The effect of dibromodulcitol on resting and dividing lymphoid cells

The effect of dibromodulcitol (DBD) on the incorporation of labelled precursors into DNA and RNA fractions of PHA-stimulated human lymphocytes and of P388F lymphoma cells at various stages of their growth was studied. Both cell systems showed sensitivity to the drug within the concentration range of 1–10 μg/ml.When DBD was added before phytohaemagglutinin (PHA), human lymphocytes showed a DNA labelling that was more affected than RNA. In contrast, by adding DBD after PHA, RNA labelling was much more inhibited than DNA. In the latter case, the decrease in DNA labelling occurred only 24 h after drug treatment whereas RNA labelling was decreased 1 h after treatment. Levels of DBD which normally produced 30% inhibition in plating efficiency of P388F lymphoma cells affected uridine-5-T incorporation to a different extent at different stages of growth of the culture. Enhanced RNA labelling occurred in early exponential stage while at later stages of growth, RNA synthesis was depressed.     

Inhibition of RNA- and DNA-synthesis by citrinin and its effects on DNA precursor-metabolism in V79-E cells.

1. The RNA synthesis of V79-E cells was inhibited by the mycotoxin citrinin time- and concentration-dependently. 2. Among the different RNA species mainly the rRNA synthesis was found to be inhibited by 200 microM citrinin. 3. At different precursor concentrations DNA synthesis was inhibited by citrinin after 30 min at least whereas labelling of the acid soluble fractions was found to be 3-fold higher than in untreated cells. 4. Remarkable perturbation of the DNA precursor metabolism, including release of precursor into the medium, was found to occur during citrinin treatment.     

Calcium thresholds in the activation of DNA and RNA synthesis in cultured planarian cells: relationship with hormonal and DB cAMP effects

DNA and RNA syntheses were reduced to a basal level in dissociated planarian cells grown for 48 h in a Ca2+-free medium. These syntheses could be triggered anew by raising the Ca2+ concentration in the medium. Serotonin could be substituted for Ca2+ in stimulating DNA synthesis by Ca2+-depleted cells, while dopamine greatly enhanced RNA synthesis in these cells. When Ca2+ concentration was raised in hormone-treated cultures, DNA synthesis was again slightly increased but RNA synthesis was depressed. Both hormonal effects were completely inhibited by the anticalmodulin drug trifluoperazine. As serotonin and dopamine are both known to stimulate the adenylate cyclase system, it was further investigated whether the hormonal effects were mediated by cAMP. Indeed, a DB cAMP concentration of 1 microM increased DNA labelling when applied for 8 h to Ca2+-depleted cultures. However, when Ca2+ was present, the 8-h treatment with 1 microM DB cAMP was inhibiting. A 4-h pulse with 1 microM DB cAMP just after Ca2+ addition was a condition for a high stimulation of DNA labelling. The other DB cAMP concentrations used, 0.1 and 10 microM, reduced DNA labelling. In the absence of Ca2+, RNA labelling was only slightly increased by 0.1 microM DB cAMP, but was highly stimulated by a 4-h treatment of 1 microM DB cAMP in the presence of Ca2+. The noted effects with 1 or 0.1 microM DB cAMP on DNA or RNA labelling corresponded to true changes in synthesis rather than alterations of the specific activity of the nucleotide pool by DB cAMP. Besides, it was precluded that these effects were due to butyrate issued from DB cAMP degradation. It was further shown that DB cAMP at 1 microM increased Ca2+ uptake in planarian cells, whereas the other concentration reduced it. This observation might explain the stimulating effect on nucleic acid synthesis of 1 microM DB cAMP applied at the appropriate moment. Based on these results it seems that, for triggering RNA synthesis, the threshold value of Ca2+ was lower than for DNA synthesis. These Ca2+ thresholds might be reached, in the absence of Ca2+ in the medium, by treatments with DB cAMP or hormones at the appropriate doses and periods. This interpretation is in agreement with the succession of biochemical events described in regenerating planarians and suggests that these events might be causally related.     

