Genome-wide association studies have identified thousands of loci for common diseases, but, for the majority of these, the mechanisms underlying disease susceptibility remain unknown. Most associated variants are not correlated with protein-coding changes, suggesting that polymorphisms in regulatory regions probably contribute to many disease phenotypes. Here we describe the Genotype-Tissue Expression (GTEx) project, which will establish a resource database and associated tissue bank for the scientific community to study the relationship between genetic variation and gene expression in human tissues

Understanding the functional consequences of genetic variation, and how it affects complex human disease and quantitative traits, remains a critical challenge for biomedicine. We present an analysis of RNA sequencing data from 1641 samples across 43 tissues from 175 individuals, generated as part of the pilot phase of the Genotype-Tissue Expression (GTEx) project. We describe the landscape of gene expression across tissues, catalog thousands of tissue-specific and shared regulatory expression quantitative trait loci (eQTL) variants, describe complex network relationships, and identify signals from genome-wide association studies explained by eQTLs. These findings provide a systematic understanding of the cellular and biological consequences of human genetic variation and of the heterogeneity of such effects among a diverse set of human tissues.

Protein interaction mapping using large-scale two-hybrid analysis has been proposed as a way to functionally annotate large numbers of uncharacterized proteins predicted by complete genome sequences. This approach was examined in Caenorhabditis elegans, starting with 27 proteins involved in vulval development. The resulting map reveals both known and new potential interactions and provides a functional annotation for approximately 100 uncharacterized gene products. A protein interaction mapping project is now feasible for C. elegans on a genome-wide scale and should contribute to the understanding of molecular mechanisms in this organism and in human diseases.

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Aging is one of the most important biological processes and is a known risk factor for many age-related diseases in human. Studying age-related transcriptomic changes in tissues across the whole body can provide valuable information for a holistic understanding of this fundamental process. In this work, we catalogue age-related gene expression changes in nine tissues from nearly two hundred individuals collected by the Genotype-Tissue Expression (GTEx) project. In general, we find the aging gene expression signatures are very tissue specific. However, enrichment for some well-known aging components such as mitochondria biology is observed in many tissues. Different levels of cross-tissue synchronization of age-related gene expression changes are observed, and some essential tissues (e.g., heart and lung) show much stronger “co-aging” than other tissues based on a principal component analysis. The aging gene signatures and complex disease genes show a complex overlapping pattern and only in some cases, we see that they are significantly overlapped in the tissues affected by the corresponding diseases. In summary, our analyses provide novel insights to the co-regulation of age-related gene expression in multiple tissues; it also presents a tissue-specific view of the link between aging and age-related diseases.

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.

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...

A human tRNALys gene was converted to an amber suppressor by site-specific mutagenesis of the anticodon. The mutated tRNALys gene directed synthesis of a tRNA that suppressed the UAG amber nonsense mutation in beta O thalassemia mRNA. Such genes may be used to detect other nonsense mutations in mammalian cells and may provide an approach to gene therapy for beta O thalassaemia due to nonsense mutations.