Nagasaki University
Home>Activities>Seminar>The 16th Seminar of the Global Strategic Center for Radiation Health Risk
The 16th Seminar

Application of Chemical Genetics Approach to DT40 Cells

Professor Shunichi Takeda
Kyoto University

The chicken B lymphocyte line DT40 is widely used for reverse genetics, because the cells exhibit targeted integration with efficiencies that are orders of magnitude higher than those observed in any mammalian cells. I will present two topics about the combined application of chemistry and genetics to studies using DT40 cells.

The first topics is the phenotypic analysis of cyclin-dependent kinase (CDK). Deletion of CDK is lethal to cells. Moreover, the function of CDK varies during the cell cycle. Thus, to understand the role of CDK in a given phase of the cell cycle, you need to quickly inactivate CDK. Vertebrate Cdk1 is required for initiation of mitosis, while S phase functions of this kinase remain unclear. We generated chicken DT40 mutants, in which an analog sensitive mutant cdk1as replaces the endogenous Cdk1, allowing us to specifically inactivate Cdk1 using bulky ATP analogs. We found that Cdk1 activity is essential for initiation of DNA replication and centrosome duplication in cells lacking Cdk2. The presence of a single Cdk2 allele rendered S-phase progression independent of Cdk1, suggesting a complete overlap of these kinases in S-phase control. Moreover, we found that inhibition of Cdk1 did not induce re-licensing of replication origins in G2 phase. Conversely, inhibition of Cdk1 during mitosis caused rapid activation of endoreplication, depending on proteolysis of the licensing inhibitor geminin. This study demonstrates essential functions of Cdk1 in the control of S-phase, and exemplifies a chemical genetics approach to target cyclin dependent kinases in vertebrate cells.

The second talk is about our study to introduce a new genetic toxicity based bioassay for common environmental contaminants using mutant chicken DT40 B cell lines deficient of various DNA repair pathways. To detect the genotoxicity of chemicals, the Ames test, a bacterial reverse mutation assay is most frequently used, though it was developed more than 40 years ago. We established a new screening method by comparing the growth kinetics between wild-type and DNA repair deficient mutants at 48 hours exposure. To optimize this method, we analyzed cellular sensitivity to killing by _-ray and ultraviolet light (UV) as positive control, and compared to conventional colony formation assay. We then collaborate with Chemical Genomics Center and set up high-throughput screening to detect genotoxicity of a large number of chemicals. I will show data that demonstrate that our genetic toxicology based bioassay technique is useful to efficiently detect genotoxicity of chemicals including common environmental contaminants and their mechanisms of action. It can also be used for a high throughput-screening tool for environmental genotoxicants as well as new chemicals with potential industrial applications.
  International Radiation Health Sciences Research
    Atomic Bomb Disease Medicine Research
    Radiation Basic Life Sciences Research
    Workshop / Special Meeting
    Academic Exchange
    Reports on Overseas' Conferences and Meetings
    Cooperation with WHO
Publicity Activities
  Truth of radiology (in Japanese)
  Radiation Q&A (in Japanese)
    Introduction of Global Strategic Center for Radiation Health Risk Control
(in Japanese) [PDF 9MB]
    Visiting Report of Chelnobyl
Yasuyuki Taira, Graduate Student
Related Links