Computational analyses of biological sequences -applications to antibody-based proteomics and gene family characterization
Following the completion of the human genome sequence, post-genomic efforts have shifted the focus towards the analysis of the encoded proteome. Several different systematic proteomics approaches have emerged, for instance, antibody-based proteomics initiatives, where antibodies are used to function...
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| Format: | Doctoral Thesis |
| Language: | English |
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KTH, Skolan för bioteknologi (BIO)
2005
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| Online Access: | http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-527 http://nbn-resolving.de/urn:isbn:91-7178-186-2 |
| Summary: | Following the completion of the human genome sequence, post-genomic efforts have shifted the focus towards the analysis of the encoded proteome. Several different systematic proteomics approaches have emerged, for instance, antibody-based proteomics initiatives, where antibodies are used to functionally explore the human proteome. One such effort is HPR (the Swedish Human Proteome Resource), where affinity-purified polyclonal antibodies are generated and subsequently used for protein expression and localization studies in normal and diseased tissues. The antibodies are directed towards protein fragments, PrESTs (Protein Epitope Signature Tags), which are selected based on criteria favourable in subsequent laboratory procedures. This thesis describes the development of novel software (Bishop) to facilitate the selection of proper protein fragments, as well as ensuring a high-throughput processing of selected target proteins. The majority of proteins were successfully processed by this approach, however, the design strategy resulted in a number ofnfall-outs. These proteins comprised alternative splice variants, as well as proteins exhibiting high sequence similarities to other human proteins. Alternative strategies were developed for processing of these proteins. The strategy for handling of alternative splice variants included the development of additional software and was validated by comparing the immunohistochemical staining patterns obtained with antibodies generated towards the same target protein. Processing of high sequence similarity proteins was enabled by assembling human proteins into clusters according to their pairwise sequence identities. Each cluster was represented by a single PrEST located in the region of the highest sequence similarity among all cluster members, thereby representing the entire cluster. This strategy was validated by identification of all proteins within a cluster using antibodies directed to such cluster specific PrESTs using Western blot analysis. In addition, the PrEST design success rates for more than 4,000 genes were evaluated. Several genomes other than human have been finished, currently more than 300 genomes are fully sequenced. Following the release of the tree model organism black cottonwood (Populus trichocarpa), a bioinformatic analysis identified unknown cellulose synthases (CesAs), and revealed a total of 18 CesA family members. These genes are thought to have arisen from several rounds of genome duplication. This number is significantly higher than previous studies performed in other plant genomes, which comprise only ten CesA family members in those genomes. Moreover, identification of corresponding orthologous ESTs belonging to the closely related hybrid aspen (P. tremula x tremuloides) for two pairs of CesAs suggest that they are actively transcribed. This indicates that a number of paralogs have preserved their functionalities following extensive genome duplication events in the tree’s evolutionary history. === QC 20101021 |
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