Differential modes of chemical decay for early and late lambda messenger RNA.

In the presence of rifampicin, chemical decay of pulse-labeled phage λ “early” messenger RNA occurred exponentially, with a half-life of 2.2 minutes at 42 °C, compared to 1.3 minutes for the host mRNA. In contrast, mRNA synthesized late during λ development exhibited a characteristic complex decay curve not previously observed for microbial mRNA; initial slow decay of about 25% during 4 to 4.5 minutes was followed by rapid exponential decay of the remainder with a half-life of 1.5 minutes. Sucrose density-gradient analyses of the RNA revealed that during the phase of slow decay, the λ-specific mRNA was progressively fragmented.     

A conditional lethal mutation in an Escherichia coli strain with a longer chemical lifetime of messenger RNA.

We have analysed an Escherichia coli temperature-sensitive mutant with altered messenger RNA stability, and it was found that: (1) the unstable fraction of pulse-labelled RNAs decayed with a half-life at 42 °C of about two minutes in the parent strain PA3092; the half-life was 11 to 12 minutes in the mutant HAK75. Puromycin enhanced the decay rate about twofold in both PA3092 and HAK75; the addition of chloramphenicol inhibited the degradation significantly in both strains. The rate of ribosomal RNA accumulation in the mutant cells at 42 °C did not differ from that in the wild-type cells. (2) Sedimentation analysis by sodium dodecyl sulphate/sucrose density-gradient centrifugation of bulk mRNA as well as tryptophan mRNA of the wild-type strain showed the expected rapid reduction in the size and level of those mRNA molecules at three minutes and five minutes respectively, after addition of rifampicin at 42 °C. In contrast, the cells of HAK75 retained almost full-length trp mRNA and bulk mRNA at 5 to 12 minutes after the addition of rifampicin at 42 °C, even though the total level of radioactivity in the mRNA fraction had decreased to about 60 to 75% of the initial activity. (3) Even though mRNA molecules were chemically protected at the non-permissive temperature in the mutant, the functional decay of both β-galactosidase and tryptophan synthetase occurred at a rate comparable to that in the parental strain. (4) We isolated temperature-resistant revertants from the mutant at a frequency of 5 × 10^?8, and these revertants (TR1 and TR2) had the normal decay rate of unstable RNA.     

Early to late switch in bacteriophage T7 development: functional decay of T7 early messenger RNA

Infection of ultraviolet light-irradiated Escherichia coli with T7 phage in the presence of chloramphenicol results in synthesis of T7 early messenger RNA but not late mRNA. T7 early mRNA accumulates in terms of acid-insoluble, T7 DNA-hybridizable RNA. However, messenger activity of the same RNA decays rapidly with a half-life of about 6.5 minutes at 30 °C when tested for the ability to direct in vitro protein synthesis. This functional decay of T7 early mRNA is attributable to a loss of structural integrity of the RNA. Polyacrylamide-agarose gel electrophoresis shows that T7 early mRNAs are cleaved, generating smaller-size RNAs. Kinetics of the appearance of T7-specific RNA polymerase, one of the early gene products, during normal T7 infection show that the capacity of the cells to produce the enzyme decays very rapidly when early mRNA synthesis is terminated either by rifampicin or by a natural mechanism programmed by T7. Preferential synthesis of late proteins in the presence of chemically stable early mRNA late in T7 infection may be explained by the observed functional decay of early mRNA.     

Characterization of the Rapidly Labeled Hybridizable RNA Synthesized in L5178Y Mouse Leukemic Cells: Hybridization Lifetime Studies

An attempt is made to characterize the rapidly labeled hybridizable RNA of L5178Y mouse leukemic cells which has been shown to have similar base sequences when synthesized in two different stages of the cell cycle. The size of rapidly labeled RNA molecules was heterogeneous. For labeling times of 20 min or less, the per cent of hybridization was maximal. With longer labeling times, the per cent of hybridization decreased as radioactivity appeared in long-lived species of low hybridization efficiency; the radioactivity profile resembled the optical density profile in sucrose gradients. The lifetime of newly synthesized hybridizable RNA was studied by pulse labeling exponentially growing cells and then “chasing” with nonradioactive uridine. The per cent of hybridization was studied as a function of chase time. Three RNA groups, which comprised different proportions of rapidly labeled hybridizable RNA, were distinguished. The short-lived group had a half-life of 10 min, much less than the values reported in the literature for messenger RNA of mammalian cells. The half-life of 1-1½ hr observed for a medium-lived group more closely corresponds to that of messenger RNA. A long-lived group had a half-life of approximately 20 hr. Specific activity measurements during chase indicate the presence of a “pool” of labeled uridine derivatives. The uridine of this pool appears to be nonexchangeable with but dilutable by exogenous uridine. A nontoxic concentration of actinomycin D was added to the chase media in an attempt to block the “pool effect”. A rapidly degradable RNA was demonstrable both by specific activity and per cent of hybridization measurements.     

