Core Hunter: an algorithm for sampling genetic resources based on multiple genetic measures

Background  

Existing algorithms and methods for forming diverse core subsets currently address either allele representativeness (breeder's preference) or allele richness (taxonomist's preference). The main objective of this paper is to propose a powerful yet flexible algorithm capable of selecting core subsets that have high average genetic distance between accessions, or rich genetic diversity overall, or a combination of both.     

Hermansky-Pudlak Syndrome Protein Complexes Associate with Phosphatidylinositol 4-Kinase Type II �� in Neuronal and Non-neuronal Cells

The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting.Disease is triggered by defects in any of 15 mouse gene products, which arepart of five distinct cytosolic molecular complexes: AP-3, homotypic fusionand vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecularassociations of these complexes, we used in vivo cross-linkingfollowed by purification of cross-linked AP-3 complexes and mass spectrometricidentification of associated proteins. AP-3 was co-isolated with BLOC-1,BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits;clathrin; and phosphatidylinositol-4-kinase type II α (PI4KIIα).We previously reported that this membrane-anchored enzyme is a regulator ofAP-3 recruitment to membranes and a cargo of AP-3 (Craige, B.,Salazar, G., and Faundez, V. (2008) Mol. Biol.Cell19,1415-1426). Using cells deficientin different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1,but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIα inclusion intoAP-3 complexes. BLOC-1, PI4KIIα, and AP-3 belong to a tripartitecomplex, and down-regulation of either PI4KIIα, BLOC-1, or AP-3complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1complex modulates the association of PI4KIIα with AP-3. These resultssuggest that AP-3 and BLOC-1 act, either in concert or sequentially, tospecify sorting of PI4KIIα along the endocytic route.Membranous organelles along the exocytic and endocytic pathways are eachdefined by unique lipid and protein composition. Vesicle carriers communicateand maintain the composition of these organelles(2). Consequently defining themachineries that specify vesicle formation, composition, and delivery arecentral to understanding membrane protein traffic. Generally vesiclebiogenesis uses multiprotein cytosolic machineries to select membranecomponents for inclusion in nascent vesicles(2,3). Heterotetrameric adaptorcomplexes (AP-1 to AP-4) are critical to generate vesicles of specificcomposition from the different organelles constituting the exocytic andendocytic routes(2-4).The best understood vesicle formation machinery in mammalian cells is theone organized around the adaptor complex AP-2(5). This complex generatesvesicles from the plasma membrane using clathrin. Our present detailedunderstanding of AP-2 vesicle biogenesis mechanisms and interactions emergedfrom a combination of organellar and in vitro binding proteomicsanalyses together with the study of binary interactions in cell-free systems(5-9).In contrast, the vesicle biogenesis pathways controlled by AP-3 are far lessunderstood. AP-3 functions to produce vesicles that traffic selected membraneproteins from endosomes to lysosomes, lysosome-related organelles, or synapticvesicles(10-13).AP-3 is one of the protein complexes affected in the Hermansky-Pudlak syndrome(HPS;³ OnlineMendelian Inheritance in Man (OMIM) 203300). So far, mutations in any of 15mouse or eight human genes trigger a common syndrome. This syndromeencompasses defects that include pigment dilution, platelet dysfunction,pulmonary fibrosis, and occasionally neurological phenotypes(14,15). All forms of HPS showdefective vesicular biogenesis or trafficking that affects lysosomes,lysosome-related organelles (for example melanosomes and platelet densegranules), and, in some of them, synaptic vesicles(11-13).Most of the 15 HPS loci encode polypeptides that assemble into five distinctmolecular complexes: the adaptor complex AP-3, HOPS, and the BLOC complexes 1,2, and 3 (14). Recently binaryinteractions between AP-3 and BLOC-1 or BLOC-1 and BLOC-2 suggested thatarrangements of these complexes could regulate membrane protein targeting(16). Despite the abundance ofgenetic deficiencies leading to HPS and genetic evidence that HPS complexesmay act on the same pathway in defined cell types(17), we have only a partialpicture of protein interactions organizing these complexes and how they mightcontrol membrane protein targeting.In this study, we took advantage of cell-permeant and reversiblecross-linking of HPS complexes followed by their immunoaffinity purificationto identify novel molecular interactions. Cross-linked AP-3 co-purified withBLOC-1, BLOC-2, HOPS, clathrin, and the membrane protein PI4KIIα. Wepreviously identified PI4KIIα as a cargo and regulator of AP-3recruitment to endosomes (1,18). Using mutant cellsdeficient in either individual HPS complexes or a combination of them, wefound that BLOC-1 facilitates the interaction of AP-3 and PI4KIIα. Ourstudies demonstrate that subunits of four of the five HPS complexes co-isolatewith AP-3. Moreover BLOC-1, PI4KIIα, and AP-3 form a tripartite complexas demonstrated by sequential co-immunoprecipitations as well as by similarLAMP1 distribution phenotypes induced by down-regulation of components of thistripartite complex. Our findings indicate that BLOC-1 complex modulates therecognition of PI4KIIα by AP-3. These data suggest that AP-3, either inconcert or sequentially with BLOC-1, participates in the sorting of commonmembrane proteins along the endocytic route.     

