Puromycin Treatment Research Articles

Ribosome formation from ribonucleic acid accumulated during puromycin treatment: Influence of proflavin

1. Escherichia coli 15T − treated with puromycin accumulates RNA in the absence of protein synthesis. The accumulated RNA is incorporated into new ribosome particles for at least 45 min following removal of puromycin. 2. If proflavin is present during recovery from puromycin treatment, very little formation of new ribosomes takes place. 3. More than 50 % of the accumulated RNA (23-S, 16-S and 4-S RNA) is degraded to trichloroacetic acid-soluble material during the first 30 min after proflavin addition. A study of the breakdown reveals that the 23-S RNA is most labile in the presence of proflavin followed by the 16-S material. Most of these two components have disappeared after 30 min incubation with proflavin. Puromycin does not prevent degradation of the accumulated RNA when proflavin is present.

Effect of puromycin on hepatic glycogen phosphorylase activity.

SummaryLiver glycogen phosphorylase activity was studied in mice at early times subsequent to in vivo administration of puromycin, and following puromycin addition to incubations of rat liver slices. No effect of puromycin on phosphorylase could be detected following injection of unanesthetized mice. Puromycin treatment of pentobarbital-anesthetized mice, however, resulted in an enhancement of the phosphorylase activity as compared to control values. Phosphorylase activity in rat liver slices incubated with puromycin was greater than those incubated without puromycin. These results indicate that puromycin can elevate liver phosphorylase activity, a finding consistent with the hepatic glycogenolytic action of the antibiotic in vivo.

Ribosome formation by puromycin-treated Bacillus subtilis

Puromycin causes an unbalanced synthesis of RNA in Bacillus subtilis, while net increase in protein of the cell is prevented. Under these conditions, an accumulation of abnormal particles (between 18 S and 25 S) containing newly formed ribosomal RNA (16 S and 23 S) occurs. Experiments on the fate of the RNA contained in these particles have provided the following information. 1. 1. The ribosomal RNA contained in the abnormal particles is unstable, as indicated by its degradation to acid-soluble material when synthesis of RNA de novo is blocked by actinomycin D. 2. 2. When puromycin is removed from the culture, most of the pre-formed ribosomal RNA is incorporated into ribosomes by combining with ribosomal protein synthesized preferentially by the recovering cells. 3. 3. The preferential synthesis of ribosomal protein during recovery from puromycin treatment can also occur in the presence of actinomycin D, resulting in incorporation of some of the pre-formed RNA directly into ribosomes. An accumulation of the information for ribosomal protein during puromycin treatment is indicated.

RNA synthesis and ribosome production in puromycin-treated cells

1. 1. Puromycin is an effective inhibitor of protein synthesis in Escherichia coli 15T when treatment takes place in a medium lacking magnesium. Although incorporation of labelled amino acid is prevented in this system, incorporation of nucleic acid precursors continues. RNA, from cells treated with puromycin, labelled with [8- 14C]adenine and separated on a sucrose density gradient exhibits the same sedimentation profile as the RNA from normal cells. The RNA accumulated during treatment with puromycin is stable after removal of the inhibitor, provided glucose is added to the medium in which the bacteria are resuspended. The ribosomal RNA, furthermore, can be incorporated into ribosomal particles during recovery from puromycin treatment. 2. 2. During this recovery phase there appears to be a preferential synthesis of ribosomal protein. Thus, the simultaneous sysnthesis of ribosomal RNA and ribosomal protein is not a necessary requirement for the formation of ribosome particles. 3. 3. In resting cell cultures (absence of glucose) nucleic acid formation, as measured by [8- 14C]adenine incorporation is increased by the addition of puromycin to the incubation medium.

The effects of ultraviolet irradiation and inhibitors of protein synthesis on the initiation of deoxyribonucleic acid synthesis in mammalian cells in culture II. Effects on the phosphorylation of thymidine

DNA synthesis in mouse strain-L fibroblasts was inhibited following exposure to ultraviolet light or puromycin. The effect produced with small doses of ultraviolet light was temporary, maximum inhibition being observed 5–8 h after irradiation. The data obtained following puromycin treatment are compatible with the hypothesis that protein synthesis is a prerequisite for DNA synthesis in L cells. Despite the marked depression in intracellular DNA synthesis observed 5–8 h after these treatments, virtually no inhibition of the phosphorylation of thymidine to thymidine triphosphate or the activity of DNA polymerase could be detected at the same time. The phosphorylation of thymidine to thymidine triphosphate does not appear to be of primary importance in the regulation of DNA synthesis in these cells.

The effects of ultraviolet irradiation and inhibitors of protein synthesis on the initiation of deoxyribonucleic acid synthesis in mammalian cells in culture II. Effects on the phosphorylation of thymidine

DNA synthesis in mouse strain-L fibroblasts was inhibited following exposure to ultraviolet light or puromycin. The effect produced with small doses of ultraviolet light was temporary, maximum inhibition being observed 5–8 h after irradiation. The data obtained following puromycin treatment are compatible with the hypothesis that protein synthesis is a prerequisite for DNA synthesis in L cells. Despite the marked depression in intracellular DNA synthesis observed 5–8 h after these treatments, virtually no inhibition of the phosphorylation of thymidine to thymidine triphosphate or the activity of DNA polymerase could be detected at the same time. The phosphorylation of thymidine to thymidine triphosphate does not appear to be of primary importance in the regulation of DNA synthesis in these cells.

An African Sea

We investigated the effects of puromycin on mouse oocyte chromosomes during meiotic maturation in vitro. Puromycin treatment for 6 hr at 100 μg/ml almost completely, but reversibly, suppressed [35S]methionine incorporation into oocyte protein at all stages of maturation tested. Nevertheless, oocytes treated at the germinal vesicle stage underwent germinal vesicle breakdown (GVBD) and chromosome condensation. These oocytes completed nuclear maturation to metaphase II (MII) if the inhibitor was withdrawn. Prolonged (24-hr) treatment, however, caused the chromsomes to degenerate. The chromosomes of oocytes treated shortly after GVBD for 6 hr remained condensed, but the oocytes failed to form a polar body. However, 24-hr treatment caused the chromosomes to decondense to form an interphase nucleus. Oocytes treated near MI for 6 hr gave off a polar body during the treatment, and their chromosomes decondensed to form a nucleus, which remained as long as the treatment was continued. However, if the puromycin was withdrawn, the chromosomes recondensed to a state morphologically similar to that at MII. Thus, the chromosome decondensation induced by protein synthesis inhibition at MI was reversible. Oocytes treated at MII, several hours after first polar body formation, also underwent chromosome decondensation to form a nucleus. In the continuous presence of puromycin, the chromosomes remained decondensed, but neither DNA synthesis nor mitosis occurred. However, following puromycin withdrawal, these occytes synthesised DNA and underwent mitosis. Thus, protein synthesis inhibition at MII, by parthenogenetically activating the oocytes, caused irreversible chromosome decondensation. Based on these observations, we discussed the roles of protein synthesis in the regulation of oocyte chromosome behaviour during meiotic maturation.