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Launched in 2001 to showcase the draft human genome assembly, the UCSC Genome Browser database (http://genome.ucsc.edu) and associated tools continue to grow, providing a comprehensive resource of genome assemblies and annotations to scientists and students worldwide. Highlights of the past year include the release of a browser for the first new human genome reference assembly in 4 years in December 2013 (GRCh38, UCSC hg38), a watershed comparative genomics annotation (100-species multiple alignment and conservation) and a novel distribution mechanism for the browser (GBiB: Genome Browser in a Box). We created browsers for new species (Chinese hamster, elephant shark, minke whale), 'mined the web' for DNA sequences and expanded the browser display with stacked color graphs and region highlighting. As our user community increasingly adopts the UCSC track hub and assembly hub representations for sharing large-scale genomic annotation data sets and genome sequencing projects, our menu of public data hubs has tripled.

The University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu) offers online public access to a growing database of genomic sequence and annotations for a wide variety of organisms. The Browser is an integrated tool set for visualizing, comparing, analysing and sharing both publicly available and user-generated genomic datasets. As of September 2012, genomic sequence and a basic set of annotation 'tracks' are provided for 63 organisms, including 26 mammals, 13 non-mammal vertebrates, 3 invertebrate deuterostomes, 13 insects, 6 worms, yeast and sea hare. In the past year 19 new genome assemblies have been added, and we anticipate releasing another 28 in early 2013. Further, a large number of annotation tracks have been either added, updated by contributors or remapped to the latest human reference genome. Among these are an updated UCSC Genes track for human and mouse assemblies. We have also introduced several features to improve usability, including new navigation menus. This article provides an update to the UCSC Genome Browser database, which has been previously featured in the Database issue of this journal.

The University of California Santa Cruz Genome Browser (http://genome.ucsc.edu) offers online public access to a growing database of genomic sequence and annotations for a wide variety of organisms. The Browser is an integrated tool set for visualizing, comparing, analyzing and sharing both publicly available and user-generated genomic data sets. In the past year, the local database has been updated with four new species assemblies, and we anticipate another four will be released by the end of 2011. Further, a large number of annotation tracks have been either added, updated by contributors, or remapped to the latest human reference genome. Among these are new phenotype and disease annotations, UCSC genes, and a major dbSNP update, which required new visualization methods. Growing beyond the local database, this year we have introduced ‘track data hubs’, which allow the Genome Browser to provide access to remotely located sets of annotations. This feature is designed to significantly extend the number and variety of annotation tracks that are publicly available for visualization and analysis from within our site. We have also introduced several usability features including track search and a context-sensitive menu of options available with a right-click anywhere on the Browser's image.

The Encyclopedia of DNA Elements (ENCODE) Consortium is entering its 5th year of production-level effort generating high-quality whole-genome functional annotations of the human genome. The past year has brought the ENCODE compendium of functional elements to critical mass, with a diverse set of 27 biochemical assays now covering 200 distinct human cell types. Within the mouse genome, which has been under study by ENCODE groups for the past 2 years, 37 cell types have been assayed. Over 2000 individual experiments have been completed and submitted to the Data Coordination Center for public use. UCSC makes this data available on the quality-reviewed public Genome Browser (http://genome.ucsc.edu) and on an early-access Preview Browser (http://genome-preview.ucsc.edu). Visual browsing, data mining and download of raw and processed data files are all supported. An ENCODE portal (http://encodeproject.org) provides specialized tools and information about the ENCODE data sets.

The first wave of personal genomes documents how no single individual genome contains the full complement of functional genes. Here, we describe the extent of variation in gene and pseudogene numbers between individuals arising from inactivation events such as premature termination or aberrant splicing due to single-nucleotide polymorphisms. This highlights the inadequacy of the current reference sequence and gene set. We present a proposal to define a reference gene set that will remain stable as more individuals are sequenced. In particular, we recommend that the ancestral allele be used to define the reference sequence from which a core human reference gene annotation set can be derived. In addition, we call for the development of an expanded gene set to include human-specific genes that have arisen recently and are absent from the ancestral set.

