ISOLATION OF CDNAS FOR PROTEIN INTERACTION NETWORKS

实现国家人类基因组计划识别ALL目标的一种方法 人类基因已经被用来分离和测序表达序列或cDNA。 CDNA通常是根据它们在 特定的组织，或它们在一个环境中的差异表达 不是另一个，但不是基于功能特征。完毕 时间，这种方法无疑将产生重大的新见解 基因的功能。然而，最初这一努力只会提供 每个新基因的序列。一种更传统的方法，持续了多年 在许多实验室中，一直在识别一个或一小部分基因 被认为参与了特定的细胞过程，然后投资 它们的功能所需的、通常是相当长的时间 在正常和疾病状态下。与基因组的目标相反 项目。，这种方法缓慢地识别新的基因。在项目中 在这里提出的，新的DNA将被快速和连续地分离使用 一种技术，它将提供有关每个已编码的 蛋白。因为细胞中的许多调节过程是由 相互作用的蛋白质网络，编码成员的新cDNA 这些网络将基于蛋白质之间的相互作用而被隔离。一个 高效的双杂交系统将被重复使用来分离新的DNA 对这些网络成员进行编码将被分离出新的基于 它们编码的蛋白质与其他蛋白质相互作用的能力。 从一小部分被认为在蛋白质中起作用的蛋白质开始 相互作用网络，新的DNA然后将被用于后续的狩猎 寻找其他新的cDNA，这个过程将继续分离cDNA 对于网络中的所有成员。该方法将提供新的cDNA 以一种易于通过测序和测试来表征的形式 编码的蛋白质是否可以与一组 400个已知蛋白质的快速相互作用交配试验。这 他们所属的信息网络。该方法提供了一种方法来 快速识别大量新的cDNA，例如在 基因组计划。所提出的方法的效率将超过1000 以这种方式分离并以一次全职和一次 兼职工人。此外，在这个项目的初始阶段， 将进一步探索该方法放大的可行性。

One approach to the National Human Genome Project goal of identifying all human genes has been to isolate and sequence expressed sequences or cDNAs. The cDNAs have generally been isolated on the basis of their expression in a particular tissue, or their differential expression in one setting and not another, but not on the basis of functional characteristics. Over time, this approach will undoubtedly yield significant new insights into gene function. Initially , however, this endeavor will only provide the sequence of each new gene. A more traditional approach, ongoing for years in many laboratories, has been to identify one or a small set of genes presumed to be involved in a specific cellular process, and then to invest the necessary, and often considerable, time characterizing their function in normal and disease states. In contrast to the goals of the Genome Project., this approach identifies new genes slowly. In the project proposed here, new cDNAs will be rapidly and successively isolated using a technique that will provide information about the function of each encoded protein. Because many regulatory processes in a cell are controlled by networks of interacting proteins, new cDNAs that encode the members of these networks will be isolated based on protein-protein interactions. An efficient two-hybrid system will be used repeatedly to isolate new cDNAs that encode the members of these networks will be isolate new cDNAs based on the ability of their encoded proteins to interact with other proteins. Beginning with a small set of proteins thought to function in protein interaction networks, new cDNAs will then be used in subsequent hunts to find other new cDNAs, and this process will be continued to isolate cDNAs for all of th members of a network. The method will provide the new cDNAs in a form that can be readily characterized by sequencing, and by testing whether the encoded proteins can interact with any members of a panel of 400 known proteins using a rapid interaction mating assay. This information networks they belong to. The approach provides a way to rapidly identify large numbers of new cDNAs such as those identified in the Genome Project. The efficiency of the method proposed will allow over 1000 cDNAs to be isolated and characterized in this way by one full-time and one part-time worker. Moreover, during the initial phase of this project, the feasibility of scaling-up the method further will be explored.