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| 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. |
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