PSYC 388 Study Guide - Midterm Guide: Circadian Clock, Circadian Rhythm, Timeless (Gene)

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Chapter 7 ` Molecular Clock: protein “tick” and RNA “tock
-Schrodinger = metaphorical cat can cat be both alive and dead at same time (cat in a box)
~What is life considered 2 issues:
1.Ability to decrease entropy (characteristic feature of living systems) with consequent increase in
order life = continual battle to maintain state of order in universe that runs towards disorders =
living systems keep climbing entropy gradient by obtaining energy from sunlight and food (second
law of thermodynamics - expression of the tendency that over time, differences in temperature,
pressure, and chemical potential equilibrate in an isolated physical system) game goes to extra time
and law gets everything at the end
2.Heredity – suggested basis for heredity must e some kind of code where specific sequences of
chemicals were written and interpreted and assumedcode-script was contained in proteins in form
of an aperiodic crystal (salt)
~ Once position of one pair of sodium and chlorine atom known = order and positions of all other atom
are defined thought chromosome was aperiodic crystal = example of basic code arranged in order
could contain vast of information (many different combinations) = lead to people working on these
-Benzer aim to link classical gene maps with new molecular mechanisms (double helix)
~ rII phage mutants experiments = showed gene itself had structure, so gene could be dissected an cut
into pieces (pieces = lengths of DNA)
~ showed correspondence between linearity of gene and section of DNA = help establish accepted view
that buried within DNA of organism is its genetic blueprint (genotype) – unique sets of instruction that
provides basic code for building an individual
-Genes direct production of specific proteins which interact with environment to determine how person
will function and phenotype, but environment can influence expression of genotype and subtly alter how
DNA blueprint is read = same genotype may have slightly different phenotype (differences in identical
-Genes don’t make proteins directly RNA (ribonucleic acid/ single strand) = protein synthesis
-Nucleotide building blocks of DNA and RNA consist of 3 smaller molecular components
1.Nitrogen base – cytosine ( c), thymine (T), adenine (A), and guanine (G); RNA = (T) replaced by
(U) uracil
~ 2 chain of nucleotides linked together by bases (C-G, T-U/A)
-DNA is Schrodinger’s aperiodic crystal and list of bases in DNA strand is genetic code
-Amino acids – building blocks’ of proteins (20) – various combinations = chains to produce different
protein and activities
-Codon - 3 bases in DNA strand ex. GGT code for particular amino acid
-DNA unravel at cell division and single strand goes to each daughter cell to act as template to form
complementary strand and re establish to double helix but most of time cell is not dividing or DNA is
-Most of cell’s time spent in protein synthesis and degradation
-Specific protein made in a cell only appropriate portion of DNA strand unzips by breaking links between
bases = one side of DNA strand act as template to make complementary strand of RNA; free nucleotides
in nucleus of cell line up along unzipped DNA strand to form RNA strand then single chain of RNA
peels away from DNA strand strand of free RNA (mRNA) strand of mRNA leaves nucleus and
enters cytoplasm of cell where it interacts with ribosome’s (large protein) and information = translated
when amino acids line up along mRNA strand to generate protein (protein synthesis)
-Sequence of bases in mRNA strand mirrors sequences of DNA strand
-Transcription - pprocess of creating a complementary RNA copy of a sequence of DNA
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-Double stranded DNA (dsDNA) unzip so single strand of DNA (ssDNA) can act as template for mRNA
transcription begins with specific transcription factors usually proteins bind to promoter region of
gene; complementary strand of RNA synthesised by RNA polymerase = leaves nucleus and enters
cytoplasm of cell and mRNA interacts with ribosome with translate mRNA, codon by codon into protein
-Proteins act as regulators, communication signals, structural elements, enzymes while genes only code
for proteins
-Central dogma: dna makes rna; rna acts as template for A.A to be linked into a chain; chains fold into
-Benzer – interested in how humans synchronised their activities by time
-Konopka – interested in genetic basis of Pittendrigh’s observations that clock regulated dawn
emergence of Drosophila (circadian clock controls timing) (emergence rhythm = entrained by light and
temperature compensated)
~ exposed flies to chemicals that cause random mutations in DNA (sperm) clock mutants
(slow/fast/arrhythmic clocks) = RESULTS: crossing mutants with wild-type flies reveal mutation linked
to behavioural change in flies’ clock was due to single gene = mutations in this gene (“period(per)
gene) altered period of fly’s circadian rhythm
~ He and Benzer tied behaviour to a gene and identified first clock gene to be found in any species
-time-sensitive emergence of adult Drosophila = behaviouralcurly wings
-initially, PER thought to function at junctions between cells, for cell –cell communication later PER
might be involved in clock function
-Siwicki and Hall detected and localized PER within individual cells of fly’s body – showed PER present
in very many different tissues of body, small group of cells in fly brain (lateral neurones) and eyes
showed 24 hour rhythms of abundance in PER protein
-Normal flies (perN), protein levels peaked early at night (8pm) then drop In middle of day; rhythms in
PER peaked early in per short (perS) mutants and late in perL mutants and no PER protein could be
detected in arrhythmic per0 flies (per mRNA peaks 4-6 hours earlier before peak in PER protein) perS
mutants peaks in per mRNa – advanced ~5 hours, perL mutants had mRNA rhythms delayed ~ 5 hrs
