Repeated adaptive introgression at a gene under multiallelic balancing selection

Recently diverged species typically have incomplete reproductive barriers, allowing introgression of genetic material from one species into the genomic background of the other. The role of natural selection in preventing or promoting introgression remains contentious. Because of genomic co-adaptation, some chromosomal fragments are expected to be selected against in the new background and resist introgression. In contrast, natural selection should favor introgression for alleles at genes evolving under multi-allelic balancing selection, such as the MHC in vertebrates, disease resistance, or self-incompatibility genes in plants. Here, we test the prediction that negative, frequency-dependent selection on alleles at the multi-allelic gene controlling pistil self-incompatibility specificity in two closely related species, Arabidopsis halleri and A. lyrata, caused introgression at this locus at a higher rate than the genomic background. Polymorphism at this gene is largely shared, and we have identified 18 pairs of S-alleles that are only slightly divergent between the two species. For these pairs of S-alleles, divergence at four-fold degenerate sites (K = 0.0193) is about four times lower than the genomic background (K = 0.0743). We demonstrate that this difference cannot be explained by differences in effective population size between the two types of loci. Rather, our data are most consistent with a five-fold increase of introgression rates for S-alleles as compared to the genomic background, making this study the first documented example of adaptive introgression facilitated by balancing selection. We suggest that this process plays an important role in the maintenance of high allelic diversity and divergence at the S-locus in flowering plant families. Because genes under balancing selection are expected to be among the last to stop introgressing, their comparison in closely related species provides a lower-bound estimate of the time since the species stopped forming fertile hybrids, thereby complementing the average portrait of divergence between species provided by genomic data.     

Doc of prophage P1 is inhibited by its antitoxin partner Phd through fold complementation

Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation. This Phd domain is intrinsically disordered in solution and folds into an alpha-helix upon binding to Doc. The details of the interactions reveal the molecular basis for the inhibitory action of the antitoxin. The complex resembles the Fic (filamentation induced by cAMP) proteins and suggests a possible evolutionary origin for the phd/doc operon. Doc induces growth arrest of Escherichia coli cells in a reversible manner, by targeting the protein synthesis machinery. Moreover, Doc activates the endogenous E. coli RelE mRNA interferase but does not require this or any other known chromosomal toxin-antitoxin locus for its action in vivo.     

Translation affects YoeB and MazF messenger RNA interferase activities by different mechanisms

Prokaryotic toxin–antitoxin loci encode mRNA cleaving enzymes that inhibit translation. Two types are known: those that cleave mRNA codons at the ribosomal A site and those that cleave any RNA site specifically. RelE of Escherichia coli cleaves mRNA at the ribosomal A site in vivo and in vitro but does not cleave pure RNA in vitro. RelE exhibits an incomplete RNase fold that may explain why RelE requires its substrate mRNA to presented by the ribosome. In contrast, RelE homologue YoeB has a complete RNase fold and cleaves RNA independently of ribosomes in vitro. Here, we show that YoeB cleavage of mRNA is strictly dependent on translation of the mRNA in vivo. Non-translated model mRNAs were not cleaved whereas the corresponding wild-type mRNAs were cleaved efficiently. Model mRNAs carrying frameshift mutations exhibited a YoeB-mediated cleavage pattern consistent with the reading frameshift thus giving strong evidence that YoeB cleavage specificity was determined by the translational reading frame. In contrast, site-specific mRNA cleavage by MazF occurred independently of translation. In one case, translation seriously influenced MazF cleavage efficiency, thus solving a previous apparent paradox. We propose that translation enhances MazF-mediated cleavage of mRNA by destabilization of the mRNA secondary structure.     

Superparamagnetic adsorbents for high-gradient magnetic fishing of lectins out of legume extracts

This work presents the development, testing, and application in high-gradient magnetic fishing of superparamagnetic supports for adsorption of lectins. Various approaches were examined to produce affinity, mixed mode, and hydrophobic charge induction type adsorbents. In clean monocomponent systems affinity supports created by direct attachment of glucose or maltose to amine-terminated iron oxide particles could bind concanavalin A at levels of up to approximately 280 mg g(-1) support with high affinity ( approximately 1 microM dissociation constants). However, the best performance was delivered by adsorbents featuring coupled tentacular dextran chains displaying a maximum binding capacity of 238 mg g(-1) and a dissociation constant of 0.13 microM. Adsorbents derivatized with mixed mode or hydrophobic charge induction ligands likewise demonstrated very high capacities for both concanavalin A and Lens culinaris agglutinin (> or = 250 mg g(-1)) with dissociation constants in the micromolar range, though neither of these systems showed any selectivity for lectins in leguminous extracts. When the affinity supports were applied to carbohydrate containing legume extracts only the dextran-linked adsorbents supplied sufficient competition to dissolved sugars to selectively bind concanavalin A in an extract of jack beans. The dextran-linked supports were employed in a high-gradient magnetic fishing experiment, in which concanavalin A was purified to near homogeneity from a crude, unclarified extract of jack beans.     

Evidence of recombination among early-vaccination era measles virus strains

Background  

The advent of live-attenuated vaccines against measles virus during the 1960'ies changed the circulation dynamics of the virus. Earlier the virus was indigenous to countries worldwide, but now it is mediated by a limited number of evolutionary lineages causing sporadic outbreaks/epidemics of measles or circulating in geographically restricted endemic areas of Africa, Asia and Europe. We expect that the evolutionary dynamics of measles virus has changed from a situation where a variety of genomic variants co-circulates in an epidemic with relatively high probabilities of co-infection of the individual to a situation where a co-infection with strains from evolutionary different lineages is unlikely.     

