Biologists require genetic as well as molecular tools to decipher genomic information and ultimately to understand gene function. The Berkeley Drosophila Genome Project is addressing these needs with a massive gene disruption project that uses individual, genetically engineered P transposable elements to target open reading frames throughout the Drosophila genome. DNA flanking the insertions is sequenced, thereby placing an extensive series of genetic markers on the physical genomic map and associating insertions with specific open reading frames and genes. Insertions from the collection now lie within or near most Drosophila genes, greatly reducing the time required to identify new mutations and analyze gene functions. Information revealed from these studies about P element site specificity is being used to target the remaining open reading frames.

The driving force behind cell motility is the actin cytoskeleton. Filopodia and lamellipodia are formed by the polymerization and extension of actin filaments towards the cell membrane. This polymerization at the barbed end of the filament is balanced by depolymerization at the pointed end, recycling the actin in a 'treadmilling' process. One protein involved in this process is cofilin/actin-depolymerizing factor (ADF), which can depolymerize actin filaments, allowing treadmilling to occur at an accelerated rate. Cofilin/ADF is an actin-binding protein that is required for actin-filament disassembly, cytokinesis and the organization of muscle actin filaments. There is also evidence that cofilin/ADF enhances cell motility, although a direct requirement in vivo has not yet been shown. Here we show that Drosophila cofilin/ADF, which is encoded by the twinstar (tsr) gene, promotes cell movements during ovary development and oogenesis. During larval development, cofilin/ADF is required for the cell rearrangement needed for formation of terminal filaments, stacks of somatic cells that are important for the initiation of ovarioles. It is also required for the migration of border cells during oogenesis. These results show that cofilin/ADF is an important regulator of actin-based cell motility during Drosophila development.

Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil–DNA glycosylase and dUTPase. Lack of the major uracil–DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200–2,000 uracil/million bases, quantified using a novel real-time PCR–based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil–DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil–DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil–DNA in this evolutionary clade.

Background / AimsClO2, the so-called “ideal biocide”, could also be applied as an antiseptic if it was understood why the solution killing microbes rapidly does not cause any harm to humans or to animals. Our aim was to find the source of that selectivity by studying its reaction-diffusion mechanism both theoretically and experimentally.MethodsClO2 permeation measurements through protein membranes were performed and the time delay of ClO2 transport due to reaction and diffusion was determined. To calculate ClO2 penetration depths and estimate bacterial killing times, approximate solutions of the reaction-diffusion equation were derived. In these calculations evaporation rates of ClO2 were also measured and taken into account.ResultsThe rate law of the reaction-diffusion model predicts that the killing time is proportional to the square of the characteristic size (e.g. diameter) of a body, thus, small ones will be killed extremely fast. For example, the killing time for a bacterium is on the order of milliseconds in a 300 ppm ClO2 solution. Thus, a few minutes of contact time (limited by the volatility of ClO2) is quite enough to kill all bacteria, but short enough to keep ClO2 penetration into the living tissues of a greater organism safely below 0.1 mm, minimizing cytotoxic effects when applying it as an antiseptic. Additional properties of ClO2, advantageous for an antiseptic, are also discussed. Most importantly, that bacteria are not able to develop resistance against ClO2 as it reacts with biological thiols which play a vital role in all living organisms.ConclusionSelectivity of ClO2 between humans and bacteria is based not on their different biochemistry, but on their different size. We hope initiating clinical applications of this promising local antiseptic.

The regulatory complex of the 26S proteasome is responsible for the selective recognition and binding of multiubiquitinated proteins. It was earlier shown that the subunit S5a/Rpn10/p54 of the regulatory complex is the only cellular protein capable of binding multiubiquitin chains in an in vitro overlay assay. The role of this subunit in substrate selection, however, is a subject of debate, following the observation that its deletion in Saccharomyces cerevisiae is not lethal and instead causes only a mild phenotype. To study the function of this subunit in higher eukaryotes, a mutant Drosophila strain was constructed by deleting the single copy gene encoding subunit S5a/Rpn10/p54. This deletion caused larval-pupal polyphasic lethality, multiple mitotic defects, the accumulation of higher multimers of ubiquitinated proteins and a huge accumulation of defective 26S proteasome particles. Deletion of the subunit S5a/Rpn10/p54 does not destabilise the regulatory complex and does not disturb the assembly of the regulatory complex and the catalytic core. The pupal lethality is a consequence of the depletion of the maternally provided 26S proteasome during the larval stages and a sudden increase in the proteasomal activity demands during the first few hours of pupal development. The huge accumulation of the fully assembled 26S proteasome in the deletion mutant and the lack of free subunits or partially assembled particles indicate that there is a highly coordinated accumulation of all the subunits of the 26S proteasome. This suggests that in higher eukaryotes, as with yeast, a feedback circuit coordinately regulates the expression of the proteasomal genes, and this adjusts the actual proteasome concentration in the cells according to the temporal and/or spatial proteolytic demands.