The effect of rifampicin on the stability of the messenger ribonucleic acid of Bacillus amyloliquefaciens as determined by DNA:RNA hybridization.

Rifampicin at a concentration of 10 mug/ml completely inhibited protein synthesis in exponential-phase cultures of Bacillus amyloliquefaciens. At this same concentration the drug was shown to have no effect on the stability of mRNA, determined as the functional and hybridizable material, when compared with hybridizable mRNA in an uninhibited system. In each case, the half-life of the mRNA had a value in the range 5 +/- 1 min, at 30 degrees C.     

Why do rotifer populations present a typical sigmoid growth curve?

To determine the underlying processes to population growth in the rotifer Brachionus plicatilis, we conducted an experiment using 1.5 ml cultures for 70 days. All individuals were transferred daily to culture media containing algae, and the number of individuals, clutch sizes and number of deaths were counted. The population dynamics showed a typical sigmoid curve. The population density increased exponentially from 10 to 682 individuals during the first 7 days (exponential growth phase), and gradually up to about 1500 individuals during the next 30 days (post-exponential growth phase). The population density then remained at a constant level with small fluctuations during the rest of the experimental period (stationary phase). Mortalities appeared from the post-exponential growth phase and were almost constant at about 2% throughout the experimental period. The clutch size decreased from 5 to 1 during the first 5 days, and afterwards females laid only one egg each. The proportion of non-reproductive females increased from 30% (exponential growth phase) to 80% (post-exponential growth phase) to 90% (stationary phase). These results suggest that the exponential growth phase resulted from the imbalance between a high birth rate and a low death rate, while the stationary phase was maintained by the compensation between low birth and death rates.     

Rifampicin Inhibition of Ribonucleic Acid and Protein Synthesis in Normal and Ethylenediaminetetraacetic Acid-Treated Escherichia coli

The kinetics of ribonucleic acid (RNA) and protein synthesis in rifampicin-inhibited normal and ethylenediaminetetraacetic acid (EDTA)-treated Escherichia coli was measured. Approximately 200-fold higher external concentrations of rifampicin were needed to produce a level of inhibition in normal cells comparable to that observed in EDTA-treated cells. The rates of RNA and protein synthesis in both kinds of cells decreased exponentially, after an initial lag phase, at all rifampicin concentrations tested. The lag phase was longer and the final exponential slope less for protein synthesis than for RNA synthesis at a given rifampicin concentration. Below certain rifampicin concentrations, both the lag phase and the subsequent exponential decrease in the rates of RNA and protein synthesis were found to be rifampicin concentration dependent. At greater concentrations only the time of the lag phase was decreased by higher rifampicin concentrations, whereas the slope of the exponential decrease in the rates of RNA and protein synthesis was unaffected. In all cases, the exponential decrease continued to at least a 99.8% inhibition of the original rate of synthesis. These in vivo results are consistent with the mode of rifampicin action determined from in vitro studies; rifampicin prevents initiations of RNA polymerase on deoxyribonucleic acid, but not its propagation, by binding the enzyme essentially irreversibly. The results also indicate the size distribution of messenger RNA molecules in E. coli under our conditions.     

Dictyostelium discoideum mRNAs developmentally regulated during spore germination have short half-lives.

mRNA decay was studied during spore germination in Dictyoselium discoideum by the use of three previously isolated cDNA clones, pLK109, pLK229, and pRK270, which are specific for mRNAs developmentally regulated during spore germination. The half-life of a constitutive mRNA, pLK125, which is present throughout germination, growth, and development, as also determined. Nogalamycin, a DNA-intercalating compound, was used to inhibit RNA synthesis. Total RNA was isolated at intervals after addition of the drug, and the decay of mRNAs specific for the cDNA clones was determined by both Northern blot and RNA dot hybridization. If nogalamycin was added immediately after activation of dormant spores, neither pLK229 nor pLK109 mRNA decayed, but pLK125 mRNA did decay. Although pLK109 mRNA did not decay under these conditions, the RNA was smaller 1 h after activation than in dormant spores, indicating that it was processed normally. At 1 h after activation, pLK229-, pLK125-specific mRNAs decayed exponentially, with half-lives of 24, 39, and 165 min, respectively. Under the same conditions, decay of pLK109-specific mRNA was biphasic. Thirty-eight percent of the mRNA decayed with a half-life of 5.5 min, and the remainder decayed with a half-life of 115 min. It seems likely that nogalamycin inhibits the synthesis of an unstable component of the mRNA degradative pathway which is needed continuously for the decay of pLK109 mRNA. By extrapolating the curve representing the rapidly decaying component, a half-life of 18 min was calculated for pLK109-specific mRNA. The mRNAs developmentally regulated during spore germination have half-lives shorter than that of the constitutive messenger and shorter than the average half-life of 3 to 4 h previously determined for total Dicyostelium polyadenylated mRNA.     

