The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame. The efficiency and flexibility of this approach enables collections of functional elements to be generated and mixed in a combinatorial fashion for the parallel assembly of numerous multi-segment constructs. The assembled constructs can be further manipulated by directing exchange of defined segments with alternate DNA segments. In this report, we demonstrate feasibility of the technology and application to the generation of fusion proteins, the linkage of promoters to genes, and the assembly of multiple protein domains. The technology has broad implications for cell and protein engineering, the expression of multidomain proteins, and gene function analysis.[Supplemental material is available online at www.genome.org.]The cloning and manipulation of DNA segments, typically encoding functional elements such as promoters, genes, protein domains, or fusion tags, are central to methods of cell engineering, protein production, and gene-function analysis. The large number of available genome sequences now makes it possible to create and apply repositories of defined functional elements to conduct high-throughput, genome-wide analyses. The Gateway Cloning Technology (Hartley et al. 2000) uses in vitro sitespecific recombination to clone and subsequently transfer DNA segments between vector backbones. This approach has been used to generate several large clone collections (Entry Clones), in some cases comprising the entire or nearly entire coding capacity of model genomes as open reading frames (ORFs). These ORFeomes include Caenorhabditis elegans (Walhout et al. 2000b;Reboul et al. 2001Reboul et al. , 2003, Pseudomonas aeruginosa (LaBaer et al. 2004), and Saccharomyces cerevisiae (G. Marsischky, pers. comm.), Arabidopsis (Yamada et al. 2003; also see Atome project http:// genoplante-info.infobiogen.fr/Databases/CT_Nouveaux_Outils/ NO2001054/), human (clones available from several commercial sources), and an incipient collection of Drosophila ORFs (http:// www.fruitfly.org/EST/gateway.shtml). A collection of sequenced, full-length Arabidopsis cDNAs in the Gateway Vector pCMV-SPORT6 will shortly be made available through INRA-Genoscope (Castelli et al. 2004). Repositories of full-length clones, some of which are in the Gateway format, are available for Xenopus (http://xgc.nci.nih.gov/), zebrafish (http://zgc.nci.nih.gov/), as well as many hu...

The eag family of K+ channels contains three known subtypes: eag, elk, and erg. Genes representing the first two subtypes have been identified in flies and mammals, whereas the third subtype has been defined only by the human HERG gene, which encodes an inwardly rectifying channel that is mutated in some cardiac arrhythmias. To establish the predicted existence of a Drosophila gene in the erg subfamily and to learn more about the structure and biological function of channels within this subfamily, we undertook a search for the Drosophila counterpart of HERG. Here we report the isolation and characterization of the Drosophila erg gene. We show that it corresponds with the previously identified seizure (sei) locus, mutations of which cause a temperature-sensitive paralytic phenotype associated with hyperactivity in the flight motor pathway. These results yield new insights into the structure and evolution of the eag family of channels, provide a molecular explanation for the sei mutant phenotype, and demonstrate the important physiological roles of erg-type channels from invertebrates to mammals.

Soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-mediated fusion of synaptic vesicles with the presynaptic-plasma membrane is essential for communication between neurons. Disassembly of the SNARE complex requires the ATPase N-ethylmaleimide-sensitive fusion protein (NSF). To determine where in the synaptic-vesicle cycle NSF functions, we have undertaken a genetic analysis of comatose (dNSF-1) in Drosophila. Characterization of 16 comatose mutations demonstrates that NSF mediates disassembly of SNARE complexes after synaptic-vesicle fusion. Hypomorphic mutations in NSF cause temperature-sensitive paralysis, whereas null mutations result in lethality. Geneticinteraction studies with para demonstrate that blocking evoked fusion delays the accumulation of assembled SNARE complexes and behavioral paralysis that normally occurs in comatose mutants, indicating NSF activity is not required in the absence of vesicle fusion. In addition, the entire vesicle pool can be depleted in shibire comatose double mutants, demonstrating that NSF activity is not required for the fusion step itself. Multiple rounds of vesicle fusion in the absence of NSF activity poisons neurotransmission by trapping SNAREs into cis-complexes. These data indicate that NSF normally dissociates and recycles SNARE proteins during the interval between exocytosis and endocytosis. In the absence of NSF activity, there are sufficient fusion-competent SNAREs to exocytose both the readily released and the reserve pool of synaptic vesicles.neurotransmitter release ͉ exocytosis ͉ synapse ͉ Drosophila T he core components of the vesicle-fusion apparatus consist of the plasma-membrane soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptors (SNAREs) syntaxin and soluble NSF attachment protein of 25 kDa (SNAP-25) and the vesicle SNARE synaptobrevin͞vesicle-associated membrane protein. Neuronal SNAREs assemble into highly stable SDS-resistant 7S SNARE complexes (1). The ATPase NSF has been implicated in numerous membrane-trafficking steps, including regulated exocytosis (2-8). NSF is recruited to the 7S complex by means of the adapter proteins ␣͞␤-and ␥-SNAP to form a larger 20S complex that is subsequently disassembled by the ATPase activity of NSF (1). Both assembly and disassembly of the SNARE complex are essential for synaptic-vesicle trafficking in vivo (6-10). NSF belongs to the AAA family of ATPases (ATPases with diverse cellular activities) and forms a hollow cylindrical hexamer when viewed with high-resolution electron microscopy (11, 12). The structure of the NSF hexamer depends on the presence or absence of bound ATP or ADP and undergoes substantial conformational changes upon ATP hydrolysis (11). Each monomer consists of three domains (13): an N-terminal domain essential for interaction with the SNAP͞SNARE complex, and two tandem ATPase domains termed D1 and D2. The D2 domain is required for hexamerization of NSF (11,(14)(15)(16). The D1 domain contains a high-affinity ATP-binding and hydrolysis sit...

A mutant Chinese hamster ovary cell line, glyB, that required exogenous glycine for survival and growth was reported previously (Kao, F., Chasin, L., and Puck, T. T. (1969) Proc. Natl. Acad. Sci. U. S. A. 64, 1284 -1291). We now report that the defect in glyB cells causative of this phenotype is a point mutation in an inner mitochondrial membrane protein required for transport of folates into mitochondria. The CHO mitochondrial folate transporter (mft) was sequenced and compared with that from glyB cells. The hamster sequence was nearly identical to that of the recently reported human mitochondrial folate transporter. The corresponding cDNA from glyB cells contained a single nucleotide change that introduced a glutamate in place of the glycine in wild-type hamster MFT at codon 192 in a predicted transmembrane domain. Transfection of the wild-type hamster cDNA into glyB cells allowed cell survival in the absence of glycine and the accumulation of folates in mitochondria, whereas transfection of the Glu-192 cDNA did not. Genomic sequence analysis and fluorescence in situ hybridization demonstrated a single mutated allele of the mft gene in glyB cells, whereas there were two alleles in CHO cells. We conclude that we have defined the cause of the glyB auxotrophy and that the glyB mft mutation identified a region of this mitochondrial folate carrier vital to its transport function.The compartmentalization of folate cofactor interconversion and folate-dependent biosynthetic reactions between cytosol and mitochondria is essential for the proper function of mammalian folate metabolism (1, 2). In mitochondria, reduced folates are required for the initiation of mitochondrial protein synthesis, the synthesis of glycine from serine, and the glycine cleavage system, which allows the generation of 10-formyltetrahydrofolate (3, 4).The transport of folates into the cytosol has been studied extensively (5, 6). Both the folate receptor (7) and the reduced folate carrier (8) have been defined as assisting in the passage of folates across the plasma membrane. In contrast, very little is known about the mechanism by which folates reach the mitochondrial matrix from the cytosol. Puck and co-workers (1, 9) isolated a CHO 1 mutant cell line that was auxotrophic for glycine and that did not accumulate folate cofactors in their mitochondria (15). A recent study in our laboratory (10) led to the isolation of the human gene encoding a protein that complemented the glyB defect. We tentatively named this protein the human mitochondrial folate transporter (MFT) in response to the fact that the structure of this protein had the characteristics of members of the inner mitochondrial membrane carrier family. The human cDNA isolated in that experiment reinstated folate uptake when transfected into glyB cells (10), strong evidence that the MFT was the endogenous protein responsible for the transport of folates into mammalian mitochondria. However, it remained possible that the complementation observed could have been through a compensatory or nonsp...