Porcine sapovirus is an enteric calicivirus in domestic pigs that belongs to the family Caliciviridae. Some porcine sapoviruses are genetically related to human caliciviruses, which has raised public health concerns over animal reservoirs and the potential cross-species transmission of sapoviruses. We report on the incidence, genetic diversity, and molecular epidemiology of sapoviruses detected in domestic pigs in a comprehensive study conducted in six European countries (Denmark, Finland, Hungary, Italy, Slovenia, and Spain) between 2004 and 2007. A total of 1,050 swine fecal samples from 88 pig farms were collected and tested by reverse transcription-PCR for sapoviruses, and positive findings were confirmed by sequencing. Sapoviruses were detected in 80 (7.6%) samples collected on 39 (44.3%) farms and in every country. The highest prevalence was seen among piglets aged 2 to 8 weeks, and there was no significant difference in the proportion of sapoviruspositive findings for healthy animals and animals with diarrhea in Spain and Denmark (the only countries where both healthy animals and animals with diarrhea were tested). On the basis of the sequence of the RNA polymerase region, highly heterogeneous populations of viruses representing six different genogroups (genogroups III, VI, VII, and VIII, including potential new genogroups IX and X) were identified, with a predominance of genogroup GIII (50.6%). Genogroup VIII, found in five of the six countries, had the highest degree of homology (up to 66% at the amino acid level) to human sapovirus strains. Sapoviruses are commonly circulating and endemic agents in swine herds throughout Europe. Highly heterogeneous and potential new genogroups of sapoviruses were found in pigs; however, no "human-like" sapoviruses were detected.

Abstract. The tumor suppressor gene overgrown hematopoietic organs-31 (oho31) of Drosophila encodes a protein with extensive homology to the Importin protein of Xenopus (50% identity), the related yeast SRP1 protein, and the mammalian hSRP1 and RCH1 proteins. A strong reduction in the expression of oho31 by a P element inserted in the 5' untranslated region of the oho31 transcript or a complete inactivation of oho31 by imprecise P element excision leads to malignant development of the hematopoietic organs and the genital disc, as shown by their growth autonomy in transplantation assays. We have cloned the oho31 gene of Drosophila melanogaster and determined its nucleotide sequence. The gene encodes a phosphoprotein of 522 amino acids made of three domains: a central hydrophobic domain of eight repeats of 42-44 amino acids each, displaying similarity to the arm motif found in junctional and nucleopore complex proteins, and flanked by two hydrophilic NH2-and COOH-terminal domains. Immunostaining revealed that the 0H031 protein is supplied maternally and rapidly degraded during the first 13 nuclear divisions. Thereafter, the 0H031 protein is predominantly expressed, albeit at reduced levels, in proliferating tissues. During the interphase of early embryonic cell cycles, the 0H031 protein is present in the cytoplasm and massively accumulates in the nucleus at the onset of mitosis in late interphase and prophase. The nuclear import of 0H031 is, however, less pronounced during later developmental stages. These results suggest that, similar to Importin, 0H031 may act as a cytosolic factor in nuclear transport. Moreover, the cell cycle-dependent accumulation of 0H031 in the nucleus indicates that this protein may be required for critical nuclear reactions occurring at the onset of mitosis.

Environmental factors act in a hierarchical manner at multiple spatial scales to influence the organisation of ecological assemblages; however, the relative influence of the different scale-related factor groups is poorly known. We evaluated the importance of catchment-scale and site-scale environmental variables, as well as the spatial context of the sampling sites, in shaping stream fish assemblages in an agriculture-dominated landscape in Hungary. Beside the variables describing spatial context (principal coordinates of a truncated distance matrix among sites), altogether 60 environmental variables were used to predict variability using a variance-partitioning procedure in redundancy analysis. Presence-absence-and relative abundance-based data were examined at two assemblage levels (entire assemblage and native assemblage) at 54 stream sites. Incorporation of spatial variables increased largely the total explained variability in case of relative abundance, but not for presence-absence data. Of the environmentally explained variance, catchment-scale variables (e.g., land cover types, patch density) were relatively more influential for the native assemblage-level analyses, than for analyses at the entire assemblage level, where site-scale variables (e.g., altitude, depth) proved to be more influential. In addition, pure catchment-and pure site-scale variables have the primary role in determining fish assemblage patterns, whereas the influence of shared variance and that of site-scale riparian variables proved to be less important. Our findings demonstrate the importance of incorporating the spatial context of the sampling sites in predicting fish assemblage patterns and the effects of channelisation (dikes) in shaping assemblage-environment relationships in this human-influenced landscape.