Defoliant Residues, Determination of Cyanamide Residues on Ginned Cottonseed

Steller, William A; Frederick, I. B.; Morgan, Paul W.

doi:10.1021/jf60140a011

Cited by 12 publications

(8 citation statements)

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“…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) (17), the end-product biuret does not interfere], urea (17), formylcyanamide and acetylcyanamide (formation of the corresponding substituted urea, measured by the decrease of the absorption at 250 nm).…”

mentioning

confidence: 99%

Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly specific for the fertilizer cyanamide and cloning of its gene.

Maier-Greiner

Obermaier-Skrobranek

Estermaier

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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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)

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confidence: 99%

Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly specific for the fertilizer cyanamide and cloning of its gene.

Maier-Greiner

Obermaier-Skrobranek

Estermaier

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…The initial CAH enzymatic activity was determined by measuring the consumption of cyanamide after incubation with CAH for 15-60 min, followed by a colorimetric assay at 530 nm (32). It was reported that A 530 nm absorption is linear with cyanamide concentrations only up to 200 M (33), but in our hands the linear relationship can be extended up to 5 mM cyanamide (Fig.…”

Section: Ddi2/3 Encodes a Member Of The Hd Domain Family Ofmentioning

confidence: 56%

Two Duplicated Genes DDI2 and DDI3 in Budding Yeast Encode a Cyanamide Hydratase and Are Induced by Cyanamide

Biss

et al. 2015

Journal of Biological Chemistry

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Background: DDI2 and DDI3 are two uncharacterized identical genes found in budding yeast. Results: They encode novel cyanamide hydratases and are massively induced by cyanamide. Their deletion causes cellular sensitivity to cyanamide. Conclusion:The two genes function in cyanamide detoxification and are tightly regulated. Significance: This is the first attempt to understand the duplicated gene cluster in budding yeast.

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“…The solution was incubated for 10 min at room temperature in the dark. Absorbance at 530 nm (Steller et al, 1965) was measured to detect reduced cyanamide concentration indicative of the presence of cyanamide hydratase.…”

Section: Methodsmentioning

confidence: 99%

Wheat Transformation Using Cyanamide as a New Selective Agent

et al. 2000

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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.

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Defoliant Residues, Determination of Cyanamide Residues on Ginned Cottonseed

Cited by 12 publications

References 1 publication

Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly specific for the fertilizer cyanamide and cloning of its gene.

Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly specific for the fertilizer cyanamide and cloning of its gene.

Two Duplicated Genes DDI2 and DDI3 in Budding Yeast Encode a Cyanamide Hydratase and Are Induced by Cyanamide

Wheat Transformation Using Cyanamide as a New Selective Agent

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