Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.     

Restriction-map variation with the yellow-achaete-scute region in five populations of Drosophila melanogaster

It has been proposed that the degree of recombination for a genomic region will affect the level of both nucleotide heterozygosity and the density of transposable elements. Both features of genomic diversity have been examined in a number of recent reports for regions undergoing relatively normal levels of recombination in Drosophila melanogaster. In this study the genomic variation associated with yellow-achaete- scute loci located at the tip of the X chromosome is examined by six- cutter restriction mapping. In this region, as usual for regions adjacent to telomeres, crossing-over is dramatically reduced, and published studies of visible mutants indicate extremely little restriction-map variation. Eight six-cutter restriction endonucleases were used to locate sequence variation in 14- and 16.5-kb regions in 109 lines sampled from North America, Africa, and Europe. The overall level of heterozygosity is estimated as 0.29%. Nine large insertions, all presumed to be transposable elements, were observed. Base-pair heterozygosity appears to be reduced compared with regions having normal levels of recombination. The estimated heterozygosity is much higher than reported in earlier studies of restriction-map variation among visible mutations in the complex. The incidence of large insertions is not elevated compared with that in other regions of the genome. This suggests that asymmetric synapsis and exchange is not an important mechanism for the elimination of transposable elements.      

Characterisation of<Emphasis Type="Italic">fusarium graminearum</Emphasis> and<Emphasis Type="Italic">F. culmorum</Emphasis> isolates by mycotoxin production and aggressiveness to wheat

A total of 27Fusarium culmorum isolates from Germany and 41F. graminearum isolates from Kenya were investigated for aggressiveness and mycotoxin production on wheat ears. In addition, ergosterol content of the kernels from ears inoculated withF. graminearum was determined and theF. culmorum isolates were tested for mycotoxin productionin vitro. For both pathogens, isolates markedly differed in aggressiveness. 59% and 37% of theF. culmorum isolates produced NIV and DON, respectively,in vivo andin vitro. The DON-producing isolates also produced 3-acDONin vitro. The more aggressive isolates produced mainly DON while the less aggressive isolates produced mainly NIV. 12% and 85% of theF. graminearum isolates produced NIV and DON, respectively. The highly aggressive isolates produced higher amounts of DON, aggressiveness being highly correlated to DON content in the kernels. NIV-producing isolates were less aggressive. Ergosterol content of kernels was moderately correlated to aggressiveness but highly correlated to DON content. Disease severity was associated with kernel weight reduction.     

GeoChip 4: a functional gene‐array‐based high‐throughput environmental technology for microbial community analysis

Micro‐organisms play critical roles in many important biogeochemical processes in the Earth's biosphere. However, understanding and characterizing the functional capacity of microbial communities are still difficult due to the extremely diverse and often uncultivable nature of most micro‐organisms. In this study, we developed a new functional gene array, GeoChip 4, for analysing the functional diversity, composition, structure, metabolic potential/activity and dynamics of microbial communities. GeoChip 4 contained approximately 82 000 probes covering 141 995 coding sequences from 410 functional gene families related to microbial carbon (C), nitrogen (N), sulphur (S), and phosphorus (P) cycling, energy metabolism, antibiotic resistance, metal resistance/reduction, organic remediation, stress responses, bacteriophage and virulence. A total of 173 archaeal, 4138 bacterial, 404 eukaryotic and 252 viral strains were targeted, providing the ability to analyse targeted functional gene families of micro‐organisms included in all four domains. Experimental assessment using different amounts of DNA suggested that as little as 500 ng environmental DNA was required for good hybridization, and the signal intensities detected were well correlated with the DNA amount used. GeoChip 4 was then applied to study the effect of long‐term warming on soil microbial communities at a Central Oklahoma site, with results indicating that microbial communities respond to long‐term warming by enriching carbon degradation, nutrient cycling (nitrogen and phosphorous) and stress response gene families. To the best of our knowledge, GeoChip 4 is the most comprehensive functional gene array for microbial community analysis.     

Reconstruction of Ancestral Metabolic Enzymes Reveals Molecular Mechanisms Underlying Evolutionary Innovation through Gene Duplication

Gene duplications are believed to facilitate evolutionary innovation. However, the mechanisms shaping the fate of duplicated genes remain heavily debated because the molecular processes and evolutionary forces involved are difficult to reconstruct. Here, we study a large family of fungal glucosidase genes that underwent several duplication events. We reconstruct all key ancestral enzymes and show that the very first preduplication enzyme was primarily active on maltose-like substrates, with trace activity for isomaltose-like sugars. Structural analysis and activity measurements on resurrected and present-day enzymes suggest that both activities cannot be fully optimized in a single enzyme. However, gene duplications repeatedly spawned daughter genes in which mutations optimized either isomaltase or maltase activity. Interestingly, similar shifts in enzyme activity were reached multiple times via different evolutionary routes. Together, our results provide a detailed picture of the molecular mechanisms that drove divergence of these duplicated enzymes and show that whereas the classic models of dosage, sub-, and neofunctionalization are helpful to conceptualize the implications of gene duplication, the three mechanisms co-occur and intertwine.     

Nucleosomes affect local transformation efficiency

Genetic transformation is a natural process during which foreign DNA enters a cell and integrates into the genome. Apart from its relevance for horizontal gene transfer in nature, transformation is also the cornerstone of today''s recombinant gene technology. Despite its importance, relatively little is known about the factors that determine transformation efficiency. We hypothesize that differences in DNA accessibility associated with nucleosome positioning may affect local transformation efficiency. We investigated the landscape of transformation efficiency at various positions in the Saccharomyces cerevisiae genome and correlated these measurements with nucleosome positioning. We find that transformation efficiency shows a highly significant inverse correlation with relative nucleosome density. This correlation was lost when the nucleosome pattern, but not the underlying sequence was changed. Together, our results demonstrate a novel role for nucleosomes and also allow researchers to predict transformation efficiency of a target region and select spots in the genome that are likely to yield higher transformation efficiency.