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Estimating Protein Function Using Protein–Protein Relationships 123P. falciparum proteins were included in the input file, and the results indicatedfunctional linkages between 993 unique proteins.The output contains information about the protein pair linked by thefusion protein, along with information about the start and stop coordinates ofthe similarity span between the queries and the database hit. “A” and “B”represent the linked proteins, whereas “RS” indicates the Rosetta stonesequence that links the proteins together. The program can be coded to producethis or any other form of output. In the author’s experience, multipleforms of the output that capture different aspects of the fusion are helpful inorganizing information. For instance, the output arranged in the followingmanner allows the user to comprehend at a glance the extent of detectedfusions:Together, the different forms of output allow efficient analysis of the results.Quality of the results can be further enhanced and the occurrence of false-positivescan be reduced by incorporating as filters, more features associated withthe HSPs. See Note 6 for additional information about enhancing result qualityand choosing possible filters.3.2.2.2. TESTING CONFIDENCE OF THE ROSETTA LINKAGESTo ensure the absence of errors, it is important to check the output of theprogram using known examples of fusion proteins. One well-known exampleof a fusion that can be used for testing data quality is the dihydrofolatereductase thymidylate synthase (DHFR-TS) protein in P. falciparum, whichrepresents a fusion of the independently encoded dihydofolate reductase andthymidylate synthase in humans, or the yeast topoisomerase II protein,which links the E. coli proteins gyrA and gyrB. Any implementation of themethod should be able to correctly identify functional links and fusion proteins,when the earlier examples are used for testing.Although most functional links identified by this method are accurate, itis likely that some false-positives will be included in the result set, especiallywhen dealing with genomes of higher-order eukaryotes. It is therefore bestto statistically determine the validity of the results, such as by using a testFig. 1. (Continued) are likely to reveal previously unknown pathways or cellular systems.P. falciparum functional linkages derived using the Rosetta stone method are available fordownload from the plasmoMAP website (http://cbil.upenn.edu/plasmoMAP/) (8). Thenetwork was generated using the LGL package (19). Some independent clusters arerepositioned for clarity.