Mlc1p Is a Light Chain for the Unconventional Myosin Myo2p in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth. We explore the role of the neck region and its associated light chains in regulating Myo2p function. Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Δ6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain. We exploit this mutant in a characterization of the light chains of Myo2p. First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p. Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p. MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency. Reduced levels of MYO2 overcome the haploinsufficiency of MLC1. The mutant MYO2-Δ6IQ is able to suppress haploinsufficiency but not deletion of MLC1. We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p. Overexpression of MYO2 is toxic, causing a severe decrease in growth rate. When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain. High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p. Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region.     

Community persistence in Broadstone Stream (U.K.) over three decades

1. Few detailed long-term data sets exist for fresh waters with which to examine large-scale temporal changes in community composition. Consequently, insight into community persistence has been restricted to a few, contingent case studies. We collated and analysed data for the aquatic macroinvertebrate community of Broadstone Stream in south-east England, spanning three decades. The pH of this naturally acid stream has risen progressively since the 1970s, and we sought to examine the potential effects of this environmental change upon the community.
2. Persistence within Broadstone was high when compared with other systems that have been analysed using similar methods. The stream was characterised by a `core' community of eight taxa that were always present, and contributed 75–97% of total invertebrate abundance, with a trailing limb of progressively rarer and more acid-sensitive taxa. There was little species turnover, although the time-series exceeded 20 generations for most species.
3. Despite this high persistence, a long-term response to rising pH was detected: species indicating profound acidity (identified a priori from independent studies) have declined since the 1970s, whereas indicators of moderate acidity increased. The structure of the community food web has also changed since the 1970s, with increased predator diversity and abundance, and a lengthening of food chains following the invasion of a new top predator.
4. These changes in the community appeared to be driven by an interaction between pH and climate. The unusually hot, dry summers characteristic of the 1990s may have raised pH during the more sensitive (i.e. early) stages of the life-cycle, and thus provided a window of opportunity for less acid-tolerant taxa to colonise and become established. Changes in pH appeared to set the boundaries of the available local species pool, within which biotic interactions ultimately shaped the community.     

Cell membrane stability and biochemical response of cultured cells of groundnut under polyethylene glycol-induced water stress

Cell membrane stability (CMS) in suspension cultures of two groundnut cultivars was studied under polyethylene glycol(PEG)-induced water stress. There was a negative relationship between PEG concentration in the medium and membrane stability measured as electrolyte leakage. The CMS values in the cell cultures correlated well with the whole plant tissue and permitted the differentiation of cultivars based on their known response to drought stress. The cell membrane stability was lower (more electrolyte leakage) when cells were grown in culture as compared to the intact plant tissue. Kadiri-3, the drought tolerant cultivar maintained higher CMS than JL-24, the drought susceptible one. With increasing PEG levels the concentration of Potassium in cultured cells declined in both cultivars. However, Kadiri-3 maintained higher K values than JL-24 accompanied with greater cell membrane stability. Total soluble sugars also increased with increasing stress in both cultivars; the increase being higher in Kadiri-3. There was no significant change in the total free amino acids but proline accumulated markedly in both varieties. However, no relationship was found between proline levels and CMS. The results demonstrated that CMS test can also be used under in vitro conditions to differentiate the drought tolerant and susceptible cultivars and the cellular K level has a positive relationship with membrane stability.     

Stably inherited killer activity in industrial yeast strains obtained by electrotransformation

Killer-sensitive strains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis were transformed by electroinjection using double-stranded RNA isolated from a superkiller strain. Various recipient strains were used: both thermo-resistant and thermo-sensitive as well as mutants of industrial strains. Conversion of respiratory competent (rho+) into respiratory deficient (rho-) strains (mutants) resulted in a significant increase of the yield of electrotransformants and/or of longterm killer stability. Electrotransformation of rho- mutants of distillery and brewery strains resulted in more than 100 clones, which exhibited weak or strong killer activity over some or all of the experimental period of 10 months.     

