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.     

The effect of temperature change on gene expression in Paramecium primaurelia

When paramecia grown at 24°C are transferred rapidly to 32°C, DNA and protein synthesis continue uninterrupted but at higher rates. Electron microscopic observations indicate that more of the macronuclear chromatin is transcribed at the elevated temperature. This interpretation is supported by hybridization experiments which show that the percentage of the macronuclear genome transcribed into poly(A)⁺ RNA is 24°C and 35% at 32°C. Kinetic analysis of cDNA-poly(A)⁺ RNA hybridizations reveals three abundance classes of poly(A)⁺ RNA and indicates that the number of genes expressing low abundance sequences is about 9000 at 24°C and 13000 at 32°C. The intermediately abundant and highly abundant classes are represented by 100–200 and 1–3 different kinds of RNA sequence, respectively. Cross hybridization shows that changes occur throughout the distribution of abundance classes of poly(A)⁺ RNA with increase in temperature.     

Differential accumulation of two size classes of poly(A) associated with messenger RNA during oogenesis in Xenopus laevis

The RNA of full-grown oocytes of Xenopus laevis contains two distinct size classes of poly(A), designated poly(A)_S and poly(A)_L, which contain 15–30 (mean = 20) and 40–80 (mean = 61) A residues, respectively. Both poly(A)_L and poly(A)_S are associated with RNA which is heterogeneous in size. The two classes of poly(A)⁺ RNA can be separated by affinity chromatography: Only poly(A)_L⁺ RNA binds to oligo(dT)-cellulose under appropriate conditions, but up to 50% of the poly(A)_S⁺ RNA can be isolated from the void fraction by binding to poly(U)-Sepharose. Both classes of poly(A)⁺ RNA are active as messenger RNA in an in vitro system and yield identical patterns of in vitro protein products. Previtellogenic oocytes contain almost exclusively poly(A)_L, which accumulates up to vitellogenesis but remains almost constant in amount (molecules/oocyte) during vitellogenesis and in the full-grown oocyte. Poly(A)_S accumulates (molecules/oocyte) from early vitellogenesis up to the full-grown oocyte. The total number of poly(A)⁺ RNA molecules per oocyte increases throughout oogenesis from 2 × 10¹⁰/previtellogenic oocyte [80–90% poly(A)_L] to 20 × 10¹⁰/full-grown oocyte (25–40% poly(A)_L). It is argued that poly(A)_S is protected from degradation in the oocyte, thus stabilizing the “maternal” poly(A)⁺ mRNA.     

The occurrence and distribution of poly(a) ribonucleic Acid in soybean

The occurrence and distribution of poly(A) sequences in the RNA of soybean (Glycine max var. Wayne) have been studied. Only one of the two species of AMP-rich RNA contains poly(A). D-RNA does not contain detectable poly(A) sequences. The TB-RNA is the poly(A) RNA in this system. At least a part (up to 50% or more) of the mRNA in polyribosomes contains a poly(A) sequence. The poly(A) RNA is heterodisperse in size but has a mean size of approximately 18S (2,000 nucleotides) in urea and formamide gels. The poly(A) fragment resulting from ribonuclease A and T₁ digestion migrates as a broad band overlapping the 4 to 5.8S regions of the gels with a mean size of somewhat greater than 5S. No evidence was found for the occurrence of a discrete oligo(A) fragment in the poly(A) RNA; however, oligonucleotides which migrate faster than the poly(A) fraction were observed in preparations which were not bound to oligo(dT) cellulose prior to electrophoresis. This oligonucleotide region was enriched in AMP (up to about 65%) as would be expected after ribonuclease A and T₁ digestion.     

