Genome and expressed sequence tag projects are rapidly cataloguing and cloning the genes of higher organisms, including humans. An emerging challenge is to rapidly uncover the functions of genes and to identify gene products with desired properties. We have developed a microarray-driven gene expression system for the functional analysis of many gene products in parallel. Mammalian cells are cultured on a glass slide printed in defined locations with different DNAs. Cells growing on the printed areas take up the DNA, creating spots of localized transfection within a lawn of non-transfected cells. By printing sets of complementary DNAs cloned in expression vectors, we make microarrays whose features are clusters of live cells that express a defined cDNA at each location. Here we demonstrate two uses for our approach: as an alternative to protein microarrays for the identification of drug targets, and as an expression cloning system for the discovery of gene products that alter cellular physiology. By screening transfected cell microarrays expressing 192 different cDNAs, we identified proteins involved in tyrosine kinase signalling, apoptosis and cell adhesion, and with distinct subcellular distributions.

Microarrays of cells expressing defined cDNAs

As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

/pdf/complete-sequencing-and-characterization-of-21-243-full-kj2n0zp8yc.pdf

Complete sequencing and characterization of 21,243 full-length human cDNAs

The Munich Information Center for Protein Sequences (MIPS-GSF), Neuherberg, Germany, provides protein sequence-related information based on whole-genome analysis. The main focus of the work is directed toward the systematic organization of sequence-related attributes as gathered by a variety of algorithms, primary information from experimental data together with information compiled from the scientific literature. MIPS maintains automatically generated and manually annotated genome-specific databases, develops systematic classification schemes for the functional annotation of protein sequences and provides tools for the comprehensive analysis of protein sequences. This report updates the information on the yeast genome (CYGD), the Neurospora crassa genome (MNCDB), the database of complete cDNAs (German Human Genome Project, NGFN), the database of mammalian protein-protein interactions (MPPI), the database of FASTA homologies (SIMAP), and the interface for the fast retrieval of protein-associated information (QUIPOS). The Arabidopsis thaliana database, the rice database, the plant EST databases (MATDB, MOsDB, SPUTNIK), as well as the databases for the comprehensive set of genomes (PEDANT genomes) are described elsewhere in the 2003 and 2004 NAR database issues, respectively. All databases described, and the detailed descriptions of our projects can be accessed through the MIPS web server (http://mips.gsf.de).

/pdf/mips-analysis-and-annotation-of-proteins-from-whole-genomes-4nsura83qm.pdf

MIPS: analysis and annotation of proteins from whole genomes

Structure of von Willebrand factor cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura

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

/pdf/the-status-quality-and-expansion-of-the-nih-full-length-cdna-1gka4v68fm.pdf

The status, quality, and expansion of the NIH full-length cDNA project: The Mammalian Gene Collection (MGC)

With the complete human genomic sequence being unraveled, the focus will shift to gene identification and to the functional analysis of gene products. The generation of a set of cDNAs, both sequences and physical clones, which contains the complete and noninterrupted protein coding regions of all human genes will provide the indispensable tools for the systematic and comprehensive analysis of protein function to eventually understand the molecular basis of man. Here we report the sequencing and analysis of 500 novel human cDNAs containing the complete protein coding frame. Assignment to functional categories was possible for 52% (259) of the encoded proteins, the remaining fraction having no similarities with known proteins. By aligning the cDNA sequences with the sequences of the finished chromosomes 21 and 22 we identified a number of genes that either had been completely missed in the analysis of the genomic sequences or had been wrongly predicted. Three of these genes appear to be present in several copies. We conclude that full-length cDNA sequencing continues to be crucial also for the accurate identification of genes. The set of 500 novel cDNAs, and another 1000 full-coding cDNAs of known transcripts we have identified, adds up to cDNA representations covering 2%–5 % of all human genes. We thus substantially contribute to the generation of a gene catalog, consisting of both full-coding cDNA sequences and clones, which should be made freely available and will become an invaluable tool for detailed functional studies.

