Troy Moore

Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.

Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkins lymphoma, is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease. We proposed that this variability in natural history reflects unrecognized molecular heterogeneity in the tumours. Using DNA microarrays, we have conducted a systematic characterization of gene expression in B-cell malignancies. Here we show that there is diversity in gene expression among the tumours of DLBCL patients, apparently reflecting the variation in tumour proliferation rate, host response and differentiation state of the tumour. We identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal centre B cells (‘germinal centre B-like DLBCL’); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells (‘activated B-like DLBCL’). Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL. The molecular classification of tumours on the basis of gene expression can thus identify previously undetected and clinically significant subtypes of cancer.

Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences.

The National Institutes of Health Mammalian Gene Collection (MGC) Program is a multiinstitutional effort to identify and sequence a cDNA clone containing a complete ORF for each human and mouse gene. ESTs were generated from libraries enriched for full-length cDNAs and analyzed to identify candidate full-ORF clones, which then were sequenced to high accuracy. The MGC has currently sequenced and verified the full ORF for a nonredundant set of >9,000 human and >6,000 mouse genes. Candidate full-ORF clones for an additional 7,800 human and 3,500 mouse genes also have been identified. All MGC sequences and clones are available without restriction through public databases and clone distribution networks (see http://mgc.nci.nih.gov).

High-quality binary protein interaction map of the yeast interactome network

Current yeast interactome network maps contain several hundred molecular complexes with limited and somewhat controversial representation of direct binary interactions. We carried out a comparative quality assessment of current yeast interactome data sets, demonstrating that high-throughput yeast two-hybrid (Y2H) screening provides high-quality binary interaction information. Because a large fraction of the yeast binary interactome remains to be mapped, we developed an empirically controlled mapping framework to produce a “second-generation” high-quality, high-throughput Y2H data set covering ∼20% of all yeast binary interactions. Both Y2H and affinity purification followed by mass spectrometry (AP/MS) data are of equally high quality but of a fundamentally different and complementary nature, resulting in networks with different topological and biological properties. Compared to co-complex interactome models, this binary map is enriched for transient signaling interactions and intercomplex connections with a highly significant clustering between essential proteins. Rather than correlating with essentiality, protein connectivity correlates with genetic pleiotropy.

Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans

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 ORFeome Collaboration: a genome-scale human ORF-clone resource

To the Editor: Here we describe the ORFeome Collaboration (OC) open reading frame (ORF) clone collection, created by the OC (http://www.orfeomecollaboration.org/), an international collaboration of academic and commercial groups committed to providing genome-scale clone resources for human genes via worldwide commercial and academic clone distributors. Proteins are the predominant functional modules determining the fate of cells, tissues and organisms. An encyclopedic understanding of cellular physiology requires protein expression for proteinprotein interaction screening, cellular functional screening, validation of knockout and knockdown phenotypes, and numerous other approaches. Performing such studies on individual proteins or at the proteome scale requires a comprehensive collection of human protein expression clones. Our collection comprises ORF clones (Supplementary Note) and covers 17,154 RefSeq and Ensembl genes, nearly 73% of human RefSeq genes (http://www.ncbi.nlm.nih.gov/refseq/rsg/) and 79% of the highly curated Consensus Coding DNA Sequence Project (CCDS) human genes (http://www.ncbi.nlm.nih.gov/CCDS/ CcdsBrowse.cgi) (Fig. 1a and Supplementary Data). The collection includes clones of transcript variants for 6,304 (37%) of those genes. All major functional categories of human genes are substantially represented (Fig. 1b). All clones are provided in the Gateway vector format (Life Technologies), permitting high-throughput, precise and directional transfer of ORFs to a large variety of vectors for protein expression in biological systems such as Escherichia coli, yeast and mammals or using cell-free protein expression1 (Supplementary Note). OC clones were generated primarily by PCR amplification of the ORF from full-length, sequence-verified human cDNA clones of the Mammalian Gene Collection2 or the German cDNA Consortium3; ORFs were also prepared by directed RT-PCR cloning4 or DNA synthesis2. All 5′ and 3′ untranslated regions were excluded, permitting direct expression of ORFs as fusions to aminoor carboxy-terminal polypeptides, or as native protein, after transfer to a Gatewayexpression vector1. The clones are designed to maintain the correct reading frame for both aminoand carboxy-fusion proteins. Among all genes represented in the OC collection, 64% of clones are without stop codons, 5% have stop codons, and 31% are present in both versions. Each OC clone was isolated from a single colony and is fully sequenced. Individual clone sequences have been deposited in the GenBank, EMBL and DDBJ databases. The OC website provides a searchable database with annotation of all OC clones, their respective genes, and clone confidence levels based on CCDS and RefSeq annotations (Supplementary Note) along with links to the UCSC and RIKEN browsers (http://genome.ucsc.edu/cgi-bin/hgGateway and http://fantom.gsc.riken.jp/zenbu/gLyphs/#config), which provide graphical representations of the gene structures and transcripts. OC clones are distributed via a good faith agreement, giving unrestricted clone access to all scientists worldwide. The OC website lists OC clone distributors. The value of the OC resource has been demonstrated in numerous studies covering a broad range of applications. These include large-scale binary protein-protein interaction mapping5, production of recombinant human proteins6, mapping of co-complex associations, fluorescent protein tagging for human protein localization in mammalian cells and microscopy-based functional screening of proteins, development of disease-specific protein interaction Figure 1 | RefSeq and Ensembl genes and functional gene categories represented in the OC. (a) Numbers of protein-coding genes represented in the OC collection from RefSeq (blue) and Ensembl (green) gene catalogs. The table summarizes these numbers, together with OC coverage for RefSeq-only and Ensembl-only genes. (b) Numbers of human RefSeq genes represented in the OC collection versus in the human genome, compared in nine functional categories; percentages of genes in the OC are presented above the bars. The methods used to calculate the gene numbers in each category are explained in the Supplementary Note and contrasted to the standard Gene Ontology categories. An expanded list of the top Gene Ontology categories is also provided in the Supplementary Note. The data underlying the graphs are provided as Supplementary Data. a

The gene number dilemma: Direct evidence for at least 19,000 protein-encoding genes in C. elegans and implications for the human genome

The gene number dilemma: direct evidence for at least 19,000 protein-encoding genes in Caenorhabditis elegans and implications for the human genome