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Die Rekombination (Neukombination) von genetischem Material ermoglicht die gezielte Bildung neuer Bakterienstamme, die Proteine in grosem Mase synthetisieren. Mit solchen Bakterien gelingt auch die Produktion bisher nicht zuganglicher Proteine. Diese konnen zum Ausgangsmaterial fur die Gewinnung von Arzneimitteln werden, welche neue Therapiemoglichkeiten eroffnen. Mehrere bakteriell erzeugte Proteine werden bereits klinisch gepruft, und die grostechnische Produktion hat fur einige begonnen. Der ubergang vom Forschungslaboratorium zur industriellen Anwendung erfolgte schneller als vielfach erwartet worden war. An den Beispielen Insulin, Somatostatin und Wachstumshormon werden die Methoden, Probleme und Aussichten bei der Gewinnung von Peptidhormonen aus Bakterien nach DNA-Rekombination dargelegt. Dabei sollen vorwiegend die molekularbiologischen Aspekte diskutiert werden.

(1990). Genetic engineering of herbicide resistance in higher plants. Critical Reviews in Plant Sciences: Vol. 9, No. 1, pp. 1-15.

A simplified procedure for the preparation of 1,4-alpha-glucan phosphorylase from Klebsiella pneumoniae is described. An 80-fold purification is achieved in two steps with an overall yield of about 50%. The specific activity of the homogeneous enzyme protein is 17.7 units/mg. Compared with glycogen phosphorylase from rabbit muscle the enzyme from K. pneumoniae shows a markedly higher stability against deforming and chaotropic agents. The 1,4-alpha-glucan phosphorylase was covalently bound to porous glass particles by three different methods. Coupling with glutaraldehyde gave the highest specific activity, i.e., 5.6 units/mg of bound protein or 133 units/g of glass with maltodextrin as substrate. This corresponds to about 30% of the specific activity of the soluble enzyme. With substrates of higher molecular weight, such as glycogen or amylopectin, lower relative activity was observed. The immobilized enzyme preparations showed pH activity profiles which were slightly displaced to higher values and exhibited an increased temperature stability.

1. A procedure is described for the isolation of d-galactose dehydrogenase from Pseudomonas fluorescens, grown on d-galactose as carbon source. A 750-fold purification was achieved with an overall yield of about 40%. The final preparation had a specific activity of about 850 μmol NADH formed per min per mg protein. 2. The enzyme has been shown to be homogeneous by ultracentrifugation, by disc gel electrophoresis, by gel isoelectric focusing and by dodecylsulfate gel electrophoresis. 3. The molecular weight of the native enzyme has been determined to be 64000 by sedimentation equilibrium studies and by gel permeation chromatography. 4. The enzyme dissociates in the presence of 0.1% sodium dodecylsulfate to polypeptide chains, whose molecular weight was determined as 32000 by electrophoresis, indicating that the native enzyme is composed of two subunits. 5. The amino acid composition of d-galactose dehydrogenase has been determined. 6. The pI of the enzyme has been shown to be 4.28. The pH optimum is between 9.1 and 9.5. At pH 9.1 and 30 °C the limiting Michaelis constant for NAD+ is Ka= 0.24 mM, for NADP+Ka= 2.3 mM and for d-galactose with NAD+ and with NADP+ as cosubstrate Kb= 0.7 mM. The dissociat on constant for NAD+ is Kia= 0.54 mM and for NADP+Kia= 6.2 mM. 7. d-Galactose dehydrogenase from Ps. fluorescens catalyzes the dehydrogenation of d-galactose by either NAD+ or NADP+. The enzyme oxidizes the following sugars arranged according to relative rates in decreasing order: d-fucose > d-galactose > l-arabinose > 2-deoxy-d-galactose ≫ 4-deoxy-d-galactose > 2-deoxy-2-amino-d-galactose. 8. The correspondence between amino acid composition, subunit size and structure and between specificity of d-galactose dehydrogenase from Ps. fluorescens and from Ps. saccharophila is discussed.

1. 1. d-Galactose dehydrogenase from Pseudomonas saccharophila (molecular weight 102 000) dissociates in 8 M urea into its subunits (molecular weight 25 000) which migrate in polyacrylamide gels, containing 8 M urea, as a single band. 2. 2. The N-terminal residue determination by the dansyl method revealed only serine. 3. 3. The C-terminal group determination with carboxypeptidase A and B indicated the sequence -Tyr-His-Leu. Leucine as the single C-terminal amino acid was confirmed by the tritiation method and by tritiation and subsequent degradation with carboxypeptidases. 4. 4. The fragmentation of d-galactose dehydrogenase (24 mol methionine per mol enzyme) by CNBr resulted in six peptides, as detected in disc electrophoresis and substantiated by end group determination, indicating the identity of the subunits. 5. 5. The treatment of d-galactose dehydrogenase (24 mol lysine and 52 mol arginine per mol enzyme) with trypsin and subsequent peptide mapping showed 21, perhaps 22 peptides, indicating a structure comprising four identical subunits.

