SUMMARYIn nearly all systems studied, ribonucleotide reductase consists of two non-identical subunits. We present here the results of our study on herpes simplex virus (HSV) ribonucleotide reductase in favour of the existence of two subunits, H1 and H2, different from the mammalian subunits, M 1 and M2. First, although the viral subunits could not be separated by Blue Sepharose chromatography (unlike mammalian subunits), they seemed to dissociate at very low protein concentration as suggested by the non-linear relationship between activity and low protein concentration. Second, pyridoxal phosphate (Pyr.P)-NaBHa treatment and 4-methyl-5-amino-l-formylisoquinoline thiosemicarbazone (MAIQ) treatment of partially purified extract of mammalian ribonucleotide reductase which inactivated M I and M2 respectively also inhibited the HSV ribonucleotide reductase. This activity could be restored by mixing Pyr.PNaBH~-treated extracts with MAIQ-treated extracts of viral ribonucleotide reductase, suggesting that each treated extract contains one active subunit. Moreover, the addition of exogenous MI or M2 subunits to one or the other of these two treated extracts did not produce any detectable reductase activity. Our interpretation of these results is that the two subunits H1 and H2 which could dissociate upon treatment did not form enzymically active hybrids with the mammalian subunits. Also, the higher degree of resistance to heat inactivation and to hydroxyurea of the viral reductase as compared to the mammalian enzyme suggests that H1 differs from M I and H2 from M2.

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance ('Ble') genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however, the highest frequency of rooted plants was obtained when transformation was carried out with the 'Sh Ble' gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.

A phosphoryl exchange reaction between fructose 1-phosphate and fructose was found to be catalyzed by a membrane preparation isolated from Bacillus subtilis. The regulation of the biosynthesis of the activity in the wild type as well as in the regulation mutants fruB closely correlates with that of the membrane-bound enzyme I1 of the phosphoenolpyruvate fructose l-phosphotransferase system which is known to mediate the transmembrane vectorial phosphorylation of fructose.The computed analysis of the kinetic data shows that the mechanism of the enzyme I1 is pingpong, i.e. that a phosphoryl-enzyme intermediate occurs in the reaction. The apparent dissociation constants of the enzyme II/fructose 1-phosphate complex and of the phosphoryl enzyme II/fructose complex are estimated. The value of the standard free energy of the hydrolysis of the bond between the phosphoryl moiety and the enzyme suggests a covalent bonding. This intermediate is assumed to occur in the physiological functioning of the enzyme which utilizes the phosphocarrier protein HPr as phosphoryl donor.The exchange reaction is competitively inhibited by high fructose concentrations : this indicates that the same site of the enzyme binds fructose and fructose 1-phosphate, this site being accessible to fructose on the external side of the membrane when the enzyme is phosphorylated.Since the discovery by Kundig et al. [l] of bacterial phosphotransferase systems, it has been shown that these systems mediate the first step of the catabolism of several carbohydrates in many bacterial species (see recent reviews by Postma and Roseman [2], and Hengstenberg [ 3 ] ) .Genetic evidence was presented that in Bacillus subtilis fructose, glucose, mannose, mannitol and sucrose phosphorylation was dependent upon such systems [4]. A detailed study of the catabolism of either intracellular or extracellular fructose [5 -71 allowed us to demonstrate that an inducible phosphoenolpyruvate fructose 1-phosphotransferase system was devoted to a physiological vectorial phosphorylation step, i.e. an inwards translocation of the sugar concomitant with the phosphorylation at the C-1 site. The system involves at least three components: enzyme I, the phosphocarrier protein HPr, and a membrane-bound enzyme 11; the overall reaction may be schematized in its simplest form, as follows: + H P r -p h o s p h a t e 3 H P r + intracellular sugar-phosphate

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