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110 Dateproducts prevents the understanding of the biology of the organisms indetail, a fact that becomes even more important when dealing with genomesof pathogenic organisms. In this regard, use of several recently introducedcomputational functional genomics methods is proving beneficial, especiallyin assigning function to genes that are difficult to characterize using homology-basedmethods alone. Herein, the implementation of two such in silicomethods—phylogenetic profiling (2,3) and identification of Rosetta stonesequences (4) is discussed, which can be used to assign function to geneproducts based on their linkages with proteins of known function.Phylogenetic profiling involves generating presence/absence profiles of proteinswith reference to a set of fully sequenced genomes. Matching profiles areindicative of functional protein–protein interactions between the correspondingentities, with functional interactions being defined as associations that canrange from direct physical contact to shared membership in the same pathwayor cellular system (2). Identification of Rosetta stone links is another means ofestablishing functional associations between protein entities. The method wasdeveloped based on the observation that independently transcribed and translatedproteins sometimes appear together as a fused protein, either in the sameorganism, or in the genome of some other organism (4). The presence of fusionproteins is likely to indicate a strong functional linkage between independentcandidates, suggesting that the pathways they are a part of are proximateenough for the occurrence of a dual function protein.Functional associations suggested by the methods describe protein–proteinrelationships, which can be used to assign putative function to uncharacterizedproteins. If a function, or more commonly, members, of a particularpathway appear to be overrepresented in a set of linked proteins, it is highlylikely that the query protein either performs a similar function, or is directlyor indirectly linked to the particular pathway. Functional linkages obtainedusing phylogenetic profile data have helped identify new pathways (5) andunderstand patterns of evolution and conservation (6,7). Similarly, besideselucidating functional relationships, Rosetta stone linkage data has beenused to reconstruct metabolic pathways in Escherichia coli (4), and has beencombined with other experimental and computational functional genomicsdata sets to generate genome-wide interaction maps of high confidence inother organisms (8,9).Protocols for constructing phylogenetic profiles and identifying Rosetta stonelinks are described below (see Methods). It is important to note that their implementationrequires the ability to write and execute computer programs, including somethat involve the creation of complex logical structures. Proficiency in computer programmingis therefore assumed, as is the knowledge of basic local alignment search