A process for the production of ectoine and poly(3-hydroxybutyrate) by <Emphasis Type=Italic>Halomonas boliviensis</Emphasis>

The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L⁻¹) at 10–15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L⁻¹) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L⁻¹, respectively, were obtained at 5–7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed—the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH₄⁺, and PO₄³⁻. In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L⁻¹ h⁻¹ and ectoine concentration, content, and volumetric productivity of 4.3 g L⁻¹, 7.2 wt.%, and 2.8 g L⁻¹ day⁻¹, respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L⁻¹ day⁻¹.     

The cotton rat model of respiratory viral infections

Development of successful vaccines against human infectious diseases depends on using appropriate animal models for testing vaccine efficacy and safety. For some viral infections the task is further complicated by the frequently changing genetic make-up of the virus, as in the case of influenza, or by the existence of the little-understood phenomenon of vaccine-enhanced disease, as in the case of respiratory syncytial virus (RSV). The cotton rat Sigmodon hispidus has been used for years as an excellent small animal model of the RSV vaccine-enhanced disease. Recently, using cotton rats, we have demonstrated that vaccination against another paramyxovirus, human metapneumovirus (hMPV), can also lead to vaccine-enhanced disease. In addition to the study of paramyxoviruses, S. hispidus presents important advantages for the study of orthomyxoviruses such as influenza. The cotton rat is susceptible to infection with unadapted human influenza strains, and heterosubtypic immunity to influenza can be evoked in S. hispidus. The mechanisms of influenza, RSV, and hMPV pathogenesis and immunity can now be investigated in the cotton rat with the development of species-specific reagents for this animal model.     

Origins of domestication and polyploidy in oca (Oxalis tuberosa; Oxalidaceae). 3. AFLP data of oca and four wild, tuber-bearing taxa

Many crops are polyploids, and it can be challenging to untangle the often complicated history of their origins of domestication and origins of polyploidy. To complement other studies of the origins of polyploidy of the octoploid tuber crop oca (Oxalis tuberosa) that used DNA sequence data and phylogenetic methods, we here compared AFLP data for oca with four wild, tuber-bearing Oxalis taxa found in different regions of the central Andes. Results confirmed the divergence of two use-categories of cultivated oca that indigenous farmers use for different purposes, suggesting the possibility that they might have had separate origins of domestication. Despite previous results with nuclear-encoded, chloroplast-expressed glutamine synthetase suggesting that O. picchensis might be a progenitor of oca, AFLP data of this species, as well as different populations of wild, tuber-bearing Oxalis found in Lima Department, Peru, were relatively divergent from O. tuberosa. Results from all analytical methods suggested that the unnamed wild, tuber-bearing Oxalis found in Bolivia and O. chicligastensis in NW Argentina are the best candidates as the genome donors for polyploid O. tuberosa, but the results were somewhat equivocal about which of these two taxa is the more strongly supported as oca's progenitor.     

