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Procedures are described for the preparation of highly purified thymidylate synthases from Escherichia coliand 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°C for 10 min resulted in the loss of 50% of the E. colithymidylate synthase activity, while 50°C for 10 min was required to obtain the same effect with the B. subtilisenzyme. In the presence of the substrate, dUMP, each protein was stabilized further by 6 to 7°C, which was increased to 9 to 10°C on addition of dihydrofolate. It was shown also that the E. colithymidylate synthase could be maintained at 4°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.

Group 1 introns, first demonstrated in the nuclear large rRNA of Tetrahymena thermophilaand subsequently in many yeast, fungal mitochondrial, and chloroplast precursor RNAs, are capable of intron excision and exon ligation in vitro, although this process occurs much more rapidly in vivo. The discovery and characterization of a similar intron in the T4 phage thymidylate synthase gene (td) led to the finding of additional group 1 introns in other T4 genes and in genes of the related T2 and T6 phages. Because protein factors are not required in the splicing of group 1 introns in vitro, it has been postulated that the precursor RNA can assume a critical conformation enabling it to undergo site‐specific autocatalytic cleavage and ligation (self‐splicing). By means of site‐directed mutation, it has been shown unequivocally that several sequence elements in the TetrahymenarRNA intron are involved in the formation of base‐paired stem structures that are essential for the self‐splicing process. These sequence elements have been demonstrated in other eukaryotic group 1 introns, as well as in the tdintron. In this brief review we shall describe the biochemical and structural properties of the tdintron in relation to other newly found phage introns. The interesting implications arising from these revelations will also be discussed.— Chu, F. K.; Maley, G. F.; Maley, F. RNA splicing in the T‐even bacteriophage. FASEB J.2: 216‐223; 1988.

The activities of six bacteriophage T2r+-induced enzymes (thymidylate synthetase, deoxycytidylate deaminase, thymidylate kinase, deoxycytidylate hydroxymethylase, deoxycytidine pyrophosphatase, and dihydrofolate reductase) were measured after dilution of phage-infected Escherichia coliB from 8 × 108to 2 × 108cells per ml. The only enzyme activity altered was that of deoxycytidylate deaminase, which increased three- to fourfold. Conversely, the rapid concentration of cells from 2 × 108to 8 × 108per ml did not result in a reduction in deaminase activity. Although an enhancement in aeration reduced the response of deoxycytidylate deaminase to cellular dilution, the influence of potential metabolic inhibitors or activators could not be shown. The change in deoxycytidylate deaminase activity appeared to be associated with an altered translational event, since the increase could not be prevented by rifampin but was blocked effectively by chloramphenicol and hydroxylamine. In addition, antibody to the T2 phage-induced deoxycytidylate deaminase demonstrated that the increase in enzyme activity was associated with a corresponding increase in radioactive leucine incorporated into the enzyme antigen.

The molecular weight of the T2r+bacteriophage-induced deoxycytidylate deaminase was determined by sedimentation equilibrium to be 124,000 ± 1,800. Sedimentation of the enzyme in 6 mguanidine hydrochloride containing 0.1 m2-mercaptoethanol yielded a homogeneous species with a molecular weight of 20,200 ± 100. Additional evidence that the enzyme is composed of six subunits, possibly identical, was obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies before and after dimethyl suberimidate treatment and also by Sephadex G-200 chromatography. The molecular weight of the deaminase obtained by the latter procedure varied directly with the protein concentration; the lowest molecular weight approximated that of the monomer and the highest that of the hexamer. Intermediate values were considered weight average quantities resulting from the rapid equilibrium of monomer and hexamer. When deoxycytidine 5′-triphosphate was included in the elution buffers, the concentration dependence of molecular weight was eliminated and only the hexamer was evident. Most of the data suggest that the hexamer is the active form of deoxycytidylate deaminase, but that conformation is also important as evidenced by the finding that 0.5 mguanidine hydrochloride can reduce the enzyme activity by 50% with little apparent change in the s20,w. Guanidine hydrochloride at concentrations of 1 m, however, effects an almost complete loss in activity accompanied by disaggregation of the enzyme protein to a form with an s20,wof 3.0. Studies on the rate and extent of deaminase reactivation following denaturation by 6 mguanidine hydrochloride reveal that the renaturation process is a complex function of many parameters.