Importing statistical measures into Artemis enhances gene identification in the <Emphasis Type="Italic">Leishmania</Emphasis> genome project

Background

Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces.

Results

Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODON USAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence.

Conclusion

An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.

     

Reorientation of aquaporin-1 topology during maturation in the endoplasmic reticulum

The topology of most eukaryotic polytopic membrane proteins is established cotranslationally in the endoplasmic reticulum (ER) through a series of coordinated translocation and membrane integration events. For the human aquaporin water channel AQP1, however, the initial four-segment-spanning topology at the ER membrane differs from the mature six-segment-spanning topology at the plasma membrane. Here we use epitope-tagged AQP1 constructs to follow the transmembrane (TM) orientation of key internal peptide loops in Xenopus oocyte and cell-free systems. This analysis revealed that AQP1 maturation in the ER involves a novel topological reorientation of three internal TM segments and two peptide loops. After the synthesis of TMs 4-6, TM3 underwent a 180-degree rotation in which TM3 C-terminal flanking residues were translocated from their initial cytosolic location into the ER lumen and N-terminal flanking residues underwent retrograde translocation from the ER lumen to the cytosol. These events convert TM3 from a type I to a type II topology and reposition TM2 and TM4 into transmembrane conformations consistent with the predicted six-segment-spanning AQP1 topology. AQP1 topological reorientation was also associated with maturation from a protease-sensitive conformation to a protease-resistant structure with water channel function. These studies demonstrate that initial protein topology established via cotranslational translocation events in the ER is dynamic and may be modified by subsequent steps of folding and/or maturation.     

A synthetic strand of cardiac muscle: its passive electrical properties

The passive electrical properties of synthetic strands of cardiac muscle, grown in tissue culture, were studied using two intracellular microelectrodes: one to inject a rectangular pulse of current and the other to record the resultant displacement of membrane potential at various distances from the current source. In all preparations, the potential displacement, instead of approaching a steady value as would be expected for a cell with constant electrical properties, increased slowly with time throughout the current step. In such circumstances, the specific electrical constants for the membrane and cytoplasm must not be obtained by applying the usual methods, which are based on the analytical solution of the partial differential equation describing a one-dimensional cell with constant electrical properties. A satisfactory fit of the potential waveforms was, however, obtained with numerical solutions of a modified form of this equation in which the membrane resistance increased linearly with time. Best fits of the waveforms from 12 preparations gave the following values for the membrane resistance times unit length, membrane capacitance per unit length, and for the myoplasmic resistance: 1.22 plus or minus 0.13 x 10-5 omegacm, 0.224 plus or minus 0.023 uF with cm-minus 1, and 1.37 plus or minus 0.13 x 10-7 omegacm-minus 1, respectively. The value of membrane capacitance per unit length was close to that obtained from the time constant of the foot of the action potential and was in keeping with the generally satisfactory fit of the recorded waveforms with solutions of the cable equation in which the membrane impedance is that of a single capacitor and resistor in parallel. The area of membrane per unit length and the cross-sectional area of myoplasm at any given length of the preparation were determined from light and composite electron micrographs, and these were used to calculate the following values for the specific electrical membrane resistance, membrane capacitance, and the resistivity of the cytoplasm: 20.5 plus or minus 3.0 x 10-3 omegacm-2, l.54 plus or minus 0.24 uFWITHcm-minus 2, and 180 plus or minus 34 omegacm, respectively.     

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single- and a double-spanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8.     

Cystic Fibrosis Transmembrane Conductance Regulator–associated ATP Release Is Controlled by a Chloride Sensor

