Replication of RNA viruses: specific binding of the Q RNA polymerase to Q RNA

The specific binding in vitro of the Qβ RNA polymerase to Qβ RNA has been detected by the formation of an enzyme-Qβ RNA complex that did not exchange bound RNA molecules and was not dissociated by 0.8 m NaCl. Formation of this nondissociating complex required GTP and two host protein factors, but not ATP, CTP, UTP, or Mg²⁺ ions. GDP, GMP, dGTP, ITP, and β,γ-methylene GTP did not replace GTP in the reaction. Complex formation at 0 °C was not observed, and the rates of the reaction at 30 °C and 25 °C were 41% and 23%, respectively, of the rate at 37 °C. The reaction occurred with intact Qβ RNA and with polycytidylic acid template but not with bacterial or other bacteriophage RNA. With limiting amounts of enzyme, the amount of Qβ RNA bound in the nondissociating complex was the same as the amount of [γ-³²P]GTP incorporated into nascent RNA chains, indicating a close relationship between complex formation and the initiation of RNA synthesis. The two reactions appear to be separate, however, because in the absence of Mg²⁺ ions, when complex formation occurred readily, no RNA synthesis could be detected either by incorporation of labeled substrate into acid-insoluble material or by formation of short RNA chains still attached to the enzyme. In the presence of factor protein and GTP, a maximum of one active enzyme molecule was bound per molecule of Qβ RNA template, as determined by a liquid polymer phase-separation procedure. These results suggest that formation of the nondissociating complex measures recognition by the Qβ RNA polymerase of a single Qβ RNA site utilized for the initiation of synthesis.     

Effect of temperature on glycerol metabolism in membranes and on phospholipases C and D of germinating pea embryos

The effect of temperature of imbibition on the synthesis and turnover of membrane phosphatidyl choline was studied. Pea seeds (Pisum sativum cv. Alaska) were imbibed in [U-¹⁴C]glycerol and then germinated. Seeds were kept constantly either at 5° or 25°, or were imbibed at one temperature and then germinated at the other one. Glycerol incorporation into phosphatidyl choline in the ER and the plasma membrane, obtained from the embryonic axes after germination, and the glycerol pool were measured. Embryos from seeds kept constantly at 25° showed a rapid incorporation of glycerol into membranes followed by a loss of label; in embryos from seeds kept at 5° incorporation was much lower. Embryos from seeds transferred from 25° to 5° behaved as if continuously kept at 25°, while the behaviour of the embryos from seeds transferred from 5° to 25° resembled embryos from seeds maintained at 5°. The glycerol content of the axes rose during imbibition and fell thereafter. The activities of phospholipases C and D also responded to the initial temperature of imbibition, but the two activities changed differently. The results are discussed in relation to the effect of transient exposure to temperature changes in the seed membranes and the possible way in which such changes are sensed.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent snucleotide pools for nucleic acid synthesis

Novikoff rat hepatoma cells (subline NlSl-67) in suspension culture incorporate ³H-5-uridine into the acid-soluble nucleotide pool more rapidly than into RNA, resulting in the accumulation of labeled UTP in the cells. When labeled uridine is removed from the medium after 20 minutes or 4.75 hours of labeling, the rate of incorporation of label from the nucleotide pool into RNA decreases to less than 10% of the original rate within five to ten minutes, in spite of the presence of a large pool of labeled UTP in the cells, and incorporation ceases completely if an excess of unlabeled uridine is present during the chase. Upon addition of ¹⁴C-uridine to ³H-uridine pulse-labeled, chased cells, the ¹⁴C begins to be incorporated into RNA without delay and at a rate predetermined by the concentration of ¹⁴C-uridine in the medium and without affecting the fate of the free ³H-nucleotides labeled during the pulse-period. The results are interpreted to indicate that uridine is incorporated into at least two different pools, only one of which serves as primary source of nucleotides for RNA synthesis. During active synthesis of RNA, the latter pool of free nucleotides is very small and rapidly exhausted when uridine is removed from the medium. However, UTP accumulates in this pool when cells are labeled at 4–6°, since at this temperature RNA synthesis is blocked while uridine is still phosphorylated by the cells, and the UTP is rapidly incorporated into RNA during a subsequent ten-minute chase at 37°. From these types of experiments it is estimated that only 20–25% of the total uridine nucleotides formed in the cells from uridine in the medium is directly available for RNA synthesis and that the remainder becomes available only at a slow rate. Evidence is presented which suggests that one uridine nucleotide pool is located in the cytoplasm and another in the nucleus and that mainly the nuclear pool supplies nucleotides for RNA synthesis. The size of the latter pool is under strict regulatory control, since preincubation of the cells with 0.5 mM unlabeled uridine has little or no effect on the subsequent incorporation of ³H-uridine, although it results in an increase of the overall cellular uridine nucleotide content to at least 5 mM. Other results indicate that adenosine is also incorporated into two independent nucleotide pools, whereas the cells normally appear to possess a single thymidine nucleotide pool.     

