High-level expression of Escherichia coli and Bacillus subtilis thymidylate synthases.

Procedures are described for the preparation of highly purified thymidylate synthases from Escherichia coli and Bacillus subtilis. The yields in each case are quite high with about 350 mg of pure protein obtained from 1 liter of cells. Basically all that is required to obtain pure enzyme is an induction step from a high-expression vector, followed by a DE-52 column elution. Both enzymes appeared to be fairly stable in that incubation at 43 degrees C for 10 min resulted in the loss of 50% of the E. coli thymidylate synthase activity, while 50 degrees C for 10 min was required to obtain the same effect with the B. subtilis enzyme. In the presence of the substrate, dUMP, each protein was stabilized further by 6 to 7 degrees C, which was increased to 9 to 10 degrees C on addition of dihydrofolate. It was shown also that the E. coli thymidylate synthase could be maintained at 4 degrees C for at least 4 months with little or no loss in activity provided that mercaptoethanol was not present. The presence of the latter led to a progressive loss in activity until little activity could be detected after 18 weeks, which was due, in part, to the formation of a disulfide bond with the active site cysteine. Addition of dithiothreitol restored the enzyme activity to its original state.

Nucleosides as a carbon source in Bacillus subtilis: characterization of the drm-pupG operon.

In Bacillus subtilis, nucleosides are readily taken up from the growth medium and metabolized. The key enzymes in nucleoside catabolism are nucleoside phosphorylases, phosphopentomutase, and deoxyriboaldolase. The characterization of two closely linked loci, drm and pupG, which encode phosphopentomutase (Drm) and guanosine (inosine) phosphorylase (PupG), respectively, is reported here. When expressed in Escherichia coli mutant backgrounds, drm and pupG confer phosphopentomutase and purine-nucleoside phosphorylase activity. Northern blot and enzyme analyses showed that drm and pupG form a dicistronic operon. Both enzymes are induced when nucleosides are present in the growth medium. Using mutants deficient in nucleoside catabolism, it was demonstrated that the low-molecular-mass effectors of this induction most likely were deoxyribose 5-phosphate and ribose 5-phosphate. Both Drm and PupG activity levels were higher when succinate rather than glucose served as the carbon source, indicating that the expression of the operon is subject to catabolite repression. Primer extension analysis identified two transcription initiation signals upstream of drm; both were utilized in induced and non-induced cells. The nucleoside-catabolizing system in B. subtilis serves to utilize the base for nucleotide synthesis while the pentose moiety serves as the carbon source. When added alone, inosine barely supports growth of B. subtilis. This slow nucleoside catabolism contrasts with that of E. coli, which grows rapidly on a nucleoside as a carbon source. When inosine was added with succinate or deoxyribose, however, a significant increase in growth was observed in B. subtilis. The findings of this study therefore indicate that the B. subtilis system for nucleoside catabolism differs greatly from the well-studied system in E. coli.

Crystal structure of thymidylate synthase A from Bacillus subtilis.

Thymidylate synthase (TS) converts dUMP to dTMP by reductive methylation, where 5,10-methylenetetrahydrofolate is the source of both the methylene group and reducing equivalents. The mechanism of this reaction has been extensively studied, mainly using the enzyme from Escherichia coli. Bacillus subtilis contains two genes for TSs, ThyA and ThyB. The ThyB enzyme is very similar to other bacterial TSs, but the ThyA enzyme is quite different, both in sequence and activity. In ThyA TS, the active site histidine is replaced by valine. In addition, the B. subtilis enzyme has a 2.4-fold greater k(cat) than the E. coli enzyme. The structure of B. subtilis thymidylate synthase in a ternary complex with 5-fluoro-dUMP and 5,10-methylenetetrahydrofolate has been determined to 2.5 A resolution. Overall, the structure of B. subtilis TS (ThyA) is similar to that of the E. coli enzyme. However, there are significant differences in the structures of two loops, the dimer interface and the details of the active site. The effects of the replacement of histidine by valine and a serine to glutamine substitution in the active site area, and the addition of a loop over the carboxy terminus may account for the differences in k(cat) found between the two enzymes.

[Subcortical structures of the brain and the behavior of white rats]. / Podkorkovye struktury mozga i povedenie belykh krys.

