A candidate taste receptor gene near a sweet taste locus.

The mechanisms underlying sweet taste in mammals have been elusive. Although numerous studies have implicated G proteins in sweet taste detection, the expected G protein-coupled receptors have not been found. Here we describe a candidate taste receptor gene, T1r3, that is located at or near the mouse Sac locus, a genetic locus that controls the detection of certain sweet tastants. T1R3 differs in amino acid sequence in mouse strains with different Sac phenotypes ('tasters' versus 'nontasters'). In addition, a perfect correlation exists between two different T1r3 alleles and Sac phenotypes in recombinant inbred mouse strains. The T1r3 gene is expressed in a subset of taste cells in circumvallate, foliate and fungiform taste papillae. In circumvallate and foliate papillae, most T1r3-expressing cells also express a gene encoding a related receptor, T1R2, raising the possibility that these cells recognize more than one ligand, or that the two receptors function as heterodimers.

Apoptosis-inducing natural products found in utero during murine pregnancy.

BACKGROUND: Hormones, lipids, vitamins and other biologically active small molecules can be removed from animal tissues by extraction with organic solvents. These compounds can have dramatic effects on cultured cells and the characterization of such compounds can lead to the discovery of new functions for known molecules, or even to the discovery of previously unknown compounds. RESULTS: Organic-soluble compounds in 17.5-day-old mouse embryos were removed with tert-butylmethylether and found to induce apoptosis in T-antigen-transformed Jurkat T cells. These embryonic extracts were fractionated and their apoptosis-inducing components were identified as a mixture of polyunsaturated fatty acids, including arachidonic, docosatetraenoic and docosahexaenoic acids. Docosatetraenoic acid was the most potent apoptosis inducer with an effective dose (ED(50)) of 30 microM. CONCLUSIONS: A family of polyunsaturated fatty acids is shown to be abundant in utero during pregnancy. Members of this family are able to induce cleavage of poly(ADP)ribose polymerase, and ultimately to induce apoptosis, in T-antigen-transformed Jurkat T cells. Free radical scavengers, including phenol and benzyl alcohol, block the apoptosis-inducing properties of these polyunsaturated fatty acids; this is consistent with a lipid peroxidation mechanism involving formation of hydroperoxy fatty acids.

Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen.

The natural product rapamycin has been used to provide temporal and quantitative control of gene expression in animals through its ability to interact with two proteins simultaneously. A shortcoming of this approach is that rapamycin is an inhibitor of cell proliferation, the result of binding to FKBP12-rapamycin-associated protein (FRAP). To overcome this limitation, nontoxic derivatives of rapamycin bearing bulky substituents at its C16-position were synthesized, each in a single step. The isosteric isopropoxy and methallyl substituents with the nonnatural C16-configuration abolish both binding to FRAP and inhibition of T cell proliferation. Binding proteins for these derivatives were identified from libraries of cDNAs encoding mutants of the FKBP12-rapamycin-binding (FRB) domain of FRAP by using a mammalian three-hybrid transcription assay. Targeting of the mutations was guided by the structure of the FKBP12-rapamycin-FRB ternary complex. Three compensatory mutations in the FRB domain, all along one face of an alpha-helix in a rapamycin-binding pocket, were identified that together restore binding of the rapamycin derivatives. Using this mutant FRB domain, one of the nontoxic rapamycin derivatives induced targeted gene expression in Jurkat T cells with an EC50 below 10 nM. Another derivative was used to recruit a cytosolic protein to the plasma membrane, mimicking a process involved in many signaling pathways.

Small molecule-dependent genetic selection in stochastic nanodroplets as a means of detecting protein-ligand interactions on a large scale.

BACKGROUND: Understanding the cellular role of a protein often requires a means of altering its function, most commonly by mutating the gene encoding the protein. Alternatively, protein function can be altered directly using a small molecule that binds to the protein, but no general method exists for the systematic discovery of small molecule ligands. Split-pool synthesis provides a means of synthesizing vast numbers of small molecules. Synthetic chemists will soon be able to synthesize natural product-like substances by this method, so compatible screening methods that detect the activity of minute quantities of molecules among many inactive ones will be in demand. RESULTS: We describe two advances towards achieving the above goals. First, a technique is described that uses a simple spray gun to create 5000-8000 droplets randomly, each having a volume of 50-200 nanoliters. The individual 'nanodroplets' contain a controlled number of cells and many also contain individual synthesis beads. As small molecules can be photochemically released from the beads in a time-dependent manner, the concentration of ligands that the cells are exposed to can be controlled. The spatial segregation of nanodroplets prevents the mixing of compounds from other beads so the effects of each molecule can be assayed individually. Second, a small molecule-dependent genetic selection involving engineered budding yeast cells was used to detect intracellular protein-ligand interactions in nanodroplets. CONCLUSIONS: The technique described here should facilitate the discovery of new cell-permeable ligands, especially when combined with a positive selection assay that detects intracellular binding of small molecules to proteins. Using 'anchored combinatorial libraries', it may be possible to screen entire libraries of natural product-like molecules against the entire collection of proteins encoded within cDNA libraries in a single experiment.