Summary A large fraction of our genome consists of mobile genetic elements. Governing transposons in germ cells is critically important, and failure to do so compromises genome integrity, leading to sterility. In animals, the piRNA pathway is the key to transposon constraint, yet the precise molecular details of how piRNAs are formed and how the pathway represses mobile elements remain poorly understood. In an effort to identify general requirements for transposon control and novel components of the piRNA pathway, we carried out a genome-wide RNAi screen in Drosophila ovarian somatic sheet cells. We identified and validated 87 genes necessary for transposon silencing. Among these were several novel piRNA biogenesis factors. We also found CG3893 (asterix) to be essential for transposon silencing, most likely by contributing to the effector step of transcriptional repression. Asterix loss leads to decreases in H3K9me3 marks on certain transposons but has no effect on piRNA levels.

With current concerns of antibiotic-resistant bacteria and biodefense, it has become important to rapidly identify infectious bacteria. Traditional technologies involving isolation and amplification of the pathogenic bacteria are time-consuming. We report a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides specific detection of as few as 10 bacterial cells per milliliter in experimental samples, with an Ϸ100-fold amplification of the signal over background in 1 h. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium, or are highly infectious, e.g., Bacillus anthracis. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are discussed.bacteriophage T7 ͉ BirA ͉ Escherichia coli ͉ water sample T he number and diversity of bacteriophages in the environment provide a promising natural pool of specific detection tools for pathogenic bacteria. Currently there are several phagebased methods for detection of pathogenic bacteria (1): a plaque assay for detection of Mycobacterium tuberculosis (2); fluorescence-labeled phage and immunomagnetic separation assay for detection of Escherichia coli O157:H7 (3, 4); phage-based electrochemical assays (5); a luciferase reporter mycobacteriophage and Listeria phage assays (6, 7); and detection of the phagemediated bacterial lysis and release of host enzymes (e.g., adenylate kinase) (8).Two limiting features when detecting pathogenic bacteria are sensitivity and rapidity. Common fluorophores (e.g., GFP and luciferase) used as reporters have two major disadvantages: low signal-to-noise ratio due to autofluorescence of clinical samples and of bacterial cells and low photostability, such as fast photobleaching. To overcome these disadvantages, we used new fluorescent semiconductor nanocrystals, quantum dots (QDs) (9). QDs are colloidal semiconductor (e.g., CdSe) crystals of a few nanometers in diameter. They exhibit broadband absorption spectra, and their emissions are of narrow bandwidth with size-dependent local maxima. The presence of an outer shell of a few atomic layers (e.g., ZnS) increases the quantum yield and further enhances the photostability, resulting in photostable fluorescent probes superior to conventional organic dyes. Recently, development in surface chemistry protocols allows conjugation of biomolecules onto these QDs to target specific biological molecules and probe nanoenvironments (10-12). The power to observe and trace single QDs or a group of bioconjugated QDs, enabling more precise quantitative biology, has been claimed to be one of the most exciting new capabilities offered to biologists today (13,14).Typically, the detection of small numbers of bacteria in environmental or clinical samples require...

The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the ␣ subunit of translation initiation factor 2 (eIF2␣) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn ؊ phenotype) by suppressing eIF2␣ phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn ؊ phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2␣ phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn ؊ phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin.

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