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

Abstract

Messenger 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 gt11 with an efficiency of cloning of approximately 10(4) 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 -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.

Transient Accumulation of Nitrite Reductase mRNA in Maize following the Addition of Nitrate

“…10 and references therein). It has been shown for both NR in several plant species (5,7,9) and for NiR in spinach and maize (3,16) (2,8).…”

“…The seedlings were grown hydroponically by floating the foam pads on the surface ofthe media. Two salt media were used: (a) minimal medium, 10 mM Mes (pH 5.5), 5 mM CaSO4; (b) complete medium (minus nitrogen), 5 For RNA blot hybridization, 20 ,g of leaf or 10 ,ug of root total cellular RNA was subjected to electrophoresis through a 1.2% agarose, 2.2 M formaldehyde gel, and the RNA was blotted onto nitrocellulose. The probe used for the hybridization was a NiR cDNA insert from the plasmid pCIB801 (16).…”

“…The aqueous phase was reextracted with 10 mL of chloroform and made 2 M with LiCl. After overnight precipitation at 4°C, the RNA was sedimented in an SW41 rotor (Beckman) at 25 K for 2 h. The RNA was resuspended in 1% SDS, 5 90,1989 response depending on what medium is used, the general regulatory pattern is the same.…”

“…[10] for a review). With the cloning of these genes, it has been possible to demonstrate that the mRNA levels for these genes increase considerably upon the addition of nitrate (3,5,7,9,16). The rate with which nitrate is taken into root tissue has also been shown to be increased in the presence of nitrate (13).…”

Phytochrome-Mediated Light Regulation of Nitrate Reductase Expression in Squash Cotyledons

“…Recent progress in cloning cDNA for NR3 demonstrated that steady state levels ofNR transcripts were increased by nitrate induction in the light (3,8,9). For corn leaves, application of nitrate to N-starved, etiolated seedlings in the dark led to small increases in NR protein and activity, but large increases were found when light was given (15).…”