The half-life of mRNA in Saccharomyces cerevisiae.

Summary The decay kinetics of mRNA was studied in a yeast temperature-sensitive mutant, ts136, which is defective in cytoplasmic RNA production at 37° C. The disappearance of the synthetic capacity of mRNA was determined by withdrawing equal volumes of ts136 cell culture and pulse-labelling with [³⁵S]methionine at various time intervals after the shift to 37° C from 23° C. The synthesized proteins were separated on a two-dimensional gel electrophoretic system and then quantitatively analyzed for their incorporated radioactivities by scintillation counting. Our results show that yeast mRNAs have divergent functional half-lives ranging from 4.5 to 41 min, with an average value of 22 min. Each mRNA exhibits a simple exponential decay with its own characteristic decay pattern. Of the approximately 500 major polypeptides made by yeast cells, which are detectable on autoradiograms of the gels, 80 were arbitrarily selected and the mRNAs coding for those polypeptides were examined for their decay kinetics.     

Functional inactivation rates of the messenger RNA molecules coding for the individual ribosomal proteins in Escherichia coli.

Summary The rates of functional decay of messenger RNA coding for total soluble, total ribosomal and individual ribosomal proteins were measured in Escherichia coli strain AS-19, at 30^o. This was accomplished by blocking RNA synthesis with the inhibitor thiolutin and measuring residual protein synthesis at various times thereafter. The data obtained expressed as a decay constant (Hartwell and Magasanik, 1963) show that both total soluble and total ribosomal protein decay with similar rates (K ₂=0.64 and 0.61 respectively) which are slightly faster than the decay rate of -galactosidse (k ₂=0.43) under these conditions. All the individual ribosomal proteins appear to comprise a population of cistrons whose individual mRNA's decay with very similar rates with the possible exception of protein L3, whose mRNA appears consistently to decay very rapidly.Additional data on the stability of the total soluble and total ribosomal proteins during thiolutin treatment (that is, proteins synthesized in the absence of concommitant ribosomal RNA synthesis) fail to demonstrate any marked difference between these two protein populations. Examination of the stability of the individual ribosomal proteins however, reveals that some are degraded up to 35% in 15 min of thiolutin exposure, some to about 15% and some appear to be completely stable. In general, a degree of correlation exists between the stability of a given protein and the observed decay rate of its messenger RNA. This observation may explain in part the spread among the rates of mRNA decay. Nevertheless, we conclude that although degradation is occurring, it is not sufficient to alter the main conclusion that the rates of functional decay of mRNA cistrons coding for the ribosomal proteins are very similar.     

Studies on ribosomal protein biosynthesis in an RNA polymerase temperature sensitive E. coli mutant.

Summary In E. coli strain XH56 the synthesis of all RNA species is blocked upon shifting the culture to the non-permissive temperature. The decay of specific messenger RNA species coding for individual ribosomal (r) proteins was followed by measuring the rate of r-protein synthesis by pulse labelling at various times after the shift. The half-lives of the average 30S r-protein and 50S r-protein mRNA species are identical (1.75 min) and shorter than those of the average messenger coding for total cell proteins (2.75 min). Most individual r-protein messengers have a half-life in the same range (1.50–2.00). Only a few r-protein messengers have significantly longer half-lives: S1 (2.80 min), S17 (3.29 min), L29 (2.30 min), L31 (2.30 min), L32 (2.33 min) and L16 (2.60 min). The results indicate that the degradation of most individual r-protein mRNA species is not specifically controlled.After a few min at the non-permissive temperature, all protein synthesis is blocked. The restart of r-protein synthesis was followed after shifting the culture back to the permissive temperature. The recovery of cell growth is very slow. During this period preferential r-protein synthesis was observed. Moreover differential rates of biosynthesis of r-proteins was obtained, it may be indicative of specific regulatory process(es).     