The relation between translation and mRNA degradation in the lacZ gene

The technique of gene fusion, in which the gene of interest, severed from its 3' end, is in-phase fused to a reporter gene--usually lacZ--is widely used to study translational regulation in Escherichia coli. Implicit in these approaches is the assumption that the activity of the ribosome binding site (RBS) fused in-phase with lacZ, does not per se modify the steady-state level of the lacZ mRNA. Herein, we have tested this hypothesis, using a model system in which the RBS of the lamB gene is fused to lacZ. Several point mutations affecting translation initiation have been formerly characterized in this RBS, and we used Northern blots to study their effect upon the lacZ mRNA pattern. Two series of constructs were assayed: in the first one, a 51-bp fragment centered around the lamB initiator codon, was inserted in front of lacZ within the natural lactose operon, whereas in the second the lacZ gene was fused to the genuine malK-lamB operon just downstream from the lamB RBS. We observed that in the first series, the concentration and average molecular weight of the lacZ mRNA dropped sharply as the efficiency of the RBS decreased. This apparently arose from a decreased stability of the message, since the mRNA patterns are equalized when the endonuclease RNase E is inactivated. We suggest that in this case the rate limiting step in the decay process is an RNase E cleavage that is outcompeted by translation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of delayed memristive neural networks with time-varying impulses

This paper addresses the stability problem on the memristive neural networks with time-varying impulses. Based on the memristor theory and neural network theory, the model of the memristor-based neural network is established. Different from the most publications on memristive networks with fixed-time impulse effects, we consider the case of time-varying impulses. Both the destabilizing and stabilizing impulses exist in the model simultaneously. Through controlling the time intervals of the stabilizing and destabilizing impulses, we ensure the effect of the impulses is stabilizing. Several sufficient conditions for the globally exponentially stability of memristive neural networks with time-varying impulses are proposed. The simulation results demonstrate the effectiveness of the theoretical results.     

Effects of Chemical Modification of Arginine Residues Outside the Active Site Cleft of Ricin A-Chain on Its RNA N-Glycosidase Activity for Ribosomes

Ricin A-chain, a protein that inactivates ribosomes by a specific RNA N-glycosidase activity, has been shown to be inactivated by chemical modification of a few arginine residues. When two or fewer arginine residues in the A-chain were modified with [¹⁴C]phenylglyoxal, arginines at positions of 193, 196, 213, and 234/235 were found to be modified, from amino acid compositions and radioactivities of the modified peptides that were obtained by cyanogen bromide cleavage followed by tryptic and chymotryptic digestion. All these arginines have side chains outside the active site cleft; the side chain of Arg213 is adjacent to the edge of the cleft, while other modified arginines are located on the opposite side of the cleft. Kinetic analysis showed that the modification of two arginine residues caused a 8-fold loss in k_cat with a 3-fold increase in K_m, suggesting that this modification mainly decrease the rate of depurination with an additional effect on the affinity for ribosomes. Neither the environment of tryptophan 211 at the bottom of the cleft nor an interaction of adenine with the cleft was changed by this modification, as judged by fluorescence spectroscopy, suggesting that a conformational change of the catalytic site does not occur upon the modification. These results, taken together with other works, suggest that some of the above arginine residues outside the active site cleft may additively contribute to the catalysis of depurination and/or the initial formation of the A-chain/ribosome complex.     

Food-Grade Expression and Characterization of a Dextranase from Chaetomium gracile Suitable for Sugarcane Juice Clarification

The microbial production of dextranase using cheap carbon sources is beneficial to solve the economic loss caused by the accumulation of dextran in syrup. A food-grade microbial cell factory was constructed by introducing the dextranase encoding gene DEX from Chaetomium gracile to the chromosome of Bacillus subtilis, and the antibiotic resistance marker gene was subsequently deleted via the Cre/loxP strategy. The dual-promoter system with a sequentially arranged constitutive P43 promoter resulted in an 85 % increase in DEX expression. Under the optimal fermentation conditions of 10 g/L maltose, 15 g/L casein, 1 g/L Na₂HPO₄, 1 g/L FeSO₄ and 8 g/L NaCl, DEX activity was increased from 2.625 to 64.34 U/mL. Recombinant DEX was purified 5.98-fold with a recovery ratio of 26.67 % and specific activity of 3935.02 U/mg. Enzyme activity was optimal at 55 °C and pH 5.0 and remained 80.34 % and 71.36 % of the initial activity at 55 °C and pH 4.0 after 60 min, respectively. The enzyme possessed high activity in the presence of Co²⁺, while Ag⁺ showed the strongest inhibition ability. The optimal substrate was 20 g/L dextran T-2000. The findings could facilitate the low-cost, large-scale production of food-grade DEX for use in the sugar industry.     

Complex behaviour in four species food-web model

A four-dimensional food-web system consisting of a bottom prey, two middle predators and a generalist predator has been developed with modified functional response. The system is well posed and dissipative. Some results on uniform persistence have been developed. The dynamics of the system is found to be chaotic for certain choice of parameters. The coexistence of all four species is possible in the form of periodic orbits/strange attractors for suitably chosen set of parameters.