Imaging and subsequent segmentation analysis in three-dimensional (3D) culture models are complicated by the light scattering that occurs when collecting fluorescent signal through multiple cell and extracellular matrix layers. For 3D cell culture models to be usable for drug discovery, effective and efficient imaging and analysis protocols need to be developed that enable high-throughput data acquisition and quantitative analysis of fluorescent signal. Here we report the first high-throughput protocol for optical clearing of spheroids, fluorescent high-content confocal imaging, 3D nuclear segmentation, and post-segmentation analysis. We demonstrate nuclear segmentation in multiple cell types, with accurate identification of fluorescently-labeled subpopulations, and develop a metric to assess the ability of clearing to improve nuclear segmentation deep within the tissue. Ultimately this analysis pipeline allows for previously unattainable segmentation throughput of 3D culture models due to increased sample clarity and optimized batch-processing analysis.

Mutations of eag, first identified in Drosophila on the basis of their leg-shaking phenotype, cause repetitive firing and enhanced transmitter release in motor neurons. The encoded EAG polypeptide is related both to voltage-gated K+ channels and to cyclic nucleotide-gated cation channels. Homology screens identified a family of eag-related channel polypeptides, highly conserved from nematodes to humans, comprising three subfamilies: EAG, ELK, and ERG. When expressed in frog oocytes, EAG channels behave as voltage-dependent, outwardly rectifying K(+)-selective channels. Mutations of the human eag-related gene (HERG) result in a form of cardiac arrhythmia that can lead to ventricular fibrillation and sudden death. Electrophysiological and pharmacological studies have provided evidence that HERG channels specify one component of the delayed rectifier, IKr, that contributes to the repolarization phase of cardiac action potentials. An important role for HERG channels in neuronal excitability is also suggested by the expression of these channels in brain tissue. Moreover, mutations of ERG-type channels in the Drosophila sei mutant cause temperature-induced convulsive seizures associated with aberrant bursting activity in the flight motor pathway. The in vivo function of ELK channels has not yet been established, but when these channels are expressed in frog oocytes, they display properties intermediate between those of EAG- and ERG-type channels. Coexpression of the K(+)-channel beta subunit encoded by Hk with EAG in oocytes dramatically increases current amplitude and also affects the gating and modulation of these currents. Biochemical evidence indicates a direct physical interaction between EAG and HK proteins. Overall, these studies highlight the diverse properties of the eag family of K+ channels, which are likely to subserve diverse functions in vivo.

The discovery of the causative gene for Huntington’s disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington’s disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington’s disease, and potentially for other neurodegenerative disorders.

Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applications. The assessment of compound activity on the hERG channel has now become an important part of the safety evaluation in the process of drug discovery. During the past decade, several in vitro assay methods have been developed and significant resources have been used to characterize hERG channel activities. However, evaluation of compound activities on hERG have not been performed for large compound collections due to technical difficulty, lack of throughput, and/or lack of biological relevance to function. Here we report a modified form of the FluxOR thallium flux assay, capable of measuring hERG activity in a homogeneous 1536-well plate format. To validate the assay, we screened a 7-point dilution series of the LOPAC 1280 library collection and reported rank order potencies of ten common hERG inhibitors. A correlation was also observed for the hERG channel activities of 10 known hERG inhibitors determined in this thallium flux assay and in the patch clamp experiment. Our findings indicate that this thallium flux assay can be used as an alternative method to profile large-volume compound libraries for compound activity on the hERG channel.