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is defective in cystic fibrosis, and has also been closely associated with ATP permeability in cells. Using a Xenopus oocyte cRNA expression system, we have evaluated the molecular mechanisms that control CFTR-modulated ATP release. CFTR-modulated ATP release was dependent on both cAMP activation and a gradient change in the extracellular chloride concentration. Activation of ATP release occurred within a narrow concentration range of external Cl⁻ that was similar to that reported in airway surface fluid. Mutagenesis of CFTR demonstrated that Cl⁻ conductance and ATP release regulatory properties could be dissociated to different regions of the CFTR protein. Despite the lack of a need for Cl⁻ conductance through CFTR to modulate ATP release, alterations in channel pore residues R347 and R334 caused changes in the relative ability of different halides to activate ATP efflux (wtCFTR, Cl >> Br; R347P, Cl >> Br; R347E, Br >> Cl; R334W, Cl = Br). We hypothesize that residues R347 and R334 may contribute a Cl⁻ binding site within the CFTR channel pore that is necessary for activation of ATP efflux in response to increases of extracellular Cl⁻. In summary, these findings suggest a novel chloride sensor mechanism by which CFTR is capable of responding to changes in the extracellular chloride concentration by modulating the activity of an unidentified ATP efflux pathway. This pathway may play an important role in maintaining fluid and electrolyte balance in the airway through purinergic regulation of epithelial cells. Insight into these molecular mechanisms enhances our understanding of pathogenesis in the cystic fibrosis lung.     

Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

The concept of typological variability of physiological individuality: I. Intrapopulation diversity of human habitual physical activity and its typological estimation

A concept of typological variability of physiological individuality is suggested. In order to substantiate it, typological characteristics of individual variation in habitual physical activity (HPA) have been analyzed, at the first stage of the research, in subjects of both sexes at different ontogenetic stages (the preschool, primary school, adolescent, adult, and elderly ages). With the results of longitudinal studies taken into consideration, it has been demonstrated that HPA is a stable individual character in each age group and, in general, remains so in the course of ontogeny, as a subject changes age groups. This has made it possible to distinguish three functional types (FTs): subjects with low, medium, and high HPAs (FT-1, FT-2, and FT-3). An integrated approach to HPA assessment (pedometer measurements, daily electrocardiographic monitoring, a physical activity diary, and calculation of the circadian index) has allowed standards of daily physical activity and some functional characteristics of the cardiovascular system to be established in subjects of different FTs and prospects for further research in the assessment of individual health and the physiologically normal state to be determined on the basis of the new concept.     

Surface structure of nerve tissue transplants growing in the anterior chamber of the rat eye (electron-microscopic study)

Two different types of the glial envelope are demonstrated around the septal cerebral area grafts (SCAG) and the hippocampal grafts (HG) developing in the anterior eye chamber of the rat during 3-4 months. In the SCAG it is multilayered, consists mainly of modified oligodendrocytes. Processes of the nervous cells perforate the surface of the envelope. In the HG the unilayered glial envelope consists of bodies and processes of the fibrous astrocytes; it is impenetrable for the nervous processes. Since both types of the cellular elements making these envelopes have been described in the norm, it is possible that the differences in the surfaces described are specific for these structures.     

Aggregatibacter actinomycetemcomitans Leukotoxin Utilizes a Cholesterol Recognition/Amino Acid Consensus Site for Membrane Association

Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β₂ integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC³³⁶ (³³³LEEYSKR³³⁹) is highly conserved among RTX toxins, whereas CRAC⁵⁰³ (⁵⁰¹VDYLK⁵⁰⁵) is unique to LtxA. A peptide corresponding to CRAC³³⁶ inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC³³⁶ and CRAC⁵⁰³ bind cholesterol, only CRAC³³⁶ competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC³³⁶ site was essential for LtxA cytotoxicity. The conservation of CRAC³³⁶ among RTX toxins suggests that this mechanism may be conserved among RTX toxins.     

Echinocampidae fam. nov., a new family of Late Jurassic-Early Cretaceous radiolarians of Arctic Siberia

When studying Late Jurassic-Early Cretaceous radiolarians from the Nordvik section (Arctic Siberia), unique morphotypes of multicyrtoid nassellarians with many horns in the apical part of the shell, which continue the rays A, V, 2l, D, and 2L of the cephalic spicule, were recorded. These morphotypes are assigned to a new family, Echinocampidae fam. nov., including three new genera (Echinocampe gen. nov., Nordvikella gen. nov., and Arctocapsula gen. nov.) and eight new species. The family Echinocampidae was probably restricted to high latitudes and belonged to the boreal fauna of the terminal Jurassic and the basal Cretaceous.