Incorporation and Degradation of C and H-labeled Thymidine by Sugarcane Cells in Suspension Culture.

Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to beta-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2-(14)C]thymidine is lost as (14)CO(2), but the label of [(3)H]methylthymidine is found in the cell as [(3)H]beta-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines.

Thymidine incorporation into Rhizobium meliloti.

Thymidine is rapidly catabolized to thymine, beta-aminoisobutyric acid, and carbon dioxide by Rhizobium meliloti cells. The incorporation of labelled thymidine into the DNA of R. meliloti cells can be enhanced by the addition of low concentrations (10-20 micrograms/mL) of deoxyadenosine or other nucleosides (adenosine, uridine, guanosine). However, at high concentrations ( greater than 50 micrograms/mL) these compounds inhibit thymidine incorporation. Conditions to obtain highly radioactive DNA of Rhizobium are described.

Uptake and release of tetracycline by cultured carrot cells.

The nature of tetracycline uptake by carrot cell suspension cultures is described. Tetracycline enters the cells by diffusion and the intracellular level of the antibiotic increases with the amount added. Exposure of carrot cells to high levels of tetracycline for a limited time (24 hr) followed by the removal of the drug and the resuspension of the cells in drug-free medium does not affect cell growth and has no inhibitory effect on protein synthesis (14C-leucine incorporation).

Deoxyribonucleic acid degradation and the lethal effect by myxin in Escherichia coli.

Exposure of Escherichia coli 15T(-) cells to the antibiotic myxin results in the inhibition of deoxyribonucleic acid (DNA) biosynthesis, degradation of intracellular DNA, and death of the cells. Each of these effects was markedly enhanced when protein synthesis was simultaneously inhibited by chloramphenicol. In the continued presence of chloramphenicol, a brief (1 min) exposure to myxin resulted in a rate of DNA degradation and cell death equivalent to that found in the continued presence of myxin alone. Single-strand breaks were present in the DNA of cells exposed to myxin, but when chloramphenicol was also present the breaks were found much earlier. Degradation of DNA in cells exposed to myxin was found to be distributed randomly in both strands and extended over the genome with no restriction to the vicinity of the replication point. There was no release of DNA from its attachment to the cellular membrane in myxin-exposed cells. The possibility that the chloramphenicol effect is due to the inhibition of repair enzyme synthesis which is stimulated by exposure of the cells to myxin is discussed. These data indicate that the extent of the lethal and metabolic damage to the cells by an exposure to myxin represents the result of competition between damage to and repair of cellular DNA.

Effect of myxin on deoxyribonucleic acid synthesis in Escherichia coli infected with T4 bacteriophage.

Exposure of Escherichia coli cells to myxin results in the almost complete inhibition of new deoxyribonucleic acid (DNA) synthesis, extensive degradation of pre-existing intracellular DNA, and a rapid loss of viability in these cells (9). After exposure to myxin for 30 min (<1% survivors and >25% degradation of DNA), infection of these cells by T4 bacteriophage results in the renewal of DNA synthesis at a rate essentially equal to that found in T4-infected cells in the absence of myxin. This DNA was characterized as T4 DNA by hybridization and by hydroxyapatite chromatography. These results suggest that the primary site of action of myxin does not involve the biochemical pathways involved in either the energy metabolism or the biosynthesis of DNA precursors in the uninfected host cell. The yield of infectious T4 particles was reduced when myxin was present during multiplication. This effect may be partly accounted for by the finding that a significant fraction of the T4 DNA synthesized in the presence of myxin is apparently not properly enclosed by the bacteriophage protein coat since it is shown to be degraded by exogenous nuclease.

Mode of action of myxin on Escherichia coli.

The effect of the new antibiotic, myxin, on the syntheses of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein in Escherichia coli (strains B and 15T(-)) was examined. Within 7 min of the addition of myxin at 5 mug/ml, the synthesis of new bacterial DNA was almost completely inhibited. This was followed by an extensive degradation of the pre-existing DNA to an acid-soluble form. All of the evidence indicated that the primary effect of the antibiotic was on cellular DNA. The synthesis of RNA was completely inhibited after 15 min of exposure to myxin (5 mug/ml), and the synthesis of protein was markedly reduced after 30 min. There was no measurable breakdown of either RNA or protein in the myxin-treated cells. A marked stimulation of (14)C-uracil incorporation was found in the presence of myxin in 15T(-) cells only. This did not result from an increased rate of RNA synthesis but was due to an increase in the proportion of exogenous uracil, relative to endogenous uracil, incorporated into cellular RNA. This probably reflected a partial inhibition of the biosynthesis of uridine monophosphate from orotate. At 4.5 mug of myxin per ml and with 0.8 x 10(8) cells per ml, 50% of the antibiotic was reduced in 15 min from the biologically active oxidized form to the biologically inactive state. Under these conditions, a maximum of 0.6% (27 mumug/ml) of the myxin was retained in the cells.