HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

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.     

Additives and Enzyme Stability

The polypeptide chain of an enzyme is folded so that the necessary functional groups are brought together in the active site. Conformational changes may disrupt this arrangement and cause loss of enzymic activity. The effect of soluble additives on the unfolding process is discussed. Additives may be classified as substrates and similar ligands, small uncharged organic molecules, specific and non-specific ionic species, and polymers.     

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.     

The lifetime of molecular (Davydov) solitons

The lifetime of Davydov solitons in a one-dimensional system is studied theoretically. The process of thermalization and the properties of solitons at finite temperature are investigated and the processes of soliton creation and disintegration are discussed.     

Screening of thermotolerant yeasts as producers of superoxide dismutase

Abstract A screening procedure for highly thermostable yeast superoxide dismutase was developed. Growth yields at various temperatures were estimated for ten mesophilic and thermotolerant strains, belonging to the genera Saccharomyces, Kluyveromyces and Pichia . Higher yields at 45°C were obtained for K. lactis 90-3 and 90-4. A correlation between the ability to grow at higher temperature and the thermostability of the superoxide dismutase enzyme synthesized was observed. A comparison of the operational stability of the superoxide dismutase of all tested strains suggests that the enzyme of K. lactis strains was more thermostable than that of the other tested microorganisms.     

Electrical stability of erythrocytes in the presence of divalent cations

Erythrocytes suspended in a medium of low ionic strength lyse under the effect of an exponential electrical pulse. The percentage of haemolysed cells decreases several-fold in the presence of divalent cations. The protective action of the ions studied increases in the following order: Ca⁺⁺, Mg⁺⁺, Zn⁺⁺. It is assumed that divalent ions bind to the negative charges of the lipid and protein molecules and reduce their electrostatic repulsion, which results in stabilization of the membranes.     

Aspects of the mineral nutrition of some native British plants — inter-site variation

Fifteen common native British plants were each sampled at a range of sites in Great Britain and green tissues analysed for several inorganic nutrients. Sampling criteria are discussed. The inter-site variation of each element within a species is assessed as a frequency distribution of raw data. Sample values of parameters including arithmetic mean, variance (coefficient of variation), skewness and kurtosis are presented. Their stability is assessed from nitrogen in sub-samples of Pteridium. This suggested sample sizes were adequate but some distributions had sufficient kurtosis to affect the variance. These parameters showed distinctions between macro- and micro-elements and between species. Some mean values sharply distinguished between species and may help to assess current theories of strategy and adaptation but a wider range of species is needed to clarify trends. Coefficients of variation are discussed and were relatively low for a nation-wide survey after allowing for sampling constraints. Coefficients of skewness and kurtosis showed two-thirds of the sample distributions were non-normal. Ecological aspects of the distributions are discussed and are relevant to studies along environmental gradients. Published hypotheses linking positive skewness to stress in the field are considered and two other postulates discussed. Distribution bounds such as those confining 95% of the values are discussed in relation to possible critical levels of nutrients.Nomenclature follows Clapham et al. (1981), Excursion flora of the British Isles. 3rd ed. University Press, Cambridge, except Chamaenerion.     

Light-driven proton transport of bacteriorhodopsin incorporated into long-term stable liposomes of a polymerizable sulfolipid

The chromoprotein bacteriorhodopsin from Halobacterium halobium has been incorporated into liposomes made of a fully synthetic, polymerizable lipid. Bacteriorhodopsin is found to be active in these polymer liposomes. The advantage in the use of such polymer systems concerning long-term stability in comparison with liposomes made of natural lipid is demonstrated.     

Site-directed mutagenesis of the CP 47 protein of photosystem II: 167W in the lumenally exposed loop C is required for photosystem II assembly and stability

The intrinsic chlorophyll-protein CP 47 is a component of photosystem II which functions in both light-harvesting and oxygen evolution. Using site-directed mutagenesis we have produced the mutant W167S which lies in loop C of CP 47. This strain exhibited a 75% loss in oxygen evolution activity and grew extremely slowly in the absence of glucose. Examination of normalized oxygen evolution traces indicated that the mutant was susceptible to photoinactivation. Analysis of the variable fluorescence yield indicated that the mutant accumulated very few functional PS II reaction centers. This was confirmed by immunoblotting experiments. Interestingly, when W167S was grown in the presence of 20 M DCMU, the mutant continued to exhibit these defects. These results indicate that tryptophan 167 in loop C of CP 47 is important for the assembly and stability of the PS II reaction center.