Comparative analysis of haplotype association mapping algorithms

Background  

Finding the genetic causes of quantitative traits is a complex and difficult task. Classical methods for mapping quantitative trail loci (QTL) in miceuse an F2 cross between two strains with substantially different phenotype and an interval mapping method to compute confidence intervals at each position in the genome. This process requires significant resources for breeding and genotyping, and the data generated are usually only applicable to one phenotype of interest. Recently, we reported the application of a haplotype association mapping method which utilizes dense genotyping data across a diverse panel of inbred mouse strains and a marker association algorithm that is independent of any specific phenotype. As the availability of genotyping data grows in size and density, analysis of these haplotype association mapping methods should be of increasing value to the statistical genetics community.     

Techniques for accurate protein identification in shotgun proteomic studies of human, mouse, bovine, and chicken lenses

Analysis of shotgun proteomics datasets requires techniques to distinguish correct peptide identifications from incorrect identifications, such as linear discriminant functions and target/decoy protein databases. We report an efficient, flexible proteomic analysis workflow pipeline that implements these techniques to control both peptide and protein false discovery rates. We demonstrate its performance by analyzing two-dimensional liquid chromatography separations of lens proteins from human, mouse, bovine, and chicken lenses. We compared the use of International Protein Index databases to UniProt databases and no-enzyme SEQUEST searches to tryptic searches. Sequences present in the International Protein Index databases allowed detection of several novel crystallins. An alternate start codon isoform of βA4 was found in human lens. The minor crystallin γN was detected for the first time in bovine and chicken lenses. Chicken γS was identified and is the first member of the γ-crystallin family observed in avian lenses.     

Sounds produced by spawning fishes

Synopsis Low frequency sounds are shown to be associated with the spawning of two Caribbean coral reef fishes: the hamlet, Hypoplectrus unicolor (Serranidae) and the striped parrotfish, Scarus iserti (Scaridae). Both fishes produce distinctive sounds while broadcasting gametes in midwater. H. unicolor produces sounds via muscle stimulation of the swimbladder. Fin movements among group spawning S. iserti produce hydrodynamic noise. Although reproductive behaviors of these two species have been previously studied in detail, the association of sounds with mating is new. The mating sounds cannot be easily detected by human hearing underwater but are recordable using a hydrophone. The sounds are distinct and recognizable enough to allow counting and acoustic mapping of mating events in these species.     

Identification and removal of proteins that co-purify with infectious prion protein improves the analysis of its secondary structure

Prion diseases are neurodegenerative disorders associated with the accumulation of an abnormal isoform of the mammalian prion protein (PrP). Fourier transform infrared spectroscopy (FTIR) has previously been used to show that the conformation of aggregated, infectious PrP (PrP(Sc) ) varies between prion strains and these unique conformations may determine strain-specific disease phenotypes. However, the relative amounts of α-helix, β-sheet and other secondary structures have not always been consistent between studies, suggesting that other proteins might be confounding the analysis of PrP(Sc) secondary structure. We have used FTIR and LC-MS/MS to analyze enriched PrP(Sc) from mouse and hamster prion strains both before and after the removal of protein contaminants that commonly co-purify with PrP(Sc) . Our data show that non-PrP proteins do contribute to absorbances that have been associated with α-helical, loop, turn and β-sheet structures attributed to PrP(Sc) . The major contaminant, the α-helical protein ferritin, absorbs strongly at 1652 cm(-1) in the FTIR spectrum associated with PrP(Sc) . However, even the removal of more than 99% of the ferritin from PrP(Sc) did not completely abolish absorbance at 1652 cm(-1) . Our results show that contaminating proteins alter the FTIR spectrum attributed to PrP(Sc) and suggest that the α-helical, loop/turn and β-sheet secondary structure that remains following their removal are derived from PrP(Sc) itself.     

Large-scale analysis of yeast filamentous growth by systematic gene disruption and overexpression

Under certain conditions of nutrient stress, the budding yeast Saccharomyces cerevisiae initiates a striking developmental transition to a filamentous form of growth, resembling developmental transitions required for virulence in closely related pathogenic fungi. In yeast, filamentous growth involves known mitogen-activated protein kinase and protein kinase A signaling modules, but the full scope of this extensive filamentous response has not been delineated. Accordingly, we have undertaken the first systematic gene disruption and overexpression analysis of yeast filamentous growth. Standard laboratory strains of yeast are nonfilamentous; thus, we constructed a unique set of reagents in the filamentous Σ1278b strain, encompassing 3627 integrated transposon insertion alleles and 2043 overexpression constructs. Collectively, we analyzed 4528 yeast genes with these reagents and identified 487 genes conferring mutant filamentous phenotypes upon transposon insertion and/or gene overexpression. Using a fluorescent protein reporter integrated at the MUC1 locus, we further assayed each filamentous growth mutant for aberrant protein levels of the key flocculence factor Muc1p. Our results indicate a variety of genes and pathways affecting filamentous growth. In total, this filamentous growth gene set represents a wealth of yeast biology, highlighting 84 genes of uncharacterized function and an underappreciated role for the mitochondrial retrograde signaling pathway as an inhibitor of filamentous growth.     