Conditioned food and defensive reflexes have been studied in white rats following bilateral injury of paleo-, archi-, neostriatum, substantia nigra and paleocortex. It has been shown that these deep structures play a role in the operational memory of animals.

[Conditioned reflexes following destruction of the deep structures of the brain]. / Uslovnye refleksy posle razrusheniia glubinnykh struktur mozga.

The role and the relative significance of paleo-, archi- and neostriatum as well as Corpus Luysii, substantia innominata, substantia nigra, nucl. ruber and the hippocampus, in the mechanism of purposeful behaviour of animals, were studied on cats and rats. It has been shown that the above structures, together with the cerebral cortex, take part in operational memory. The data obtained continue the evolutionary concept of L. A. Orbeli that in higher vertebrates with a well-developed cortex, the deeper brain structures do not lose their initial function, and together with the cortex, participate in the provision for the integrative activity of the brain.

[Participation of the basal ganglia in the mechanism of visual memory in the rat]. / Uchatie bazal'nykh gangliev v mekhanizmakh zritel'noi pamiati u krys.

Studies have been made of the effect of bilateral injury of paleo-, archi- and neostriatum, as well as that of the nucleus ruber on adaptive behaviour in albino rats. It was shown that injury of the basal structures of the brain results in disturbances of optimal visual choice of a food signal. The data obtained are discussed from a standpoint of disturbances in operative memory of visual signals. It is suggested that during evolutionary development, when the higher brain functions pass to neocortex, the basal ganglia do not loose their initial role in the higher analytical-synthetic activity.

[Effect of damage to the substantia nigra on conditioned reflex activity in animals]. / Vliianie povrezhdeniia chernoi substantsii na uslovnoreflektornuiu deiatel'nost' zhivotnykh.

Effects of the Substantia nigra injury on conditioned activities were studied on rats and cats by defensive and feeding methods, respectively. Bilateral injury of the Substantia nigra led to temporary disappearance of conditioned defensive and feeding motor reflexes, to prolongation of latency and duration of motor reaction. In cats the injury of the Substantia nigra produced a 30--40% decrease of the accuracy of food direction choice. Similar changes were observed following injury of the Nucleus caudatus head.

[Conditioned motor-alimentary reflexes in cats following ablation of the body of Luys]. / Uslovnye dvigatel'no-pishchevye refleksy pri povrezhdenii tela Liuisa u koshek.

Alimentary conditioned motor reflexes in cats were elaborated in special chambers. Subsequently, the subthalamic nucleus was damaged, first on one and then on the other side. After its unilateral destruction, temporary disturbance in the cat behaviour was observed disappearing within 2--3 weeks. Bilateral destruction of the subthalamic nucleus led to a stable disturbance in the reaction of choice of reinforcement side in conditions of uncertainty.

[The role of the pallidum in regulating cortical activity]. / Rol' palliduma v reguliatsii korkovoi aktivnosti

Bilateral ablation of the pallidum halves the duration of extinction of conditioned motor food reflexes and contributes to 30 to 50% extinction of the electro-defensive reflexes. Pallidum functional depression by potassium chloride or novacaine leads to a temporary total depression of conditioned motor food reflexes. Depending on the frequency of pallidum stimulation, synchronization or desynchronization of the cortical bioelectrical activity is observed. Ablation of the pallidum in anaesthetized cats results in a high amplitude and low-frequency cortical activity. Injection of large doses of potassium chloride into the pallidum results in a forced running forward which lasts 30 to 40 min. The pallidum is considreed as a structure controlling the cortex activity, which takes part in the mechanisms of sensory information processes in the course of adaptive behaviour.

[Conditioned alimentary motor reflexes in cats following destruction of the scorpula]. / Uslovnye dvigatel'nye pishchevye refleksy u koshek pri povrezhdenii skorpuly

Bilateral ablation of the putamen results in temporary disappearance of natural as well as artificial conditioned motor reflexes in cats. Further training is necessary for their reestablishment. Restored conditioned reflexes appear with a long latency. After partial bilateral ablation of the putamen, animals that have been trained not only to press a pedal, but also to make a choice between a right or left feeding tray, lose the latter ability. After the reestablishment of conditioned reflexes, the animal makes a correct choice of side only in 68--70% of cases. After total bilateral ablation of the putamen the animals lose artificial conditioned reflexes. The results suggest that the putamen has a close relation to the mechanisms of learning and memory.