SummaryExpression in mammalian COS cells and an efficient microtiter-based strategy for immunoselectlon was used in a novel approach to identify genes encoding plant membrane proteins. COS cells were transfected with an Arabidopsis thaliana root cDNA library constructed in a bacterial mammalian shuffle vector and screened with an antiserum raised against purified deglycosylated integral plasma membrane proteins from A. thallana roots. Antibodies directed against a prominent 27 kDa antigen led to the identification of five different genes. They comprised two subfamilies related to the major intrinsic protein (MIP) superfamily and were named plasma membrane intrinsic proteins, PIP1 and PIP2, since the cellular localization of PIP1 and most probably PIP2 proteins in the plasma membrane was independently confirmed by their cosegregation with marker enzymes during aequeous two-phase partitioning. Surprisingly, expression in Xenopus laevis oocytes revealed that all five PIP mRNAs coded for Hg2+-sensitive water transport facilitating activities. There had been no previous evidence of the existence of water channels in the plasma membrane of plant cells and the high diffusional water permeability of the lipid bilayer was considered to be sufficient for water exchange. Neverthelese, Northern and Western analyses showed that the PIP genes are constitutively and possibly even redundantly expressed from the small A. thaliana genome.

l h e PlPl subfamily of water channel proteins (aquaporins) constitute about 1 % of the plasma membrane (PM) proteins from Arabidopsis thaliana leaves. lmmunogold electron microscopy has confirmed their localization at the PM of mesophyll cells. Very high labeling density at PM invaginations known as plasmalemmasomes was observed. Therefore, we suggest that these subcellular structures are involved in water transport between the apoplast and the vacuole.

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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