Poly(A) length,cytoplasmic adenylation and synthesis of poly(A)+ RNA in early mouse embryos

The poly(A) content of early mouse embryos fluctuates widely: after a transient increase in the one-cell embryo, there is a 70% drop in the two-cell and an approximately fivefold increase between the two-cell and early blastocyst stages (L. Pikó and K. B. Clegg, 1982, Dev. Biol.89, 362–378). To shed light on the significance of these changes, we analyzed the size distribution of total poly(A) from embryos at different stages of development by gel electrophoresis and hybridization with [³H]poly(U). The number-average size of poly(A) tracts varies only slightly, from 61 to 77 nucleotides, indicating that the changes in poly(A) content are due primarily to changes in the number of poly(A) sequences, i.e., the number of poly(A)+ mRNA. From these data, the number of poly(A)+ mRNA can be estimated as follows: ovulated egg, 1.7 × 10⁷; one-cell embryo, 2.4 × 10⁷; late two-cell, 0.7 × 10⁷; late eight-cell, 1.3 × 10⁷; and early blastocyst, 3.4 × 10⁷. These results suggest the elimination of the bulk of maternal poly(A)+ mRNA at the two-cell stage, to be replaced by newly synthesized mRNA derived from the embryonic genome. To study the synthesis of poly(A)+ mRNA, we cultured mouse embryos in vitro with [³H]adenosine and analyzed the labeled poly(A)+ RNA as to molecular size, length of the poly(A) tail, and relative distribution of label in poly(A) vs internal locations. We observed an active incorporation of label into large-molecular-weight (average size about 2 kb) poly(A)+ RNA at all stages from the one-cell to the blastocyst. However, in the one-cell embryo, about 70% of the label was localized in the poly(A) tail, suggesting cytoplasmic polyadenylation, and only about 30% was localized in the remainder of the molecule, suggesting the complete new synthesis of a small amount of poly(A)+ RNA. Differences in the size distribution of the labeled poly(A) as compared with the total poly(A) in the one-cell embryo indicate that the labeling is not due to a general turnover of poly(A) tails, but rather to the polyadenylation of previously nonpolyadenylated, stored RNA. Significant new synthesis of poly(A)+ RNA is evident from the two-cell stage onward and most likely accounts for the sharp rise in the number of poly(A)+ RNA molecules by the early blastocyst stage.     

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.     

Polyadenylic acid-containing RNA and its polyadenylic acid sequences newly synthesized in isolated embryonic cells of Xenopus laevis.

Newly synthesized polyriboadenylic acid [poly(A)]-containing RNA and its poly(A) sequences were isolated and characterized in Xenopus embryonic cells. Upon sedimentation analysis, the poly(A)-containing RNA labeled for 30 min showed a very heterogeneous size distribution ranging from 9 to >40 S. After 5 hr of labeling, the profile became much less heterogeneous and the main component was distributed in the 9–28 S region. The average molecular weight of 6.5–7.0 × 10⁵ daltons was calculated for the 5-hr labeled RNA. This poly(A)-containing RNA, comprising about 10% of the total labeled RNA, was metabolically stable and accumulated linearly for 5 hr. Gel electrophoresis of the RNA revealed the presence of little or no free poly(A) sequences. Most of the poly(A) sequences, which were isolated from 30-min labeled poly(A)-containing RNA migrated as a single discrete component approximately 150 nucleotides long. In contrast, they were slightly smaller (130 nucleotides long) and more heterogeneous, when obtained from the poly(A)-containing RNA labeled for 5 hr. From these results, it may be likely that the embryonic poly(A)-containing RNA is similar in size to the steady-state population of the poly(A)-containing RNA reported to occur in vitellogenic oocytes and cultured kidney cells of the same species.     

Prey consumption by phytoseiid spider mite predators as affected by diurnal temperature variations

The consumption rate of an ectothermic predator is highly temperature-dependent and is a key driver of pest-predator population interactions. Not only average daily temperature, but also diurnal temperature variations may affect prey consumption and life history traits of ectotherms. In the present study, we evaluated the impact of temperature alternations on body size, predation capacity and oviposition rate of the predatory mites Phytoseiulus persimilis Athias-Henriot and Neoseiulus californicus McGregor (Acari: Phytoseiidae) when presented with eggs of their natural prey, the two-spotted spider mite Tetranychus urticae Koch (Acari: Tetranychidae). For both predators, mean daily temperature as well as temperature alternation had a substantial impact on the number of prey consumed. At lower average temperatures, more eggs were killed under an alternating temperature regime (20 °C/5 °C and 25 °C/10 °C) than at the corresponding mean constant temperatures (15 and 20 °C). At higher average temperatures (>25 °C), however, the opposite was observed with higher numbers of prey killed at constant temperatures than at alternating temperatures. At 25 °C, temperature variation had no effect on the predation capacity. A similar trend as for the predation rates was observed for the oviposition rates of the phytoseiids. Body size of N. californicus was affected both by average daily temperature and temperature variation, with smaller adult females emerging at alternating temperatures than at constant temperatures, whereas for P. persimilis, temperature variation had no impact on its body size. Our results demonstrate that temperature variations are likely to affect biological control of T. urticae by the studied phytoseiid predators.     

