ENCODING UROPORPHYRINOGEN DECARBOXYLASE FROM TOBACCO AND BARLEY

We have cloned and sequenced a full-length cDNA for uroporphyrinogen d ecarboxylase (UROD, EC 4.1.1.37) from tobacco (Nicotiana tabacum L.) a nd a partial cDNA clone from barley (Hordeum vulgare L.). The cDNA of tobacco encodes a protein of 43 kDa, which has 33% overall similarity to UROD sequences determined from other organisms. We propose that tob acco UROD has an N-terminal extension of 39 amino acid residues. This extension is most likely a chloroplast transit sequence. The in vitro translation product of UROD was imported into pea chloroplasts and pro cessed to ca. 39 kDa. A truncated cDNA, from which the putative transi t peptide had been deleted, was used to over-express the mature UROD i n Escherichia coli. Purified protein showed UROD activity, thus provid ing an adequate source for subsequent enzymatic characterization and i nhibition studies. Expression of UROD was investigated by northern and western blot analysis during greening of etiolated barley seedlings, and in segments of barley primary leaves grown under day/night cycles. The amount of RNA and protein increased during illumination. Maximum UROD-RNA levels were detected in the basal segments relative to the to p of the leaf.