Locational distribution of gene functional classes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

We are interested in understanding the locational distribution of genes and their functions in genomes, as this distribution has both functional and evolutionary significance. Gene locational distribution is known to be affected by various evolutionary processes, with tandem duplication thought to be the main process producing clustering of homologous sequences. Recent research has found clustering of protein structural families in the human genome, even when genes identified as tandem duplicates have been removed from the data. However, this previous research was hindered as they were unable to analyse small sample sizes. This is a challenge for bioinformatics as more specific functional classes have fewer examples and conventional statistical analyses of these small data sets often produces unsatisfactory results.     

Microinjection of Intact 200- to 500-kb Fragments of YAC DNA into Mammalian Cells

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human β-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the β-globin gene. Three cell lines were analyzed by Rec A-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.     

Cryptic Species? Patterns of Maternal and Paternal Gene Flow in Eight Neotropical Bats

Levels of sequence divergence at mitochondrial loci are frequently used in phylogeographic analysis and species delimitation though single marker systems cannot assess bi-parental gene flow. In this investigation I compare the phylogeographic patterns revealed through the maternally inherited mitochondrial COI region and the paternally inherited 7(th) intron region of the Dby gene on the Y-chromosome in eight common Neotropical bat species. These species are diverse and include members of two families from the feeding guilds of sanguivores, nectarivores, frugivores, carnivores and insectivores. In each case, the currently recognized taxon is comprised of distinct, substantially divergent intraspecific mitochondrial lineages suggesting cryptic species complexes. In Chrotopterus auritus, and Saccopteryx bilineata I observed congruent patterns of divergence in both genetic regions suggesting a cessation of gene flow between intraspecific groups. This evidence supports the existence of cryptic species complexes which meet the criteria of the genetic species concept. In Glossophaga soricina two intraspecific groups with largely sympatric South American ranges show evidence for incomplete lineage sorting or frequent hybridization while a third group with a Central American distribution appears to diverge congruently at both loci suggesting speciation. Within Desmodus rotundus and Trachops cirrhosus the paternally inherited region was monomorphic and thus does not support or refute the potential for cryptic speciation. In Uroderma bilobatum, Micronycteris megalotis and Platyrrhinus helleri the gene regions show conflicting patterns of divergence and I cannot exclude ongoing gene flow between intraspecific groups. This analysis provides a comprehensive comparison across taxa and employs both maternally and paternally inherited gene regions to validate patterns of gene flow. I present evidence for previously unrecognized species meeting the criteria of the genetic species concept but demonstrate that estimates of mitochondrial diversity alone do not accurately represent gene flow in these species and that contact/hybrid zones must be explored to evaluate reproductive isolation.     

TaqMan Real-Time PCR Assays for Single-Nucleotide Polymorphisms Which Identify Francisella tularensis and Its Subspecies and Subpopulations

Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.     

Next-Generation Site-Directed Transgenesis in the Malaria Vector Mosquito Anopheles gambiae: Self-Docking Strains Expressing Germline-Specific phiC31 Integrase

Diseases transmitted by mosquitoes have a devastating impact on global health and the situation is complicated due to difficulties with both existing control measures and the impact of climate change. Genetically modified mosquitoes that are refractory to disease transmission are seen as having great potential in the delivery of novel control strategies. The Streptomyces phage phiC31 integrase system has been successfully adapted for site-directed transgene integration in a range of insects, thus overcoming many limitations due to size constraints and random integration associated with transposon-mediated transformation. Using this technology, we previously published the first site-directed transformation of Anopheles gambiae, the principal vector of human malaria. Mosquitoes were initially engineered to incorporate the phiC31 docking site at a defined genomic location. A second phase of genetic modification then achieved site-directed integration of an anti-malarial effector gene. In the current publication we report improved efficiency and utility of the phiC31 integrase system following the generation of Anopheles gambiae self-docking strains. Four independent strains, with docking sites at known locations on three different chromosome arms, were engineered to express integrase under control of the regulatory regions of the nanos gene from Anopheles gambiae. The resulting protein accumulates in the posterior oocyte to provide integrase activity at the site of germline development. Two self-docking strains, exhibiting significantly different levels of integrase expression, were assessed for site-directed transgene integration and found to demonstrate greatly improved survival and efficiency of transformation. In the fight against malaria, it is imperative to establish a broad repertoire of both anti-malarial effector genes and tissue-specific promoters to regulate their expression, enabling those offering maximum effect with minimum fitness cost to be identified. The improved technology we describe here will facilitate comparative studies of effector transgenes, allowing informed choices to be made that potentially lead to transmission blockade.     

Chk1 Inhibits E2F6 Repressor Function in Response to Replication Stress to Maintain Cell-Cycle Transcription

1. Download : Download high-res image (184KB)
2. Download : Download full-size image

     

Estimating total knee replacement joint load ratios from kinematics

Accurate prediction of loads acting at the joint in total knee replacement (TKR) patients is key to developing experimental or computational simulations which evaluate implant designs under physiological loading conditions. In vivo joint loads have been measured for a small number of telemetric TKR patients, but in order to assess device performance across the entire patient population, a larger patient cohort is necessary. This study investigates the accuracy of predicting joint loads from joint kinematics. Specifically, the objective of the study was to assess the accuracy of internal–external (I–E) and anterior–posterior (A–P) joint load predictions from I–E and A–P motions under a given compressive load, and to evaluate the repeatability of joint load ratios (I–E torque to compressive force (I–E:C), and A–P force to compressive force (A–P:C)) for a range of compressive loading profiles. A tibiofemoral finite element model was developed and used to simulate deep knee bend, chair-rise and step-up activities for five patients. Root-mean-square (RMS) differences in I–E:C and A–P:C load ratios between telemetric measurements and model predictions were less than 1.10e–3 Nm/N and 0.035 N/N for all activities. I–E:C and A–P:C load ratios were consistently reproduced regardless of the compressive force profile applied (RMS differences less than 0.53e–3 Nm/N and 0.010 N/N, respectively). When error in kinematic measurement was introduced to the model, joint load predictions were forgiving to kinematic measurement error when conformity between femoral and tibial components was low. The prevalence of kinematic data, in conjunction with the analysis presented here, facilitates determining the scope of A–P and I–E joint loading ratios experienced by the TKR population.     

A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states.