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.

The reaction product of the ribosomal poly(A) polymerase [ATP(UTP) :RNA ucleotidyltra Sferase] is analyzed. Two systems are used in vitro: (a) isolated polyribosomes with endogenous enzyme and RNA primer and (b) purified enzyme with total polyribosomal RNA as primer. In the polyribosome system about 50 % of the [3H]AMP label is in poly(A)-containing mRNA. This RNA displays a heterogeneous size ditribution in the range of 8 -30 S with a maximum at about 14 S. Upon denaturation the maximum is shifted towards the 10-S zone. The poly(A) polymerase catalyzes the addition of 12-18 adenylate residues to pre-existing mRNA poly(A) sequences of 40-160 residues. The [3H]AMP incorporated into poly(A)-lacking RNA is mainly in a fraction with an electrophoretic mobility corresponding to 4-S RNA. In the purified enzyme system, specificity towards poly(A)-containing mRNA is lost to a considerable extent. Only 10% of the [3H]AMP label is retained by oligo(dT)-cellulose. The bulk of the product is in 18-S rRNA and heterogeneous small molecular weight RNA.We conclude that the ribosome-associated poly(A) polymerase is most likely the enzyme responsible for the cytoplasmic polyadenylation of poly(A)-containing mRNA in vivo.

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The poly(A) polymerases from the cytosol and ribosomal fractions of Ehrlich ascites tumour cells are isolated and partially purified by DEAE-cellulose and phosphocellulose column chromatography. Two distinct enzymes are identified : (a) a cytosol Mn2+-dependent poly(A) polymerase (ATP : RNA adenylyltransferase) and (b) a ribosome-associated enzyme defined tentatively as ATP(UTP) : RNA nucleotidyltransferase.The cytosol poly(A) polymerase is strictly Mn2+-dependent (optimum at 1 mM Mn2+) and uses only ATP as substrate, Poly(A) is a better primer than ribosomal RNA. The purified enzyme is free of poly(A) hydrolase activity, but degradation of [3H]poly(A) takes place in the presence of inorganic pyrophosphate. Most likely this enzyme is of nuclear origin.The ribosomal enzyme is associated with the ribosomes but it is found also in free state in the cytosol. The purified enzyme uses both ATP and UTP as substrates. The substrate specificity varies depending on ionic conditions: the optimal enzyme activity with ATP as substrate is at 1 mM Mn2+, while that with UTP as substrate is at 10-20 mM Mg2+. The enzyme uses both ribosomal RNA and poly(A) [but not poly(U)] as primers. The purified enzyme is free of poly(A) hydrolase activity.The problem of the post-transcriptionally added poly(A) sequences to the 3' end of pre-mRNA and mRNA is still controversial. This fact has stimulated the investigation of the enzymes catalyzing the addition of poly(A) tails to ribonucleotide primers, currently designated as poly(A) polymerases. Such enzymes have been detected in a variety of mammalian cells (reviews in [1,2]). The problem became more complicated when poly(A) polymerase activities were found not only in the nucleus but also in mitochondria1 [3 -51, ribosomal [6 -81 and cytosol[9 -131 fractions, although the possibility for a leakage of nuclear enzymes has to be considered in evaluating these findings [14]. Further, the terminal enzymatic addition of AMP residues to the 3' ends of poly(A)-containing RNA was found recently in the nuclei and in the cytoplasm. This reaction is apparently distinct from the de n o w poly(A) synthesis [15 -171. Thus, cellular metabolism of poly(A) sequences in pre-mRNA and mRNA seems to be more complex than inferred from current models. This paper is dedicated to Dr Fritz Lipmann on his 80th birthday.Ahbreviations. Poly(A)-containing RNA, RNA containing poly(A) sequences and retained by oligo(dT)-cellulose; poly(A)-lacking RNA, RNA not retained by oligo(dT)-cellulose.Enzymes. ATP : RNA adenylyltransferase or poly(A) polymerase (EC 2.7.7.19); ATP(UTP):RNA nucleotidyltransferase or poly(A) and poly(U) polymerase (EC 2.7.7-).In this work we report the isolation and partial purification of two distinct poly(A) polymerases from the cytoplasm of Ehrlich ascites tumour cells. The first one had an absolute requirement for Mn2+ and used only ATP as substrate. It was isolated from the cytosol but evidence was obtained that it is of nuclear origin. The second poly(A) polymerase was found associated with ribosom...

To assess the lethal doses of gamma radiation and corresponding apoptotic response in new established human melanoma cell lines we exposed exponentially growing cultures to 8- 100 Gy gamma radiation. The apoptosis and cell survival were determined by trypan blue exclusion, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction, agarose gel electrophoresis, colony forming assay, and long-term survival assay. The maximal DNA fragmentation 3 days after irradiation was observed in cultures irradiated with 20 Gy (36.9% TUNEL positive cells). The cultures irradiated with 50 and 100 Gy contained 18.7% and 16.4% TUNEL positive cells, respectively. Cultures exposed to 8 and 20 Gy gamma radiation recovered by week 3-4. Lethally irradiated (50 and 100 Gy) cultures which contained less apoptotic cells by day 3 died by week 5. A detectable increase in melanoma cell pigmentation after irradiation was also observed. The survival of human melanoma cell cultures after exposure to gamma radiation does not correlate with the level of apoptotic cells by day 3. At high radiation doses (> 50 Gy) when the radiation induced cell pigmentation is not inhibited the processes of apoptotic DNA fragmentation might be preferentially inactivated