Kroll E S, Hyland K M, Hieter P, Li J J
Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205, USA.
Genetics. 1996 May;143(1):95-102. doi: 10.1093/genetics/143.1.95.
We have devised a genetic screen, termed synthetic dosage lethality, in which a cloned "reference" gene is inducibly overexpressed in a set of mutant strains carrying potential "target" mutations. To test the specificity of the method, two reference genes, CTF13, encoding a centromere binding protein, and ORC6, encoding a subunit of the origin of replication binding complex, were overexpressed in a large collection of mutants defective in either chromosome segregation or replication. CTF13 overexpression caused synthetic dosage lethality in combination with ctf14-42 (cbf2, ndc10), ctf17-61 (chl4), ctf19-58 and ctf19-26. ORC6 overexpression caused synthetic dosage lethality in combination with cdc2-1, cdc6-1, cdc14-1, cdc16-1 and cdc46-1. These relationships reflect specific interactions, as overexpression of CTF13 caused lethality in kinetochore mutants and overexpression of ORC6 caused lethality in replication mutants. In contrast, only one case of dosage suppression was observed. We suggest that synthetic dosage lethality identifies a broad spectrum of interacting mutations and is of general utility in detecting specific genetic interactions using a cloned wild-type gene as a starting point. Furthermore, synthetic dosage lethality is easily adapted to the study of cloned genes in other organisms.
我们设计了一种基因筛选方法，称为合成剂量致死性筛选，即在一组携带潜在“靶标”突变的突变菌株中，可诱导性地过表达一个克隆的“参考”基因。为了测试该方法的特异性，我们在大量染色体分离或复制存在缺陷的突变体中过表达了两个参考基因，一个是编码着丝粒结合蛋白的CTF13，另一个是编码复制起始点结合复合体亚基的ORC6。CTF13过表达与ctf14 - 42（cbf2，ndc10）、ctf17 - 61（chl4）、ctf19 - 58和ctf19 - 26组合时会导致合成剂量致死性。ORC6过表达与cdc2 - 1、cdc6 - 1、cdc14 - 1、cdc16 - 1和cdc46 - 1组合时会导致合成剂量致死性。这些关系反映了特异性相互作用，因为CTF13过表达在动粒突变体中导致致死性，而ORC6过表达在复制突变体中导致致死性。相比之下，仅观察到一例剂量抑制情况。我们认为，合成剂量致死性筛选可识别广泛的相互作用突变，并且以克隆的野生型基因作为起点，在检测特定基因相互作用方面具有普遍用途。此外，合成剂量致死性筛选很容易应用于研究其他生物体中的克隆基因。