Defect in translation of poliovirus RNA at the restrictive temperature.

Translation of the RNA of LSc type 1 poliovirus was examined in vivo at the restrictive temperature (39 °C). During the first two hours of infection at 39 °C the levels of viral polyribosomes were 50% lower than at 35 °C (permissive temperature). During the third hour of infection at 39 °C, only 4 to 10% of the control levels of polyribosomes were observed. Three experiments indicate that the elongation of viral peptides was not occurring properly at 39 °C. First, cultures incubated at 39 °C during the third hour of infection with both [³⁵S]methionine and [³H]uridine exhibit a fourfold increase in the ratio of viral protein/viral RNA in the polyribosome region of sucrose gradients in comparison to controls kept at 35 °C. However, at both temperatures the relative size distribution of polyribosomes was similar. Second, the ratios of released protein/nascent protein after 90-second and 5-minute pulses with [³⁵S]methionine indicate that elongation of peptide chains was inhibited at 39 °C. Third, when initiation of synthesis of viral protein was blocked with 150 mM-NaCl, the polyribosomes disaggregated four to five times more rapidly at 35 °C than at 39 °C. The data indicate that translation of viral RNA is inhibited at the restrictive temperature because of a reduced rate of elongation of viral proteins. The reduced rate of peptide chain elongation at 39 °C was fully reversible when cultures were shifted to 35 °C in the presence of 150 mm-NaCl. The latter finding indicates a conformational change in viral protein at 39 °C.     

Thermolabile analogue proteins and the entry of HeLa and CHO cells into division

Entry of HeLa and CHO-10 cells into mitosis can be inhibited by incorporation of p-fluorophenylalanine at certain temperatures, 37 °C for the former cell type and 39.5 °C for the latter. At lower temperatures, 32 °C in the former and 37 °C in the latter, the analogue does not inhibit entry of cells into mitosis. The possibility that the analogue is not incorporated at the permissive temperatures has been ruled out; indeed incorporation is relatively greater at the permissive temperatures. The results suggest that the physiological properties of analogue protein molecules differ depending on the temperature at which they are synthesized; the higher the temperature the more likely they are to malfunction.     

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10 °C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1 week at low temperature, followed by a further decrease after 2 weeks. The enzyme activity was not changed during 25 °C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10 °C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25 °C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.     

Influence of different temperatures on the tachinid parastite,Sturmiopsis inferens [Dip.]

The influence of constant temperatures of 27, 29, 31 and 33°C and alternating temperature of 31/33°C (18/6 h) onSturmiopsis inferens Townsend was studied during 12 successive generations. The larval and pupal periods for male parasites were 13.5±0.5 and 11.0±0.3 days respectively and for female 12.8±0.5 and 11.1±0.3 days respectively in the 1st generatioin at 27°C. It decreased progressively with increase in temperature. Survival of females, fertility and fecundity were adversely affected at higher temperatures. A temperature range of 27–29°C appeared to be optimum for mass rearing of the parasite in the laboratory. The higher premature mortality observed at a constant 33°C was not observed at temperatures fluctuating between 31/33°C. Presumably under field conditions, where temperature is constantly fluctuating, the flies will be able to withstand a comparatively higher temperature.     

