Genome-enabled hitchhiking mapping identifies QTLs for stress resistance in natural Drosophila

Identification of genes underlying complex traits is an important problem. Quantitative trait loci (QTL) are mapped using marker-trait co-segregation in large panels of recombinant genotypes. Most frequently, recombinant inbred lines derived from two isogenic parents are used. Segregation patterns are also studied in pedigrees from multiple families. Great advances have been made through creative use of these techniques, but narrow sampling and inadequate power represent strong limitations. Here, we propose an approach combining the strengths of both techniques. We established a mapping population from a sample of natural genotypes, and applied artificial selection for a complex character. Selection changed the frequencies of alleles in QTLs contributing to the selection response. We infer QTLs with dense genotyping microarrays by identifying blocks of linked markers undergoing selective changes in allele frequency. We demonstrated this approach with an experimental population composed from 20 isogenic strains. Selection for starvation survival was executed in three replicated populations with three control non-selected populations. Three individuals per population were genotyped using Affymetrix GeneChips. Two regions of the genome, one each on the left arms of the second and third chromosomes, showed significant divergence between control and selected populations. For the former region, we inferred allele frequencies in selected and control populations by pyrosequencing. We conclude that the allele frequency difference, averaging approximately 40% between selected and control lines, contributed to selection response. Our approach can contribute to the fine scale decomposition of the genetics of direct and indirect selection responses, and genotype by environment interactions.     

Characterization of multiple myeloma vesicles by label‐free relative quantitation

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. The role that extracellular vesicles (EVs), microvesicles and exosomes, released by MM cells have in cell‐to‐cell communication and signaling in the bone marrow is currently unknown. This paper describes the proteomic content of EVs derived from MM.1S and U266 MM cell lines. First, we compared the protein identifications between the vesicles and cellular lysates of each cell line finding a large overlap in protein identifications. Next, we applied label‐free spectral count quantitation to determine proteins with differential abundance between the groups. Finally, we used bioinformatics to categorize proteins with significantly different abundances into functional groups. The results illustrate the first use of label‐free spectral counting applied to determine relative protein abundances in EVs.     

Substantial deletion overlap among divergent <Emphasis Type=Italic>Arabidopsis</Emphasis> genomes revealed by intersection of short reads and tiling arrays

Identification of small polymorphisms from next generation sequencing short read data is relatively easy, but detection of larger deletions is less straightforward. Here, we analyzed four divergent Arabidopsis accessions and found that intersection of absent short read coverage with weak tiling array hybridization signal reliably flags deletions. Interestingly, individual deletions were frequently observed in two or more of the accessions examined, suggesting that variation in gene content partly reflects a common history of deletion events.     

Quantitative Trait Loci for Lipid Content in Drosophila melanogaster

Recombinant inbred lines derived from a natural population were used to investigate natural genetic variation for lipid abundance, protein abundance, and weight of Drosophila melanogaster. Females were heavier and contained more lipid and soluble protein than males. Lipid and protein abundance were genetically correlated with female weight, but male weight was not correlated with lipid or protein. Lipid and protein abundance were genetically correlated in males, but not in females. Quantitative trait loci (QTLs) for weight and protein abundance were predominantly on the X chromosome, whereas QTLs for lipid abundance were found on the second and third chromosomes. QTLs for lipid proportion (lipid abundance normalized by weight or protein abundance) were present on all chromosomes; a lipid proportion QTL on the third chromosome correlated with a QTL for starvation resistance observed in a previous study using the same set of recombinant inbred lines, suggesting that it might underlie both traits. Candidate genes are discussed in relationship to lipid abundance, lipid proportion, and starvation resistance.     

Oocyte maturation and ovariole number in lines of Drosophila melanogaster selected for postponed senescence

1. Postponed senescence lines of Drosophila melanogaster have previously been generated by selection for delayed female reproduction.
2. This study addresses the question of whether the selection regime has differentiated lines with respect to oocyte maturation or ovariole number.
3. Oocyte stage and ovariole number were characterized in replicate postponed senescence lines (O) and in replicate control lines (B).
4. Oocyte maturation is delayed in O females.
5. The delay in oocyte maturation in O lines corresponds to reduced early age fecundity.
6. Selection may have resulted in an increased number of ovarioles in O females.     

EVOLUTION OF STARVATION RESISTANCE IN DROSOPHILA MELANOGASTER: ASPECTS OF METABOLISM AND COUNTER-IMPACT SELECTION

An artificial selection experiment for increased female starvation resistance employed five selected lines and five control lines of Drosophila melanogaster. Females responded to selection within the first five generations, but a substantial male response was not observed until starvation resistance was assessed at generation 15. By measuring respiration rate in selected and control lines, it was possible to test the hypothesis that reduced metabolic rate is a general mechanism for stress resistance. There was no association between starvation resistance and respiration rate and thus no support for the hypothesis. Studies using vertebrates have shown that starvation causes a decrease in intermediary metabolism enzyme activity, but this relationship is not well documented in invertebrates. In the present study, intermediary metabolism enzyme activities decreased in response to starvation in control-line females and males, and in selected-line males. However, the selected females showed no overall decrease in enzyme activities in response to starvation. One interpretation is that selected females evolved to resist the phenotypic impact of stress. The concept of “counter-impact selection” is discussed in relationship to the use of phenotypic manipulations for the study of evolution.     

Staphylococcal alpha-toxin: a study of membrane penetration and pore formation

Cell lysis by staphylococcal alpha-toxin, a potent virulence factor of most pathogenic strains of Staphylococcus aureus, follows a three-step sequence: binding of toxin to the membrane, leaking of ions caused by membrane injury, and rupturing of the membrane caused by osmotic swelling. The membrane injury step is composed of two separate events, membrane penetration and membrane perturbation. The membrane penetration event involves conversion of the soluble toxin monomer into an amphipathic molecule, which inserts into the lipid bilayer of the membrane. The membrane perturbation event involves association of the toxin monomers, in the plane of the membrane, to form hexameric transmembrane pores. In this study, we demonstrate that, in an asolectin liposome system, controlling the pH of the external buffer permits these two events to be temporally resolved. Using Controlled-Pore Glass bead-purified alpha-toxin, four events are measured as a function of pH: (a) release of potassium from prelabeled asolectin vesicles, (b) conversion of the toxin to a globally hydrophobic molecule, (c) binding of detergent by the toxin, and (d) labeling of the toxin with photoactivable, radiolabeled, hydrophobic probes. Two of these events, potassium release and conversion to a net hydrophobic state, are paired in that, for the event to occur, each requires a pH of 4.6 or less. In contrast, photolabeling with the membrane probes PC I and PC II (where PC represents phosphatidylcholine) is easily detectable at pH values as high as 5.0 and 6.0. These results demonstrate that, as the pH is lowered, two distinct changes in the physical properties of alpha-toxin occur. The first, which occurs under mild acidic conditions, converts the toxin from a water-soluble molecule into an amphipathic molecule. The second, requiring relatively more acidic conditions, converts the amphipathic toxin molecule into a globally hydrophobic molecule. Correlated with these physical changes in the alpha-toxin molecule is the acquisition of two new biological properties. The conversion of alpha-toxin into an amphipathic conformation correlates with the acquisition of the biological property of the reversible penetration into the bilayer of the asolectin liposome membrane, as evidenced by labeling with the photoactivable probes. At lower pH, the conversion of the toxin into a globally hydrophobic molecule correlates with the biological property of causing damage to the cell membrane, as measured by the release of internal potassium ions, presumably by the formation of transmembrane hexamer pores.(ABSTRACT TRUNCATED AT 400 WORDS)