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241 Cards in this Set

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LECTURE 23
Lecture 23
what is the signal that the cell receives?
Ligand
What receives this signal?
Cell surface receptors
The ligand activates a ? in the ? and what does it do.
allosteric change; alters the shape and therefore function of the Intercellular domain
An allosteric change is an example of ? signaling
Direct
Indirect signaling is done through?
signal transduction pathways
When the ligand binds the cell sueface receptor, what happens?
It brings together two receptors
When these two receptors are brought together, what hapens?
Their kinases on their ends phosphorlates sequences in the other receptor
What happens to these phosphorlated sites?
they are recognized by STAT proteins
What happens to these STAT proteins?
They are phosphorlated by the kinase causing them to dimer (come together)
What does this newly formed STAT dimer do next?
moves into the nucleus and binds specific DNA sites and starts transcription.
What binds the cell surface receptors in this case?
Signal molecule
once the signal molecule binds, what happens?
two receptors are brought together
When the two recpetors are brought together what do they do?
Phosphorlate each other other.
This phosphorlyation of the recpetors does what?
recruits Grb2
Grb2 does what
associates with SOS
the association with SOS helps Grb2 to..
attach to the Ras kinase
How do we activate the Ras kinase
GDP on the Ras is changed to GTP
Once activated, what does the Ras kinase do?
activates the MAPk pathway
What does the MAPk pathway do then?
phosphorlates transcription factors (Jun)
Where do these transcription factors go and what do they do?
into the nucleus and activate transcription
Eukaryotes are not activated by ? and require ?
Direct ligand to regulator contact; communication of the ligand through signal transduction pathways
Three ways signaling helps modify transcription
-Unmasking an activating region
-Transport into and out of the nucleus (STAT)
- Brings together activator/repressor pieces
first way unmasking and activating region is done
a change in the DNA bound activator, revelaing a perviously buried activating region
Second way
release of a protein that was bound, blocking the activating region
how Transport into and out of the nucleus signals transcription
When not active, regulators are found in the cytoplasm. The signaling ligand causes them to move into the nucleus where they regulate transcription
how activators and receptors some times come in pairs activates transcription
in the absence of its ligand glucocorticord receptor (GR) is found in the cytoplasm. when its ligand is bound it moves into the nucleus and binds GRE sites
What does transcriptional scilencing mean?
that the silencing is a position effect
What does the position effect mean?
a gene is silienced because of where it is located, not in response to a specific signal
What does it mean to say the repression of gene expression can be global?
OVer very long stretches of DNA
Most common form of silencing is associated with ?
Heterochromatin (denser)
where is heterchromatin usually found?
in the telomeres/centromeres
what happens if a gene is moed into the telomeres or centromeres?
it is turned off
another way transcription can be silienced?
Methylation
what does DNA methylation do?
in hibits the binding of proteins. including transcriptional machinery and therefore blocking gene expression
chromatin at the telomere is ? acetylated
less. (more methylated)
Gene silencing in yeast is mediated by ? and ?
deacetylation and methylation
First step in this process
DNA binding protein RAP 1 binds the telomere
Then Rap1 recruites ?
SIR proteins
How many proteins make up the Sir?
three
What does the Sir2 protein do?
It is the histone deacetylase enzyme and it begins to deacetylate nearby nucleosomes
once Sir2 has deacetylated the nucleosomes what happens?