Interpretation of intramolecular stacking effect on the fluorescence intensity decay of 3-methylbenzimidazolyl(5′-5′)guanosine dinucleotides using a model of lifetime distribution

Time-resolved fluorescence of 3-methylbenzimidazole (m³B) was used to study stacking interaction between base moieties in di-, tri- and tetra-phosphate analogues of 3-methylbenzimidazolyl(5′-5′)guanosine (m³Bp _n G, n = 2, 3, 4), using 5′-triphosphate of 3-methylbenzimidazole riboside (m³BTP) as reference. Fluorescence intensity decays of all compounds cannot be satisfactory fitted with single-exponential function. Although an increase of a number of exponents led to better fits, interpretation of the individual exponential terms, i.e. pre-exponential amplitudes and fluorescence lifetimes, cannot be adequately characterized. We show that these fluorescence decays are best fitted by power-like function derived from physically justified distribution of the fluorescence lifetimes, and characterized by the mean value of the excited-state lifetime and relative variance of lifetime fluctuations around the mean value. The latter led to the parameter of heterogeneity and number of decay paths, which depend on the factors responsible for non-radiative decay of the excited state, including base–base stacking interaction. This was studied by means of changes of temperature and the number of phosphate groups in dinucleotides. It was shown that the strongest effect of stacking interactions, characterized by lowest values of both fluorescence mean decay time and relative variance, occurs in the case of m³Bp₃G containing the same number of phosphates as natural mRNA cap. The possible importance of these results for interpretation of the mechanism of function of the mRNA cap structure is discussed.     

Fractional exponential decay in the capture of ligands by randomly distributed traps in one dimension

In many biophysical and biochemical experiments one observes the decay of some ligand population by an appropriate system of traps. We analyse this decay for a one-dimensional system of randomly distributed traps, and show that one can distinguish three different regimes. The decay starts with a fractional exponential of the form exp[−(t/t ₀)^1/2], which changes into a fractional exponential of the form exp[−(t/t ₁)^1/3] for long times, which in its turn changes into a pure exponential time dependence, i.e. exp[−t/t ₂] for very long times. With these three regimes, we associate three time scales, related to the average trap density and the diffusion constant characterizing the motion of the ligands.     

Stability of rapidly labelled messenger ribonucleic acid in Bacillus amyloliquefaciens during the phases of minimum and maximum extracellular enzyme formation.

The stability of rapidly labelled hybridizable messenger RNA in both exponential and post-exponential phase cells of Bacillus amyloliquefaciens was measured in terms of the rate of loss of its radioactivity. In the exponential phase, where 96% of the mRNA was specific for cell proteins and only 4% was exoprotein mRNA, the label was lost exponentially from the rapidly labelled hybridizable mRNA fraction with a half-life of six minutes at 30 °C. The antibiotic rifampicin, at a concentration of 10 μg/ml, had no effect on the characteristics of decay of this exponential-phase mRNA. In the post-exponential phase, where there were equal amounts of cell protein and exoprotein-specific mRNA, rapidly labelled hybridizable mRNA decayed exponentially in the presence of rifampicin (10 μg/ml), with a half-life of six minutes at 30 °C. In the absence of rifampicin the characteristics of decay were more complex. The evidence available suggested that this was due to the superimposition of a component attributable to reincorporation of degradation products of radioactive RNA on the characteristic exponential decay pattern of the post-exponential mRNA.Measurement of the stability of active mRNA, by studying the loss of ability to incorporate l-[¹⁴C]leucine into protein in the presence of rifampicin (10 μg/ml), gave half-lives of 4.5 minutes and six minutes, respectively, for exponential and post-exponential material.     

The ribonucleases J1 and J2 are essential for growth and have independent roles in mRNA decay in Streptococcus pyogenes

The paralogous ribonucleases J1 and J2, recently identified in Bacillus subtilis, have both endoribonucleolytic and 5′‐to‐3′ exoribonucleolytic activities and participate in degradation and regulatory processing of mRNA. RNases J1 and J2 have partially overlapping target specificities, but only RNase J1 is essential for B. subtilis growth. Because mRNA decay is important in regulation of virulence factors of Streptococcus pyogenes (the group A streptococcus, GAS), we investigated the role of these newly described RNases in GAS. We found that conditional mutants for both RNases J1 and J2 require induction for growth, so we conclude that, unlike the case in B. subtilis, both of these RNases are essential for GAS growth, and therefore their functions are not redundant. We compared decay of representatives of the two classes of messages we had previously identified: Class I, which decay rapidly in exponential and stationary phase of growth (hasA and gyrA), and Class II, which are stable in stationary phase and exhibit a biphasic decay curve in exponential phase (sagA and sda). We report that RNases J1 and J2 affect the rate of decay of Class I messages and the length of the first phase in decay of Class II messages.     

