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.     

Common evolutionary origin of the central portions of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti T-DNAs of Agrobacterium tumefaciens

Analysis of published sequences for Ri TL-DNA (root-inducing left-hand transferred DNA) of Agrobacterium rhizogenes revealed several unsuspected structural features. First, Ri TL-DNA genes are redundant. Using redundancy as a criterion, three regions (left, middle and right) were discerned. The left one, ORFs (open reading frames) 1–7, contains no detectable redundancy. In the middle region a highly diverged gene family was detected in ORFs 8, 11, 12, 13 and 14. The right region contains an apparently recent duplication (ORF 15 =18+17). We interpret the phenomenon of redundancy, particularly in the central region that encodes the transformed phenotype, to be an adaptation that ensures function in a variety of host species. Comparison of Ri TL-DNA and Ti T-DNAs from Agrobacterium tumefaciens revealed common structures, unpredicted by previous nucleic acid hybridization studies. Ri TL-DNA ORF 8 is a diverged Ti T-DNA tms1. Both Agrobacterium genes consist of a member of the diverged gene family detected in the central part of the Ri TL-DNA, but fused to a sequence similar to iaaM of Pseudomonas savastonoi. Other members of this gene family were found scattered throughout Ti T-DNA. We argue that the central region of Ri and the part of Ti T-DNA including ORFs 5–10 evolved from a common ancestor. We present the hypothesis that the gene family encodes functions that alter developmental plasticity in higher plants.     

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.     

Sinistral snail shells in the sea: developmental causes and consequences

The paucity of sinistral (left-coiling) relative to dextral (right-coiling) species of gastropods in the marine realm is an enigma. In Conus , one of the most diverse marine animal genera, sinistral shell coiling has evolved as a species-wide character only once. Fossils of this species, Conus adversarius , are found in Upper Pliocene and lowermost Pleistocene deposits in the southeastern USA. Conus adversarius had nonplanktonic larval development; this may have been a critical factor for the early establishment of the species, as well as sinistral marine species in other clades. Notably, most specimens of aberrantly sinistral modern Conus are derived from typically dextral species that have nonplanktonic development. If C. adversarius was reproductively isolated from dextral conspecifics, then this species may provide an example of nearly instantaneous sympatric speciation in the fossil record. Furthermore, the common and widespread – while geologically short-lived – fossil shells of C. adversarius show large amounts of variability in form and this variation may be related, at least in part, to a pleiotropic effect associated with the reversed coiling direction of this species.     

Within-tree spatial variation in the leaf monoterpenes of Sequoia sempervirens

Location within a tree was analyzed as a source of variation in Sequoia sempervirens leaf monoterpenes. No differences were found for quantitative composition or total yield/dry wt among lower, middle and upper canopy positions. The awlshaped, spirally arranged leaves of vigorous upper shoots showed small quantitative compositional differences, but not differences in total yield. The intermediate leaf form of young sprouts had the most different monoterpene quantitative composition and about three times the total yield of the above two leaf forms. Analysis of a clonal ring of 17 adult trees resulted in coefficients of variation similar to those for samples collected from different canopy levels of the same shoot. Results revealed the sources and magnitudes of experimental error in comparative studies of this species' leaf monoterpenes, and did not support the concept that somatic mutation provides an important source of variation in a large, long-lived organism such as coast redwood.     

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.     

The dynamics of two-species allelopathic competition with optimal harvesting

This paper analyses a bionomic model of two competitive species in the presence of toxicity with different harvesting efforts. An interesting dynamics in the first quadrant is analysed and two saddle-node bifurcations are detected for different bifurcation parameters. It is noted that under certain parametric restrictions, the model has a unique positive equilibrium point that is globally asymptotically stable whenever it is locally stable. It is also noted that the model can have zero, one or two feasible equilibria appearing through saddle-node bifurcations. The non-existence of a limit cycle in the interior of the first quadrant is also discussed using the Poincare–Dulac criteria. The saddle-node bifurcations are studied using Sotomayor's theorem. Numerical simulations are carried out to validate the analytical findings. The conditions for the existence of bionomic equilibria are discussed and an optimal harvesting policy is derived using Pontryagin's maximum principle.     

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.