The full-ORF clone resource of the German cDNA Consortium

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing.

Unbiased deep sequencing offers the potential for improved adventitious virus screening in vaccines and biotherapeutics. Successful implementation of such assays will require appropriate control materials to confirm assay performance and sensitivity. A common reference material containing 25 target viruses was produced and 16 laboratories were invited to process it using their preferred adventitious virus detection assay. Fifteen laboratories returned results, obtained using a wide range of wet-lab and informatics methods. Six of 25 target viruses were detected by all laboratories, with the remaining viruses detected by 4-14 laboratories. Six non-target viruses were detected by three or more laboratories. The study demonstrated that a wide range of methods are currently used for adventitious virus detection screening in biological products by deep sequencing and that they can yield significantly different results. This underscores the need for common reference materials to ensure satisfactory assay performance and enable comparisons between laboratories

Expression Profiling Of Drosophila Mitochondrial Genes Via Deep Mrna Sequencing.

Mitochondria play an essential role in several cellular processes. Nevertheless, very little is known about patterns of gene expression of genes encoded by the mitochondrial DNA (mtDNA). In this study, we used next-generation sequencing (NGS) for transcription profiling of genes encoded in the mitochondrial genome of Drosophila melanogaster and D. pseudoobscura. The analysis of males and females in both species indicated that the expression pattern was conserved between the two species, but differed significantly between both sexes. Interestingly, mRNA levels were not only different among genes encoded by separate transcription units, but also showed significant differences among genes located in the same transcription unit. Hence, mRNA abundance of genes encoded by mtDNA seems to be heavily modulated by post-transcriptional regulation. Finally, we also identified several transcripts with a noncanonical structure, suggesting that processing of mitochondrial transcripts may be more complex than previously assumed.377509-1

Gene expression profiling by massively parallel sequencing

Massively parallel sequencing holds great promise for expression profiling, as it combines the high throughput of SAGE with the accuracy of EST sequencing. Nevertheless, until now only very limited information had been available on the suitability of the current technology to meet the requirements. Here, we evaluate the potential of 454 sequencing technology for expression profiling using Drosophila melanogaster. We show that short (< ∼80 bp) and long (> ∼300–400 bp) cDNA fragments are under-represented in 454 sequence reads. Nevertheless, sequencing of 3′ cDNA fragments generated by nebulization could be used to overcome the length bias of the 454 sequencing technology. Gene expression measurements generated by restriction analysis and nebulization for fragments within the 80- to 300-bp range showed correlations similar to those reported for replicated microarray experiments (0.83–0.91); 97% of the cDNA fragments could be unambiguously mapped to the genomic DNA, demonstrating the advantage of longer sequence reads. Our analyses suggest that the 454 technology has a large potential for expression profiling, and the high mapping accuracy indicates that it should be possible to compare expression profiles across species

Protein engineering of lantibiotics

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases he a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibioties were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.

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

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 2.