Gerald M. Rubin

Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.

Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.

The Gene Ontology (GO) database and informatics resource.

The Gene Ontology (GO) project (http://www. geneontology.org/) provides structured, controlled vocabularies and classifications that cover several domains of molecular and cellular biology and are freely available for community use in the annotation of genes, gene products and sequences. Many model organism databases and genome annotation groups use the GO and contribute their annotation sets to the GO resource. The GO database integrates the vocabularies and contributed annotations and provides full access to this information in several formats. Members of the GO Consortium continually work collectively, involving outside experts as needed, to expand and update the GO vocabularies. The GO Web resource also provides access to extensive documentation about the GO project and links to applications that use GO data for functional analyses.

Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http://mgc.nci.nih.gov).

Molecular characterization of the drosophila trp locus: A putative integral membrane protein required for phototransduction

Recent studies suggest that the fly uses the inositol lipid signaling system for visual excitation and that the Drosophila transient receptor potential (trp) mutation disrupts this process subsequent to the production of IP3. In this paper, we show that trp encodes a novel 1275 amino acid protein with eight putative transmembrane segments. Immunolocalization indicates that the trp protein is expressed predominantly in the rhabdomeric membranes of the photoreceptor cells.

Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase

We have conducted a genetic screen for mutations that decrease the effectiveness of signaling by a protein tyrosine kinase, the product of the Drosophila melanogaster sevenless gene. These mutations define seven genes whose wild-type products may be required for signaling by sevenless. Four of the seven genes also appear to be essential for signaling by a second protein tyrosine kinase, the product of the Ellipse gene. The putative products of two of these seven genes have been identified. One encodes a ras protein. The other locus encodes a protein that is homologous to the S. cerevisiae CDC25 protein, an activator of guanine nucleotide exchange by ras proteins. These results suggest that the stimulation of ras protein activity is a key element in the signaling by sevenless and Ellipse and that this stimulation may be achieved by activating the exchange of GTP for bound GDP by the ras protein.

Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death

Apoptotic cell death is a mechanism by which organisms eliminate superfluous or harmful cells. Expression of the cell death regulatory protein REAPER (RPR) in the developing Drosophila eye results in a small eye owing to excess cell death. We show that mutations in thread (th) are dominant enhancers of RPR-induced cell death and that th encodes a protein homologous to baculovirus inhibitors of apoptosis (IAPs), which we call Drosophila IAP1 (DIAP1). Overexpression of DIAP1 or a related protein, DIAP2, in the eye suppresses normally occurring cell death as well as death due to overexpression of rpr or head involution defective. IAP death-preventing activity localizes to the N-terminal baculovirus IAP repeats, a motif found in both viral and cellular proteins associated with death prevention.

Structures of P transposable elements and their sites of insertion and excision in the Drosophila melanogaster genome

We have isolated and characterized several members of the P transposable element family from a Drosophila melanogaster P strain. Large 2.9 kb elements are present as multiple highly conserved copies together with smaller (0.5-1.6 kb), heterogeneous elements. The complete DNA sequences of the 2.9 kb element and four small elements (previously isolated from hybrid-dysgenesis-induced mutations of the white locus) have been determined. Each small element appears to have arisen from the 2.9 kb element by a different internal deletion. P elements have 31 bp perfect inverse terminal repeats and upon insertion duplicate an 8 bp sequence found only once at the site of insertion. Three of the insertions into the white locus occurred at the same nucleotide, indicating a high degree of local site specificity for insertion. The basis of this specificity has been investigated by DNA sequence analysis of the sites where 18 P elements are found. A revertant of one of the white locus mutants has been found to result from precise excision of the P element, restoring the wild-type DNA sequence.

Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays

To identify new Drosophila genes involved in the immune response, we monitored the gene expression profile of adult flies in response to microbial infection by using high-density oligonucleotide microarrays encompassing nearly the full Drosophila genome. Of 13,197 genes tested, we have characterized 230 induced and 170 repressed by microbial infection, most of which had not previously been associated with the immune response. Many of these genes can be assigned to specific aspects of the immune response, including recognition, phagocytosis, coagulation, melanization, activation of NF-κB transcription factors, synthesis of antimicrobial peptides, production of reactive oxygen species, and regulation of iron metabolism. Additionally, we found a large number of genes with unknown function that may be involved in control and execution of the immune response. Determining the function of these genes represents an important challenge for improving our knowledge of innate immunity. Complete results may be found at http://www.fruitfly.org/expression/immunity/.

The activities of two Ets-related transcription factors required for drosophila eye development are modulated by the Ras/MAPK pathway

We show that the activities of two Ets-related transcription factors required for normal eye development in Drosophila, pointed and yan, are regulated by the Ras1/MAPK pathway. The pointed gene codes for two related proteins, and we show that one form is a constitutive activator of transcription, while the activity of the other form is stimulated by the Ras1/MAPK pathway. Mutation of the single consensus MAPK phosphorylation site in the second form abrogates this responsiveness. yan is a negative regulator of photoreceptor determination, and genetic data suggest that it acts as an antagonist of Ras1. We demonstrate that yan can repress transcription and that this repression activity is negatively regulated by the Ras1/MAPK signal, most likely through direct phosphorylation of yan by MAPK.

Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome

A major challenge in interpreting genome sequences is understanding how the genome encodes the information that specifies when and where a gene will be expressed. The first step in this process is the identification of regions of the genome that contain regulatory information. In higher eukaryotes, this cis-regulatory information is organized into modular units [cis-regulatory modules (CRMs)] of a few hundred base pairs. A common feature of these cis-regulatory modules is the presence of multiple binding sites for multiple transcription factors. Here, we evaluate the extent to which the tendency for transcription factor binding sites to be clustered can be used as the basis for the computational identification of cis-regulatory modules. By using published DNA binding specificity data for five transcription factors active in the early Drosophila embryo, we identified genomic regions containing unusually high concentrations of predicted binding sites for these factors. A significant fraction of these binding site clusters overlap known CRMs that are regulated by these factors. In addition, many of the remaining clusters are adjacent to genes expressed in a pattern characteristic of genes regulated by these factors. We tested one of the newly identified clusters, mapping upstream of the gap gene giant (gt), and show that it acts as an enhancer that recapitulates the posterior expression pattern of gt.