Daily Biological Clocks Circadian Rhythms circa about dies one day

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Daily Biological Clocks Circadian Rhythms
(circa about dies one day)
Properties of Circadian Rhythms

• They continue to oscillate in constant
  conditions with a period of approximately 24
  hours (therefore, there is an endogenous
  biological clock inside of us).
• These internal clocks run at about the same speed
  at different temperatures, even in cold-blooded
  animals and plants.
• Normally, these internal clocks are synchronized
  to the daily light/dark cycle of exactly 24 hours.

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Cell Division Cycles versus Circadian Cycles
The crucial difference between these biological
timing circuits
The cdc must wait for the completion of DNA
repair and replication, and for adequate
growth. Therefore checkpoints are essential
On the other hand, the purpose of the circadian
cycle is to provide an internal estimate of
external time. Therefore, the circadian clock
cant wait (the sun waits for nothing), but must
be a precise, self-sustained oscillator that is
relatively unaffected by what is happening in the
cell
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Circadian control of the timing of cell division
in two unicellular organisms
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Numerous tissues show daily rhythms of DNA
synthesis in mice with different peak times
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Sensitivity to drugs, poisons, and chemotherapy
exhibits daily rhythmicity
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Principle of Circadian Chemotherapy
Timed treatment
Timing of treatment may allow fewer side effects,
thereby lessening discomfort and allowing
higher dosage. Timed chemotherapy can be
administered automatically using programmable, ind
welling pumps.
side effects
sensitivity to drug
Time of day
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Dissecting the ClockworkIsolation of Circadian
Clock Genes
From mutagenesis screens
Other methods

• Drosophila per, tim
• Neurospora frq
• Cyanobacteria kaiA, kaiB, kaiC
• Arabidopsis toc1, ztl
• Mice mClock

• Homology to fly per mPer1, mPer2, mPer3
• Homology to plant cryptochromes mCry1, mCry2
• From protein interaction assays Bmal1

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Mutagenesis Screening Using the Simplest
System Cyanobacteria
Advantages of the System

• Unicellular bacteria that are easy to grow in
  liquid culture or as colonies on agar plates
• Prokaryotic gene organization single circular
  chromosome
• Genome is smaller than that of E.coli
• Mutations can be easily complemented by
  conjugation with E.coli or by targeting
  constructs to neutral sites on the chromosome
  by homologous recombination
• Luciferase reporter system for gene expression
  patterns

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Cyanobacterial Rhythm of Luminescence
Luminescence
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Luminescence Rhythm is Independent of Growth
Phase Circadian Rhythm of Cell Division
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Overexpression of FtsZ inhibits division, but
does not affect the circadian clock
Luminescence
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Over 150 different period mutants
of cyanobacteria have been isolated by
luminescence rhythm screening.
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So, how do these clocks tick?Conceptual models
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Cyanobacterial Clockworks Model circa-1998
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Mammalian Central Clock Proteins
Positive Elements CLOCK, BMAL1
Negative Elements PER1, PER2, CRY1, CRY2
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Mammalian Clockwork
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Phototransduction Path to the SCN
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The Current Model of the Mammalian Clock. . .

• CAN EXPLAIN
• a closed loop biochemical circuit
• the path by which light signals set the phase of
  the clock

• CANNOT EXPLAIN
• temperature compensation
• why and how the circadian clock can have such a
  long period (24 h) and still be very precise

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Luminescence
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Rhythms of gene expression in fibroblasts in vitro
5 serum
Fibroblast cells at 100 confluence
50 serum
for 2 h to synchronize the circadian clocks
within the cells in the population
0 serum
mRNA collection over a time course
RNase Protection assay
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Rhythms of gene expression in fibroblasts in vitro
0 1 4 8 12 16 20 24 28 32 36 40 44 48 52 56
60646872
Rev- Erb?
DBP
rPer2
TBP
Balsalobre, A. et al. (1998) Cell 93, 929