Hybridizable ribonucleic acid of rat brain

1. Cerebral RNA of adult and newborn rats was labelled in vivo by intracervical injection of [5-³H]uridine or [³²P]phosphate. Hepatic RNA of similar animals was labelled by intraperitoneal administration of [6-¹⁴C]orotic acid. Nuclear and cytoplasmic fractions were isolated and purified by procedures involving extraction with phenol and repeated precipitation with ethanol. 2. The fraction of pulse-labelled RNA from cerebral nuclei that hybridized to homologous DNA exhibited a wide range of turnover values and was heterogeneous in sucrose density gradients. 3. Base composition of the hybridizable RNA was similar to that of the total pulse-labelled material; both were DNA-like. 4. Pulse-labelled cerebral nuclear RNA hybridized to a greater extent than cytoplasmic RNA for at least a week after administration of labelled precursor. This finding suggested that cerebral nuclei contained a hybridizable component that was not transferred to cytoplasm. 5. The rates of decay of the hybridizable fractions of cerebral nuclei and cytoplasm were faster in the newborn animal than in the adult. Presumably a larger proportion of labile messenger RNA molecules was present in the immature brain. 6. Cerebral nuclear and cytoplasmic RNA fractions from newborn or adult rats, labelled either in vivo for periods varying from 4min. to 7 days or in vitro by exposure to [³H]-dimethyl sulphate, uniformly hybridized more effectively than the corresponding hepatic preparation. These data suggested that a larger proportion of RNA synthesis was oriented towards messenger RNA formation in brain than in liver.     

Functional half-lives of bacteriophage m13 gene 5 and gene 8 messages.

The half-lives of the M13 gene 5 and gene 8 messages were determined by measuring the decay in the rate of synthesis of the gene 5 and gene 8 proteins after inhibition of new RNA chain initiations with rifampin. The gene 5 and gene 8 messages decay with half-lives of approximately 2.5 and 5 min, respectively. We found no evidence of a functional M13 message with a half-life as long as that reported for hybridizable mRNA.     

Regulation of protein synthesis in mammalian cells. V. Further studies on the effect of actinomycin D on translation control in HeLa cells

The rate of protein synthesis in HeLa cells appears to be regulated, in part, by a factor which promotes the association of ribosomes with messenger RNA and whose production is inhibited by actinomycin. The decline in protein synthesis after the administration of actinomycin is not primarily due to a decay of available messenger RNA but, rather, is a result of a decrease in the rate of ribosomal association with message.The decay of protein synthesis in actinomycin can be varied over a wide range by altering the temperature of cell incubation. Thus the half-life of protein synthesis decay ranges from eight hours at 34 °C to two hours at 41°C. The rapid decline of protein synthesis at 41 °C is not accompanied by a corresponding decay of the messenger RNA. Polyribosomes decrease in size, but they can be restored to normal sedimentation distributions by low levels of cycloheximide, suggesting that messenger RNA remains functional. The translation rate at 41 °C is unaltered. The dose-response of protein synthesis inhibition by actinomycin was measured and a half-maximum inhibition was found to be effected by 0·1 μg of the drug/ml.Another important aspect of the regulation of translation in HeLa cells is the response of cells to depressed rates of protein synthesis. At 42 °C, protein synthesis is severely inhibited, due to a failure in the association of ribosomes with messenger RNA. Prolonged incubation at the elevated temperature results in a significant repair of the lesion. This repair is inhibited by actinomycin. The half-maximum inhibition is achieved at levels of from 0·05 to 0·1 μg of the drug/ml.The cell response to depressed rates of protein synthesis can also be demonstrated using the drug cycloheximide. Prolonged incubation in the drug results in a response which then can promote protein synthesis at 42 °C. Here again, the half-maximum inhibition of the response to cyclohemixide is achieved by 0·1 μg of actinomycin/ml. These experiments suggest, but do not prove, that the cellular response may be mediated through the synthesis of RNA that promotes the initiation of translation and does not involve the subsequent production of protein.     