Pyrimidine Deoxyribonucleosides Are Phosphorylated to Triphosphates at Low Temperatures Too,But Are Not Incorporated into DNA or Phospholipids.

Abstract

Thymidine (Thd) was phosphorylated to dTTP also at 0°C, both in Ehrlich ascites tumor cells and human tonsillar lymphocytes, but was not incorporated into DNA. The uptake and phosphorylation of ¹⁴C-Thd into the pool showed regular kinetics (Km 6, 6 uM), and the main metabolite was dTTP (75–84%) both at 0°C, and 37°C. Similarly, deoxycytidine (dCyd) was also transported and phosphorylated to nucleotides (76%) at low temperature, but no incorporation into DNA and phospholipid precursor liponucleotides could be detected at 0°C. Under the same conditions, at 37°C, when lymphocytes were labeled with 5-³H-dCyd, 51% of the total pool radioactivity was found in liponucleotides. Transport and phosphorylation of deoxynucleosides seem to be tightly coordinated at both temperatures, which processes are directly coupled to membrane-phospholipid and DNA biosynthesis, but only at physiological temperature while they seem “uncoupled” at low temperature. The fact that nucleoside phosphorylation occures also at low temperature has implications for several experimental techniques used in cell biology.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

The influence of the thermal environment and other early life events on growth rate of piglets during lactation

The effects of early life events on average daily weight gain from birth to day 21 (ADG) of suckling pigs kept at different room temperatures (15°C, 20°C and 25°C) from birth to weaning were investigated. Data were collected from litters born by 61 sows in a loose housing system. The ADG for piglets with low birth weight (estimated for birth weights below the 10% percentile) was estimated to be 20 to 30 g higher per day at room temperature 20°C to 25°C compared with 15°C. In contrast, the ADG during the lactation period decreased for larger piglets (estimated for birth weights above the 10% percentile) by 28 g/day at room temperature 25°C compared with 15°C. Thus, high ambient temperatures (20°C to 25°C) are favourable for the growth in smaller piglets during lactation. Neither latency to first suckle nor birth-induced hypoxia, measured as concentration of umbilical cord lactate, affected the growth rate of the piglets. Lowest rectal temperature during the first 24 h after birth had a long-term negative effect on ADG (P<0.05), so that piglets with a lowest rectal temperature of 32.8°C (10% percentile) had an ADG which was on average 19 g lower per day than piglets with a rectal temperature of 37.3°C (90% percentile). Our results showed that hypothermia at birth, low birth weight and high number of suckling piglets lead to reduced ADG during the suckling period. The results suggest that keeping the room temperature at 20°C during lactation to some extent could compensate for the otherwise negative effects of low birth weight on ADG in piglets without decreasing the ADG of high birth weight piglets. However, to avoid hypothermia in the smallest piglets it may be beneficial to increase the room temperature above 20°C during the farrowing period of loose housed sows.     

The Impact of Temperature Stress on DNA and RNA Synthesis in Potentially Toxic Dinoflagellates <Emphasis Type="Italic">Prorocentrum minimum</Emphasis>

Biomarkers of temperature stress were studied as major characteristics crucial for the understanding complex processes that underlie the response of marine planktonic microorganisms to environmental factors and their sublethal effects. Using the potentially toxic dinoflagellates Prorocentrum minimum as a model object, the impact of temperature stress on viability, cell cycle, RNA synthesis and DNA replication in these protists was evaluated. It was shown by flow cytometry that stress evoked by a temperature increase from 25°C (control) to 37 or 42°C during 15 to 60 min did not cause any considerable alterations in the cell cycle, while cell death rate increased from ≤ 1% (control) to 2–12% at 37°C and 4–22% at 42°C. Along with a relatively low cell death rate, following a temperature increase to 37 and/or 42°C, P. minimum displayed the ability to boost the synthesis of DNA (1.7–1.9 and 1.2–1.6 times, respectively) and especially RNA (2.5–3.1 and 1.7–2.8 times, respectively) during the first 15–30 min after stress. At certain stages of the life cycle, this effect can be critical for maintaining the viability and normal development of the P. minimum population. The obtained results demonstrate that a significantly elevated synthesis of nucleic acids can serve as an indicator (biomarker) of sublethal environmental stress.     