The messenger ribonucleic acid content of Bacillus subtilis 168

Bacillus subtilis 168 messenger RNA was determined by DNA-RNA hybridization techniques, with denatured DNA immobilized upon cellulose nitrate membrane filters. The following results were obtained. (1) Cultures of B. subtilis, growing exponentially in enriched glucose-salts medium at 37 degrees , incorporated [5-(3)H]uracil into both ribosomal and messenger RNA fractions without the kinetic delay expected from the presence of the intracellular nucleotide pools. (2) However short the time of labelling with exogenous labelled uracil (down to 7sec.), 32-36% of the rapidly labelled RNA was messenger RNA and 68-64% was an RNA with the hybridization characteristics of ribosomal RNA. Analysis of the apparent nucleotide base composition of total (32)P-labelled rapidly labelled RNA and the two RNA fractions separated by hybridization at a DNA/RNA ratio 5:1 confirmed this finding. Of the rapidly labelled RNA, 31% readily hybridized with DNA at low DNA/RNA ratios and had an apparent base composition like that of the DNA, whereas 69% was hybridized only at low efficiency at low DNA/RNA ratios and had a composition identical with that of ribosomal RNA. (3) In cultures dividing every 48min. at 37 degrees , kinetic analysis of RNA labelled over a 20min. period showed that the average life-time of messenger RNA was 2.7-3.0min. and that its amount was 3.0% of the total RNA. (4) The hybridization of (3)H-labelled randomly labelled RNA with DNA at a DNA/RNA ratio 5:1 showed that 2.9% of the randomly labelled RNA had the characteristics of messenger RNA. (5) Experiments carried out as described by Pigott & Midgley (1968) indicated that hybridization at low DNA/RNA ratios (5:1) effectively accounted for all the messenger RNA in a given specimen. The efficiency coefficient of RNA hybridization lay within the range of 90-95% input, if an excess of DNA sites was offered for RNA binding. (6) These measurements are compared with other results obtained by different methods, and reasons for any major disagreement are suggested.     

N4-hydroxycytidine-a new mutagen of a base analogue type

N⁴-hydroxycytidine (N⁴-OHcyd)¹ is incorporated into nucleic acids of a cytidine-requiring strain of S.typhimurium 1045 and can act mutagenically. The reversion frequency of pyrG^? → pyrG⁺ is 10–20 fold higher than the spontaneous background. N⁴OHcyd-induced revertants show a strong inhibitory effect in the presence of N⁴OHcyd. The influence of N⁴OHcyd on cytidine metabolism is discussed.     

SELECTIVE RETENTION AND FILTRATION OF BRAIN NUCLEIC ACIDS IN AGAROSE GELS

Abstract— Total nucleic acids of rat brain have been separated by agarose gel chromatography at 2 m -NaCl into DNA. transfer RNA plus low molecular weight RNA. and high molecular weight RNA fractions. The DNA fraction contained less than 1 per cent RNA by weight judged by either short-term or long-term labelling with ortho[³²P]phosphate. The high molecular weight RNA fraction contained 28 s and 18 s ribosomal RNAs and a heterogeneous population of 20-60 s RNAs, apparent after short-term labelling and characterized by a high content of nearest-neighbour-labelled uridylic acid. The rapidly sedimenting (>30 s ) portion of these RNAs could be largely separated from ribosomal RNAs by gel filtration using 4% agarose. The ribosomal RNAs could be fully resolved into 28 s and 18 s components by agarose gel chromatography at 0.5 m -0.6 m -NaCl, as shown by analysis of their sedimentation and nucleotide composition.     

Selective effect of hormones on nucleic acid metabolism during germination of pear embryos

1. The effect of hormones on (32)P incorporation into various RNA fractions in germinating pear embryos was studied by fractionation on methylated albumin-kieselguhr columns. Abscisic acid inhibited labelling of soluble RNA, DNA-RNA hybrid and light-ribosomal RNA fractions with (32)P and this effect was reversed by both kinetin and gibberellic acid. 2. Kinetin reversed the inhibition by abscisic acid of (32)P incorporation into total ribosomal RNA and appeared to promote labelling of heavy-ribosomal RNA. Gibberellic acid was more active than kinetin in reversing the inhibition by abscisic acid of labelling of the DNA-RNA hybrid fraction with (32)P, but in contrast with kinetin appeared to increase further the inhibition by abscisic acid of labelling of total ribosomal RNA. 3. The percentage of radioactivity in various RNA fractions showed marked variation in response to hormones. 4. The pattern of labelling of RNA in pear embryos during reversal of inhibition by abscisic acid with a combination of kinetin and gibberellic acid was similar to that after cold-treatment of dormant pear embryos. This is suggestive of hormonal interplay in dormancy release by cold-treatment in pear embryos.     

