TOBACCO CYTOCHROME B(5) - CDNA ISOLATION, EXPRESSION ANALYSIS AND IN-VITRO PROTEIN TARGETING

A full-length clone encoding cytochrome b(5) has been isolated from a tobacco leaf cDNA library in lambda gt11 by PCR using degenerate prime rs. This cDNA encodes a protein of 139 residues which exhibits a high degree of homology to other cytochrome b(5)s, the message for which is expressed predominantly in developing seeds and in pigmented flower t issue. In the developing tobacco seed the mRNA is abundant at very ear ly stages (<10 days after flowering). Southern analysis indicated that more than one gene encodes cytochrome b(5) in the tobacco genome. In vitro transcription and translation studies of the cDNA indicated that the protein inserts into the ER membrane by a non-SRP-mediated pathwa y and that the C-terminus of the protein is required for targeting and insertion.