Complexity and characterization of polyadenylated RNA in the mouse brain

The complexity of nuclear RNA, poly(A)hnRNA, poly(A)mRNA, and total poly(A)RNA from mouse brain has been measured by saturation hybridization with nonrepeated DNA. These DNA populations were complementary, respectively, to 21, 13.5, 3.8, and 13.3% of the DNA. From the RNA Cot required to achieve half-sturation, it was estimated that about 2.5–3% of the mass of total nuclear RNA constituted most of the complexity. Similarly, complexity driver molecules constituted 6–7% of the mass of the poly(A)hnRNA. 75–80% of the poly(A)mRNA diversity is contained in an estimated 4–5% of the mass of this mRNA. Poly(A)hnRNA constituted about 20% of the mass of nuclear RNA and was comprised of molecules which sedimented in DMSO-sucrose gradients largely between 16S and 60S. The number average size of poly(A)hnRNA determined by sedimentation, electron microscopy, or poly(A) content was 4200–4800 nucleotides. Poly(A)mRNA constituted about 2% of the total polysomal RNA, and the number average size was 1100–1400 nucleotides. The complexity of whole cell poly(A)RNA, which contains both poly(A)hnRNA and poly(A)mRNA populations, was the same as poly(A)hnRNA. This implies that cytoplasmic polyadenylation does not occur to any apparent qualitative extent and that poly(A)mRNA is a subset of the poly(A)hnRNA population. The complexity of poly(A)hnRNA and poly(A)mRNA in kilobases was 5 × 10⁵ and 1.4 × 10⁵, respectively. DNA which hybridized with poly(A)mRNA renatures in the presence of excess total DNA at the same rate as nonrepetitive tracer DNA. Hence saturation values are due to hybridization with nonrepeated DNA and are therefore a direct measure of the sequence complexity of poly(A)mRNA. These results indicate that the nonrepeated sequence complexity of the poly(A)mRNA population is equal to about one fourth that observed for poly(A)hnRNA.     

Effects of constant and fluctuating incubation temperatures on hatching success and hatchling traits in the diamondback terrapin (Malaclemys terrapin) in the context of the warming climate

As global temperatures continue to rise, so too will the nest temperatures of many species of turtles. Yet for most turtle species, including the estuarine diamondback terrapin (Malaclemys terrapin), there is limited information on embryonic sensitivity to elevated temperature. We incubated eggs of M. terrapin at three, mean temperatures (31, 34, 37 °C) under two thermal exposure regimes (constant or semi-naturally fluctuating temperature) and measured hatching success, developmental rate, and hatchling size. Hatching success was 100% at 31 °C and 67% at 34 °C, respectively; at 37 °C, all eggs failed early in the incubation period. These values were unaffected by exposure regime. The modeled LT₅₀ (temperature that was lethal to 50% of the test population) was 34.0 °C in the constant and 34.2 °C in the fluctuating thermal regime, reflecting a steep decline in survival between 33 and 35 °C. Hatchlings having been incubated at a constant 34 °C hatched sooner than those incubated at 31 °C under either constant or fluctuating temperature. Hatchlings were smaller in straight carapace length (CL) and width after having been incubated at 34 °C compared to 31 °C. Larger (CL) hatchlings resulted from fluctuating temperature conditions relative to constant temperature conditions, regardless of mean temperature. Based upon recent temperatures in natural nests, the M. terrapin population studied here appears to possess resiliency to several degrees of elevated mean nest temperatures, beyond which, embryonic mortality will likely sharply increase. When considered within the mosaic of challenges that Maryland's M. terrapin face as the climate warms, including ongoing habitat losses due to sea level rise and impending thermal impacts on bioenergetics and offspring sex ratios, a future increase in embryonic mortality could be a critical factor for a population already experiencing ecological and physiological challenges due to climate change.     