The University of California, Santa Cruz Genome Browser (http://genome.ucsc.edu) offers online access to a database of genomic sequence and annotation data for a wide variety of organisms. The Browser also has many tools for visualizing, comparing and analyzing both publicly available and user-generated genomic data sets, aligning sequences and uploading user data. Among the features released this year are a gene search tool and annotation track drag-reorder functionality as well as support for BAM and BigWig/BigBed file formats. New display enhancements include overlay of multiple wiggle tracks through use of transparent coloring, options for displaying transformed wiggle data, a ‘mean+whiskers’ windowing function for display of wiggle data at high zoom levels, and more color schemes for microarray data. New data highlights include seven new genome assemblies, a Neandertal genome data portal, phenotype and disease association data, a human RNA editing track, and a zebrafish Conservation track. We also describe updates to existing tracks.

The University of California, Santa Cruz (UCSC) Genome Browser website (http://genome.ucsc.edu/) provides a large database of publicly available sequence and annotation data along with an integrated tool set for examining and comparing the genomes of organisms, aligning sequence to genomes, and displaying and sharing users' own annotation data. As of September 2009, genomic sequence and a basic set of annotation 'tracks' are provided for 47 organisms, including 14 mammals, 10 non-mammal vertebrates, 3 invertebrate deuterostomes, 13 insects, 6 worms and a yeast. New data highlights this year include an updated human genome browser, a 44-species multiple sequence alignment track, improved variation and phenotype tracks and 16 new genome-wide ENCODE tracks. New features include drag-and-zoom navigation, a Wiki track for user-added annotations, new custom track formats for large datasets (bigBed and bigWig), a new multiple alignment output tool, links to variation and protein structure tools, in silico PCR utility enhancements, and improved track configuration tools.

For the past 15 years, the UCSC Genome Browser (http://genome.ucsc.edu/) has served the international research community by offering an integrated platform for viewing and analyzing information from a large database of genome assemblies and their associated annotations. The UCSC Genome Browser has been under continuous development since its inception with new data sets and software features added frequently. Some release highlights of this year include new and updated genome browsers for various assemblies, including bonobo and zebrafish; new gene annotation sets; improvements to track and assembly hub support; and a new interactive tool, the “Data Integrator”, for intersecting data from multiple tracks. We have greatly expanded the data sets available on the most recent human assembly, hg38/GRCh38, to include updated gene prediction sets from GENCODE, more phenotype- and disease-associated variants from ClinVar and ClinGen, more genomic regulatory data, and a new multiple genome alignment.

The goal of the Encyclopedia Of DNA Elements (ENCODE) Project is to identify all functional elements in the human genome. The pilot phase is for comparison of existing methods and for the development of new methods to rigorously analyze a defined 1% of the human genome sequence. Experimental datasets are focused on the origin of replication, DNase I hypersensitivity, chromatin immunoprecipitation, promoter function, gene structure, pseudogenes, non-protein-coding RNAs, transcribed RNAs, multiple sequence alignment and evolutionarily constrained elements. The ENCODE project at UCSC website (http://genome.ucsc.edu/ENCODE) is the primary portal for the sequence-based data produced as part of the ENCODE project. In the pilot phase of the project, over 30 labs provided experimental results for a total of 56 browser tracks supported by 385 database tables. The site provides researchers with a number of tools that allow them to visualize and analyze the data as well as download data for local analyses. This paper describes the portal to the data, highlights the data that has been made available, and presents the tools that have been developed within the ENCODE project. Access to the data and types of interactive analysis that are possible are illustrated through supplemental examples.

This correspondence is a primer for the zebrafish research community on zebrafish tracks available in the UCSC Genome Browser at http://genome.ucsc.edu based on Sanger's Zv4 assembly. A primary capability of this facility is comparative informatics between humans (as well as many other model organisms) and zebrafish. The zebrafish genome sequencing project has played important roles in mutant mapping and cloning, and comparative genomic research projects. This easy-to-use genome browser aims to display and download useful genome sequence information for zebrafish mutant mapping and cloning projects. Its user-friendly interface expedites annotation of the zebrafish genome sequence.

Cancer cells are capable of serum- and anchorage-independent growth, and focus formation on monolayers of normal cells. Previously, we showed that RACK1 inhibits c-Src kinase activity and NIH3T3 cell growth. Here, we show that RACK1 partially inhibits v-Src kinase activity, and the serum- and anchorage-independent growth of v-Src transformed cells, but has no effect on focus formation. RACK1-overexpressing v-Src cells show disassembly of podosomes, which are actin-rich structures that are distinctive to fully transformed cells. Together, our results demonstrate that RACK1 overexpression in v-Src cells partially reverses the transformed phenotype of the cells. Our results identify an endogenous inhibitor of the oncogenic Src tyrosine kinase and of cell transformation.

