Extra Credit
Due 5 pm, February 26
Exam Questions need to be:
• Multiple choice (a-e)
• Factually correct (no conceptual errors)
• Test understanding (how things work) rather than
memorization; no fill in the blank, no definitions
• Understandable (no long essays, clear writing)
• Must cover a topic discussed in this class (no exam
questions from other classes)
• Must be original (no plagiarism)
Submit 2 questions only, not on the same topic
Last Time
I am not advocating Anorexia!!!
(1) It is true that the average American has a caloric intake
that is too high
(2) It is true that the recommended caloric intake itself is
on the high side
(3) BUT, a caloric reduction of 30% from the
recommendation for a given height is fairly drastic
(4) There would be a cost to reproduction (shunting of
resources away from reproduction and toward maintenance
and longevity—ie you’d become sterile)
Caloric Restriction
Caloric restriction is one of the few reliable ways to
promote somatic maintenance and increase lifespan
(1) The key is to figure out what physiological pathways
are induced by caloric restriction
(2) There are compounds that mimic caloric restriction
without the cost to reproduction, such as resveratrol
Caloric
Restriction
• The physiological responses and patterns of
gene expression following caloric restriction are
not completely understood
•But there are two general models, in response to
moderate and severe caloric restriction
Activate Sirtuins
Upregulate AMPK and
downregulate TOR genes
•Compounds such as resveratrol mimic DR, but not clear what it is
doing
•There is a debate as to whether resveratrol induces the response of
moderate DR or severe DR
• The more popular view is that it induces the pathway of moderate DR
Activate Sirtuins
Upregulate AMPK and
downregulate TOR genes
• It is known that reducing IGF/insulin signaling
pathway increases longevity
• Caloric restriction appears to reduce
IGF/insulin signaling pathway
• The link between caloric restriction and the
IGF/insulin signaling pathway is not clear
• Not clear why reducing the IGF/insulin
signaling pathway promotes longevity
• The paper that came out yesterday
• A population of short Ecuadorians have a
mutation in a growth hormone receptor
• So that they cannot take up the growth hormone,
and have a deficiency (making them short)
• This hormone makes IGF. IGF/insulin signaling
goes down
• Not clear why reducing the IGF/insulin signaling
pathway promotes longevity
Molecular Regulation of
Circadian Rhythm
Molecular Regulation of
Circadian Rhythm
Example of genes, proteins, and their regulation in a system
Earth Rotation: Photoperiod
Photoperiod: Period from
sunrise to sunset
Circadian Clock
• Definition: an endogenous time-keeping
mechanism, on a roughly 24-h cycle, that
coordinates physiological processes
• The circadian period is generated at the level of
the cell
• Organisms of all phyla show circadian rhythms in
gene expression and physiological processes
Circadian Clock
Free running clock: internal biological oscillator
Entrainment by external environmental cues
(light, temperature, food)
Latitude
Parallel
• Definition: The angular
distance between a
point on the Earth’s
surface and the equator
• Values increase North/South
of Equator
Latitude
• Degrees/Minutes/Seconds.
Maximum value: 90o
Seasonal variation in
photoperiod increases
with greater distance from
the equator
The mammalian pacemaker is located in the
suprachiasmatic nucleus (SCN) of the hypothalamus
Regulatory Control of Circadian Rhythm
Pacemaker cells in the
suprachiasmatic nucleus
in the hypothalamus
Mechanism of circadian clock
regulation for the fly, Drosophila
melanogaster
• At midday, clock (CLK) and cycle
(CYC) proteins induce the
expression of period (PER) and
timeless (TIM)
• However, the PER protein is
rendered unstable by doubletime
(DBT), through phosphorylation
CLK and CYC are
transcription factors
(induce transcription)
Wager-Smith and Kay. 2000 Nature
doubletime (DBT) regulates PER stability
through phosphorylation
In Humans stability of
PER sets the clock
speed
Increased
phosphorylation of period
by doubletime or CK1 (in
humans) destabilizes
period and slows down
the clock, as period
cannot accumulate, and
the clock cannot proceed
as quickly
Increased period stability
speeds up the clock
Wager-Smith and Kay. 2000 Nature
• At the same time, the CLK:CYC
complex represses the CLK promoter
• Once night falls, TIM can accumulate
and interfere with the action of DBT
and allow PER to accumulate
Wager-Smith and Kay. 2000 Nature
•In the middle of the night, PER:TIM
dimers translocate into the nucleus
(oval) and impede the functions of
CLK:CYC (negative feedback)
Wager-Smith and Kay. 2000 Nature
•This brings about a cessation of
PER and TIM mRNA production, and
a simultaneous increase in Clk
expression
• The perception of light at dawn by
cryptochrome (CRY) leads to
degradation of timeless (TIM)
• As the PER:TIM complex declines
in the nucleus and no longer
impedes the activity of the CLK:CYC
complex
• The increase in CLK:CYC then
induces the transcription
(expression) of PER and TIM and
the cycle starts all over
In humans, there are 10 “clock genes”
• Period:
Per1, 2, 3
• Cryptochrome:
Cry1, 2
• BMAL:
Bmal 1
= Brain and Muscle Aryl hydrocarbon receptor nuclear translocator (ARNT)-Like 1
(analogous to Cycle in Drosophila)
• Clock:
Clock
• casein kinase 1 delta/epsilon:
CK1
(analogous to Doubletime in Drosophila)
• Timeless has been identified in mammals, but function is unknown
• Given the mechanisms shown on how circadian
rhythm is regulated, what are some of the ways
in which circadian rhythm could evolve?
