A protein was purified from crude extracts of the soil fungus Myrothecium verrucaria by gel filtration and hydrophobic chromatography to homogeneity; this protein catalyzed the stoichiometric hydration of the fertilizer cyanamide to urea with high substrate specificity. This cyanamide hydratase (urea hydro-lyase; EC 4.2.1.69) contained zinc and consisted of six identical subunits with Mr = 27,700. It was partially sequenced. The protein was detectable only when the fungus was grown on cyanamide as the sole nitrogen source. Genomic DNA from the fungus was cloned, and the gene encoding the enzyme was mapped with an oligonucleotide probe derived from the amino acid sequence within a 25,800-base-pair DNA region. The subunit of the enzyme is encoded by a 795-base-pair DNA sequence containing a 63-base-pair intron. A cDNA clone containing the intronless gene with an open reading frame encoding a sequence of 244 amino acids expressed the enzyme in active form in Escherichia coli with excellent yield.Cyanamide (H2N-G-N) in aqueous solution or in the form of its calcium salt is used as a fertilizer in agriculture. It provides ammonia to the soil by its metabolic conversion. It has the additional advantage of acting as an effective herbicide. Therefore it has to be applied prior to sowing. In view of the present interest in the ecological effects of widely used chemicals, we became interested in the metabolic conversion of this not naturally occurring compound. Chemically, cyanamide belongs to the class of nitriles. In spite of the relatively rare occurrence in nature of compounds containing the nitrile group, enzymes that hydrate this group to the correspondihg amide (nitrile hydratases) have been frequently found in various bacteria (1-5) and also in plants (6). In 1973 Stransky and Amberger (7) EXPERIMENTAL PROCEDURES Growth of Fungi. M. verrucaria (DSM no. 2087) was obtained from the Deutsche Sammlung von Mikroorganismen (Braunschweig, F.R.G.) and grown as described (7) but scaled up. From 4 liters of medium, 10-25 g (fresh weight) of mycelium was harvested after 10 days. The rapidly filtered mycelium was stored at -70°C.Cyanamide Hydratase Assay. The assay is based on the decrease in cyanamide concentration during incubation with the enzyme (7). The enzyme (0.05-0.2 units) was incubated with 20mM cyanamide in 5 mM sodium phosphate buffer (pH 7.7) (total volume = 0.25 ml) at 37°C for 15-60 min, depending on the activity of the enzyme. The cyanamide concentration was determined by a colorometric assay at 530 nm in 20 Al of the incubation mixture before and after enzyme addition (8). One unit of cyanamide hydratase metabolizes 1 ,umol of substrate per min under these conditions. Purification of Cyanamide Hydratase. Twenty grams of the frozen mycelium, after addition of 10%6 (vol/vol)
There is a general need for additional selectable marker genes for plant transformation. Only a few have been reported in wheat (Triticum aestivum L.) transformation experiments, some of which are under patent restriction or have other disadvantages. A new selectable marker gene was identified which can be used to select resistant callus in tissue culture and regenerate transgenic wheat plants. A gene from the soil fungus Myrothecium verrucaria (Albertini & Schwein.) Ditmar:Fr., coding for the enzyme cyanamide hydratase which converts cyanamide into urea, was previously described. In our wheat transformation experiments, the gene conferred resistance to cyanamide at a tissue culture level and therefore cyanamide could be used to select for transformants. At the whole plant level, progeny of transformed wheat plants showed resistance to cyanamide, whereas sensitive plants expressed a lethal necrosis and yellowing when cyanamide was applied. This gene has several potential advantages when compared with other selectable marker genes. Transformed wheat plants can be selected at the tissue culture level and may be able to convert cyanamide into a useful nitrogen compound (fertilizer). The selectable marker gene could be introduced with other genes for value‐added traits in wheat and might also be applicable in other transformation systems.
The incorporation of the gene for cyanamide hydratase can make transgenic tobacco plants resistant to the herbicide cyanamide H2NCN. The herbicide is degraded by the expressed gene to the physiologically harmless urea H2NCONH2. In the presence of the ubiquitous plant enzyme urease, which degrades urea, the hydrolysis product NH can, in fact, serve as nitrogen source. The efficiency of this new approach is impressively demonstrated.
Durch Einbau des Gens für Cyanamid‐Hydratase können transgene Tabakpflanzen resistent gegenüber dem Herbizid Cyanamid H2NCN gemacht werden. Das Herbizid wird durch das exprimierte Gen zu physiologisch unbedenklichem Harnstoff H2NCONH2 abgebaut. In Gegenwart des Harnstoff‐abbauenden Enzyms Urease, das in vielen Pflanzen vorkommt, kann das Hydrolyseprodukt NH+4 sogar noch als Stickstoffquelle für die Pflanzen dienen. Die Effizienz dieses neuen Ansatzes wird eindrucksvoll belegt.
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