Pseudogenes are degraded fossil copies of genes. Here, we report a comparison of pseudogenes spanning three phyla, leveraging the completed annotations of the human, worm, and fly genomes, which we make available as an online resource. We find that pseudogenes are lineage specific, much more so than protein-coding genes, reflecting the different remodeling processes marking each organism’s genome evolution. The majority of human pseudogenes are processed, resulting from a retrotranspositional burst at the dawn of the primate lineage. This burst can be seen in the largely uniform distribution of pseudogenes across the genome, their preservation in areas with low recombination rates, and their preponderance in highly expressed gene families. In contrast, worm and fly pseudogenes tell a story of numerous duplication events. In worm, these duplications have been preserved through selective sweeps, so we see a large number of pseudogenes associated with highly duplicated families such as chemoreceptors. However, in fly, the large effective population size and high deletion rate resulted in a depletion of the pseudogene complement. Despite large variations between these species, we also find notable similarities. Overall, we identify a broad spectrum of biochemical activity for pseudogenes, with the majority in each organism exhibiting varying degrees of partial activity. In particular, we identify a consistent amount of transcription (∼15%) across all species, suggesting a uniform degradation process. Also, we see a uniform decay of pseudogene promoter activity relative to their coding counterparts and identify a number of pseudogenes with conserved upstream sequences and activity, hinting at potential regulatory roles.

The University of California Santa Cruz (UCSC) Genome Browser (http://genome.ucsc.edu) offers online public access to a growing database of genomic sequence and annotations for a large collection of organisms, primarily vertebrates, with an emphasis on the human and mouse genomes. The Browser's web-based tools provide an integrated environment for visualizing, comparing, analysing and sharing both publicly available and user-generated genomic data sets. As of September 2013, the database contained genomic sequence and a basic set of annotation 'tracks' for ∼90 organisms. Significant new annotations include a 60-species multiple alignment conservation track on the mouse, updated UCSC Genes tracks for human and mouse, and several new sets of variation and ENCODE data. New software tools include a Variant Annotation Integrator that returns predicted functional effects of a set of variants uploaded as a custom track, an extension to UCSC Genes that displays haplotype alleles for protein-coding genes and an expansion of data hubs that includes the capability to display remotely hosted user-provided assembly sequence in addition to annotation data. To improve European access, we have added a Genome Browser mirror (http://genome-euro.ucsc.edu) hosted at Bielefeld University in Germany.

Conversion of arterial macrophages into foam cells is a key process involved in both the initiation and progression of atherosclerotic lesions. Foam cell formation involves the progressive accumulation and storage of lipoprotein-derived cholesteryl esters. The resulting imbalance in cholesterol metabolism in arterial foam cells may be due in part to an inadequately low level of cytoplasmic neutral cholesteryl ester hydrolase (NCEH) activity. In this study, we have demonstrated that hormone-sensitive lipase (HSL) mRNA is expressed at very low levels in macrophage-derived foam cells, using the unique approach of extracting mRNA from macrophage-derived foam cells purified from human and rabbit atherosclerotic plaques coupled with reverse transcriptase polymerase chain reaction (RT-PCR). We also demonstrate that macrophage-derived foam cells isolated from rabbit atherosclerotic lesions exhibit a resistance to high density lipoprotein (HDL)-mediated cholesterol efflux along with reduced levels of NCEH activity compared to lipid-loaded mouse peritoneal macrophages. Thus, low level expression of HSL may partially account for the reduced NCEH activity observed in arterial foam cells isolated from atherosclerosis-susceptible species.

The Consensus Coding Sequence (CCDS) project (http://www.ncbi.nlm.nih.gov/CCDS/) is a collaborative effort to maintain a dataset of protein-coding regions that are identically annotated on the human and mouse reference genome assemblies by the National Center for Biotechnology Information (NCBI) and Ensembl genome annotation pipelines. Identical annotations that pass quality assurance tests are tracked with a stable identifier (CCDS ID). Members of the collaboration, who are from NCBI, the Wellcome Trust Sanger Institute and the University of California Santa Cruz, provide coordinated and continuous review of the dataset to ensure high-quality CCDS representations. We describe here the current status and recent growth in the CCDS dataset, as well as recent changes to the CCDS web and FTP sites. These changes include more explicit reporting about the NCBI and Ensembl annotation releases being compared, new search and display options, the addition of biologically descriptive information and our approach to representing genes for which support evidence is incomplete. We also present a summary of recent and future curation targets.

