Our findings suggest that each taste sensillum represents a discrete, functional unit expressing at least one Gr receptor and that most Gr genes are expressed in spatially restricted domains of the gustatory system. These observations imply the potential for high taste discrimination of the Drosophila brain.

A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification ( To understand the function of a protein, it is crucial to characterize its physical environment: what other proteins is it interacting with under various conditions? Traditionally, this question has been addressed by biochemical fractionation of cell extracts under mild conditions and subsequent identification of the members of a purified protein complex by immunoblotting or peptide sequencing.Primed by the dawning of the post-genomic era, genomewide yeast two-hybrid interaction screens (1, 2) and protein chip-based methods (3) have supplemented traditional purification and identification techniques, allowing broader insight into the interaction networks that constitute a functional cell. Both of these approaches require the creation and maintenance of libraries of tagged proteins and in the case of protein chips the daunting task of purifying and spotting them under conditions that preserve their activity. The potential for detecting nonphysiological protein-protein interactions and the necessity to piece together interaction networks from a catalog of resulting binary interactions further complicate these approaches.Developed in parallel with two-hybrid and protein chip technologies, mass spectrometry of protein complexes purified through single or tandem affinity steps eliminates the need for complex-specific immunochemicals and enables analysis of very small amounts of sample on a proteome-wide scale (4,5). This approach can be performed under more physiological conditions and substitutes whole-complex analysis for the reconstruction of interaction networks from binary interaction data. However, the Gavin et al. (4) and Ho et al. (5) studies employed SDS-PAGE to separate affinity-purified protein mixtures prior to mass spectrometric analysis, thereby encountering the problems linked to this technique including: limitations of dynamic range of detection, considerable sample parallelization, variable elution efficiency of peptides from the polyacrylamide matrix, and potential selection against proteins with properties that impede analysis by SDS-PAGE (e.g. unusually high or low molecular mass, diffuse migration, comigration with contaminants, and poor binding to stain).To circumvent these problems, McCormack et al. (6) demo...

It has been shown in several organisms that multiple cis-regulatory modules (CRMs) of a gene locus can be active concurrently to support similar spatiotemporal expression. To understand the functional importance of such seemingly redundant CRMs, we examined two CRMs from the Drosophila snail gene locus, which are both active in the ventral region of pre-gastrulation embryos. By performing a deletion series in a ∼25 kb DNA rescue construct using BAC recombineering and site-directed transgenesis, we demonstrate that the two CRMs are not redundant. The distal CRM is absolutely required for viability, whereas the proximal CRM is required only under extreme conditions such as high temperature. Consistent with their distinct requirements, the CRMs support distinct expression patterns: the proximal CRM exhibits an expanded expression domain relative to endogenous snail, whereas the distal CRM exhibits almost complete overlap with snail except at the anterior-most pole. We further show that the distal CRM normally limits the increased expression domain of the proximal CRM and that the proximal CRM serves as a `damper' for the expression levels driven by the distal CRM. Thus, the two CRMs interact in cis in a non-additive fashion and these interactions may be important for fine-tuning the domains and levels of gene expression.