SYMBIODINIUM (DINOPHYTA) DIVERSITY AND STABILITY IN AQUARIUM CORALS1

Indo‐Pacific reef corals growing for years in closed‐system aquaria provide an alternate means to investigate host–symbiont specificity and stability. The diversity of dinoflagellate endosymbionts (Symbiodinium spp.) from coral communities in private and public aquaria was investigated using molecular‐genetic analyses. Of the 29 symbiont types (i.e., species) identified, 90% belonged to the most prevalent group of Symbiodinium harbored by Indo‐Pacific reef corals, Clade C, while the rest belonged to Clade D. Sixty‐five percent of all types were known from field surveys conducted throughout the Pacific and Indian oceans. Because specific coral–dinoflagellate partnerships appear to have defined geographic distributions, correspondence of the same symbionts in aquarium and field‐collected specimens identifies regions where particular colonies must have been collected in the wild. Symbiodinium spp. in clade D, believed to be “stress‐tolerant” and/or “opportunistic,” occurred in a limited number of individual colonies. The absence of a prevalent, or “weedy,” symbiont suggests that conditions under which aquarium corals are grown do not favor competitive replacements of their native symbiont populations. The finding of typical and diverse assemblages of Symbiodinium spp. among aquarium corals living many years under variable chemical/physical conditions, artificial and natural light, while undergoing fragmentation periodically, indicates that individual colonies maintain stable, long‐term symbiotic associations.     

Scarlet-Rz1, an EMS-generated hexaploid wheat with tolerance to the soilborne necrotrophic pathogens <Emphasis Type=Italic>Rhizoctonia solani</Emphasis> AG-8 and <Emphasis Type=Italic>R. oryzae</Emphasis>

The necrotrophic root pathogens Rhizoctonia solani AG-8 and R. oryzae cause Rhizoctonia root rot and damping-off, yield-limiting diseases that pose barriers to the adoption of conservation tillage in wheat production systems. Existing control practices are only partially effective, and natural genetic resistance to Rhizoctonia has not been identified in wheat or its close relatives. We report the first genetic resistance/tolerance to R. solani AG-8 and R. oryzae in wheat (Triticum aestivum L. em Thell) germplasm ‘Scarlet-Rz1’. Scarlet-Rz1 was derived from the allohexaploid spring wheat cultivar Scarlet using EMS mutagenesis. Tolerant seedlings displayed substantial root and shoot growth after 14 days in the presence of 100–400 propagules per gram soil of R. solani AG-8 and R. oryzae in greenhouse assays. BC₂F₄ individuals of Scarlet-Rz1 showed a high and consistent degree of tolerance. Seedling tolerance was transmissible and appeared to be dominant or co-dominant. Scarlet-Rz1 is a promising genetic resource for developing Rhizoctonia-tolerant wheat cultivars because the tolerance trait immediately can be deployed into wheat breeding germplasm through cross-hybridization, thereby avoiding difficulties with transfer from secondary or tertiary relatives as well as constraints associated with genetically modified plants. Our findings also demonstrate the utility of chemical mutagenesis for generating tolerance to necrotrophic pathogens in allohexaploid wheat. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. P. A. Okubara and C. M. Steber contributed equally to this work.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

Telomere length alterations in microsatellite stable colorectal cancer and association with the immune response

Telomeres are repetitive sequences (TTAGGG) located at the end of chromosomes. Telomeres progressively shorten with each cell replication cycle, ultimately leading to chromosomal instability and loss of cell viability. Telomere length anomaly appears to be one of the earliest and most prevalent genetic alterations in malignant transformation. Here we aim to estimate telomere length from whole-exome sequencing data in colon tumors and normal colonic mucosa, and to analyze the potential association of telomere length with clinical factors and gene expression in colon cancer.Reads containing at least five repetitions of the telomere sequence (TTAGGG) were extracted from the raw sequences of 42 adjacent normal-tumor paired samples. The number of reads from the tumor sample was normalized to build the Tumor Telomere Length Ratio (TTLR), considered an estimation of telomere length change in the tumor compared to the paired normal tissue. We evaluated the associations between TTLR and clinical factors, gene expression and copy number (CN) aberrations measured in the same tumor samples.Colon tumors showed significantly shorter telomeres than their paired normal samples. No significant association was observed between TTLR and gender, age, tumor location, prognosis, stromal infiltration or molecular subtypes. The functional gene set enrichment analysis showed pathways related to immune response significantly associated with TLLR.By extracting a relative measure of telomere length from whole-exome sequencing data, we have assessed that colon tumor cells predominantly shorten telomeres, and this alteration is associated with expression changes in genes related to immune response and inflammation in tumor cells.