Insights into the fate of a 13C labelled phenol pulse for stable isotope probing (SIP) experiments

Stable isotope probing (SIP) using DNA or RNA as a biomarker has proven to be a useful method for attributing substrate utilisation to specific microbial taxa. In this study we followed the transfer of a (13)C(6)-phenol pulse in an activated sludge micro-reactor to examine the resulting distribution of labelled carbon in the context of SIP. Most of the added phenol was metabolically converted within the first 100 min after (13)C(6)-phenol addition, with 49% incorporated into microbial biomass and 6% respired as CO(2). Less than 1% of the total (13)C labelled carbon supplied was incorporated into microbial RNA and DNA, with RNA labelling 6.5 times faster than DNA. The remainder of the added (13)C was adsorbed and/or complexed to suspended solids within the sludge. The (13)C content of nucleic acids increased beyond the initial consumption of the (13)C-phenol pulse. This study confirms that RNA labels more efficiently than DNA and reveals that only a small proportion of a pulse is incorporated into nucleic acids. Evidence of continued (13)C incorporation into nucleic acids suggests that cross-feeding of the SIP substrate was rapid. This highlights both the benefits of using a biomarker that is rapidly labelled and the importance of sampling within appropriate timescales to avoid or capture the effects of cross-feeding, depending on the goal of the study.     

Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin.

A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100-400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 X 10(-18) mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.     

Studies on the kinetics of the RNA metabolism in the prostate of normal and castrated rats (author's transl)]

In the prostate of adult Wistar rats the RNA/DNA quotient of the whole organ as well as the amount of RNA and DNA in the nucleus was measured at different times after castration. Furthermore the half-life time for the turnover of the RNA in the nucleus and the cytoplasm was determined for normal and castrated rats with the aid of pulse labelling using [5(-3)H]uridine. A mathematical model was developed to analyze the experimental results. This model enabled us to make differentiated statements on the heterogeneous nuclear RNA (hmRNA) and the remaining RNA in the nucleus. The evaluation of the experimental values gave the following results: 1. By deprivation of androgens the uptake of [3H]uridine into the prostate is lowered. 2. The amount of DNA in the morphologically intact nucleus remains constant at least up to the 12th day after castration. 3. 6 days after castration the amount of hmRNA decreases to 1/10 and that of cytoplasmic RNA to 1/4. 4. The half-life time for the decrease of the whole nuclear RNA is 3.7 d and that of the cytoplasmic RNA 1.7 d. 5. The half-life time for the turnover of hmRNA is 16 min and that of cytoplasmic RNA about 2 days. 6 days after castration the half-life times are unchanged. The experimental results suggest that the observed decrease of nuclear RNA following castration can mainly be attributed to a reduced synthesis of hnRNA, while the decrease of cytoplasmic RNA is first of all caused by an increase in RNA degradation.     

Preferential alkylation of mitochondrial deoxyribonucleic acid by N-methyl-N-nitrosourea

The reaction of the carcinogen N-methyl-N-nitrosourea with mitochondrial DNA from various rat tissues was examined in vivo and in vitro. After incubation of isolated mitochondria or cell nuclei with N[(14)C]-methyl-N-nitrosourea in vitro and subsequent isolation and purification of the DNA the specific radioactivity of the mitochondrial DNA was 3-7 times that of the nuclear DNA. The incorporation of (14)C into embryonic mitochondrial DNA in vitro was about twice that into the liver mitochondrial DNA. Identical incorporation rates, however, were found for the reaction of isolated mitochondrial DNA or nuclear DNA respectively with N[(14)C]-methyl-N-nitrosourea. After intraperitoneal injection of 43.3-58.5mg of N[(14)C]-methyl-N-nitrosourea/kg body wt. to adult rats the labelling of the mitochondrial DNA was on average 5 times that of the nuclear DNA. A smaller specific labelling was observed for the ribosomal RNA, transfer RNA, and mitochondrial RNA as well as for the mitochondrial protein as compared with the mitochondrial DNA. After hydrolysis of the alkylated nucleic acids with hydrochloric acid, fractionation was carried out on Dowex 50 cation-exchange columns. In most experiments 70-80% of the input (14)C radioactivity was eluted in the 7-methylguanine fraction. The preferential alkylation of the mitochondrial DNA by N-methyl-N-nitrosourea in situ is discussed in connexion with the cytoplasmic-mutation hypothesis of carcinogenesis.     

