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Takao Kondo Professor |
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Takao Kondo (Professor)
Physiology of circadian clocks
Tokitaka Oyama (Assistant Professor)
Molecular genetics of circadian clocks
Yoko Kitayama (Assistant Professor)
Physiology of circadian clocks |
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Most organisms on the earth live by the 24-hour day night cycle,
although human beings might be somewhat of an exception. The chronobiology
laboratories aim to understand how organisms adapt to this environmental
alteration. People always wear wristwatches. If you confirm how dependent
you are on your watch, the merit of having a clock should be obvious.
Therefore, it is also evident that animals, plants, and bacteria must
adjust to the time of day. It is thought that living organisms have
evolved a cellular clock mechanism of approximately 24-hour period
(the circadian clock).
To understand the molecular mechanisms of the circadian clock and
how it makes the lives of organisms efficient, we have been studying
cyanobacteria, the simplest organism to have the circadian clock.
We are also studying the photoperiodic flower induction of a higher
plant, duckweed. Cyanobacteria are photoautotrophic organisms. Among
them, Synechococcus elongatus PCC7942 stain is amenable to
genetic engineering. We introduced a bacterial luciferase reporter
system into this cyanobacterium so that it would be possible to monitor
gene expression by bioluminescence. Using forward molecular genetic
approaches, we cloned three clock genes, kaiA, kaiB, and kaiC,
that encode a central component of the cyanobacterial clock.
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| Fig1. KaiA, KaiB, KaiC, and ATP were mixed
in a test tube and KaiC was analyzed by SDS-gel electrophoresis.
The phosphorylation of KaiC appeared as dual bands that changed
over a 24-hour period. |
How do the three proteins, KaiA, KaiB, and KaiC, that are encoded
by the kai genes tell time? Surprisingly, we were able to reconstitute
stable circadian oscillation by mixing the three proteins and ATP
(Fig. 1). KaiB and KaiA regulate the phosphorylation state of KaiC
over a 24-hour period even in a test tube. The KaiC phosphorylation
cycle regulates the gene expression of the cyanobacteria. We would
like to address the molecular mechanisms that generate the oscillation
and how the phosphorylation cycle regulates the cellular metabolism
(Fig. 2).
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| Fig.2 KaiA, KaiB, KaiC, and ATP were mixed
in a test tube and KaiC was analyzed by SDS-gel electrophoresis.
The phosphorylation of KaiC appeared as dual bands that changed
over a 24-hour period. |
Another interesting point of the cyanobacterial clock system is that
the circadian clock regulates almost all the genes in the genome.
To address the mechanism of global regulation by the clock and to
understand cellular physiology as a temporally integrated system,
we analyzed gene expression using DNA chips and real-time monitoring
of whole sets of individual gene.
As cyanobacteria are simple prokayotes, studies of the circadian clock
can be more easily advanced than in most of the higher organisms.
At the same time, these studies are important for understanding the
molecular mechanisms in higher eukaryotes, because the principles
of circadian clocks should be consistent, while the clock system proteins
seem to differ. Additionally, studies of duckweed will contribute
to understanding the molecular mechanisms of photoperiodism, a protocol
that allows living organisms to choose the best season for reproduction.
Most of the research in our laboratory is molecular biology. However,
physiological approaches with custom-made automated assay systems
are also essential for our research. Collaboration with physics, chemistry,
mathematics, and system engineering groups will be also important.
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| Fig3. Monitoring the clock gene expression
by bio luminescence |
Fig4. Sampling from a continuous culture of
cyanobacteria |
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References
- Ishiura, M. et al. (1998) Science 281: 1519-1523
- Kitayama Y. et al. (2003) EMBO J 22: 2127-2134
- Nakahira Y. et al. (2004) Proc. Natl. Acad. Sci. 101:881-5
- Nishiwaki T et al. (2004) Proc. Natl. Acad. Sci. 101: 13927-32
- Tomita, J, et al. (2005) Science 307: 251-254
- Nakajima M, et al. (2005) Science 308, 414-5
- Kageyama T, et. al. (2006) Mol. Cell 23:161-171
- Takai, N.,et. al., (2006) Proc. Natl. Acad. Sci. 103: 12109-12114
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i n d e x |
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 Biological
Rhythm Group (Chronobiology)
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Molecular
Neurobiology Group