the unacetylated tails then bind Sir 3 and Sir 4
What do Sir3 and Sir 4 do?
They recruit more Sir complex
When does methylation stop?
When there is no more Rap1 protein
What is the stop signal for Rap1
histone methlyation
When a gene can be leaky and not completely turned off, what happens?
The DNA is methlyated
after the DNA is methlyated, what happens?
Proteins bind the methlyated groups
What do these bound proteins do next?
They recruit Histone deacytalase and chromatin remodeling proteins
What does these histone deacetylase and chromatin remodeling proteins do?
bind the DNA structure and condense it to make it super tight (heterochromatin)
Impritting is...
When out of two alleles, only one is expressed.
If the chromosome is unmethlyated...
its insulator site will bind CTCF
when this happens, ? is turned on, and ? is turned off
H19= on
Igf2=off
If the chromosome is methlyated...
its insulator site will be blocked
When this happens ? is turned on, and ? is turned off
Igf2 = on
H19 = off
DNA methlyation is maintained through cell division by...
Maintenance methlyase
Maintenance methlyase does what?
Recognizes the hemi-methlyated cytosin after replication and methlyates it
This is an example of epigenetics and it shows that gene expression can be inherited across ...
generations, not just a single cell division
LECTURE 24
Lecture 24
Three ways cells express genes in development
-mRNA localization
- Cell to cell contact
- diffusion of secreted signaling molecules
How does mRNA localization help to distinguish between identical cells?
by asymmetrically localizing mRNA
Where does this process happen?
In the cytoplasm
How do the mRNA locoalize?
By hitching a ride on actin filaments
How does it hitch a ride?
an adaptor protein binds the UTR of the mRNA
Then what binds the adaptor protein
The myosin motor protein
What do these motor proteins do?
They walk along the polarized actin filament and bring the mRNA with them
Which direction in the complex moving?
Towards the + end
CASE STUDY FOR mRNA localization
mother and daughter cells swtiching mating types
A ? cell can switch mating types, but a ? cell cannot
Mother
Daughterq\
Mating type swtiching is controlled by what protein?
HO
If the HO is expressed?
If the HO is not expressed?
It can switch mating types
I cannot switch mating types
What represses the HO protein?
Ash1
What do the mother cells do to the ash1 and what effect does it have>
The mother cells transcribe ash1 and it is localized to the daughter cell so then the HO protein is repressed in the daughter cell
A cell can influence which genes are expressed in neighboring cells by
Cell to cell contact
How do these cells come in contact?
Through ligands and receptors
when the ligand and receptors contect, what happens next>
it makes a kinase
What does the kinase then do?
causes the phosphorlyation of DNA bind proteins in the nucleus
What does this phosphorlation cause the DNA binding proteins to do?
activate/repress transcription for certain genes
What happens to the receptor?
It is cleaved and the intracytoplasmic domain moves into the nucleus
What can this cleaved domain do?
Regulate transcription of sets of genes
CASE STUDY: Cell to cell contact
nerve-skin cell formation
Developing nuerons produce a cell surface signaling molecule called
Delta
What does delta do?
binds its receptor Notch
What happens to the Notch?
Its intracytoplasmic portion is cleaved and goes into the nucleus
What does the Notch do inside the nucleus?
Binds with Su(H)
What does the Notch-Su(H) complex do?
they activate the expression of neuronal gene
What does this lead to?
It makes the nueron cells become epidermal cells
Diffusion of a secreted signaling molecule ..
Helps decide where what will form in the cell
Cells located at the front (anterior) portion of the embryo will form..
Portions of the head
how is positional information distributed?
by the extracellular gradient
Where do these signaling molecules come from?
The morphogen source
The closer a cell is to the morphogen source...