A series of novel sterols was synthesised as probes for the enzymatic and cellular functions of two important enzymes of intracellular cholesterol metabolism, acyl-CoA:cholesterol acyltransferase (ACAT) and cholesterol 7 alpha-hydroxylase. The compounds were 6-fluoro-5-cholesten-3 beta-ol (6-fluorocholesterol), 7-cholesten-3 beta-ol (7-cholestenol), 6 beta-fluorocholestan-3 beta-ol (6 beta-fluorocholestanol), 3-acetoxy-6-fluorocholestan-3 beta-ol (3-acetoxy-6-fluorocholestanol) and 7-methoxy-5-cholesten-3 beta-ol (7-methoxycholesterol). They were designed to reveal the effect of small changes in sterol structure, particularly reactivity of certain parts of the ring structure and polarity, on enzyme activity and intracellular cholesterol metabolism. The 3 beta-hydroxy group was essential for interaction with both enzymes since 3-acetoxy-6-fluorocholestanol did not affect any of the enzyme-catalysed reactions. 6-Fluorocholesterol and 7-cholestenol had no effect on cholesterol esterification but did inhibit the hydroxylation of cholesterol, as did the other compounds with groups that could influence the 7 position, namely 6 beta-fluorocholestanol and 7-methoxycholesterol. The fluorocholestanols were all competitive substrates for cholesterol esterification in cell-free and cellular assays of ACAT activity. 7-Methoxycholesterol was a surprisingly effective inhibitor of ACAT for a simple sterol. However, 6-fluorocholesterol did not have any effect on ACAT, suggesting that interactions between the enzyme and the region around C-6 and C-7 of the sterol are important. These results show that it is possible to dissect components of cholesterol metabolism using simple, specifically substituted sterols and thus define a new approach to studying the relationships between the various enzymes that catalyse intracellular cholesterol metabolism.

The Encyclopedia of DNA Elements (ENCODE), http://encodeproject.org, has completed its fifth year of scientific collaboration to create a comprehensive catalog of functional elements in the human genome, and its third year of investigations in the mouse genome. Since the last report in this journal, the ENCODE human data repertoire has grown by 898 new experiments (totaling 2886), accompanied by a major integrative analysis. In the mouse genome, results from 404 new experiments became available this year, increasing the total to 583, collected during the course of the project. The University of California, Santa Cruz, makes this data available on the public Genome Browser http://genome.ucsc.edu for visual browsing and data mining. Download of raw and processed data files are all supported. The ENCODE portal provides specialized tools and information about the ENCODE data sets.

BACKGROUND: Pseudogenes have long been considered as nonfunctional genomic sequences. However, recent evidence suggests that many of them might have some form of biological activity, and the possibility of functionality has increased interest in their accurate annotation and integration with functional genomics data. RESULTS: As part of the GENCODE annotation of the human genome, we present the first genome-wide pseudogene assignment for protein-coding genes, based on both large-scale manual annotation and in silico pipelines. A key aspect of this coupled approach is that it allows us to identify pseudogenes in an unbiased fashion as well as untangle complex events through manual evaluation. We integrate the pseudogene annotations with the extensive ENCODE functional genomics information. In particular, we determine the expression level, transcription-factor and RNA polymerase II binding, and chromatin marks associated with each pseudogene. Based on their distribution, we develop simple statistical models for each type of activity, which we validate with large-scale RT-PCR-Seq experiments. Finally, we compare our pseudogenes with conservation and variation data from primate alignments and the 1000 Genomes project, producing lists of pseudogenes potentially under selection. CONCLUSIONS: At one extreme, some pseudogenes possess conventional characteristics of functionality; these may represent genes that have recently died. On the other hand, we find interesting patterns of partial activity, which may suggest that dead genes are being resurrected as functioning non-coding RNAs. The activity data of each pseudogene are stored in an associated resource, psiDR, which will be useful for the initial identification of potentially functional pseudogenes.

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome., National Human Genome Research Institute (U.S.), National Institutes of Health (U.S.)

