Per Hellung-Larsen

Small molecular weight RNA components in Ehrlich ascites tumor cells

Abstract Ehrlich ascites tumor cells contain five small molecular weight nuclear RNA components (small nRNA) (L, A, C, D, and F) which migrate slower than 5-S RNA by electrophoresis in 10 % polyacrylamide gels and three components (H components) which migrate between 5-S RNA and tRNA. The small nRNA components are specifically localized in the nucleus and they account for 0.2 to 0.7 % of the total cellular RNA and 0.7 to 2.9 % of the nuclear RNA. Apart from one H component, which shows high rate of labeling with [ 3 H]uridine, the other small nRNA components are labeled with rates which are similar or somewhat less than tRNA and 5-S RNA, but 2–3 times higher than rRNA. When Ehrlich cells which have been labeled in vivo with [ 3 H]uridine were incubated in vitro the radioactivity remained in the small nRNA components in the nucleus whereas the labeling in 5-S RNA and rRNA disappeared from the nucleus. Inhibition experiments divide the small nRNA components into two groups: One group consisting of the H components which are inhibited like tRNA and one group consisting of A, C, and D which are inhibited differently. The H-components are inhibited less by actinomycin and more by 3′-deoxyadenosine than component A, C and D. [ 3 H]Methyl groups from [ Me - 3 H]methionine are incorporated more into component C than into A and D. The incorporation of [ 3 H]methyl groups in small nRNA components is higher than in rRNA, but much less than in tRNA. The corresponding small nRNA components from Ehrlich, Yoshida and L 5178 Y ascites cells have identical mobilities on 10 % polyacrylamide gels. Differences in mobilities of small nRNA components from HeLa cells and Ehrlich cells were found for component A and the H components. Furthermore component L was present in relatively larger amounts in Ehrlich cells than in HeLa cells whereas HeLa cell components B and K are absent in Ehrlich cells.

CHEMOATTRACTION IN TETRAHYMENA: ON THE ROLE OF CHEMOKINESIS

Chemoattraction of Tetrahymena pyriformis, strain GL, was measured during starvation and under different growth conditions. Log phase cells starved in buffer are attracted by certain amino acids, peptides, and proteins. Cysteine, methionine, and phenylalanine are attractants at i0@ M. The peptides in proteose peptone (PP) and yeast extract (YE) are active at 10_6M. Epidermal growth factor (EGF) is active at >3 X i0@ M. Among the proteins, platelet derived growth factor (PDGF) is the most active (3 X 108 M). Cells growing in defined medium are attracted by PP, YE, and some proteins (PDGF). Swimming speed was measured for starved cells with and without added attractants or repellents. With addition of PP the swimming speed increases from 0.42 to 0.51 mm/s., but for PDGF it is unchanged. The swimming speed of starved cells increases when the cells approach a solidified attractant (PP) as measured by the speed at a given distance. The speed of cells moving towards the attractant is higher than that of cells moving away from it. In conclusion, certain amino acids, peptides, and proteins are chemoattractants for Tetrahymena. Chemokinesis likely plays a considerable role in the case of PP (and YE), since they increase swimming speeds, whereas attraction by PDGF may involve chemotaxis.

Metabolic studies of small molecular weight nuclear RNA components in BHK-21 cells

Abstract The metabolism of small molecular weight homodisperse nuclear RNA components (snRNA) has been studied in Baby Hamster Kidney (BHK-21) cells. 1. 1. Actinomycin D in concentrations which totally inhibits the synthesis of rRNA has little or no effect on the synthesis of snRNA. Increasing the concentration of actinomycin shows that the synthesis of the snRNA components K and L is much less sensitive than that of A, C and D. 2. 2. 3′-Deoxyadenosine inhibits the synthesis of snRNA less than that of rRNA but no difference in sensitivity within the snRNA components is observed. 3. 3. Labelling with [ Me - 3 H]methionine shows incorporation into components A, C and D but not into K and L. 4. 4. Cytosine arabinoside used in concentrations which blocked DNA synthesis has no preferential inhibitory effect on the synthesis of the snRNA components. 5. 5. The electrophoretic mobilities on polyacrylamide gels of components K and L are changed as a result of heat treatment. 6. 6. The presence of Mg 2+ during phenol extraction increases the amount of component K. 7. 7. Components L, A, C and D have a metabolic half-life of 5–7 days in logarithmically growing cells.