Defensin-rich granules of human neutrophils: characterization of secretory properties

The various granule subtypes of the human neutrophil differ in propensity for exocytosis. As a rule, granules formed at late stages of myelopoiesis have a higher secretory potential than granules formed in more immature myeloid cells. Neutrophils contain four closely related alpha-defensins, which are stored in a subset of azurophil granules. These defensin-rich azurophil granules (DRG) are formed later than defensin-poor azurophil granules, near the promyelocyte/myelocyte transition. In order to characterize the secretory properties of DRG, we developed a sensitive and accurate ELISA for detection of the neutrophil alpha-defensins HNP 1-3. This allowed us to quantify the exocytosis of alpha-defensins and markers of azurophil (myeloperoxidase), specific (lactoferrin) and gelatinase (gelatinase) granules from neutrophils stimulated with different secretagogues. The release pattern of alpha-defensins correlated perfectly with the release of myeloperoxidase and showed no resemblance to the exocytosis of lactoferrin or gelatinase. This finding was substantiated through subcellular fractionation experiments. In conclusion, despite a distinct profile of biosynthesis, DRG are indistinguishable from defensin-poor azurophil granules with respect to exocytosis. Thus, in contrast to peroxidase-negative granules, azurophil granules display homogeneity in their availability for extracellular release.     

Development and characterization of a reconstituted yeast translation initiation system

To provide a bridge between in vivo and in vitro studies of eukaryotic translation initiation, we have developed a reconstituted translation initiation system using components from the yeast Saccharomyces cerevisiae. We have purified a minimal set of initiation factors (elFs) that, together with yeast 80S ribosomes, GTP, and initiator methionyl-tRNA, are sufficient to assemble active initiation complexes on a minimal mRNA template. The kinetics of various steps in the pathway of initiation complex assembly and the formation of the first peptide bond in vitro have been explored. The formation of active initiation complexes in this system is dependent on ribosomes, mRNA, Met-tRNAi, GTP hydrolysis, elF1, elF1A, elF2, elF5, and elF5B. Our data indicate that elF1 and elF1A both facilitate the binding of the elF2 x GTP x Met-tRNAi complex to the 40S ribosomal subunit to form the 43S complex. elF5 stimulates a step after 43S complex formation, consistent with its proposed role in activating GTP hydrolysis by elF2 upon initiation codon recognition. The presence of elF5B is required for the joining of the 40S and 60S subunits to form the 80S initiation complex. The step at which each of these factors acts in this reconstituted system is in agreement with previous data from in vivo studies and work using reconstituted mammalian systems, indicating that the system recapitulates fundamental events in translation initiation in eukaryotic cells. This system should allow us to couple powerful yeast genetic and molecular biological experiments with in vitro kinetic and biophysical experiments, yielding a better understanding of the molecular mechanics of this central, complex process.     

Annexin-V binds to the intracellular part of the beta(5) integrin receptor subunit

Bovine lactadherin binds to the alpha(v)beta(3) and alpha(v)beta(5) integrins in an RGD-dependent manner and also to anionic phospholipids. During the affinity purification of lactadherin binding receptors, a 35-kDa protein persistently coeluted with the alpha(v)beta(5) integrin receptor. Subsequently, peptide mapping, amino acid sequencing, and mass spectrometry analysis identified this protein as bovine annexin-V. Annexin-V accompanied the integrin receptor eluted with either RGD peptide or with EDTA suggesting that annexin-V bound specifically to the alpha(v)beta(5) integrin. To further investigate this putative interaction of annexin-V with the alpha(v)beta(5) integrin receptor, human annexin-V and intracellular domains of the human alpha(v)beta(5) integrin subunits were used in ligand blotting assays. Radiolabeled annexin-V showed weak binding to the intracellular part of beta(5) integrin subunit. However, by adding the aminophospholipid, phosphatidyl serine, the interaction with the beta(5) cytoplasmic peptide was enhanced many fold. Furthermore, the interaction was shown to be independent of phosphorylation, as annexin-V bound to unphosphorylated beta(5) peptide at a similar level to the phosphorylated peptide. Since binding of annexin-V to the alpha(v) integrin subunit tail was not detected, annexin-V was shown to associate specifically with the beta(5) cytoplasmic tail. Together these findings suggest a novel link between annexins and the integrin receptor family.     

A combination of two antibodies recognizing non‐overlapping epitopes of HER2 induces kinase activity‐dependent internalization of HER2

The human epidermal growth factor receptor 2 (HER2/ErbB2) is overexpressed in a number of human cancers. HER2 is the preferred heterodimerization partner for other epidermal growth factor receptor (EGFR) family members and is considered to be resistant to endocytic down‐regulation, properties which both contribute to the high oncogenic potential of HER2. Antibodies targeting members of the EGFR family are powerful tools in cancer treatment and can function by blocking ligand binding, preventing receptor dimerization, inhibiting receptor activation and/or inducing receptor internalization and degradation. With respect to antibody‐induced endocytosis of HER2, various results are reported, and the effect seems to depend on the HER2 expression level and whether antibodies are given as individual antibodies or as mixtures of two or more. In this study, the effect of a mixture of two monoclonal antibodies against non‐overlapping epitopes of HER2 was investigated with respect to localization and stability of HER2. Individual antibodies had limited effect, but the combination of antibodies induced internalization and degradation of HER2 by multiple endocytic pathways. In addition, HER2 was phosphorylated and ubiquitinated upon incubation with the antibody combination, and the HER2 kinase activity was found to be instrumental in antibody‐induced HER2 down‐regulation.