Pandinus imperator scorpion venom blocks voltage-gated potassium channels in GH3 cells

We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.     

Cerebellar neurones: Differentiation and modulation of sensitivity to excitotoxic treatment

The neurite outgrowth and adhesion complex (NOAC), isolated from rabbit sera has been dissociated in its major components by reverse-phase chromatography in HPLC by using a C₁₈ column. SDS-PAGE analisys of the active fractions revealed the presence of three major bands of approximately 100, 70 and 50 kDa. Studies on the biological activity of NOAC were carried out on rat cerebellar granule cells. NOAC-cultured cells exhibit a marked resistance to excitotoxic stimuli carried by glutamate.     

Comparative evolutionary analysis of rDNA ITS regions in Drosophila

The internal transcribed spacer (ITS) of the ribosomal DNA is generally considered to be under low functional constraint, and it is therefore often treated as a typical nonfunctional spacer sequence. We have analyzed the ITS regions of five species from the Drosophila melanogaster subgroup, two Drosophila species from outside this group (D. pseudoobscura and D. virilis), as well as from the more distantly related dipteran fly Musca domestica. The sequence comparisons show a distinctive conservation/divergence pattern, indicating that some regions are more conserved than others. Moreover, secondary-structure calculations indicate several conserved structural elements within the ITS regions. On the other hand, a statistical test that allows us to estimate the fraction of sites that are not under selective constraint suggests that more than half of the spacer is apparently free to diverge and evolves with a rate that is close to the neutral rate of sequence evolution in Drosophila. The ITS sequences can be used to derive a molecular phylogeny for the species under study. We find that the ITS tree is largely in line with the so-far-known phylogeny of this group of species, with one difference. The species most distant within the D. melanogaster subgroup is D. yakuba, rather than D. orena, as is normally assumed.      

High nucleotide sequence variation in a region of low recombination in Drosophila simulans is consistent with the background selection model

We surveyed nucleotide sequence variation at glucose dehydrogenase (Gld), in a region of low recombination on chromosome 3R, from a population sample of Drosophila simulans. The levels of nucleotide variation were surprisingly high. There was no departure from the expectation of a neutral model for the level of polymorphism, indicating no evidence of a selective sweep in this region. There was a significant deficiency of singleton polymorphisms according to the Fu and Li test, although Tajima and Hudson, Kreitman, and Aguade (HKA) tests do not provide evidence of a significant elevation of variation due to balancing selection. Genetic map data for the D. simulans third chromosome were used to calculate expected values of pi for Gld under a current model of background selection, varying the values for the parameter sh (selection coefficient against deleterious mutations). We show that the recombinational landscape of D. simulans is sufficiently different from that of D. melanogaster that we expect higher variation under the background selection model, even when effective population sizes are assumed to be equal. The data for Gld were tested against the predictions using computer simulations of the distribution of the number of segregating sites conditioned on pi. Background selection alone can explain our observations as long as sh is larger than 0.005 and species-level effective population size is assumed to be several- fold larger than in D. melanogaster. Alternatively, the deleterious mutation rate may be smaller in D. simulans, or balancing selection may be acting nearby, thereby reducing the effect of background selection.      

Development of species diagnostic SNP markers for quality control genotyping in four rice (<Emphasis Type="Italic">Oryza</Emphasis> L.) species

Species misclassification (misidentification) and handling errors have been frequently reported in various plant species conserved at diverse gene banks, which could restrict use of germplasm for correct purpose. The objectives of the present study were to (i) determine the extent of genotyping error (reproducibility) on DArTseq-based single-nucleotide polymorphisms (SNPs); (ii) determine the proportion of misclassified accessions across 3134 samples representing three African rice species complex (Oryza glaberrima, O. barthii, and O. longistaminata) and an Asian rice (O. sativa), which are conserved at the AfricaRice gene bank; and (iii) develop species- and sub-species (ecotype)-specific diagnostic SNP markers for rapid and low-cost quality control (QC) analysis. Genotyping error estimated from 15 accessions, each replicated from 2 to 16 times, varied from 0.2 to 3.1%, with an overall average of 0.8%. Using a total of 3134 accessions genotyped with 31,739 SNPs, the proportion of misclassified samples was 3.1% (97 of the 3134 accessions). Excluding the 97 misclassified accessions, we identified a total of 332 diagnostic SNPs that clearly discriminated the three indigenous African species complex from Asian rice (156 SNPs), O. longistaminata accessions from both O. barthii and O. glaberrima (131 SNPs), and O. sativa spp. indica from O. sativa spp. japonica (45 SNPs). Using chromosomal position, minor allele frequency, and polymorphic information content as selection criteria, we recommended a subset of 24 to 36 of the 332 diagnostic SNPs for routine QC genotyping, which would be highly useful in determining the genetic identity of each species and correct human errors during routine gene bank operations.     

Mating pattern and pollen dispersal in the wild olive tree (<Emphasis Type="Italic">Olea europaea</Emphasis> subsp. <Emphasis Type="Italic">cuspidata</Emphasis>)

In this study, the mating system, contemporary pollen flow, and landscape pollen connectivity of the wild olive tree (Olea europaea subsp. cuspidata) were analyzed in a fragmented landscape of less than 4-km diameter located in north-western Ethiopia. Four remnant populations of different sizes were investigated. Eight highly polymorphic microsatellite markers were used to genotype 534 adults and 704 embryos. We used contrasting sampling schemes and different methodological approaches to analyze the pollen flow. We observed a lower rate of inbreeding and correlated mating in the fragmented vs. the non-fragmented subpopulation. Using parentage analysis, we detected a bidirectional pollen movement across subpopulations. Pollen flow was found to be directed towards small subpopulations based on parentage and anisotropic analysis. Pollen immigration amounted to more than 50%. Although most pollination occurred within a distance of less than 200 m, longer distance pollen movements of more than 3 km were also detected. Pollen dispersal in the large and dense subpopulation was reduced, and a smaller number of effective pollen sources were detected compared to a smaller fragmented subpopulation. We obtained consistent estimates for the number of effective pollen donors (approximately 6 per mother tree) using three different methods. The average pollen dispersal distance at the landscape level amounted to 276 m while at the local level, 174 m was estimated. Bigger trees were better pollen contributors than smaller trees. We showed here for the first time that pollen dispersal in wild olive follows a leptokurtic distribution.     

Molecular evolution of chloroplast DNA sequences

Comparative data on the evolution of chloroplast genes are reviewed. The chloroplast genome has maintained a similar structural organization over most plant taxa so far examined. Comparisons of nucleotide sequence divergence among chloroplast genes reveals marked similarity across the plant kingdom and beyond to the cyanobacteria (blue-green algae). Estimates of rates of nucleotide substitution indicate a synonymous rate of 1.1 x 10(-9) substitutions per site per year. Noncoding regions also appear to be constrained in their evolution, although addition/deletion events are common. There have also been evolutionary changes in the distribution of introns in chloroplast encoded genes. Relative to mammalian mitochondrial DNA, the chloroplast genome evolves at a conservative rate.