compared to normal flies; per 0 flies = no rhythms in per mRNA + no detectable PER protein
-PER rhythmic in eyes and lateral neurones, patterns of protein abundance mirrored circadian behaviour
rhythms of per mRNA drove rhythmic production of PER protein (flies collected at different times in
day and night then frozen by liquid nitrogen = place din mesh sieve and shaken to separate heads from
bodies = per mRNA measured = per m RNA showed 24 hour cycle)
-Hardin & Hall proposed PER protein was involved in negative feedback loop where it inhibit its own
production for rhythmic expression of PER (made sense because PER synthesised in cytoplasm and high
levels of PER protein concentrated in nucleus) = proposed per mRNA transcribed form per gene = allow
production of PER protein so as PER levels build in cytoplasm = protein enters nucleus and inhibits
production of further per mRNA by binding with promoter = reduced levels of per mRNA transcription
resulting in reduced levels of PER protein production so all PER translation stops = PER protein broken
down then releases per gene from inhibition, per mRNa can now transcribed and cycle restart 
proposal don’t answer how this generates stable rhythm (negative feedback = keep system in stable
equilibrium but oscillation is different; oscillation – system in regular manner moves away form
equilibrium before returning)
-Simple “per alone model – first model for circadian clock in Drosophila based on
transcriptional/translational feedback loop of PER protein and per mRNA = PER protein inhibit further
transcription and hence translation of PER= half-life of protein and mRNA would determine 24 hour
dynamics of system
~ Major problem = dPER playing role of transcriptional regulator because don’t have features expected
of a protein that would bind promoter region of a gene = need a partner
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-To oscillate on 24hr cycle, inhibitory effects of PER on per transcription need to be turned off, per
mRNA production needs to be turned back on again, has to be delay of many hours in feedback process
-Tension between positive element of loop = promote transcription and translation of PER and negative
element inhibit this transcription = rhythm
-Sehgal & Price – identify new Drosophila circadian mutanttimeless (tim)” – analysis of tim gene
suggested tim and per = act similar = flies lacking tim gene (tim0) are arrhythmic, mutations in tim can
alter period length of circadian behaviour, TIM protein accumulates in same cell nuclei as PER protein,
tim and per mRNA oscillate with same circadian phase, both peaking 4-6 hours before protein levels,
PER protein never decreased in cell nuclei of tim0, TIM never detected in cell nuclei of per0 flies
-PER rapidly degraded in absence of TIM protein = PER and TIM proteins bind to each other using PAS
domains PER/TIM model for circadian clock in Drosophila
~ PER/TIM interaction required for nuclear localisation of both proteins = enter nucleus PER/TIM
complex able to suppress per and tim gene transcription= stability of PER/TIM protein complex will
determine duration of transcriptional suppression of per and tim genes = rate of entry of PER/TIM
complex into nucleus and how long PER/TIM complex can persist before its degraded in nucleus, will
determine extent of negative feedback on per and tim transcription and hence period of circadian
~ cycles in per and tim mRNA transcription are regulated by cycles of nuclear localisation of a PER/TIM
protein complex
~ explains generation and entrainment of molecular clock but problem =PER alone can’t bind to DNA or
TIM alone or PER & TIM complex; feedback loop didn’t close
-TIM protein stable in dark but rapidly degraded by light any time in cycle suggested link between
oscillation of internal circadian rhythm and entrainment by light
-Example, pulse in early night will normally delay clock because TIM needed for entry of PER/TIM into
nucleus, loss of cytoplasmic TIM during first part of night due to degradation by light will result in
delayed build-up of PER/TIM and delayed entry of PER/TIM complex into nucleus causing delay in
suppression of per and tim mRNA = net result is delay in whole molecular feedback loop
-Ex. Light during later night (reverse effect)= advance circadian behaviour = TIM degraded rapidly, but
this time TIM is in nucleus bound in TIM/PER complex = breakdown of complex realises transcriptional
inhibition of per and tim genes earlier = resulting in advance of molecular cycle [ degrade TIM to levels
that would normally occur later in molecular cycle = TIM degraded rapidly and release transcriptional
inhibition of per and tim genes in advance of normal time reusl.itn gin advance cycle]
-PER/TIM model = explain generation and entrainment of molecular clock, but problem = PER alone
can’t bind to DNA or TIM alone, or complex of PER and TIM so can’t act as direct transcription
inhibitor of per and timgenes (this feedback loop don’t close)
-Takahashi & Pinto study --> mice exposed to mutagen so offspring of mutagenised mice were studied
for circadian abnormalities – mutant mouse with long circadian period (circadian locomotor output
cycles KaputClock”) = first mammalian clock gene
~ mice = DD =clock mutants with one copy of defective gene show lengthened circadian period, those
with 2 defective copies showed longer circadian period for few days then arrhythmic
~ led to new approach = identified gene of interest by screening for behaviour, isolating desired mutation
then cloning gene responsible
-CLOCK protein has necessary features to bind DNA directly and possess PAS domain = act as direct
transcriptional regulator and probably performs it in partnership with another
-Drosophila Clock gene protein (dCLOCK) binds (via PAS region) to another protein that also possess
DNA binding region (gene coding for this protein – “cycle”)
-dclock and cycle” closed loop of drosophila clock
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