A selective temperature-sensitive defect in viral RNA expression in cells infected with a ts transformation mutant of murine sarcoma virus

We investigated the nature of the defect in the temperature-sensitive mutant of Moloney murine sarcoma virus (Mo-MuSV), termed ts110. This mutant has a temperature-sensitive defect in a function required for maintenance of the transformed state. A nonproducer cell clone, 6m2, infected with ts110 expresses P85 and P58 at 33°C, the transformed temperature, but only P58 is detected at the restrictive temperature of 39°C. Shift-up (33°C → 39°C) and in vitro experiments have established that P85 is not thermolabile for immunoprecipitation. Previous temperature-shift experiments (39°C → 33°C) have shown that P85 synthesis resumes after a 2–3 hr lag period. Temperature shifts (39°C → 33°C) performed in the presence of actinomycin D prevented the synthesis of P85, whereas P58 synthesis did not decline for 5 hr, suggesting that P58 and P85 are translated from different mRNAs. The shift-up experiments also indicated that, once made, the RNA coding for P85 can function at the restrictive temperature for several hours. MuSV-ts110-infected cells superinfected with Mo-MuLV produced a ts110 MuSV-MuLV mixture. Sucrose gradient analysis of virus subunit RNAs revealed a ～28S and a ～35S peak. Electrophoresis of the ～28S poly(A)-containing RNA from ts110 virus in methyl mercuric hydroxide gels resolved two RNAs with estimated sizes of 1.9 × 10⁶ and 1.6 × 10⁶ daltons, both smaller than the wild type MuSV-349 genomic RNA (2.2 × 10⁶ daltons). RNA in the ～28S size class from virus preparations harvested at 33°C was found to translate from P85 and P58, whereas, the ～35S RNA yielded helper virus Pr63^gag. In contrast, virus harvested at 39°C was deficient in P85 coding RNA only. Peptide mapping experiments indicate that P85 contains P23 sequences, a candidate Moloney mouse sarcoma virus src gene product. Taken together, these results suggest that two virus-specific RNAs are present in ts 110-infected 6m2 cells and rescued ts110 pseudotype virions at 33°C, one coding for P85, whose expression can be interfered with by shifting the culture to 39°C; the other coding for P58, whose expression is unaffected by temperature shifts. P85 is a candidate gag-src fusion protein, while P58 contains gag sequences only.     

Synthesis of mitochondrial RNA in disaggregated embryos of Xenopus laevis

Mitochondrial RNA synthesis was studied during early Xenopus laevis development using disaggregated embryos. By gel electrophoresis, the labeled mitochondrial RNA consisted of three discrete species. Transfer RNA was the only mitochondrial RNA species which was methylated. Incorporation of ³²P into mitochondrial RNA was detectable as early as the blastula stages. The level of incorporation was found to increase with increasing developmental age. All species of mitochondrial RNA appeared to be labeled at a constant rate relative to one another. Partial analysis of the acid-soluble nucleotide pool indicated that the observed incorporation probably represents a net increase in the synthetic rate for mitochondrial RNA as development proceeds.     