The advent of the human genome project and subsequent projects to sequence genomes of other species and multiple individuals has driven the need for tools that can visualize vast amounts of genomics data. Software for genome browsing has had a vast impact in the arenas of human medical and genetics research, enabling researchers to process and integrate different data types from a large variety of sources. Three major genome browsers are freely accessible online — the University of California, Santa Cruz (UCSC) Genome Browser, the Wellcome Trust Sanger Institute (WTSI)/European Bioinformatics Institute (EBI) Ensembl browser and the National Center for Biotechnology Information (NCBI) MapViewer. The UCSC Genome Browser is a key part of the UCSC Genome Bioinformatics suite of integrated tools that facilitate data mining, together with allowing users to visualize and query their own data in the context of the existing Genome Browser annotations. The chapter provides an overview of the types of annotation data displayed by the Genome Browser, as well as step-by-step examples illustrating how to create custom tracks and query both the Genome Browser and Table Browser. The Genome Browser offers links to several programs: BLAT for performing fast sequence alignment to genomes; the In Silico PCR tool for aligning primers to the genome, and liftOver for converting genomic coordinates from one assembly to another. Other tools in the suite include the Gene Sorter for sorting genes based on their relationships such as expression profiles and genomic proximity; the Proteome Browser, which shows protein-related information in graphical form and links out to external protein-related sites; and Genome Graphs, which allows the user to display genome-wide datasets such as those from SNP association studies, linkage studies and homozygosity mapping. The suite of UCSC Genome Bioinformatics tools, data downloads, extensive documentation and links to further training materials can be found at http://genome.ucsc.edu.

Since its start, the Mammalian Gene Collection (MGC) has sought to provide at least one full-protein-coding sequence cDNA clone for every human and mouse gene with a RefSeq transcript, and at least 6200 rat genes. The MGC cloning effort initially relied on random expressed sequence tag screening of cDNA libraries. Here, we summarize our recent progress using directed RT-PCR cloning and DNA synthesis. The MGC now contains clones with the entire protein-coding sequence for 92% of human and 89% of mouse genes with curated RefSeq (NM-accession) transcripts, and for 97% of human and 96% of mouse genes with curated RefSeq transcripts that have one or more PubMed publications, in addition to clones for more than 6300 rat genes. These high-quality MGC clones and their sequences are accessible without restriction to researchers worldwide.

The Human and Vertebrate Analysis and Annotation (HAVANA) group at the Wellcome Trust Sanger Institute produced the manually annotated geneset for the Encyclopedia of DNA Elements (ENCODE) pilot project and, as part of the Gencode subgroup, are reprising this role in the scale up to cover the whole human genome. Our manual annotation is checked computationally and validated experimentally. Loci and transcripts predicted to be absent from the initial annotation are identified by comparison with a number of state-of-the-art algorithms for identifying exons, splice sites, transcripts and pseudogenes. Where novel features are confirmed the annotation is updated. Annotated coding transcripts are analysed to assess their coding potential by investigating patterns of conservation within the coding sequence (CDS) and comparing predicted secondary structures of annotated CDSs to similar proteins with solved structures. Annotated coding transcripts are also checked against the current set of human Consensus CDSs (CCDS) to check agreement with other participating centres (EBI, NCBI, & UCSC).An initial round of annotation and analysis of chromosomes 21 and 22 has shown that while HAVANA annotation is both comprehensive and robust, it has benefitted from computational review. 13 novel non-coding loci, 27 novel splice variants and 6 extensions to existing variants were identified, many of which were found using supporting EST/mRNA sequences that were not present at the time of initial annotation. Fewer than 10 annotated CDSs required reclassification, no CCDS sequences required updating and 26 novel pseudogene were added. The annotation of human chromosome 2 is complete and we are currently annotating chromosomes 3 and 7. Data from all members of Gencode is distributed via DAS and is now visible in our Zmap annotation interface, allowing assessment of computational predictions contemporaneous with first-pass gene annotation.