[The sequence data described in this paper have been submitted to the EMBL database under the accession nos. given in Table ​Table22.]



Table 2

Functional Classification of Individual cDNAsa






The recent past has witnessed major advances in the determination of the sequence of the human genome (Dunham et al. 1999; Hattori et al. 2000). Although the whole genomic sequence will be completely unraveled in the near future (Collins et al. 1998), the identification of genes and the deciphering of gene structures will extend for a prolonged time, and cDNA sequences will continue to be invaluable tools for this adventure, especially in view of alternative splicing. The primary focus will shift to the functional analysis of the genes and their protein products to finally understand the molecular basis of human life. Current estimates vary between 29,000 and >70,000 genes to constitute the protein coding repertoire of the human genome (Fields et al. 1994; Ewing and Green 2000; Liang et al. 2000; Roest Crollius et al. 2000). However, thus far only some 11,000 cDNA sequences have been deposited in public databases, which are supposed to contain the complete protein coding open reading frame (ORF). The majority of the respective cDNA clones are most likely not accessible. The generation of a physical clone set representing all human genes that should be made freely accessible is consequently regarded to have an extremely high impact (Schuler 1997; Pruitt et al. 2000). This would permit the establishment of a catalog of clones to provide the resources needed in the proteomics era where the functions of proteins, their action in pathways, and the possible disease relation are deciphered.

Until recently, the long-cDNA sequencing project carried out at the Kazusa Institute (Nomura et al. 1994; Nagase et al. 2000) Consortium had been the only systematic full-length cDNA sequencing project with a significant output of novel sequence information. The initiation of a new large-scale cDNA sequencing project has been announced lately that is coordinated by the National Institute of Health (Strausberg et al. 1999). We founded a cDNA Consortium in 1997 as part of the German Genome Project and aim at the characterization of the complete sequences of novel human transcripts at the cDNA level.

Here, we report the sequences and analysis of 500 novel human cDNAs that all contain the complete protein coding region. These cDNAs constitute the most valuable essence of 30,000 clones that have been EST sequenced and 3630 fully sequenced cDNAs. Over 1000 cDNAs that cover the complete coding sequence of already known transcripts have been identified in the EST-sequenced clone set. All clones are made available through the Resource Center of the German Genome Project (RZPD).

/pdf/toward-a-catalog-of-human-genes-and-proteins-sequencing-and-40jfrwnhnt.pdf

Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs

With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis Gene and ORF coverage with full-length ORF clones thus needs to be extended In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen) A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci 177 splice variants were cloned representing 121 of these genes The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community

https://link.springer.com/content/pdf/10.1186%2F1471-2164-8-399.pdf

The full-ORF clone resource of the German cDNA Consortium

Several disorders of the small intestine are associated with disturbances in villus architecture. Thus, an understanding of the molecular mechanisms associated with the differentiation of villi represents an important step in the improvement of the understanding of small intestinal pathology. Screening of antibodies from a hybridoma library led to the identification of an acyl-CoA synthetase 5-specific monoclonal antibody. Protein synthesis, mRNA expression, and the enzyme activity of acyl-CoA synthetase 5 were studied by several methods in human small intestinal tissues with Crohn's disease or coeliac disease, respectively. Acyl-CoA synthetase 5 mRNA and protein levels were substantially reduced in injured small intestinal mucosa. Moreover, impaired synthesis of the acyl-CoA synthetase 5 protein was reflected by a decrease in intramucosal enzyme activity. Subtle changes of the acyl-CoA synthetase 5 pattern correlate with conversion of intestinal epithelial cells to a gastric phenotype. These results suggest that deranged acyl-CoA synthetase 5 expression, synthesis, and activity are closely related to the state of villus architecture and epithelial homeostasis in human small intestine.

Birgit Ottenwälder

Papers

Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs

The full-ORF clone resource of the German cDNA Consortium

Expression of acyl-CoA synthetase 5 reflects the state of villus architecture in human small intestine.