Minimal levels of cold-adaptation in postsynaptic currents of a subantarctic teleost, <Emphasis Type="Italic">Notothenia rossii</Emphasis> (Perciformes: Notothenioidei: Nototheniidae)

As a part of the ICEFISH04 project on the RVIB Nathaniel B. Palmer, miniature end plate currents (MEPCs) were recorded from the extraocular muscles of Notothenia rossii captured at King Edward Point, South Georgia. A total of 1,176 MEPCs were recorded from the inferior oblique extraocular muscles of four specimens, over a temperature range of 1–12°C. The MEPCs were normal in form, with a rapid quasi-linear increase in inward current (typically <500 μs), followed by a slower exponential decay of the inward current to baseline. Exponential decay rates were calculated for individual MEPCs by linear regression of the log-transformed data, and converted to exponential time constants (τ). Only those MEPCs that fit the exponential model well, with r ² ≥ 0.95 (or in some cases r ² ≥ 0.99) were used for further calculations. At temperatures between 1 and 2°C, τ ranged from about 2,000 to 4,000 μs, similar to values extrapolated for temperate teleosts at the same temperature, but significantly longer than τ from MEPCs of high-latitude Antarctic nototheniids. Between 11 and 12°C, τ values for the N. rossii MEPCS were mainly between 1,100 and 1,700 μs, giving a Q ₁₀ of 2.05. An Arrhenius plot and linear regression were used to describe the effect of changing temperature on the decay phase of the N. rossii MEPCs: −ln τ = 27.887−6078/K, yielding an Arrhenius temperature coefficient (μ or apparent E _a) of −50.5 ± 2.9 (95% CL) kJ mol⁻¹ deg⁻¹. When compared with other nototheniids, these results showed that the neuromuscular junctions of N. rossii are compensated for low temperature, but not to the same degree as those of high Antarctic species. The ICEFISH Cruise (International Collaborative Expedition to collect and study Fish Indigenous to Sub-antarctic Habitats) was conducted on board the RVIB Nathaniel B. Palmer in May to July 2004. For further information, please visit .     

Brownian Dynamics Simulations of Colloidal Liquids: Hydrodynamics and Stress Relaxation

Abstract

We describe the statistical mechanics background and additional algorithmic features of a recently proposed simple mean-field Brownian Dynamics algorithm formulated to include many-body hydrodynamics, using a local density approximation for the friction coefficient. We show that the equations of motion satisfy the incompressibility of phase space. We make further developments to the model, computing the hydrodynamic effects on the shear stress relaxation function. We show that stress relaxation takes place over two well-defined regimes, in both cases with and without mean field hydrodynamics, MFH. At short times ta ²/D ₀ < 10^?3, where a is the radius of the colloidal particle and D ₀ is the self-diffusion coefficient at infinite dilution, decay of the stress autocorrelation function, C_s(t) is essentially independent of volume fraction and does not fit to a simple analytic form. At longer times than ta ²/D ₀ < 10^?2 the decay has the fractional exponential form ～exp(-t ^β) with β ? 1. The transition between these two regimes coincides with a rapid fall in the time-dependent diffusion coefficient from the so-called short-time to long-time values. We do not find any evidence for power law decay in the C_s(t) as predicted by recent mode-coupling based analytical expansions.     

Construction and characterization of a clostripain-like protease-deficient mutant of <Emphasis Type="Italic">Clostridium perfringens</Emphasis> as a strain for clostridial gene expression

The inherent difficulty of expressing clostridial AT-rich genes in a heterologous host has limited their biotechnological application. We previously reported a plasmid for high-level expression of clostridial genes in Clostridium perfringens (Takamizawa et al., Protein Expr Purif 36:70–75, 2004). In this study, we examined the extracellular proteases of C. perfringens strain 13. Zymographic analysis and caseinase assaying of a culture supernatant showed that it contained a protease activated by dithiothreitol and Ca²⁺, suggesting that clostripain-like protease (Clp) is the most likely candidate for the major extracellular protease. Disruption of the clp gene by homologous recombination markedly decreased the level of caseinase activity in the culture supernatant. Analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Clp⁻ mutant but not the wild type strain increased the levels of many polypeptides in the culture supernatant after the late exponential growth phase. Such polypeptides included both cytoplasmic and secretory proteins, suggesting proteins secreted or released into the medium were degraded by Clp. To assess the effects of Clp on the productivity and stability of recombinant proteins, 74-kDa NanI sialidase was expressed in the two strains. The mutant strain produced a higher level of NanI activity than the wild type strain. Furthermore, under the conditions where Clp was activated, NanI was degraded easily in the latter culture but not in the former one. These results indicate that the Clp⁻ mutant could serve as a useful strain for efficiently expressing and preparing protease-free clostridial proteins.     