Insertion of a MalE β-Galactosidase Fusion Protein into the Envelope of Escherichia coli Disrupts Biogenesis of Outer Membrane Proteins and Processing of Inner Membrane Proteins

The synthesis of a membrane-bound MalE β-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.     

Hierarchical recruitment by AMPA but not staurosporine of pro-apoptotic mitochondrial signaling in cultured cortical neurons: evidence for caspase-dependent/independent cross-talk

Excitotoxicity mediated via the ( S )-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of receptor for l -glutamate contributes to various neuropathologies involving acute brain injury and chronic degenerative disorders. In this study, AMPA-induced neuronal injury and staurosporine (STS)-mediated apoptosis were compared in primary neuronal cultures of murine cerebral cortex by analyzing indices up- and downstream of mitochondrial activation. AMPA-mediated apoptosis involved induction of Bax, loss of mitochondrial transmembrane potential (ΔΨ_m), early release of cytochrome c (cyt c ), and more delayed release of second mitochondrial activator of caspases (SMAC), Omi, and apoptosis-inducing factor (AIF) with early calpain and minor late activation of caspase 3. STS-induced apoptosis was characterized by a number of differences, a more rapid time course, non-involvement of ΔΨ_m, and relatively early recruitment of SMAC and caspase 3. The AMPA-induced rise in intracellular calcium appeared insufficient to evoke ΔΨ_m as release of cyt c preceded mitochondrial depolarization, which was followed by the cytosolic translocation of SMAC, Omi, and AIF. Bax translocation preceded cyt c release for both stimuli inferring its involvement in apoptotic induction. Inclusion of the broad spectrum caspase inhibitor zVAD-fmk reduced the AMPA-induced release of cyt c , SMAC, and AIF, while only affecting the redistribution of Omi and AIF in the STS-treated neurons. Only AIF release was affected by a calpain inhibitor (calpastatin) which exerted relatively minor effects on the progression of cellular injury. AMPA-mediated release of apoptogenic proteins was more hierarchical relative to STS with its calpain activation and caspase-dependent AIF redistribution arguing for a model with cross-talk between caspase-dependent/independent apoptosis.     

Seasonal abundance of stable flies and filth fly pupal parasitoids (Hymenoptera: Pteromalidae) at Florida equine facilities

Beginning in November 2007 and continuing until December 2009, weekly stable fly, Stomoxys calcitrans (L.), surveillance was conducted at four equine facilities near Ocala, FL, by using alsynite sticky traps for adults and by searching immature developmental sites for pupae. Adult stable fly trap captures were highly variable throughout the year, ranging from 0 to 1,400 flies per trap per farm. The greatest adult stable fly activity was observed during the spring months of March and April, with weekly three-trap means of 121 and 136 flies per farm, respectively. The importance of cultural control measures was most apparent on the only farm with no reported insecticide use and the lowest stable fly trap captures, where an intense daily sanitation and composting program was conducted. A survey of on-site filth fly pupae revealed that 99.9% of all parasitoids recovered were Spalangia spp., consisting of Spalangia cameroni Perkins (56.5%), Spalangia nigroaenea Curtis (34.0%), Spalangia endius Walker (5.8%), and Spalangia nigra Latreille (3.7%). The implications of these findings are discussed.     

Water mass-specificity of bacterial communities in the North Atlantic revealed by massively parallel sequencing

Bacterial assemblages from subsurface (100 m depth), meso- (200-1000 m depth) and bathy-pelagic (below 1000 m depth) zones at 10 stations along a North Atlantic Ocean transect from 60°N to 5°S were characterized using massively parallel pyrotag sequencing of the V6 region of the 16S rRNA gene (V6 pyrotags). In a dataset of more than 830,000 pyrotags, we identified 10,780 OTUs of which 52% were singletons. The singletons accounted for less than 2% of the OTU abundance, whereas the 100 and 1000 most abundant OTUs represented 80% and 96% respectively of all recovered OTUs. Non-metric Multi-Dimensional Scaling and Canonical Correspondence Analysis of all the OTUs excluding the singletons revealed a clear clustering of the bacterial communities according to the water masses. More than 80% of the 1000 most abundant OTUs corresponded to Proteobacteria of which 55% were Alphaproteobacteria, mostly composed of the SAR11 cluster. Gammaproteobacteria increased with depth and included a relatively large number of OTUs belonging to Alteromonadales and Oceanospirillales. The bathypelagic zone showed higher taxonomic evenness than the overlying waters, albeit bacterial diversity was remarkably variable. Both abundant and low-abundance OTUs were responsible for the distinct bacterial communities characterizing the major deep-water masses. Taken together, our results reveal that deep-water masses act as bio-oceanographic islands for bacterioplankton leading to water mass-specific bacterial communities in the deep waters of the Atlantic.