C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression.

To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.

Value of Wellness Ratings and Countermovement Jumping Velocity to Monitor Performance.

This study examined the relationship between subjective ratings of overall wellness and neuromuscular performance throughout a 6-week intensive offseason strength and conditioning program. Thirty experienced NCAA Division II baseball players completed all phases of the program. A comprehensive wellness rating and 5 countermovement jumps (CMJ5) were measured and averaged for 4 phases of training. Pre- and post-testing measures of strength and speed also were evaluated. Internal consistency of the wellness rating for each phase ranged α = 0.77-0.92, and CMJ5 velocities had decent consistency (ICCα = 0.88, TE = 0.19 m·s-1, CV = 5.90%). The training program evoked significant (p < 0.01) improvements in front squats (d = 0.55), trap bar deadlifts (d = 0.62), chin ups (d = 0.39), 30-yd dash (d = 0.39), with no change in the 300-yd dash (p > 0.05), where d is the treatment effect size. Average CMJ5 velocities (m·s-1) were similar for the preparation phase (1.90 ± 0.25), eccentric phase (1.91 ± 0.28), strength & power phase (1.91 ± 0.24), and recovery phase (1.91 ± 0.30; F = 0.04, p = 0.99, η p 2 = 0.001). No significant correlations were observed for pre-or post-testing measures of wellness ratings in comparison to any performance measures, including a composite standardized score from each performance test at pre-testing (r = 0.22, p = 0.26). The CMJ5 exhibited too high of a typical error to determine a change in neuromuscular status. Additionally, the wellness rating did not reflect changes in relation to performance.

Interaction between the glutamate transporter GLT1b and the synaptic PDZ domain protein PICK1.

Synaptic plasticity is implemented by the interaction of glutamate receptors with PDZ domain proteins. Glutamate transporters provide the only known mechanism of clearance of glutamate from excitatory synapses, and GLT1 is the major glutamate transporter. We show here that GLT1 interacts with the PDZ domain protein PICK1, which plays a critical role in regulating the expression of glutamate receptors at excitatory synapses. A yeast two-hybrid screen of a neuronal library using the carboxyl tail of GLT1b yielded clones expressing PICK1. The GLT1b C-terminal peptide bound to PICK1 with high affinity (K(i) = 6.5 +/- 0.4 microM) in an in vitro fluorescence polarization assay. We also tested peptides based on other variants of GLT1 and other glutamate transporters. GLT1b co-immunoprecipitated with PICK1 from rat brain lysates and COS7 cell lysates derived from cells transfected with plasmids expressing PICK1 and GLT1b. In addition, expression of GLT1b in COS7 cells changed the distribution of PICK1, bringing it to the surface. GLT1b and PICK1 co-localized with each other and with synaptic markers in hippocampal neurons in culture. Phorbol ester, an activator of protein kinase C (PKC), a known PICK1 interactor, had no effect on glutamate transport in rat forebrain neurons in culture. However, we found that exposure of neurons to a myristolated decoy peptide with sequence identical to the C-terminal sequence of GLT1b designed to block the PICK1-GLT1b interaction rendered glutamate transport into neurons responsive to phorbol ester. These results suggest that the PICK1-GLT1b interaction regulates the modulation of GLT1 function by PKC.

A cell-based PDE4 assay in 1536-well plate format for high-throughput screening.

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.

ORFeome cloning and systems biology: standardized mass production of the parts from the parts-list.

Together with metabolites, proteins and RNAs form complex biological systems through highly intricate networks of physical and functional interactions. Large-scale studies aimed at a molecular understanding of the structure, function, and dynamics of proteins and RNAs in the context of cellular networks require novel approaches and technologies. This Special Issue of Genome Research features strategies for the high-throughput construction and manipulation of complete sets of protein-encoding open reading frames (ORFeome), gene promoters (promoterome), and noncoding RNAs, as predicted from genome and transcriptome sequences. Here we discuss the use of a recombinational cloning system that allows efficiency, adaptability, and compatibility in the generation of ORFeome, promoterome, and other resources.

TSG101 interaction with HRS mediates endosomal trafficking and receptor down-regulation.

Down-regulation of mitogenic signaling in mammalian cells relies in part on endosomal trafficking of activated receptors into lysosomes, where the receptors are degraded. These events are mediated by ubiquitination of the endosomal cargo and its consequent sorting into multivesicular bodies that form at the surfaces of late endosomes. Tumor susceptibility gene 101 (tsg101) recently was found to be centrally involved in this process. Here we report that TSG101 interacts with hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), an early endosomal protein, and that disruption of this interaction impedes endosomal trafficking and endocytosis-mediated degradation of mitogenic receptors. TSG101/HRS interaction occurs between a ubiquitin-binding domain of TSG101 and two distinct proline-rich regions of HRS, and is modulated by a C-terminal TSG101 sequence that resembles a motif targeted in HRS. Mutational perturbation of TSG101/HRS interaction prevented delivery of epidermal growth factor receptor (EGFR) to late endosomes, resulted in the cellular accumulation of ubiquitinated EGFR in early endosomes, and inhibited ligand-induced down-regulation of EGFR. Our results reveal the TSG101 interaction with HRS as a crucial step in endocytic down-regulation of mitogenic signaling and suggest a role for this interaction in linking the functions of early and late endosomes.

Feasibility of genome-scale construction of promoter::reporter gene fusions for expression in Caenorhabditis elegans using a multisite gateway recombination system.

The understanding of gene function increasingly requires the characterization of DNA segments containing promoters and their associated regulatory sequences. We describe a novel approach for linking multiple DNA segments, here applied to the generation of promoter::reporter fusions. Promoters from Caenorhabditis elegans genes were cloned using the MultiSite Gateway cloning technology. The capacity for using this system for efficient construction of chimeric genes was explored by constructing promoter::reporter gene fusions with a gfp reporter. The promoters were found to provide appropriate expression of GFP upon introduction into C. elegans, demonstrating that the short Gateway recombination site between the promoter and the reporter did not interfere with transcription or translation. The recombinational cloning involved in the Gateway system, which permits the highly efficient and precise transfer of DNA segments between plasmid vectors, makes this technology ideal for genomics research programs.

Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones.

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.