Characterization of a model cyanobacterium Synechocystis sp. PCC 6803 autotrophic growth in a flat‐panel photobioreactor

We characterized the photoautotrophic growth of glucose‐tolerant Synechocystis sp. PCC 6803 in a flat‐panel photobioreactor running on a semicontinuous regime under various lights, temperatures, and influx carbon dioxide concentrations. The maximum reached growth rate was 0.135 h^?1, which corresponds to a doubling time of 5.13 h—a growth speed never reported for Synechocystis before. Saturating red light intensity for the strain was 220–360 μmol(photons) m^?2 s^?1, and we did not observe any photoinhibition up to 660 μmol(photons) m^?2 s^?1. Synechocystis was able to grow under red light only; however, photons of wavelengths 405–585 and 670–700 nm further improved its growth. Optimal growth temperature was 35°C. Below 32°C, the growth rates decreased linearly with temperature coefficient (Q₁₀) 1.70. Semicontinuous cultivation is known to be efficient for growth characterization and optimization. However, the assumption of correct growth rates calculation—culture exponential growth—is often not fulfilled. The semicontinuous setup in this study was operated as a turbidostat. Accurate online OD measurements with high time‐resolution allowed fast and reliable growth rates determination. Repeating diluting frequencies (up to 18 dilutions per day) were essential for rapid growth stability evaluation. The presented setup provides improvement to previously published semicontinuous characterization strategies by decreasing experimental time requirements and maintaining the culture in exponential growth phase throughout the entire characterization procedure.     

Effect of Cultivation Conditions on Growth and Adhesion of Bacillus licheniformis

Adhesion to glass of actively growing cells of the thermophilic Bacillus licheniformis, isolated from the Medyaginskaya test borehole (Yaroslavl' oblast), was studied. The reversible adhesion (RA) manifests itself in a decline of cell density (without cell lysis) in the liquid culture over the first 20–40 min of growth followed by normal exponential growth. The RA is minimal under favorable growth conditions but increases when cells are transferred to a new medium, especially one with a pH, temperature, salinity, or concentration of Ca²⁺ ions nonoptimal for the given species. Under unfavorable growth conditions, the adhesion becomes irreversible. The obtained data suggest that RA represents an adaptation mechanism important for population survival.     

RNA synthesis and coding capacity of polyadenylated and nonpolyadenylated mRNA from cultures of differentiating Drosophila melanogaster myoblasts

We have investigated the synthesis and coding capacity of RNA isolated from cultures of differentiating Drosophila embryonic muscle cells. We find that following muscle cell fusion, the sedimentation profile of newly synthesized polyadenylated RNA becomes somewhat lighter. In vitro translation products analyzed by two-dimensional gel electrophoresis indicate that the coding capacity of translatable myogenic mRNA changes during differentiation. A group of several muscle-specific proteins (including the contractile proteins) is translated only from mRNA isolated after the initiation of fusion. This pattern coincides with proteins synthesized in vivo during differentiation. Additionally, we find that polyadenylated and nonpolyadenylated myogenic mRNA from a given developmental stage in culture have extremely similar coding potentials.     

Effects of temperature and glycerol and methanol‐feeding profiles on the production of recombinant galactose oxidase in Pichia pastoris

Optimization of protein production from methanol‐induced Pichia pastoris cultures is necessary to ensure high productivity rates and high yields of recombinant proteins. We investigated the effects of temperature and different linear or exponential methanol‐feeding rates on the production of recombinant Fusarium graminearum galactose oxidase (EC 1.1.3.9) in a P. pastoris Mut⁺ strain, under regulation of the AOX1 promoter. We found that low exponential methanol feeding led to 1.5‐fold higher volumetric productivity compared to high exponential feeding rates. The duration of glycerol feeding did not affect the subsequent product yield, but longer glycerol feeding led to higher initial biomass concentration, which would reduce the oxygen demand and generate less heat during induction. A linear and a low exponential feeding profile led to productivities in the same range, but the latter was characterized by intense fluctuations in the titers of galactose oxidase and total protein. An exponential feeding profile that has been adapted to the apparent biomass concentration results in more stable cultures, but the concentration of recombinant protein is in the same range as when constant methanol feeding is employed. © 2014 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 30:728–735, 2014     

