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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.

Two /3-1,4-endoglucanases were isolated in nearly pure form from the culture supernatant of Thermomonospora fusca strain YX. The purification procedures included gel chromatography, ion-exchange chromatography, hydroxylapatite chromatography, and preparative gel electrophoresis. These enzymes and their proteolytic breakdown products account for at least 90% of the total extracellular carboxymethylcellulase activity, although at least one other @-1,4-endoglucanase is present. Even though these enzymes are essential for the hydrolysis of filter paper by the extracellular fraction, they are not sufficient. The specific activity of the starting material on filter paper was 4 times that of the most active purified enzyme. Even though they are both endoglucanases, these two enzymes differ in their molecular weights, substrate specificities, and carbohydrate contents and are immunologically distinct proteins. The most active enzyme (E,) has a specific activity greater than 700 IU/mg on carboxymethylcellulose (CMC) when assayed at 55 OC in 0.05 M potassium phosphate buffer, pH 6.5. Its molecular weight is 94000 determined by both sodium dodecyl sulfate gel electrophoresis and glycerol gradient centrifugation, showing that it is a monomeric enzyme. It contains less than 1% carbohydrate and has an isoelectric point at pH 3.5. Its temperature optimum is 74 OC, and it has a broad pH optimum centered at pH 6. The products of cellulose hydrolysis catalyzed by E, are mainly cellobiose with a small amount of glucose. Its K, for C M C is 360 pg/mL; it is stimulated

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An NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.24) was cloned from the ruminal ciliate protozoan, Entodinium caudatum. The gene had high sequence similarity to GDH genes from the Bacteroides (class)--a class of bacteria which is highly represented in the rumen. When expressed in Escherichia coli the enzyme had a high affinity for ammonia and alpha-ketoglutarate (apparent K(m) of 2.33 and 0.71 mM, respectively) and a low affinity for glutamate (apparent K(m) of 98 mM). GDH activity and GDH mRNA concentration were increased by incubating washed E. caudatum cells with ammonia and antibiotics. These results suggest that the GDH is an anabolic enzyme catalysing the assimilation of ammonia by E. caudatum in the rumen and that the gene was probably acquired by lateral gene transfer from a ruminal bacterium.