Effective use of the human and mouse genomes requires reliable identification of genes and their products. Although multiple public resources provide annotation, different methods are used that can result in similar but not identical representation of genes, transcripts, and proteins. The collaborative consensus coding sequence (CCDS) project tracks identical protein annotations on the reference mouse and human genomes with a stable identifier (CCDS ID), and ensures that they are consistently represented on the NCBI, Ensembl, and UCSC Genome Browsers. Importantly, the project coordinates on manually reviewing inconsistent protein annotations between sites, as well as annotations for which new evidence suggests a revision is needed, to progressively converge on a complete protein-coding set for the human and mouse reference genomes, while maintaining a high standard of reliability and biological accuracy. To date, the project has identified 20,159 human and 17,707 mouse consensus coding regions from 17,052 human and 16,893 mouse genes. Three evaluation methods indicate that the entries in the CCDS set are highly likely to represent real proteins, more so than annotations from contributing groups not included in CCDS. The CCDS database thus centralizes the function of identifying well-supported, identically-annotated, protein-coding regions., National Human Genome Research Institute (U.S.) (Grant number 1U54HG004555-01), Wellcome Trust (London, England) (Grant number WT062023), Wellcome Trust (London, England) (Grant number WT077198)

The UCSC Genome Browser Database (GBD, http://genome.ucsc.edu) is a publicly available collection of genome assembly sequence data and integrated annotations for a large number of organisms, including extensive comparative-genomic resources. In the past year, 13 new genome assemblies have been added, including two important primate species, orangutan and marmoset, bringing the total to 46 assemblies for 24 different vertebrates and 39 assemblies for 22 different invertebrate animals. The GBD datasets may be viewed graphically with the UCSC Genome Browser, which uses a coordinate-based display system allowing users to juxtapose a wide variety of data. These data include all mRNAs from GenBank mapped to all organisms, RefSeq alignments, gene predictions, regulatory elements, gene expression data, repeats, SNPs and other variation data, as well as pairwise and multiple-genome alignments. A variety of other bioinformatics tools are also provided, including BLAT, the Table Browser, the Gene Sorter, the Proteome Browser, VisiGene and Genome Graphs.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view about chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function.

A key component of the ongoing ENCODE project involves rigorous comparative sequence analyses for the initially targeted 1% of the human genome. Here, we present orthologous sequence generation, alignment, and evolutionary constraint analyses of 23 mammalian species for all ENCODE targets. Alignments were generated using four different methods; comparisons of these methods reveal large-scale consistency but substantial differences in terms of small genomic rearrangements, sensitivity (sequence coverage), and specificity (alignment accuracy). We describe the quantitative and qualitative trade-offs concomitant with alignment method choice and the levels of technical error that need to be accounted for in applications that require multisequence alignments. Using the generated alignments, we identified constrained regions using three different methods. While the different constraint-detecting methods are in general agreement, there are important discrepancies relating to both the underlying alignments and the specific algorithms. However, by integrating the results across the alignments and constraint-detecting methods, we produced constraint annotations that were found to be robust based on multiple independent measures. Analyses of these annotations illustrate that most classes of experimentally annotated functional elements are enriched for constrained sequences; however, large portions of each class (with the exception of protein-coding sequences) do not overlap constrained regions. The latter elements might not be under primary sequence constraint, might not be constrained across all mammals, or might have expendable molecular functions. Conversely, 40% of the constrained sequences do not overlap any of the functional elements that have been experimentally identified. Together, these findings demonstrate and quantify how many genomic functional elements await basic molecular characterization.

Background Since the early stages of tumorigenesis involve adhesion, escape from immune surveillance, vascularization and angiogenesis, we devised a strategy to study the expression profiles of all publicly known and putative secreted and cell surface genes. We designed a custom oligonucleotide microarray containing probes for 3531 secreted and cell surface genes to study 5 diverse human transformed cell lines and their derivative xenograft tumors. The origins of these human cell lines were lung (A549), breast (MDA MB-231), colon (HCT-116), ovarian (SK-OV-3) and prostate (PC3) carcinomas. Results Three different analyses were performed: (1) A PCA-based linear discriminant analysis identified a 54 gene profile characteristic of all tumors, (2) Application of MANOVA (Pcorr < .05) to tumor data revealed a larger set of 149 differentially expressed genes. (3) After MANOVA was performed on data from individual tumors, a comparison of differential genes amongst all tumor types revealed 12 common differential genes. Seven of the 12 genes were identified by all three analytical methods. These included late angiogenic, morphogenic and extracellular matrix genes such as ANGPTL4, COL1A1, GP2, GPR57, LAMB3, PCDHB9 and PTGER3. The differential expression of ANGPTL4 and COL1A1 and other genes was confirmed by quantitative PCR. Conclusion Overall, a comparison of the three analyses revealed an expression pattern indicative of late angiogenic processes. These results show that a xenograft model using multiple cell lines of diverse tissue origin can identify common tumorigenic cell surface or secreted molecules that may be important biomarker and therapeutic discoveries.