Electron tunneling in proteins: role of the intervening medium

Analysis of electron-transfer (ET) kinetics data obtained from experiments on Ru-modified proteins (cytochrome c, azurin, myoglobin) reveals that distant donor-acceptor electronic couplings depend upon the secondary structure of the intervening polypeptide matrix. Rates of Fe²⁺→Ru³⁺ ET reactions in cytochrome c decay exponentially with tunneling-pathway length (decay constant 0.73?Å^–1); these rates also decay exponentially with Ru-Fe distance (decay constant 1.1?Å^–1). In azurin, a β-sheet protein, Cu⁺→Ru³⁺ rates exhibit an exponential Cu-Ru distance dependence with a decay constant of 1.1?Å^–1. Comparison of distant couplings in azurin and myoglobin suggests that hydrogen bonds are better mediators across β sheets than through α helices.     

西藏色季拉山急尖长苞冷杉原始林粗木质残体空间格局分析

粗木质残体（Coarse woody debris,CWD）的空间格局反映了森林群落的死亡格局和干扰格局,在一定程度上体现了群落内林木的死亡过程。采用相邻网格法对色季拉山急尖长苞冷杉（Abies georgei var.smithii）原始林1 hm²固定样地内CWD进行调查,从CWD类型、腐烂等级、径级3个方面对CWD空间分布格局进行分析。结果表明：样地内CWD总密度为582株/hm²,倒木占55.33%,是CWD的主要输入形式。CWD密度在腐烂等级上的分布可用多项式拟合（R²=0.9973）,在径级上的分布可用指数衰减模型拟合（R²=0.9746）,且在不同类型、腐烂等级及径级上的分布差异较大。在50 m尺度内,CWD整体表现为小尺度的集群分布和中、大尺度的随机分布。在3种CWD分类中,仅有大枯枝、Ⅰ级腐烂、径级ⅠCWD在小尺度或中尺度表现为较强的集群分布,其余则均以随机分布为主,只是在个别尺度达到或接近集群分布。不同类型CWD间整体关联不显著,只有枯立木与大枯枝在0-21 m尺度内达到显著负关联。CWD空间分布格局是急尖长苞冷杉原始林的重要结构特征,在很大程度上决定着林下植物群落及林型自然更新格局。     

Alexandrium ostenfeldii growth and spirolide production in batch culture and photobioreactor

Growth and spirolide production of the toxic dinoflagellate Alexandrium ostenfeldii (Danish strain CCMP1773) were studied in batch culture and a photobioreactor (continuous cultures). First, batch cultures were grown in 450 mL flasks without aeration and under varying conditions of temperature (16 and 22 °C) and culture medium (L1, f/2 and L1 with addition of soil extract). Second, cultures were grown at 16 °C in 8 L aerated flat-bottomed vessels using L1 with soil extract as culture medium. Finally, continuous cultures in a photobioreactor were conducted at 18 °C in L1 with soil extract; pH was maintained at 8.5 and continuous stirring was applied.This study showed that A. ostenfeldii growth was significantly affected by temperature. At the end of the exponential phase, maximum cell concentration and cell diameter were significantly higher at 16 °C than at 22 °C. In batch culture, maximum spirolide quota per cell (approx. 5 pg SPX 13-desMeC eq cell⁻¹) was detected during lag phase for all conditions used. Spirolide quota per cell was negatively and significantly correlated to cell concentration according to the following equation: y = 4013.9x^−0.858. Temperature and culture medium affected the spirolide profile which was characterized by the dominance of 13,19-didesMeC (29–46%), followed by SPX-D (21–28%), 13-desMeC (21–23%), and 13-desMeD (17–21%).Stable growth of A. ostenfeldii was maintained in a photobioreactor over two months, with maximum cell concentration of 7 × 10⁴ cells mL⁻¹. As in batch culture, maximum spirolide cell quota was found in lag phase and then decreased significantly throughout the exponential phase. Spirolide cell quota was negatively and significantly correlated to cell concentration according to the equation: y = 12,858x^−0.8986. In photobioreactor, spirolide profile was characterized by higher proportion of 13,19-didesMeC (60–87%) and lower proportions of SPX-D (3–12%) and 13-desMeD (1.6–10%) as compared to batch culture.