Recombination of genetic material enables the creation of new bacterial strains which can synthesize specific proteins in large amounts. Such bacteria permit the production of previously inaccessible proteins. They can therefore be used as starting materials for the production of drugs which will open up new paths for therapy. Several proteins produced by bacteria after DNA recombination are presently undergoing clinical trials while others are already being produced on a large scale. Thus, in the area of recombinant DNA techniques the transition from the research laboratory to industrial exploitation has occurred much faster than was anticipated several years ago. The methods, possibilities and problems encountered in the synthesis of peptide hormones by bacteria after DNA recombination are outlined, using insulin, somatostatin, and growth hormone as examples. Great emphasis is placed on the molecular biological aspects of this approach.

Today there are no longer any basic problems involved in isolating and characterizing plant genes. Our knowledge of the structure and function of the genes so far sequenced provides a beginning understanding of gene regulation in plants. In connection with the recently developed methods of gene transfer to plant cells, the most important application is the production of plants with new characteristics through genetic engineering. Since the genes can be transferred from any kind of plant—indeed even from any organism—completely new possibilities are opened up for plant breeding.

Biologie de la cellule vegetale: techniques de cultures cellulaires, utilisation de protoplastes, selection de mutants in vitro, importance de la biologie de la cellule pour le genie genetique. Transfert d'ADN: systeme utilisant Agrobacterium tumefaciens, transformation directe de cellules avec du DNA isole. Expression genique chez les plantes. Applications du genie genetique: obtention de plantes resistantes, modifications de la composition des substances vegetales, manipulation des processus physiologiques, fixation de l'azote. Problemes et risques

We have obtained transgenic tobacco plants overexpressing the enzyme glutamine synthetase (GS) by fusing an alfalfa GS gene to the cauliflower mosaic virus 35S promotor and integrating it into Nicotiana tabacum var. W38 plants by Agrobacterium tumefaciens mediated gene transfer. The amount of RNA specific to alfalfa GS was about 10 times higher in transgenic tobacco plants than in alfalfa. The alfalfa GS produced by these transgenic plants was identified by Western blotting and represented 5% of total soluble protein in the transformed plants, amounting to a 5-fold increase in specific GS activity and in a 20-fold increase in resistance to the GS inhibitor L-phosphinothricin in vitro. Tissue from GS overproducing plants showed a sevenfold lower amount of free NH3. The amino acid composition of the plant tissue was not altered significantly by GS overproduction. GS overproducing plants were fertile and grew normally. These data show that a high level of expression of a key metabolic enzyme such as glutamine synthetase does not interfere with growth and fertility of plants.

Growth inhibition of plants and bacteria by the experimental herbicide Hoe 704 (2-methylphosphinoyl-2-hydroxyacetic acid) was alleviated by the addition of the branched-chain amino acids to growth media. Hoe 704 caused a massive accumulation of acetoin and acetolactate, indicating its direct interference with the branched-chain amino acid biosynthetic pathway. The second enzyme of this pathway, acetolactate reductoisomerase (EC 1.1.1.86), was found to be subject to strong inhibition by Hoe 704. The inhibition was time-dependent and competitive with the enzyme's substrate, acetolactate. This report establishes acetolactate reductoisomerase as a new target for a herbicidal compound.

1 d-Galactose dehydrogenase from Pseudomonas saccharophila has been purified over 700-fold with an over-all yield of 27%. The purified enzyme exhibits a specific activity of about 200 μmol NADH formed × min−1× mg protein−1 and has been shown to be homogeneous by ultracentrifugation and by gel electrophoresis. 2 The amino acid composition of the enzyme has been determined. 3 The native enzyme has a sedimentation coefficient (S20,w0) of 5.94 S and a diffusion coefficient (D20,w0) of 5.60 F. The experimentally determined partial specific volume (v) is 0.744 ml/g. The molecular weight calculated from these data is 103000, in good agreement with the value of 102000 estimated by gel permeation chromatography. 4 The enzyme dissociates into polypeptide chains in the presence of 5 M guanidine hydrochloride or 0.1% sodium dodecylsulfate. The molecular weight of the polypeptide chains has been found to be 25000 by ultracentrifugation (S20,w0= 1.35 S, D20,w0= 5.28 F) and by dodecylsulfate-polycrylamide electrophoresis. The native enzyme is composed of four polypeptide chains. 5 The pI of d-galactose dehydrogenase is 5.1. The pH-optimum is between pH 8–9 in Tris-HCI buffer. AT PH 8.6 and 30°C the limiting michaelis constant for NAD+ is Ka= 0.14 mM, the limiting michaelis constant for d-galactose is Kb= 1.0 mM and the dissociation constant for NAD+ is Kia= 0.48 mM. 6 The enzyme is specific for NAD and uses NADP only to a minor extent as a coenzyme. It oxidizes the following sugars arranged according to relative rates, in decreasing order: d-FUCOSE > d-galactose > 3-deoxy-d-galactose > 2-deoxy-d-galactose > 4-deoxy-d-galactose > l-arabinose > 2-deoxy-2-amino-d-galactose.