Functional analysis of conserved hypothetical proteins in prokaryotes

目前已经完成了100多种细菌基因组的测序，但其中约三分之一的基因没有已知的功能。我建议通过系统的双杂交筛选来识别梅毒螺旋体的1,000个蛋白质之间的所有物理相互作用来解决这个问题。未表征蛋白质的相互作用将提示这些蛋白质的可能功能。虽然苍白球T编码了750种蛋白质，这些蛋白质在其他细菌中是保守的，但我建议把重点放在-180种保守的假设蛋白质上。我们的筛选不仅将提供对这些蛋白质的第一个功能见解，而且这些功能信息可以自信地转移到更容易处理的模型物种(如E。大肠杆菌，我们的预测可以通过实验验证，例如通过产生突变。T. pallidum的基因组比E. pallidum的基因组小四倍。杆菌. .因此，我们的项目将比大肠杆菌的可比项目小一个数量级以上，因为实验的数量随着基因的数量呈指数增长。在我们的。使用几种鞭毛蛋白的实验室已经表明，所提出的策略(1)高效且信息丰富，(2)提供了表征和非表征蛋白质的新信息，(3)做出正确的功能（实验可验证）预测，(4)将设计出可以应用于其他基因组的技术，包括真核生物基因组。

Currently more than 100 bacterial genomes have been completely sequenced but about one third of their genes have no known function. I suggest to address this problem by identifying all physical interactions among the -1,000 proteins of Treponema pattidum by systematic two-hybrid screens. Interactions of uncharacterized proteins will suggest possible functions for these proteins. Although T, pallidum encodes about 750 proteins which are conserved in other bacteria, I suggest to concentrate on -180 conserved hypothetical proteins. Our screen will not only provide first functional insights into these proteins but this functional information can be confidently transferred to homologous proteins in more tractable model species such as E.. coli, in which our predictions can be experimentally verified, e.g. by generating mutations. The genome of T. pallidum is four times smaller, than the genome of E.. coli.. Thus our project will be more than one order of magnitude smaller than a comparable project in E. coli because the number of experiments grows exponentially with the number of genes. Pilot screens in our. laboratory using several flagellar proteins have shown that the proposed strategy is (1) highly efficient and informative, (2) provides novel information on both characterized and uncharacterized proteins, (3) makes correct functional (experimentally verifiable) predictions, and (4) will devise technologies that can be applied to other genomes as well, including eukaryotic ones.