The differential inhibitory effect of α-amanitin on the synthesis of low molecular weight RNA components in BHK cells

Vertebrate cells contain a number of low molecular weight RNA (LMW RNA) components apart from t-RNA, 5 S RNA and 5.5 S RNA (reviewed [l-3]). The major components are called D (U-l), C (U-2), A (U-3) and L. The function of these components is unknown although information is available about their metabolism [4-71, subcellular localization [4-81 , occurrence in different species [9] and formation from unstable precursors [ lo]. Studies on the primary structures of U-l and U-2 [ 1 l-141 show that the S’ends have a cap structure similar to mRNAs. An interesting question is which polymerase synthesizes LMW RNA? We have tried to answer this question by using the differential inhibitory effect of cw-amanitin. The DNA-dependent RNA-polymerases from mammalian cells can be distinguished from each other on the basis of their sensitivity towards a-amanitin. The nucleolar RNA polymerase I is insensitive to o-amanitin, the nucleoplasmic RNA polymerase II is sensitive to low concentration (0.1-l .O pg/ml) and the nucleoplasmic RNA polymerase III is inhibited only by high concentrations of a-amanitin (about 200 /,&ml) [ 15-211. When Chinese hamster ovary cells are treated with o-amanitin the synthesis of nucleoplasmic heterodisperse RNA is strongly inhibited during time periods where ribosomal RNA synthesis is not inhibited [22] . We have used similar conditions in the present investigation. The results show that cu-amanitin has a differential inhibitory effect on the synthesis of the nucleoplasmic heterodisperse RNA and the low molecular weight RNA

Small molecular weight RNA components in Tetrahymena pyriformis.

Abstract Several procedures have been used for the extraction of 32 P- or 3 H-labeled RNA from whole cells, nuclei, cytoplasm and ribosomes of Tetrahymena pyriformis . The RNA has been separated on 10 % polyacrylamide gels mainly. Extracts of whole cells and nuclei contain, apart from tRNA and 5-S RNA, at least three small molecular weight nuclear RNA components (snRNA). These components (T 1 , T 2 and T 3 ) which constitute 0.04–0.15 % of the cellular RNA are found in the same amount in exponentially as well as synchronously dividing cultures. The T components are labeled as rapidly as tRNA. None of the snRNA components of Tetrahymena have electrophoretic migration similar to the snRNA components of Ehrlich ascites cells. A component, T 0 is found to be associated with ribosomal RNA, but is liberated by phenol extraction at 55°. T 0 is associated with 25-S rRNA and constitutes about 1.5 % of 25-S rRNA. The 25-S rRNA-associated T 0 has a smaller electrophoretic mobility than Ehrlich ascites 28-S rRNA-associated 5.5-S RNA.

Liquid scintillation counting of 3H and 32P RNA in slices of polyacrylamide gels

Abstract 3H- and 32P-RNA in slices of polyacrylamide gels cross-linked with bisacrylamide were digested with NCS and counted in a new dioxanebased scintillation liquid. Reproducible and stable high-efficiency measurements were obtained. The method is superior to procedures reported so far with respect to reproducibility, efficiency, and economy.

Effects of Pluronic F-68 on Tetrahymena cells: protection against chemical and physical stress and prolongation of survival under toxic conditions

The effects of the non-ionic surfactant Pluronic F-68 (0.01% w/v) on Tetrahymena cells have been studied. A marked protection against chemical and physical stress was observed. The chemical stress effects were studied in cells suspended in buffer (starvation) or in buffers with added ingredients from a chemically defined medium (Ca2+, Mg2+, Na+, K+, trace metal ions). The physical stress was due to mechanical stress or hyperthermia. The data show that Pluronic: (a) prolongs the survival of low concentration cell suspensions during starvation; (b) prevents the cell death caused by low concentrations of Ca2+ (70 microM); (c) prolongs the survival of cells exposed to higher ion concentrations (10 mM Ca2+, or Na+ or K+); (d) postpones the death caused by trace metal ions like Zn2+, Fe3+ and, Cu2+; (e) protects cells from the death caused by shearing forces; and (f) prolongs the survival of cells exposed to hyperthermia (43 degrees C). The cellular survival is increased at reduced temperatures (e.g. 4 degrees C instead of 36 degrees C) and at increased cellular concentrations (e.g. 100 cells ml(-1) instead of 25 or 10 cells ml(-1)). There is no effect of pre-incubation with Pluronic. The protective effect of Pluronic towards Tetrahymena is observed for concentrations in the range from 0.001 to 0.1% w/v.