PATTERNS AND LABELLING CHARACTERISTICS IN NEURONAL AND GLIAL RNA

Abstract— Rabbit cortex slices were incubated in a medium containing [³H]-uridine for various periods of time. Following incubation, neuronal and glial cell fractions were prepared on a discontinuous Ficoll-sucrose gradient. RNA was extracted from neurons and glia with a tris-sodium dodecyl sulphate-phenol solution and fractionated on a composite agarose-polyacrylamide gel. The stained gel showed major bands corresponding to 28 s , 18 s , 5 s and 4 s fractions and additional minor bands at the position of 24 s , 21 s and 13 s. Neuronal and glial RNA had the same general RNA pattern but the 5 s fraction was more pronounced in neuronal RNA and 4 s more pronounced in glial RNA. After 30 min labelling both neuronal and glial RNA had maximum activities in fractions higher than 28 s with a peak corresponding to 45 s . In the lower mol. wt. region the labelling was essentially poly-disperse. With increasing incubation time, peaks corresponding to 38 s and 32 s appeared as well as to ribosomal and soluble fractions. Incorporation of activity into total RNA expressed as d.p.m/ μ g of nucleic acids, showed similar labelling in neurons and glia after 30 and 60 min and a 3-4 times higher incorporation into neuronal RNA after 180 min. The possible implications of these results are discussed.     

Selective Interruption of High Molecular Weight RNA Synthesis in HeLa Cells by Camptothecin

THE study of macromolecular metabolism in eukaryotic cells has depended to a large extent on the use of selective inhibitors. Camptothecin is a potent, rapidly acting inhibitor of both DNA and RNA synthesis^1–3 which first came to attention as a potential anti-tumour agent⁴ and which has since been shown to have the remarkable property of inducing breaks in cellular DNA⁵; it also has unusual effects on RNA synthesis possibly as a result of DNA breakage. We show that the drug causes aberrant synthesis of high molecular weight nuclear RNA, but has a much smaller effect on the labelling of 4S RNA and does not affect 5S RNA synthesis. If the effect on RNA synthesis is due to DNA breakage, the results suggest that the breaks are induced at specific points.     

DNA synthesis in Hela cells at low temperature

It has been proved that ³H-thymidine is incorporated into DNA of HeLa cells cultured at 4 °C and its labelling distribution in DNA is homogeneous. This incorporation of ³H-thymidine increased with the duration of incubation and only 30% of the cell population was labelled after 12 h. When synchronous cell populations were used, the rate and extent of DNA synthesis at 4 °C was proportional to the relative number of cells in S phase at that temperature. Thus, cellular labelling at 4 °C does not result from a non-specific absorption phenomenon, but indicates a DNA synthesis process.     

Interrelationship between protein synthesis and mRNA metabolism in rat liver cells]

It is demonstrated that RNA isolated from polyribosomes and postmitochondrial fraction of rat liver cells and bound to nitrocellulose filters (Milliport) represent mRNA. RNA taken from the nitrocellulose filters sedimented in sucrose concentration gradient with a wide peak within the range of 18--6S, attaining a maximum at 12S. The (A+U)/(G+C) ratio of this RNA was equal to 1.04. On the other hand, the same ratio for rRNA was 0.64. Specific radioactivity of polysomal mRNA containing poly-A sequences, was significantly lower at 14-hour labelling with 14C-orotate than at 4-hour labelling (control). Inhibitors (cycloheximide, puromycin, ethionine, actinomycin D) stabilized polysomal mRNA. Specific radioactivity of postmitochondrial fraction mRNA was higher at 14-hour labelling than at 4-hour labelling. Specific radioactivity of postmitochondrial fraction mRNA during protein synthesis blocking by different inhibitors was comparable to those of control animals. It is hypothesized that active translation is necessary for the initiation of rat liver mRNA degradation.     

Metabolic effects of nucleosides in rat thymus cells in vitro

The incorporation of pyrimidine nucleosides into deoxyribonucleotides by rat thymocytes in vitro was decreased by the addition of any one of several nucleosides. In cells incubated with deoxyguanosine, the decrease was apparently caused by interference with the ribonucleotide reductase reaction, and at least part of the effect of deoxyadenosine was produced in the same way. While ara-C also produced a decrease in labelling of deoxynucleotides, this was quantitatively less than the effect on DNA synthesis, and may have resulted from an indirect effect on the reductase by a deoxyribonucleoside triphosphate which accumulated due to a direct effect of an ara-C derivative on the DNA polymerase. Cells incubated in the presence of adenosine showed a decreased labelling of deoxynucleotides and DNA due to inhibition of earlier steps in utilization of the labelled precursor, its uptake and phosphorylation. Guanosine, or a derivative, apparently reduced labelling of DNA and RNA even at concentrations which produced no alteration in uptake and phosphorylation of the precursor. An effect on the reductase was indicated. Uptake and phosphorylation of the pyrimidine ribonucleosides seem to be separate processes, since each can vary independently of the other when cells are incubated in the presence of various concentrations of exogenous nucleosides.     

