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.     

Sequence heterogeneity and anomalous electrophoretic mobility of kinetoplast minicircle DNA from Leishmania tarentolae

Several unit-length minicircles from the kinetoplast DNA of Leishmania tarentolae were cloned into pBR322 and into M13 phage vectors. The complete nucleotide sequences of three different partially homologous minicircles were obtained. The molecules contained a region of approx. 80% sequence homology extending for 160–270 bp and a region unique to each minicircle. A 14-mer was found to be conserved in all kinetoplast minicircle sequences reported to date. The frequency distributions of various minicircle sequence classes in L. tarentolae were obtained by quantitative gel electrophoresis and by examination of the “T ladder” patterns of minicircles randomly cloned into M13 at several sites. By these methods we could assign approx. 50% of the total minicircle DNA into a minimum of five sequence classes. A sequence-dependent polyacrylamide gel migration abnormality was observed with several minicircle fragments both cloned and uncloned. The abnormality was dependent on the presence of a portion of the conserved region of the minicircle.     

Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

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.     

Horizontal transfer of a mitochondrial plasmid

Direct evidence for horizontal transfer of a mitochodnrial plasmid from the discomyceteAscobolus immersus to the pyrenomycetePodospora anserina is presented. Southern blot hybridisation analysis, polymerase chain reaction (PCR) amplification, and DNA sequencing demonstrate transmission of a linear plasmid upon hyphal contact. DNA extraction from isolated organelles indicates a mitochondrial localisation for the plasmid inP. anserina. This is the first report of horizontal gene transfer among unrelated fungi. These results have important evolutionary implications for plasmid propagation in fungi.     

Plasmid-coded degradation of ethylbenzene and 1-phenylethanol in Pseudomonas fluorescens

Abstract Pseudomonas fluorescens EB carries genes for the catabolism of ethylbenzene and 1-phenylethanol on a plasmid. The size of the plasmid as measured by analysis of agarose electrophoresis gels after restriction endonuclease hydrolysis, was 253–267 kb. By treatment with Mitomycin C, mutants of EB strain were obtained bearing a plasmid which had undergone an extensive deletion of about 80 kb. These mutants have lost the ability to grow on ethylbenzene and 1-phenylethanol as well as to synthesize meta-cleavage enzymes.     

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.