Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens
Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens
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DOI：
10.1038/ng0797-277
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发表时间：
1997-07-01
期刊：
影响因子：
30.8
通讯作者：
Legrain, P
中科院分区：
文献类型：
--
作者：
FromontRacine, M;Rain, JC;Legrain, P
酿酒酵母的基因组现已完全测序1。尽管近年来成功的遗传工作，60%的酵母基因没有指定的功能，其中一半编码与已知蛋白质没有任何同源性的推定蛋白质2。基因分析，如抑制或合成致死筛选，已经提出了许多基因产物之间的功能联系，其中一些已被生物化学手段证实。总而言之，这些方法导致了相当广泛的知识定义的生化途径。然而，在活细胞中的复杂性的背景下，这些途径的整合仍有待完成。应用于酵母基因组的双杂交方法可能允许表征酵母蛋白质之间的相互作用网络，从而更好地理解细胞功能3。这样的分析已经进行了噬菌体T7基因组编码55蛋白4和果蝇细胞周期调节剂5。然而，如果没有具体的方法改进，目前可用的双混合方法不适合大规模项目。特别是，必须首先大大提高筛选的穷尽性和选择性。我们构建了一个新的酵母基因组文库，并开发了一个高选择性的双杂交程序，适用于酵母基因组的穷举筛选。对于每个诱饵，我们选择了一组有限的相互作用的猎物，我们分类在不同的启发式值的类别。考虑到这一分类，在猎物中选择新的诱饵，并反过来用于第二轮筛选。重复这一过程几次，就得到了一个相互作用网络的特征。使用已知的前mRNA剪接因子作为初始诱饵，我们能够表征已知剪接因子之间的新相互作用，鉴定新的酵母剪接因子，包括人SF1和SAP 49的同源物，并揭示细胞通路之间的新的潜在功能联系。使用不同的细胞通路作为锚点，这种新的策略使我们能够设想构建酵母蛋白质组的相互作用图谱。此外，这种双杂交策略可以应用于其他基因组，并可能有助于解决人类蛋白质连锁图谱。
The genome of the yeast Saccharomyces cerevisiae is now completely sequenced1. Despite successful genetic work in recent years, 60% of yeast genes have no assigned function and half of those encode putative proteins without any homology with known proteins2. Genetic analyses, such as suppressor or synthetic lethal screens, have suggested many functional links between gene products, some of which have been confirmed by biochemical means. Altogether, these approaches have led to a fairly extensive knowledge of defined biochemical pathways. However, the integration of these pathways against the background of complexity in a living cell remains to be accomplished. The two-hybrid method applied to the yeast genome might allow the characterization of the network of interactions between yeast proteins, leading to a better understanding of cellular functions3. Such an analysis has been performed for the bacteriophage T7 genome that encodes 55 proteins4and forDrosophilacell cycle regulators5. However, the currently available two-hybrid methodology is not suitable for a large-scale project without specific methodological improvements. In particular, the exhaustivity and selectivity of the screens must first be greatly improved. We constructed a new yeast genomic library and developed a highly selective two-hybrid procedure adapted for exhaustive screens of the yeast genome. For each bait we selected a limited set of interacting preys that we classified in categories of distinct heuristic values. Taking into account this classification, new baits were chosen among preys and, in turn, used for second-round screens. Repeating this procedure several times led to the characterization of a network of interactions. Using known pre-mRNA splicing factors as initial baits, we were able to characterize new interactions between known splicing factors, identify new yeast splicing factors, including homologues of human SF1 and SAP49, and reveal novel potential functional links between cellular pathways. Using different cellular pathways as anchor points, this novel strategy allows us to envision the building of an interaction map of the yeast proteome. In addition, this two-hybrid strategy could be applied to other genomes and might help to resolve the human protein linkage map.