• Genetic disorders (or genetic variation) can
offer some examples
• Such “disorders” represent genetic variation
upon which natural selection could act
“Early to bed and early to rise makes
a man healthy, wealthy, and wise.”
Benjamin Franklin
The perception above fails to account for
genetic variation incircadian rhythm in human
and other populations
Genetic Variation in Circadian
Rhythm within Populations
• Delayed Sleep Phase Syndrome
(DSPS; “owls”)
• Advanced Sleep Phase
Syndrome (ASPS; “early birds”)
• Non-24-h sleep–wake syndrome
(N-24)
Advanced Sleep Phase
Syndrome (ASPS)
“Early Bird Syndrome”
• Pacemaker runs too fast, but is still modulated by light
cycle
• Free-running clock is less than 24 hours
• 0.2% of the population go to sleep ~3 h earlier than the
mean (9:30 or earlier)
• Higher rate of depression
Delayed Sleep Phase
Syndrome (DSPS)
“Night-Owl Syndrome”
• Estimated ~4.5% of the population prefer go to sleep ~3h or
later than the mean (3:30 am or later)
• Ontogenetic shift: In humans, 7% of adolescents are thought
to have DSPS
• High incidence of depression, heart disease, is noted when
individuals are forced to comply with a diurnal schedule
• Chronic fatigue in adolescents that are forced to comply with
a diurnal schedule
• Night owls can remain alert longer because of a longer
running clock
Examples of Mutational Targets that
would alter Circadian Rhythm
Mutations that destabilize (phosphorylate) Period will
lengthen the circadian clock (Period takes longer to
accumulate, and the circadian clock slows down)
• Mutations in period at phosphorylating site (where CK1
binds)
• Mutations in CK1 (doubletime in Drosophila) at the site
that phosphorylates period
• Mutations in timeless that prevent it from stabilizing
period
• Mutations that alter transcription of timeless or period
Ebisawa, 2007. Journal of Pharmacological Science. 103:150-154
• Many of the mutations affect the phosphorylation status of
the Period proteins
Advanced Sleep Phase
Syndrome
“Early Bird Syndrome”
Examples of mutations in humans:
• Mutation in the PER2 gene
– Mutation in PER2 gene, at the phosphorylation site within the
casein kinase I (CKI)-binding domain
Serine --> Glycine at the phosphorylation site
(Toh et al. Science 2001)
• Mutation in the Casein Kinase I epsilon (CK1-T44A) gene:
– This enzyme is similar to Drosophila doubletime and phosphorylates
PER. The mutation within CKI decreases enzymatic activity--> more
stable period
• These mutations shorten the circadian cycle
Delayed Sleep Phase
Syndrome
“Night-Owl Syndrome”
Examples of mutations in humans:
• Length variation in the PER3 gene: 75% of individuals
DSPS carry two copies of the “short Per3” gene
• Allelic Variation (Amino Acid substitutions): a particular
allele (H4 haplotype) of the Per3 gene may confer
susceptibility to DSPS in ∼15% of affected people
• Many other mutations found (see Ebisawa), such as in CK1
Ebisawa 2007
Christina Schmidt et al. 2009. Homeostatic sleep pressure and
sustained attention in the suprachiasmatic area. Science. 324:5
http://www.sciencemag.org/cgi/content/full/324/5926/516
• Basic conclusion: Night owls remain alert longer (longer clock)
• Higher performance in Night owls 10.5 hrs after awakening
Selection potential in Populations
• Long term fitness of a species relies on variation
upon which selection can act, leading to adaptations
to environmental change
• Genetic variation in populations must exist for
photoperiodic response to evolve
• Polymorphism in circadian rhythm could allow
populations to evolve in their photoperiodic
response (nocturnal vs diurnal, or seasonal)
Genetic Variation among Populations
• There is known genetic variation in circadian rhythm
within human populations (night-owls, early birds, etc.)
• Anecdotal observations suggest variation among human
populations: those closer to the equator tend to have
higher % of night owls, and higher % early birds toward
the poles
• However, a shift in allelic frequency with latitude has not
been confirmed in human populations
• A latitudinal shift has been found in Drosophila (fruit
flies)
• Drosophila populations toward the poles
(higher latitude) tend to have greater mid-day
activity
Planet X is larger than earth and has a longer
daily photoperiod. Which type of mutation
would LEAST LIKELY lead to a shift to
accommodate the photoperiod on this
planet?
(a) Increase activity of CK1 (in humans) or
Doubletime (in Drosophila)
(b) Increase phosphorylation of Period
(c) Increased stability of Period
(d) Decreased accumulation of Timeless
Which of the following is MOST LIKELY to lead to Advance
Sleep Phase Syndrome (ASPS)?
(a) A mutation in CK1
(b) A mutation in Period
(c) A mutation in Timeless
(d) It depends on where the mutation is located and how it
affects the conformation of the proteins above
Which of the following is LEAST LIKELY to lead to Advance
Sleep Phase Syndrome (ASPS)?
(a) Period is effectively phosphorylated
(b) Period is allowed to accumulate
(c) Timeless accumulates more quickly
(d) Reduced activity of CK1
You are a manager for a radio station, and people are
complaining that your late night DJs are falling asleep on
the job. Which of the following might help alleviate your
problem?
(a) Hire a DJ who has a mutation in the hPER2 gene, at the
phosphorylation site within the Casein Kinase I (CK1) binding
domain
(b) Hire a DJ who has a mutation which causes increased gene
expression of PER and TIM
(c) Hire a DJ with a mutation in TIM that increases stabilization
of PER
(d) Hire a DJ with a mutation in the Casein Kinase I epsilon
(CKI-T44A) gene