Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants

Abstract

SummaryMessenger RNAs encoding the nitrate reductase apoenzyme from tobacco can be translated in a cell-free system. Poly(A)+ mRNA fractions from the 23-32 S area of a sucrose gradient were used to build a cDNA library in the expression vector λgt11 with an efficiency of cloning of approximately 104 recombinants/ng mRNA. Recombinant clones were screened with a rabbit polyclonal antibody directed against the corn nitrate reductase, which cross reacts specifically with the nitrate reductases from dicotyledons. Among 240000 recombinant plaques, eight clones were isolated containing inserts of sizes ranging from 1.6 kb to 2.1 kb and sharing sequence homologies. Seven of these clones contained a common internal 1.6 kb EcoRI fragment. The identity of these clones was confirmed as follows. A fusion protein of 170 kDa inducible by IPTG and recognized by the rabbit nitrate reductase antibody was expressed by a lysogen derived from one of the recombinants. The antibodies binding the fused protein were eluted and shown to be inhibitory to the catalytic activity of tobacco nitrate reductase. Two monoclonal antibodies directed against nitrate reductase were also able to bind the hybrid protein. The 1.6 kb EcoRI fragment was sequenced by the method of Sanger. The open reading frame corresponding to a translational fusion with the β-galactosidase coding sequence of the vector shared strong homology at the amino acid level with the heme-binding domain of proteins of the cytochrome b5 superfamily and with human erythrocyte cytochrome b5 reductase. When the 1.6 kb EcoRI fragment was used as a probe for Northern blot experiments a signal corresponding to a 3.5 kb RNA was detected in tobacco and in Nicotiana plumbaginifolia mRNA preparations but no cross-hybridization with corn mRNAs was detected. The probe hybridized with low copy number sequences in genomic blots of tobacco DNA.