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).T he gene content of the mammalian genome is a topic of great interest. While draft sequences are now available for the human (1, 2), mouse (www.ensembl.org͞Mus musculus), and rat (http:͞͞hgsc.bcm.tmc.edu͞projects͞rat) genomes, the challenge remains to correctly identify all of the encoded genes. Difficulty in deciphering the anatomy of mammalian genes is due to several factors, including large amounts of intervening (noncoding) sequence, the imperfection of gene-prediction algorithms (3), and the incompleteness of cDNA-sequence resources, many of which consist of gene tags of variable length and quality. Full-length cDNA sequences are extremely useful for determining the genomic structure of genes, especially when analyzed within the context of genomic sequence. To facilitate geneidentification efforts and to catalyze experimental investigation, the National Institutes of Health (NIH) launched the Mammalian Gene Collection (MGC) program (4) with the aim of providing freely accessible, high-quality sequences for validated, complete ORF cDNA clones. In this article, we describe our progress toward the goal of identifying and accurately sequencing at least one full ORF-containing cDNA clone for each human and mouse gene, as well as making these fully sequenced clones available without restriction. Materials and MethodscDNA Library Production. MGC cDNA libraries were prepared from a diverse set of tissues and cell lines, in several different vector systems, by using a variety of methods. Vector maps and details of library construction are available at http:͞͞mgc. nci.nih.gov͞Info͞VectorMaps. The complete sequences for each of the MGC vectors can be found at http:͞͞image.llnl.gov͞ image͞html͞vectors.shtml. The catalog of MGC cDNA libraries can be accessed at http:͞͞mgc.nci.nih.gov.

Pseudoachondroplasia (PSACH) is a well characterized dwarfing condition mapping to chromosome 19p12-13.1. Cartilage oligomeric matrix protein (COMP), a cartilage specific protein, maps to the same location within a contig that spans the PSACH locus. Using single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing we have identified COMP mutations in eight familial and isolated PSACH cases. All mutations involve either a single base-pair change or a three base-pair deletion in exon 17B. Six mutations delete or change a well conserved aspartic acid residue within the calcium-binding type 3 repeats. These results demonstrate that mutations in the COMP gene cause pseudochondroplasia.

We report the generation of 319,311 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' and 3' ends of 194,031 human cDNA clones. Our goal has been to obtain tag sequences from many different genes and to deposit these in the publicly accessible Data Base for Expressed Sequence Tags. Highly efficient automatic screening of the data allows deposition of the annotated sequences without delay. Sequences have been generated from 26 oligo(dT) primed directionally cloned libraries, of which 18 were normalized. The libraries were constructed using mRNA isolated from 17 different tissues representing three developmental states. Comparisons of a subset of our data with nonredundant human mRNA and protein data bases show that the ESTs represent many known sequences and contain many that are novel. Analysis of protein families using Hidden Markov Models confirms this observation and supports the contention that although normalization reduces significantly the relative abundance of redundant cDNA clones, it does not result in the complete removal of members of gene families.

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

Genes expressed specifically in malignant tissue may have potential as therapeutic targets but have been difficult to locate for most cancers. The information hidden within certain public databases can reveal RNA transcripts specifically expressed in transformed tissue. To be useful, database information must be verified and a more complete pattern of tissue expression must be demonstrated. We tested database mining plus rapid screening by fluorescent-PCR expression comparison (F-PEC) as an approach to locate candidate brain tumor antigens. Cancer Genome Anatomy Project (CGAP) data was mined for genes highly expressed in glioblastoma multiforme. From 13 mined genes, seven showed potential as possible tumor markers or antigens as determined by further expression profiling. Now that large-scale expression information is readily available for many of the commonly occurring cancers, other candidate tumor markers or antigens could be located and evaluated with this approach.[The expression data described in this paper have been submitted to the NCBI SAGEmap database under library name SAGE_Duke_GBM_H1110, SAGE_pooled_GBM, SAGE_BB542_whitematter, and SAGE_normal_pool(6th).]