Cytoplasmic ribosomes of Ehrlich ascites tumour cells were isolated under conditions where ribonuclease contamination is minimized. These ribosomes displayed poly(A) and poly(U) polymerase activity with [3H]ATP or [3H]UTP as substrates, while incorporation of [3H]GTP or [3H]CTP was not observed. Poly(A) polymerase is Mn2+ ‐dependent with an optimum at 1 mM, while poly(U) polymerase is activated by Mg2+ with a broad optimum at 5–20 mM. Both enzyme activities remained associated with the two ribosomal subparticles upon EDTA treatment and sucrose density gradient fractionation. The product of poly(U) polymerase was bound to endogenous 28‐S and 18‐S rRNA. About 40% of the poly(A) polymerase product was retained by oligo(dT)‐cellulose and the radioactivity distributed in 28‐S and 18‐S rRNA as well as in 4–16‐S RNA material. Addition of poly(A) stimulates markedly the activity of poly(A) polymerase, but addition of poly(U) is without effect on either enzyme. The poly(U) polymerase of the small and the large subparticle did not display a preferential use of endogenous versus exogenous RNA as primer. The same was true for poly(A) polymerase of the small subparticle. However, the poly(A) polymerase of the large subparticle showed a marked preference for exogenous cytoplasmic 18‐S RNA before or after purification through oligo(dT)‐cellulose. This effect was not observed with 18‐S rRNA obtained from previously isolated small ribosomal subparticles. It is suggested that ribosome‐associated poly(A) polymerase may display a preference for mRNA lacking poly(A) as primer.

Ribonucleic acid (RNA) synthesis in the sorbitol-dependent, fragile yeast mutant VY1160 (Venkov et al., 1974) is rapidly inhibited by rifampin. The growth of the mutant cells and protein synthesis are more slowly affected by the antibiotic, apparently as secondary phenomena. Lower doses of rifampin (50 to 100 μg/ml) preferentially inhibit ribosomal RNA synthesis in comparison to that of messenger RNA and transfer RNA. Transcription and translation of messenger RNA continues in the presence of low doses of rifampin, as evidenced by the unimpaired induction of α-glucosidase. Partially purified RNA polymerase II from this mutant, in contrast to that from the parental strain, is strongly inhibited by low concentrations (1 μg/ml) of rifampin, whereas RNA polymerase I from the two strains is similar in behavior. The mutant may be useful for the study of regulatory mechanisms of transcription in eukaryotes.

The synthesis and processing of RNA by isolated HeLa cell nuclei was studied at low ionic strength in the presence of alpha-amanitin. The RNA polymerase reaction, with endogenous template and enzyme, rapidly reaches a plateau dependent on the amount of nuclei. Evidence is presented that incorporation of [(3)H]UMP proceeds only in growing RNA chains, whereas initiation of new RNA chains is arrested. The product formed contains all the main components of the 45S pre-rRNA (precursor of rRNA) maturation pathway (45S, 32S and 20S pre-rRNA; 28S and 18S rRNA). Most of the labelled material is in the mature rRNA components and their immediate precursors, even at very short times of incubation (2min). Small, but definite, 5S and 4S RNA peaks are also observed. At shorter incubation times a substantial amount of [(3)H]UMP is incorporated into RNA molecules in the 24S and 10-16S zones. This RNA material is considered to represent the non-conserved segments of 45S pre-rRNA in the process of nucleolytic degradation. A model for the tracer study of the topology of 45S pre-rRNA, on arrest of rRNA initiation, is discussed. The experimental evidence obtained supports the following structure of 45S pre-rRNA: 5'-end-28S rRNA unit-18S rRNA unit-nonconserved segment-3'-end.

The cell death and survival of proliferating (clonogenic) cells were investigated in two human melanoma cell lines to assess the optimal conditions for preparation of apoptotic bodies from melanoma cells. After 50 J/m2 UVB+UVC the maximal levels of apoptotic cells assayed by Trypan blue staining, nucleosomal DNA fragmentation, MTT, and TUNEL tests were observed within 2-3 d of radiation. In 100 Gy gamma-irradiated cultures these apoptosis indicators were delayed for up to 3 weeks. In addition, clonogenic cells were observed only in exponentially growing cultures irradiated with UV at high cell density but not in gamma-irradiated cultures. The response of melanoma cultures after high UV radiation doses contrasted to the response in lethally gamma-irradiated cultures. UV-irradiated melanoma cultures were recovered within two weeks. Most of the clonogenic cells in the recovered colonies contained micronuclei. ROS levels determined by DCF fluorescence and a modified MTT test were also normalized obviously due to the extensive antioxidant defense system of melanoma cells. UV radiation of tumor cells might be the preferential method for preparation of apoptotic bodies. The presence of clonogenic cells in the suspension of apoptotic bodies from melanoma cells used for pulsing of dendritic cells with tumor antigens might compromise this protocol for preparation of cell vaccines.