Fractionation of Chromatin by thermal precipitation in phosphate buffer.

The stability of sonicated rat liver chromatin in sodium phosphate buffer, pH 6.8 was studied as a function of buffer concentration (0.012 to 0.16 m) and temperature (20 to 98 °C). It was found that as the temperature was increased a stepwise precipitation of chromatin took place which was revealed by the presence of three plateaux (20 to 50 °C, 70 to 75 °C and above 90 °C) and two transitional zones (55 to 70 °C and 75 to 90 °C) on the A₃₂₀ curves and on the percentage precipitated nucleoprotein versus temperature curves as well.This permitted the fractionation of chromatin in 0.08 m-phosphate buffer into three fractions by a stepwise heating at 50 °C (50 °C-pellet) and 98 °C (50–98 °C-pellet and post 98 °C-supernatant). DNA isolated from these fractions was characterized in respect to sedimentation velocity and hybridization with heterogeneous nuclear RNA. The hybridization studies showed a different ability of these three DNA preparations in binding nuclear heterogeneous RNA: 16%, 8% and 30% for DNA isolated from 50 °C-pellet, 50–98 °C-pellet and post 98 °C-supernatant, respectively. The results are discussed in terms of chromatin structure and function.     

Evidence for a nuclease in Saccharomyces cerevisiae that affects the cell-free translation of natural messenger RNA.

A postpolysomal extract of $\text{Saccharomyces}\text{cerevisiae}$, treated with micrococcal nuclease to remove endogenous mRNAs, translates exogenous natural and synthetic mRNA templates actively and accurately at 20°C. When the temperature of incubation is 30°C or higher, protein synthesis with yeast poly(A)⁺ mRNA is markedly reduced, but synthesis of polyphenyl-alanine with poly (U) is only slightly affected. The protein synthesizing activity of the extract is decreased 50% in 30 minutes at 37°C, while the ability of yeast mRNA to template for protein synthesis is decreased 50% in 5 to 7 minutes when it is incubated with the postpolysomal fraction at 37°C. The release of radioactivity from isotopically-labeled yeast mRNA, into the acid-soluble form, is also much greater at 37°C than at 20°C. Thus, at the elevated temperatures, the loss of mRNA templating activity and RNA hydrolysis occur more rapidly than the loss of activity of the translational apparatus. The evidence suggests that the failure of the extract to catalyze translation at 30°C or higher, as compared to 20°C, is due to a temperature-stimulated nuclease that degrades mRNA.     

Binding properties of Ru(II) polypyridyl complexes with poly(U)·poly(A)*poly(U) triplex: the ancillary ligand effect on third-strand stabilization

The binding properties of [RuL₂(mip)]²⁺ {where L is 1,10-phenanthroline (phen) or 4,7-dimethyl-1,10-phenanthrollne (4,7-dmp) and mip is 2′-(3″,4″-methylenedioxyphenyl)imidazo[4′,5′-f][1,10]phenanthroline} with regard to the triplex RNA poly(U)·poly(A)*poly(U) were investigated using various biophysical techniques and quantum chemistry calculations. In comparison with [Ru(4,7-dmp)₂(mip)]²⁺, remarkably higher binding affinity of [Ru(phen)₂(mip)]²⁺ for the triplex RNA poly(U)·poly(A)*poly(U) was achieved by changing the ancillary ligands. The stabilization of the Hoogsteen-base-paired third strand was improved by about 10.9 °C by [Ru(phen)₂(mip)]²⁺ against 6.6 °C by [Ru(4,7-dmp)₂(mip)]²⁺. To the best of our knowledge, [Ru(phen)₂(mip)]²⁺ is the first metal complex able to raise the third-strand stabilization of poly(U)·poly(A)*poly(U) from 37.5 to 48.4 °C. The results reveal that the ancillary ligands have an important effect on third-strand stabilization of the triplex RNA poly(U)·poly(A)*poly(U) when metal complexes contain the same intercalative ligands.     