Stability of cytoplasmic ribonucleic acid in a mouse myeloma: estimation of the half-life of the messenger RNA coding for an immunoglobulin light chain

This paper describes experiments in which the half-lives of a number of cytoplasmic RNA species have been estimated in a mouse myeloma (MOPC 21) without resort to metabolic inhibitors. Partial purification of the messenger RNA coding for immunoglobulin light chains enabled an estimate of the stability of this species to be made. The procedure chosen was that of a conventional pulse-chase following uniform labelling of cells with [³H]uridine. Centrifugation of the uniformly labelled cells and resuspension in 0·1 mm-uridine resulted in a 75% drop in the specific activity of the UTP pool within 2 hours, followed by a logarithmic decay with a half-life of about 3·5 hours. Exposure of P3K cells to uridine causes them to swell appreciably and centrifugation at the end of the pulse period is followed by a lag phase of 3 hours before the cells re-enter logarithmic growth. Since all chase conditions had certain disadvantages, a comparison of experiments using different chase conditions was undertaken. The stability of the various RNA species did not vary greatly under the different chase conditions. The half-life of the light-chain mRNA is estimated to be 12 to 14 hours, although a value in the range of 5 to 20 hours cannot be excluded. An RNA fraction including the heavy-chain mRNA behaves similarly. Half-lives determined for other RNA species were: 18 S ribosomal RNA (40 to 60 h); 12 S mitochondrial ribosomal RNA (28 to 32 h). Poly(A)-containing RNA from free polyribosomes decays rapidly in the first 5 hours with a half-life of 20 to 30 hours, subsequently.     

Cellular Changes Accompanying the Transition from Minimal to Maximal Rate of Extracellular Enzyme Secretion by Bacillus amyloliquefaciens

S ummary . During exponential growth of Bacillus amyloliquefaciens in a maltose–L-amino acids medium at 30°, cellular protein, RNA and DNA increased in parallel. After the exponential phase, extracellular protein, including α-amylase, was secreted into the medium at a quarter of the maximum rate of total cellular protein synthesis. The free amino acid pool for protein and the 'nucleotide'pool for RNA both increased fourfold during the transition to the post-exponential phase to 4.0 and 1.6%, respectively, of the cellular dry weight. Subsequently, the nucleotide pool did not change significantly whilst the free amino acid pool was reduced to 2/3 of its maximum size. When post-exponential phase, exoprotein-secreting bacteria were transferred to fresh culture medium, growth was re-established and there was a 4–5 fold reduction in nucleotide pool size accompanied by a loss of exoenzyme-forming ability.     

The estimation of turnover of spermidine in Anacystis nidulans.

The turnover of spermidine in Anacystis nidulans was studied using [2-¹⁴C]methionine to prelabel intracellular spermidine. It was found that there is essentially neither excretion nor degradation of spermidine in exponentially growing Anacystis nidulans. Spermidine was degraded rapidly in stationary phase cells. The half-life of specific activity of spermidine in exponential phase was 8.3 h, a period similar to that of the doubling time (7.5 h) of the bacterium. The rate of synthesis of spermidine was calculated to be 0.04 nmol/10⁸ cells/h.     

Messenger RNA turnover in mouse L cells

The turnover of polyadenylic acid-containing messenger RNA and histone messenger RNA, which lacks poly(A), was studied in exponentially growing mouse L cells by measuring the kinetics of approach to steady-state uridine labeling. Constant specific activity of precursor pools was verified by showing that the data for stable RNA components, like ribosomal RNA and transfer RNA, follow theoretically predictable curves. In agreement with a previous report by Greenberg (1972), the data for poly(A)-containing mRNA (poly(A)(+)mRNA) follow theoretical curves for a class of molecules turning over with first-order (stochastic) kinetics. Cells growing with doubling times of 13·5 hours at 37 °C and 41 hours at 30 °C exhibited mean lifetimes for their poly(A)(+)mRNA of 15 hours and 42 hours, respectively, suggesting a parallelism between growth and turnover rates. The kinetic data for histone mRNA are not indicative of a stochastic process. Rather, they suggest an age-dependent decay or a zero-order (ordered) turnover with a mean lifetime of about six hours. One model, which gave a good fit to the data, considers that the histone messages persist for a fixed duration of the cell cycle, e.g. the DNA synthetic phase, and are then destroyed in a “sensitive period” after this phase. These results are discussed with regard to the possible implications of the poly(A) sequences in messenger RNA aging.