the more signaling molecules it recieves
the more signaling molecules it recieves...
the more activated surface receptors it has
more activated receptors...
More genes expressed
LECTURE 25
LECTURE 25
Once an egg is fertilized, the nucleus of the embryo undergoes
Multiple rounds of mitosis without cell division
The bag of cytoplasm with many nuclei inside is called the
syncytium
how rounds of nuclear divison does the cell go through?
14
What happens after the 14th round
the nuclei migrate to the outter edge of the cell and start to form cell membranes around the nucleus
Cells in the posterior region form
pole cells
How is the dorsal-ventral patterning in the early drosphilla controlled?
by a protein called Dorsal
How is Dorsal initially distributed in the cell?
evenly
When can Dorsal enter the cell?
Once the nuelci have made their way to the outter portion of the cell
How is the transportation of Dorsal into the nucleus monitored?
Spatzle
Where on the cell is the Dorsal allowed to move into the nucleus?
The ventral region (bottom)
Where is spatzle most concentrated?
Ventral region
Once the cell is fertilized
Spatzle binds toll receptors
After binding and activating the toll receptors
the toll receptors activates Tube
Tube activates
Pelle
Pelle does what
It destroys the cattus protein on the Dorsal
attacking the cactus protein by Pelle does wat
Allows Dorsal to move into the nucleus
The three genes ? ? ? are turned on by ?
Twist
Rhomboid
Sog
Dorsal
Twist is turned on only when concentrations of Dorsal are
High (low affinity)
Rhomboid is turned on when concentrations of Dorsal are
moderate-to-high
sog is turned on when concentrations of dorsal are
low (high affinity)
Highest dorsal leverl
mesoderm
medium dorsal level
Ventral NE
low dorsal levels
dorsal NE
What does Snail do?
prevents the expression of rhombiod and sog in the mesoderm
snail is only expressed in the
mesoderm
how does snail stop the expression of rhomboid and sog?
it binds the dorsal binding site for rhomboid and sog in the mesoderm and therefore prevents dorsal from turning on expression of these genes
Therefore rhomboid and sog are only expressed
in lateral regions that formthe neurogenic ectoderm (NE)
At fertilization the drosphilla egg contains two localized mRNA called
bicoid mRNA
oscar mRNA
the bicoid mRNA is found
at the anterior pole
the oscar mRNA is found
at the posteior pole
the oscar mRNA encodes for a RNA binding protein that
assembles polar grandules
these polar grandules are responsible for
the development of the posterior embryp
Where is the oscar mRNA first deposited?
in the anterior region
This anterior region is called
oocyte
when the oocyte starts to get bigger
the oscar mRNA get transported to the posterior end
How does this relocation occur?
the oocyte is very polarized so when it goes, microtubles from the nucleus extend to the + end and the oscar mRNA interact with the + end of the microtubles
The cells that inherit these oskar mRNA and become fertilized form
pole cells
these pole cells help to
develop the posterior side of the embryo
the UTR of the bicoid and oskar mRNA play a crucial role by
moving the mRNA to where they need to be
If the UTR of a bicoid mRNA was switched to an oskar mRNA
the oscar mRNA would stay in the anterior end and cause formation of pole cells in abnormal places
LECTURE 26
LECTURE 26
Bicoid regulatory proteins diffuse how
from anterior to posterior
this diffusion forms a
bicoid protein gradient
Does this bicoid gradient need to utilize cell surface receptor? why?
no because it diffuses freely
High levels of bicoid are required where
in the anterior region to produce structure like the head
high levels of bicoid control expression of
Orthodenticle gene (low affinity)
High/Medium levels of bicoid activate
Hunchback gene (high affinity)
The hunchback gene is required for development of the
thorax
Hunchback is transcribed from two different promotors
-one activated by the bicoid gradient