Characteristics of dividing and non-dividing Tetrahymena cells at different physiological states

Eight defined physiological states of Tetrahymena pyriformis are described. For dividing cells the states comprise: 1. Exponentially growing cells, 2. Cells at late exponential growth phase, 3. Cells kept at a high cell concentration, 4. Cells shifted up or down by change of medium, temperature or degree of aeration. For non-dividing cells the states are: 5. Cells at stationary phase, 6. Cells during starvation, 7. Cells during shift-up after long-term starvation, 8. Cells at self-induced hypoxia. The different cellular states are described by one or more of the following characteristics: growth rate, volume, swimming speed, oxygen consumption and by the oxygen saturation and the pH in the medium. The results show that T. pyriformis grows equally well in proteose-peptone (PY) medium from 1 cell ml(-1) to 10(3) cells ml(-1) as from - e.g. - 10(2) to 10(5) cells ml(-1). The maximum cell concentration obtained depends on the medium and the availability of oxygen. At shift-down by decrease of temperature the cells grow slower and obtain a considerable oversize. Single cells tolerate starvation for 12 days. The cell volume (electronically determined) decreased from about 7000 μm(3) to about 200 μm(3). Long-term starved cells may be upshifted. Thereby growth without cell division can be studied until the cell volume approaches 2100 μm(3) which is the minimum volume of division competence. Under certain conditions cells may grow into self-induced hypoxia leading to growth arrest. These cells will attain an oversize. The swimming speed at 28°C of exponentially growing cells is 0.33 to 0.59 mm sec(-1) depending on the medium. At lower temperature the swimming speed is decreased. In PY-medium the values are: 28°C (0.57), 16°C (0.50), 9°C (0.37). During starvation the swimming speed decreases from about 0.6 to about 0.1 mm sec(-1) (after 6 days). The oxygen consumption is for state 1 cells: 3.9 μl O(2)/10(6) cells min(-1) (maximal value). The value of hypoxic cultures is 2.1, for cells kept at high concentration 0.4, and for starved cells (24 h) 0.2.

Metabolic studies on small molecular weight nuclear RNA components in human lymphocytes.

Abstract Some metabolic properties of small molecular weight nuclear RNA (snRNA) components have been studied in human lymphocytes cultured with PHA. Pulse-labelling experiments with 3H-uridine in 3 h-intervals around the onset of DNA synthesis showed no qualitative or quantitative differences in the snRNA labelling pattern. Long labelling experiment with 3H-methionine demonstrated the following relative degrees of methylation: tRNA (1.0), 5S RNA (0), D (0.3), 5.5S RNA (0.2), C (0.6), A (0.2), L (0) and rRNA (0.2). Chase-experiments with 3H-methionine showed that the snRNA components D, C and A are metabolically stable with half-lives of not less than 30 h. Actinomycin D (0.05 μg/ml) reduced markedly the synthesis of rRNA and 5 S RNA whereas the synthesis of D, C, A and L was unaffected or only slightly affected. Actinomycin D at a concentration of 0.25 μg/ml inhibited the synthesis of D, C and A. Cycloheximide (0.19 μg/ml) reduced the synthesis of D, C and rRNA to about 50% of control whereas 5S RNA synthesis was only slightly inhibited and tRNA synthesis was unaffected.

Surface mediated death of unconditioned Tetrahymena cells: effect of physical parameters, growth factors, hormones, and surfactants.

A new form of cell death has been observed. The death occurs at liquid‐air interfaces when Tetrahymena cells are grown in a chemically defined medium (CDM) at low inocula. The cells die by lysis at the liquid‐air interface (medium surface), which they reach due to negative gravitaxis as well as positive aerotaxis. When the cells are grown in a closed compartment, with no liquid‐air interface, the death is not observed, and the cells proliferate. Cloning of cells in CDM is thus possible. The addition of effectors such as NGF (10−11 M), EGF (10−10 M), PDGF (10−10 M), and insulin (10−10 M) to cells in CDM prevents the surface mediated death. Since detergents/surfactants like SDS (7 × 10−5 M), NP‐40 (2 × 10−5 M), Tween 80 (10−4% w/v), Pluronic F‐68 (10−7 M), and the biosurfactant surfactin (10−6 M) have the same effect, we suggest that the effectors act by stimulating the cells to exudate surfactant(s) of their own. Furthermore, lyzed cells and exudates from living cells (pre‐conditioned medium) prevent the death. In conditions with liquid‐air interfaces, certain physical parameters are of great importance for the survival of cells at low inocula. The parameters are the distance to the surface, the temperature, and the inoculum. By increasing the height of the medium, lowering the temperature, and increasing the inoculum of the culture, the survival can be greatly enhanced. There is no evidence for programmed cell death (PCD) or apoptosis.