The Altitudinal Limit of Beech Woods in the Northern Apennines (Italy). Its Spatial Pattern and Some Thermal Inferences

The spatial pattern of the altitudinal limit of beech woods in the Northern Apennines and its correlation with the Fagus sylvatica thermal requirements were studied. The study area was the whole northern side of the Northern Apennines (latitude 44° N), including a timberline. The pattern was described on a scale of 1:25,000, using digitized phytosociological vegetation maps. The timberline elevation ranges from 1,200 to 1,825 m a.s.l., with the highest range at 1,525 to 1,725 m and a peak (13%) at 1,600–1,625 m. As suggested by the known beech thermal requirements, the following thermal parameters were considered: mean annual temperature, mean of the coldest month (January), mean of the warmest month (July); days with maximum temperatures >10°C and summer mean (June to September). The values were calculated using data for 1951–2002 from six weather stations. The present upper timberline (1,825 m) corresponds to the following: i) mean annual temperature of 4.5°C; ii) coldest month mean temperature of ?2.3°C; iii) summer mean of 11.5°C; iv) warmest month mean temperature of 13°C; v) 139 days with maximum temperatures of 10°C or higher. The mean temperature of the warmest month corresponds to the July mean temperature in maritime mountains, such as the Appalachians and the New Zealand Alps (13°C). The geographical location of the Northern Apennines close to the Mediterranean Sea seems to indicate that such a conclusion is reliable. With reference to the elevation variability of the upper timberline, the multiple regression shows that the annual mean is the best explaining thermal parameter (P?<?0.05). Our thermal data do not take into account the atmospheric phenomena that can affect the temperature lapse rate with altitude, such as the cloudiness of the chain, and site factors, such as wind-exposed topography and snow cover duration, which play a crucial role in the Northern Apennine local climate.     

Multiple oligo(A) tracts associated with inactive sea urchin maternal mRNA sequences

Maternal RNA of sea urchin eggs and embryos was analyzed for short poly(A) sequences by digesting hybrids formed between [³H]poly(U) and poly(A) with RNase at 4°C. When the undigested [³H]poly(U) is precipitated with CTAB, all (A)_n tracts longer than 6 nucleotides are detected. This assay revealed a poly(A) content severalfold higher than is obtained with a similar assay using RNase at higher temperatures. On polyacrylamide gel electrophoresis, most of the previously undetected (A)_n tracts ran as a peak of oligo(A) of less than 20 nucleotides which accumulated at the dye front. The oligo(A) sequences were resolved into a single peak of (A)₁₀ when sized on Sephadex G100. These (A)₁₀ sequences were associated with large mRNA-sized molecules of about 3000 nucloetides average length which comprised 0.5 to 2% of the total maternal RNA. However, the (A)₁₀ sequences were not in mRNA molecules containing 3′-terminal poly(A) of 50–120 nucleotides nor did they remain in RNA that entered polysomes upon fertilization. However, hybridization studies showed that all sequences represented in the maternal poly(A)-containing RNA appeared to be present in the RNA molecules containing only (A)₁₀ sequences. The results suggest that the (A)₁₀-containing RNA might be incompletely processed mRNA precursor-like molecules.     

A Novel PHB Depolymerase from a Thermophilic Streptomyces Sp.

A novel PHB depolymerase from a thermophilic Streptomyces sp. MG was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. The molecular mass of the purified enzyme was 43 kDa as determined by size exclusion chromatography and 41 kDa by SDS-PAGE. The optimum pH and temperature were 8.5 and 60 °C respectively. The enzyme was stable at 50 °C and from pH 6.5–8.5. The enzyme hydrolyzed not only bacterial polyesters, i.e. poly(3-hydroxybutyric acid and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), but also synthetic, aliphatic polyesters such as polypropiolactone, poly(ethylene adipate) and poly(ethylene succinate). Revisions requested 9 November 2005; Revisions received 12 December 2005