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Target Gene Notebook was developed to enable more efficient linking of genetic associations to functional biological information. This process is essential to translating genetic insights into therapeutic hypotheses and, eventually, drug discovery. Although many public databases provide access to unfiltered genome annotations and genetic results, there was no existing tool to maintain group curation and integration with proprietary experimental data. We provide Target Gene Notebook freely via the MIT open-source license for the purposes of assisting therapeutic target evaluation and the creation of durable institutional and public knowledge bases. Implemented as a Java backend serving mainly Javascript content derived from gene-specific SQLite databases, Target Gene Notebook enables automated access to the most widely used sources of genetic association, expression and protein QTL data, provides intuitive interfaces to credible set and colocalization information, and enables comprehensive literature review and annotation by multiple users simultaneously to create a consistent target knowledgebase within an organization or across a consortium. TargetGeneNotebook is freely available from GitHub https://github.com/targetgenenotebook/tgn.git under the MIT open-source end-user license agreement and a live version of the interface is provided at http://tgn.broadinstitute.org/.

SummaryAminoglycoside antibiotics, such as gentamicin and kanamycin, directly target the ribosome, yet the mechanisms by which these bactericidal drugs induce cell death are not fully understood. Recently, oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here, we use systems-level approaches and phenotypic analyses to provide insight into the pathway whereby aminoglycosides ultimately trigger hydroxyl radical formation. We show, by disabling systems that facilitate membrane protein traffic, that mistranslation and misfolding of membrane proteins are central to aminoglycoside-induced oxidative stress and cell death. Signaling through the envelope stress-response two-component system is found to be a key player in this process, and the redox-responsive two-component system is shown to have an associated role. Additionally, we show that these two-component systems play a general role in bactericidal antibiotic-mediated oxidative stress and cell death, expanding our understanding of the common mechanism of killing induced by bactericidal antibiotics.

Protein interaction maps can reveal novel pathways and functional complexes, allowing 'guilt by association' annotation of uncharacterized proteins. To address the need for large-scale protein interaction analyses, a bacterial two-hybrid system was coupled with a whole genome shotgun sequencing approach for microbial genome analysis. We report the first large-scale proteomics study using this system, integrating de novo genome sequencing with functional interaction mapping and annotation in a high-throughput format. We apply the approach by shotgun sequencing and annotating the genome of Rickettsia sibirica strain 246, an obligate intracellular human pathogen among the Spotted Fever Group rickettsiae. The bacteria invade endothelial cells and cause lysis after large amounts of progeny have accumulated. Little is known about specific Rickettsial virulence factors and their mode of pathogenicity. Analysis of the combined genomic sequence and protein-protein interaction data for a set of virulence related Type IV secretion system (T4SS) proteins revealed over 250 interactions and will provide insight into the mechanism of Rickettsial pathogenicity.

The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.

The complete 1,751,377-bp sequence of the genome of the thermophilic archaeon Methanobacterium thermoautotrophicum deltaH has been determined by a whole-genome shotgun sequencing approach. A total of 1,855 open reading frames (ORFs) have been identified that appear to encode polypeptides, 844 (46%) of which have been assigned putative functions based on their similarities to database sequences with assigned functions. A total of 514 (28%) of the ORF-encoded polypeptides are related to sequences with unknown functions, and 496 (27%) have little or no homology to sequences in public databases. Comparisons with Eucarya-, Bacteria-, and Archaea-specific databases reveal that 1,013 of the putative gene products (54%) are most similar to polypeptide sequences described previously for other organisms in the domain Archaea. Comparisons with the Methanococcus jannaschii genome data underline the extensive divergence that has occurred between these two methanogens; only 352 (19%) of M. thermoautotrophicum ORFs encode sequences that are >50% identical to M. jannaschii polypeptides, and there is little conservation in the relative locations of orthologous genes. When the M. thermoautotrophicum ORFs are compared to sequences from only the eucaryal and bacterial domains, 786 (42%) are more similar to bacterial sequences and 241 (13%) are more similar to eucaryal sequences. The bacterial domain-like gene products include the majority of those predicted to be involved in cofactor and small molecule biosyntheses, intermediary metabolism, transport, nitrogen fixation, regulatory functions, and interactions with the environment. Most proteins predicted to be involved in DNA metabolism, transcription, and translation are more similar to eucaryal sequences. Gene structure and organization have features that are typical of the Bacteria, including genes that encode polypeptides closely related to eucaryal proteins. There are 24 polypeptides that could form two-component sensor kinase-response regulator systems and homologs of the bacterial Hsp70-response proteins DnaK and DnaJ, which are notably absent in M. jannaschii. DNA replication initiation and chromosome packaging in M. thermoautotrophicum are predicted to have eucaryal features, based on the presence of two Cdc6 homologs and three histones; however, the presence of an ftsZ gene indicates a bacterial type of cell division initiation. The DNA polymerases include an X-family repair type and an unusual archaeal B type formed by two separate polypeptides. The DNA-dependent RNA polymerase (RNAP) subunits A', A", B', B" and H are encoded in a typical archaeal RNAP operon, although a second A' subunit-encoding gene is present at a remote location. There are two rRNA operons, and 39 tRNA genes are dispersed around the genome, although most of these occur in clusters. Three of the tRNA genes have introns, including the tRNAPro (GGG) gene, which contains a second intron at an unprecedented location. There is no selenocysteinyl-tRNA gene nor evidence for classically organized IS elements, prophages, or plasmids. The genome contains one intein and two extended repeats (3.6 and 8.6 kb) that are members of a family with 18 representatives in the M. jannaschii genome.

We have developed and are currently using a first generation fully automated hybridizer and sequence imager for multiplex sequencing. This instrument processes four multiplex membranes concurrently with a five hour cycle time, yielding a projected throughput of more than 50 million bases per year. We have also begun development of a second generation instrument which will be capable of outputs in excess of 1.5 billion raw bases per year by processing up to one hundred multiplex membranes concurrently. After direct transfer electrophoresis, each DNA containing membrane is placed in a flat tray for processing. Solutions are automatically added to and removed from the independently processed trays/membranes. In order to achieve short cycle times, a non-radioactive detection method is utilized in which oligonucleotide probes are conjugated to an enzyme and then hybridized to each of the multiplexed sequencing reaction products on the membrane. Chemiluminescent substrate is added and converted by the enzyme into an unstable product which emits light upon decay, thus revealing the sequence pattern. In the first generation instrument, the emitted light is collected at an imaging station consisting a single scientific CCD camera mounted on linear slides. The second generation instrument will increase throughput by utilizing a stationary array of cameras. By capturing segments and then tiling to form an image of the entire membrane, high collection efficiency and high resolution optics may be used to detect the extremely low light levels associated with chemiluminescence.

A major challenge in drug discovery is to distinguish the molecular targets of a bioactive compound from the hundreds to thousands of additional gene products that respond indirectly to changes in the activity of the targets. Here, we present an integrated computational-experimental approach for computing the likelihood that gene products and associated pathways are targets of a compound. This is achieved by filtering the mRNA expression profile of compound-exposed cells using a reverse-engineered model of the cell's gene regulatory network. We apply the method to a set of 6 whole-genome Escherichia coli expression profiles at different time points after treatment with the antibiotic Norfloxacin. We show that the algorithm can correctly identify the known drug targets and associated pathways.