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.

     

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.     

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.      

Kinetic study of the mechanisms of eliminating substrate inhibition of lactate dehydrogenase by anions and pH

The dependence of lactate dehydrogenase inhibition at high pyruvate concentrations on pH and neutral salt anions was studied. It was shown that Cl- anions compete with the substrate within the ternary inhibitory complex, ENADpyr in equilibrium ENADCl-, as a result of which the pyruvate-induced inhibition is eliminated. The KD values for Cl- (50 mM) and I- (27 mM) were calculated from the substrate velocity curves at high concentrations of pyruvate. It was supposed that pyruvate inhibition elimination by OH- proceeds via the same kinetic mechanism. The pK value (7.1 +/- 0.1) calculated from this model corresponds to pKn of essential His-195. The additivity of OH- and Cl- function was demonstrated.     

PROROCENTRUM BIMACULATUM SP. NOV. (DINOPHYCEAE,PROROCENTRALES), A NEW BENTHIC DINOFLAGELLATE SPECIES FROM KUWAIT (ARABIAN GULF)1

A new benthic dinoflagellate species, Prorocentrum bimaculatum sp. nov., is studied from Kuwait’s marine sediments, based on detailed morphological and molecular data. Cells are large, oblong oval in shape. They are 49.9–55.3 μm long and 38.4–43.2 μm wide. The ornamentation of this new species is peculiar, and characterized by smooth valves with large pores (0.32–0.50 μm) scattered on their surface, except in two circular patches of ～15 μm in diameter, devoid of ornamentation and located on both sides of the valve centers. The periflagellar area is widely triangular, located in a moderate excavation of the right valve, and comprises nine platelets. The intercalary band of P. bimaculatum is smooth. The molecular phylogenetic position of this new taxon was inferred from SSU and LSU rDNA genes. In both phylogenetic analyses, P. bimaculatum branched with high support with Prorocentrum consutum and formed a clade sister to the one including P. lima and related species such as P. arenarium, P. belizeanum, P. hoffmannianum, and P. maculosum. From the phylogenetic study, since most species related to P. bimaculatum are known for their toxic effects and production of okadaic acid, this new species can be considered as a potential toxin producer, but this has to be analyzed.     

Temperature stability of lactate dehydrogenase in complex with anionic polyelectrolyte poly(styrenesulfonate)

The temperature stability of the cytoplasmic enzyme of glycolysis, lactate dehydrogenase from pig muscle (isoenzyme M4) in complex with anionic polyelectrolyte poly(styrenesulfonate) has been investigated by the methods of adiabatic differential scanning microcalorimetry, own protein fluorescence, and circular dichroism. Calorimetric investigations of the complex of lactate dehydrogenase with poly(styrenesulfonate) in 50 mM phosphate buffer at pH 7.0 have shown that the temperature of the transition and enthalpy of lactate dehydrogenase thermal denaturation sharply decreases with growing weight ratio poly(styrenesulfonate)/lactate dehydrogenase, though at 20°C the enzyme activity of lactate dehydrogenase remains unchanged for several hours irrespective of the addition of poly(styrenesulfonate). The addition of phosphate ions to the solution enhances the resistance of lactate dehydrogenase to both thermal denaturation and inactivation by polyelectrolyte. The data obtained are interpreted from the viewpoint of a special role of two anion-binding centers in intersubunits contacts of lactate dehydrogenase, which enhance its resistance to both thermal denaturation and destruction by polyelectrolyte.     

Identification and characterization of a prevacuolar compartment in stigmas of nicotiana alata

The stigmas of the ornamental tobacco plant Nicotiana alata accumulate large quantities of a series of 6-kD proteinase inhibitors (PIs) in the central vacuole that are derived from a 40-kD precursor protein, Na-PI. The sorting information that directs Na-PI to the vacuole is likely to reside in a C-terminal propeptide domain of 25 amino acids that forms an amphipathic alpha helix. Using cell fractionation techniques, we have examined transit of Na-PI through the endomembrane system and have identified a prevacuolar compartment that contains Na-PI with an intact targeting signal. In contrast, the targeting signal is not present on the predominant form of Na-PI in the vacuole. The prevacuolar compartment is marked by the presence of homologs of both the t-SNARE, PEP12p, and the putative vacuolar sorting receptor BP-80. Cross-linking and affinity precipitation studies revealed that Na-PI associates with BP-80 within this compartment, providing in vivo evidence for the function of BP-80 as a sorting receptor for a protein with a C-terminal vacuolar targeting signal.     

Self-similar properties of the spatial structure of intertidal macro- and microbenthic communities

Spatial distribution (SD) of White Sea intertidal soft-bottom communities was studied at scales from decimetres to dozens of kilometres on the basis of an extensive dataset (464 samples of macrofauna, 349 samples of ciliates, and 333 samples of diatoms). We used the information index of structural heterogeneity D(I) (Azovsky et al., 2000 // Mar. Biol. 136 (3): 581-590) to characterize spatial variability in the species composition of the communities at different extent (total area surveyed) and grain (finest spatial resolution). The type of distribution was determined via the relation between D(I) and parameters of the spatial scale (extent and grain). At small scale (in terms of extent), all the communities were distributed randomly (mosaic SD). At larger scales, the estimated spatial variability depended neither on extent nor grain, exclusively on their ratio, i.e., was scale-invariant. This means that at some scale the spatial patterns of communities display self-similar properties (fractal SD). Such SD was found at a rather wide range of scales scales: 10(1)-10(4) m for the macrofauna, 10(0)-10(3) m for the ciliates, and 10(-1)-10(2) m for the diatoms. At still greater scales, patchy or gradient patters were observed. Thus, the ranges of fractal distribution were proportional to the average size of the organisms (approximately 10(4)-10(7) times the body size). We suppose that such spatial pattern reflects community self-organization in a relatively homogeneous environment and may be the most efficient way to realize the highest structural diversity on the basis of pre-formed complexes of predominant species. We also suppose that fractal-like patterns may be a general feature of the spatial organization of communities.     

The Structural and Functional Characteristics of Rat Soleus Endplates under Short-Term Disruption of Motor Activity

Biophysics - Abstract—In this study we analyzed the distribution of nicotinic acetylcholine receptors, the degree of their fragmentation, endplate area, and membrane localization of the...