Survival factors play critical roles in regulating cell growth in normal and cancer cells. We designed a genetic screen to identify survival factors which protect tumor cells from apoptosis. A retroviral expression library of random cDNA fragments was constructed from cancer cells and used to transduce the colon carcinoma cell line HCT116. Recipient cells were functionally selected for induction of caspase 3-mediated apoptosis. Analyses of over 10,000 putative genetic suppression elements (GSEs) sequences revealed cognate gene candidates that are implicated in apoptosis. We further analysed 26 genes encoding cell surface and secreted proteins that can potentially serve as targets for therapeutic antibodies. Tetracycline-inducible GSEs from several gene candidates induced apoptosis in stable HCT 116 cell lines. Similar phenotypes were caused by RNAi derived from the same genes. Our data suggest requirement for the cell surface targets IGF2R, L1CAM and SLC31A1 in tumor cell growth in vitro, and suggests that IGF2R is required for xenograft tumor growth in a mouse model.

RACK1 is one of a group of PKC-interacting proteins collectively called RACKs (Receptors for Activated C-Kinases). Previously, we showed that RACK1 also interacts with the Src tyrosine kinase, and is an inhibitor of Src activity and cell growth. PKC activation induces the intracellular movement and co-localization of RACK1 and Src, and the tyrosine phosphorylation of RACK1. To determine whether RACK1 is a Src substrate, we assessed phosphorylation of RACK1 by various tyrosine kinases in vitro, and by kinase-active and inactive mutants of Src in vivo. We found that RACK1 is a Src substrate. Moreover, Src activity is necessary for both the tyrosine phosphorylation of RACK1 and the binding of RACK1 to Src's SH2 domain that occur following PKC activation. To identify the tyrosine(s) on RACK1 that is phosphorylated by Src, we generated and tested a series of RACK1 mutants. We found that Src phosphorylates RACK1 on Tyr 228 and/or Tyr 246, highly-conserved tyrosines located in the sixth WD repeat that interact with Src's SH2 domain. We think that RACK1 is an important Src substrate that signals downstream of growth factor receptor tyrosine kinases and is involved in the regulation of Src function and cell growth.

The effect of the membrane environment of acyl-CoA:cholesterol acyl transferase (ACAT), an important intracellular enzyme of cholesterol metabolism, on the properties of a range of inhibitors of varying potencies was studied. ACAT activity from rat liver was solubilised with 3% deoxycholate (97% solubilised activity). After dilution into cholesterol/phosphatidylcholine liposomes (molar ratio 0.35), the assay of this reconstituted system showed linearity with protein and time. Saturation with oleoyl-CoA was achieved at 10 microM. Comparison of the potency of the ACAT inhibitors in the reconstituted assay and in a microsomal assay revealed a relationship between the lipid content of the assay and the inhibitory activity for potent inhibitors of ACAT (CI976, CL277,082, YMI7E and DuP128). This relationship was unrelated to lipophilicity of the drugs. Octimibate, lovastatin and progesterone, none of which is a potent ACAT inhibitor but which have all been described as ACAT inhibitors in the literature, all had low potencies in both assay systems. These results suggest that the lipid concentration must be taken into account when comparing potencies of ACAT inhibitors. The present data also indicate that some compounds which inhibit cholesterol esterification may do so by an indirect mechanism.

The Consensus Coding Sequence (CCDS) collaboration involves curators at multiple centers with a goal of producing a conservative set of high quality, protein-coding region annotations for the human and mouse reference genome assemblies. The CCDS data set reflects a 'gold standard' definition of best supported protein annotations, and corresponding genes, which pass a standard series of quality assurance checks and are supported by manual curation. This data set supports use of genome annotation information by human and mouse researchers for effective experimental design, analysis and interpretation. The CCDS project consists of analysis of automated whole-genome annotation builds to identify identical CDS annotations, quality assurance testing and manual curation support. Identical CDS annotations are tracked with a CCDS identifier (ID) and any future change to the annotated CDS structure must be agreed upon by the collaborating members. CCDS curation guidelines were developed to address some aspects of curation in order to improve initial annotation consistency and to reduce time spent in discussing proposed annotation updates. Here, we present the current status of the CCDS database and details on our procedures to track and coordinate our efforts. We also present the relevant background and reasoning behind the curation standards that we have developed for CCDS database treatment of transcripts that are nonsense-mediated decay (NMD) candidates, for transcripts containing upstream open reading frames, for identifying the most likely translation start codons and for the annotation of readthrough transcripts. Examples are provided to illustrate the application of these guidelines. DATABASE URL: http://www.ncbi.nlm.nih.gov/CCDS/CcdsBrowse.cgi.