EFFECTS OF CORTICOSTEROIDS ON THE BIOCHEMICAL MATURATION OF RAT BRAIN: POSTNATAL CELL FORMATION

(1) Treatment with cortisol acetate (0.2 mg daily during the first 4 days after birth) reduced the rate of growth in the rat: at 35 days of age the body weight was reduced by 50 per cent and the brain weight, depending on the region, by up to 30 per cent. (2) In the brain the normal increase in cell number was severely inhibited during the period of cortisol treatment; this resulted in a final deficit in cell number of about 20 per cent in the cerebrum and 30 per cent in the cerebellum. (3) To determine whether cortisol affected primarily cell formation or cell destruction the labelling of brain DNA was studied 1 h after a subcutaneous injection of 20 Ci/100 g [2-¹⁴C]thymidine. In the controls the amount of labelled DNA increased by a factor of two in the cerebrum and seven in the cerebellum during the period 2-13 days, and it decreased to 40 and 27 per cent of the peak values in the cerebrum and cerebellum respectively in the following 7 days. The results indicated that mitotic activity is higher in the cerebellum than in the cerebrum in the 2nd week of life. It would appear that in the cerebrum appreciable cell death accompanies new cell formation, especially during the period 13-35 days of age. (4) Cortisol treatment affected cell division rather than cell destruction in the brain since it strongly inhibited the incorporation of [2-¹⁴C]thymidine into DNA. The inhibition was severe during the period of treatment but it did not result in a lasting fall in mitotic activity. At the age of 13 days the amount of labelled DNA formed approached the normal level and it was twice that in controls at 20 days, indicating a tendency for compensating cell deficit by an accelerated mitotic activity. Nevertheless, massive cell proliferation ceased at about the same age as in normals; the labelling of DNA decreased markedly between 13 and 20 days after birth, and the DNA content did not increase after the age of 20 days. (5) In contrast to the marked effect on cell number, cortisol treatment did not influence significantly the maturational changes related to average cell size (DNA concentration) or the chemical composition of cells (RNA/DNA and protein/DNA).     

Binding of androgens to a nuclear-envelope fraction from the rat ventral prostate.

A nuclear-envelope fraction was isolated from the rat ventral prostate which is virtually free of DNA and contains little RNA or plasma membrane. Isolation of this nuclear-envelope fraction after incubation of purified nuclei with radioactive dihydrotestosterone results in labelling of the membrane. More binding of dihydrotestosterone is observed after incubations at 22 degrees C for 17 h than at 4 degrees C for 17 h or at 22 degrees C for 60 min. Scatchard analysis revealed a class of binding sites with KD 8.4 nM. Dihydrotesterone and testosterone were almost equally effective as competitors of labelled dihydrotestosterone binding on the purified nuclear-envelope fraction, whereas diethylstilboestrol was less effective and dexamethasone did not compete well. When the outer membrane of the nuclei was removed with Triton X-100, a 24% decrease in specific binding of androgens was observed. Castration 24 h before preparation of nuclei resulted in loss of the androgen binding to the membrane.     

Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]

The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids.     

Basal-cell subpopulations and cell-cycle kinetics in human epidermal explant cultures

Cultured human epidermal cells were studied by cell sorting and autoradiography after different 3H-thymidine (3H-dThd)-labelling procedures and after labelling with DNA precursors that are incorporated via salvage or de novo pathways. It was shown that 3H-dThd incorporation was the best measure of the rate of DNA replication. Dose-response experiments with pulse and continuous labelling revealed that all S- and G2-phase cells were cycling, whereas some 20% of the cells stayed in G1-phase for long periods of time. Most, if not all of these cells were probably non-proliferating differentiated keratinocytes. At least two subpopulations of S-phase cells could be discriminated on the basis of the rate of incorporation of DNA precursors. The difference in precursor incorporation did not seem to be caused by differences in nucleotide metabolism but rather to reflect true differences in the rate of DNA replication. Continuous labelling experiments showed that these subpopulations also were apparent in the G1- and G2-phases. Studies of the grain-count distribution revealed that cells that appeared to move rapidly through the S-phase moved slowly through the G2-phase, and vice versa. Cells stained with acridine orange were subjected to a two-parameter analysis in the cell sorter by simultaneous measurement of the DNA and RNA fluorescence. Autoradiography of sorted cells revealed that, on average, cells with low RNA contents incorporated 3H-dThd at a higher rate than cells with high RNA contents.