Isolation of ribosomal RNA precursors from Physarum polycephalum

Ribosomal RNA synthesis in Physarum polycephalum was studied by labeling intact microplasmodia with [³H]uridine. Labeled, high-molecular-weight RNA species were found in a 30,000 S structure released by phenol extraction at room temperature. RNA was released from the structure by further phenol extraction at 65–70 °C. If the labeling period was 15 min or longer, the labeled RNA was seen by polyacrylamide gel electrophoresis to be of two major types, a heterodisperse collection of 45-35 S molecules and a 26 S species. If the labeling was carried out for 30 min in the presence of cycloheximide, the major labeled species had an electrophoretic mobility corresponding to 40 S. Studies of the labeling kinetics, methylation, and base composition of these RNA molecules indicate that they are precursors to ribosomal RNA. The molecular weights of the homogeneous 40 and 26 S precursors are 3.0 × 10⁶ and 1.45 × 10⁶ daltons, respectively, in comparison with molecular weights of 1.29 × 10⁶ and 0.68 × 10⁶ daltons for the completed ribosomal RNA's.     

The effect of temperature on the growth,development and survival of Macropetasma africanus (Balss) (Penaeoidea:Penaeidae) larvae reared in the laboratory

Macropetasma africanus (Balss) has been successfully spawned and its larvae reared under controlled laboratory conditions. The relationship between egg number (E) and female total length (L) was E = 18.59 L^2.11. An experiment was designed to test the effect of temperature on larval development, survival and growth. Temperature effected larval development time, from 13–15 days at 25°C, to 25 days at 15°C (nauplius 1 to post-larva). Mortality was low for the naupliar stages at 25, 22 and 18°C, while at 15°C only 52% of the larvae reached nauplius 6. Mortality was highest from nauplius 6 to protozoea 1 (17, 21, and 18% at 25, 22, and 18°C, respectively), but decreased considerably for all temperatures once the mysis stage was reached. Overall survival rates from nauplius 1 to post-larva decreased with decreasing temperature (65, 54, 48, and 39% at 25, 22, 18, and 15°C respectively). Temperature also significantly affected larval growth. At 25°C mean total length was significantly (P < 0.05) larger than at 15°C (protozoea 2 to post-larva), while from protozoea 3 to post-larva total length differences were significantly different (P < 0.05) between 18 and 25°C. M. africanus has a major spawning peak in summer, suggesting that there may be a selective advantage to reproducing during the warmer months.     

Temperature dependence-independence of antifreeze turnover in Eurosta solidaginis (Fitch)

Temperature effects on antifreeze metabolism were investigated in two populations (northern and southern) of the golden rod gallfly, Eurosta solidaginis. Sorbitol production was temperature dependent and was triggered by short-term exposure to < +10°C. The maximal rate sorbitol synthesis occurred at 0°C. For both populations, sorbitol was rapidly catabolized during warm acclimation at +20°C. During the first 12 h of warm acclimation, sorbitol levels decreased by 36% (19.7 ± 0.6) to 12.6 ± 1.2 μg/mg) and by 83% (to 3.3 ± 1.7 μg/mg) after 48 h in the northern population. The southern population decreased sorbitol levels 64% (11.8 ± 0.69 to 4.2 ± 0.62) after 48 h. The southern population resynthesized more sorbitol than did the northern population upon re-exposure to 0°C. Glycerol levels increased linearly during the experimental period independent of temperature.     

The influence of temperature variations and thermal pollution on various aspects of the biology of the prawn Palaemon pacificus (Stimpson)

The influence of different temperatures 10, 15, 20, and 25°C on the food consumption, growth, moulting rate, and respiration of Palaemon pacificus (Stimpson) from Langebaan Lagoon, west coast of South Africa, was studied under laboratory conditions. At 10°C mortality was high so that food consumption and moulting rate could not be determined as these were very low. At higher temperatures, food consumption was found to be temperature dependent, the rate at 25°C being twice that at 15°C. Growth rate was most favourable at 25°C. At 28°C growth rate was lower than at 20°C but higher than at 15°C. The intermoult period was 17 days at 15°C, and 11 and 10 days at 20, and 25°C, respectively. It seems that from an energetic point of view, 25°C is the most favourable temperature for P. pacificus. Several indices of growth efficiency at different temperatures are presented. The appearance of this prawn in South African west coast localities such as Langebaan during the summer and its disappearance during winter, can be explained by its temperature preferences. The possibility that thermal pollution from a nuclear power station may be beneficial to this prawn, is discussed.