The number of coding genes in the human genome is still under debate [1]. Here, we present a proposal to define the human reference gene set that takes into account the inter-individual differences in gene numbers arising from gene inactivation events, such as premature termination or aberrant splicing due to nonsense SNPs or SNPs at essential splice sites respectively. We have analyzed SNPs (specifically nonsense SNPs and SNPs affecting essential splice sites) from 23 personal genomes and exomes. We see a wide range in numbers of SNPs in each of the categories surveyed. A large fraction of these SNPs are singletons. Using a data set of high-confidence SNPs obtained by intersecting SNPs from dbSNP and the personal genomes, we identify a common set of 279 genes predicted to be pseudogenic (non-functional) in some individuals and functional in others. We focused on two key questions arising from these considerations: (i) Which criteria should be used for inclusion and exclusion of genes from the reference set? (ii) What sequence should be used as the reference for genes that are non-functional in some humans? For the first question, we propose to include all genes that are functional even in one individual to produce a maximally-inclusive set of genes. For the second, we propose the use of the ancestral allele as the reference allele. This will provide a uniform basis for gene annotation and ensure that the reference gene set and sequence will be relatively stable as more individual genomes are sequenced. In the few cases where an ancestral state assignment is unavailable or ambiguous, we propose that genes be annotated as the functional allele.

This article is part of the supplement: Beyond the Genome: The true gene count, human evolution and disease genomics, Boston, MA, USA. 11-13 October 2010., The GENCODE consortium is a sub group of the ENCODE consortium. Its aim is to provide complete annotation of genes in the human genome including protein-coding loci, non-coding loci and pseudogenes, based on experimental evidence. The final aim is for the HAVANA team to manually annotate the complete genome. This is a time-consuming process which will be completed over the course of the ENCODE project. Currently, to provide a set of annotation covering the complete genome, rather than just the regions that have been manually annotated, a merge of manual annotation from HAVANA with automatic annotation from the Ensembl automatically annotated gene set is created. This process also adds unique full-length CDS predictions from the Ensembl protein coding set into manually annotated genes, to provide the most complete up to date annotation of the genome possible. Also included in the set are short and long ncRNA genes predicted by the Ensembl prediction pipelines and a consensus set of pseudogene predictions agreed between Havana, Yale and UCSC. The CCDS set is also fully represented within the GENCODE set. The GENCODE set is the default annotation available in Ensembl and is also available in the UCSC genome browser. All the annotation is tagged as to whether it is produced by manual annotation alone, automatic annotation alone, or by both approaches. We are currently working to provide confidence levels for annotation, based on depth and type of evidence supporting it.

Correction to : Oncogene (2002) 21, 7619–7629. doi:10.1038/sj.onc.1206002 Due to a typesetting error, the wrong version of Figure 5 was published in the above paper. Please see the correct version below.

The GENCODE Consortium aims to identify all gene features in the human genome using a combination of computational analysis, manual annotation, and experimental validation. Since the first public release of this annotation data set, few new protein-coding loci have been added, yet the number of alternative splicing transcripts annotated has steadily increased. The GENCODE 7 release contains 20,687 protein-coding and 9640 long noncoding RNA loci and has 33,977 coding transcripts not represented in UCSC genes and RefSeq. It also has the most comprehensive annotation of long noncoding RNA (lncRNA) loci publicly available with the predominant transcript form consisting of two exons. We have examined the completeness of the transcript annotation and found that 35% of transcriptional start sites are supported by CAGE clusters and 62% of protein-coding genes have annotated polyA sites. Over one-third of GENCODE protein-coding genes are supported by peptide hits derived from mass spectrometry spectra submitted to Peptide Atlas. New models derived from the Illumina Body Map 2.0 RNA-seq data identify 3689 new loci not currently in GENCODE, of which 3127 consist of two exon models indicating that they are possibly unannotated long noncoding loci. GENCODE 7 is publicly available from gencodegenes.org and via the Ensembl and UCSC Genome Browsers., National Institutes of Health (U.S.) (Grant 5U54HG004555), Wellcome Trust (London, England) (Grant WT098051)