- one in the developing oocyte
When hunchback is activated by the developing oocyte
RNA is distributed evenly
What and where are Nanos proteins
Nanos proteins bind hunchback mRNA in the posterior region
What does this do?
Inhibits translation of the Hunchback mRNA in the posterior region
What forms because of this?
a steep Hunchback gradient
Hunchback protein is a
repressor
Since it is a repressor, it limits the expression of
gap genes
the three gap genes are
Kruppel
knirps
giant
High levels of Hunback repress
Kruppel (low affinity)
High/Medium levels of Hunchback repress
knirps (moderate affinity)
Low levels of hunchback repress
giant (high affinity)
gap genes are
repressors
what genes do these gap genes repress?
pair rule genes
pair rule genes are also called
even-skipped genes (eve)
eve is expressed in
a series of alternating stripes along the embryo
each eve stripe is controlled by
an enhancer
ASK FOR HELP ON THE EVE STRIPE QUESTIONS
ask for help eve stripe questions
LECTURE 27
LECTURE 27
3 ways GENE EXPRESSION can be altered during evolution
- a gene that helps determine body patterning can be expressed in different patterns

- a gene that helps determine body can be altered so it develops new functions

- Target genes of a specific patterning gene can acquire new DNA regulatory system
Pax6 is..
a gene that yields for normal eye development
Completely remove Pax6 ...
No eyes
Misexpress Pax6 in cells that don;t normally express it,,
Get development of eyes where they aren't usually expressed
Antp is ..
patterning gene that controls the development of the mesothorax (LEGS)
the mesothorax develops
midlegs and wings
the metathorax develops
hindlegs and halteres
Ubx is the gene that controls..
development of of the metathorax (HALTERES)
The Ubx represses
Antp in the metathorax
Ubx allows expression of
hindlegs and halteres
Mutation in the Ubx allows..
Antp to be expressed in the metathorax, so we get midlegs and wings in the metathorax
Mutation in the Ubx regulatory sequence can cause..
Ubx expression in the mesothorax, so we get hindlegs and halteres in the mesothorax
Pax6 expresses
eyes
Antp expresses
Wings and midlegs
Ubx expresses
Hindlegs and halteres
LECTURE 28
LECTURE 28
RNAi stands for
RNA interface
What does RNAi do
silences genes using small regulatory RNA
3 ways RNAi silences genes
-trigger the destruction of an mRNA

- Blocking translation of an mRNA

-Inducing chromatin modifications of a target gene
RNAi is found in
all eukaryotes
the two important types of small regulatory RNA
siRNA
miRNA
how is siRNA produced?
produced in the lab from dsRNA
how is miRNA produced?
from a precursor mRNA expressed in the cell
What is this precursor mRNA called?
primary RNA of pri-RNA
What is inside this pri-mRNA
hairpin like pre-mRNA
where can these pre-mRNAs appear
anywhere. coding or noncoding regions
Once pre-mRNA hairpins have formed on pri-mRNAs, what happens
the pre-mRNAs are cleaved from the pri-mRNA
what does this cleaving?
Drosha and Pasha
once the pri-mRNA is cleaved by Drosha and Pasha, what happens next
it is exported to the cytom plasm
Once the pre-mRNA is in the cytoplasm, what happens?
its terminal (loop) end is cleaved
what does the cleaving, and what is the outcome?
Dicer. it creast a 22 bp dsRNA
this newly formed dsRNA is called
miRNA
Once siRNA is introduced to the cytoplasm, what happens?
Dicer cleaves the dsRNA
What is the result of this cleavage on the newly introduced dsRNA?
siRNA is formed
What allows Dicer to recognize and bind RNA?
its PAZ domain
What allows Dicer to cleave the RNA?
RNAse III domains
Once siRNA and miRNA are formed, what happens
They are unwound to produce two ssRNA
What are these two ssRNAs called and which one is important
Guide and Passenger

Guide
What is the job of the guide RNA?
to lead the RISC complex to the target mRNA
What is the central component of the RISC complex
Argonaute
what does Argonaute do
it slices the ssRNA-mRNA hybrid in the middle of the quide (ssRNA)
What is Argonaute also called
Slicer
what does Argonaute have that allows it recognize and bind RNA
PAZ domain
How can we use siRNA to knock down genes?
introduce dsRNA into the cell that is complementary to a gene of interest
How do we monitor if the gene expression has been knocked down?
Immunoblotting
What is the first step to immunoblotting
all the protein is extracted from the cell
Once the protein is extracted, what happens?
it is denatured
What does this denaturng and how?
SDS and BME. reduces the disulfide bonds within proteins
What is the result of treating the proteins with SDS
it gives them a negative charge
Once these proteins have a negative charge, what happens?
They are ran on electrophoresis to separate them based on size. (smaller ones go further)
What allows us to see the proteins on the gel?
treating them with coomassie blue
What happens next
the proteins are transferred to a membrane
once the proteins are on the membrane what happens
the are treated with antibodies
what do they antibodies do
bind specific proteins
after the antibodies have bound a specific proteins what happens
a chemical is added that is processed by an enzyme in the antibody that produces a color
LECTURE 29
LECTURE 29
What does it mean that the RNAi pathway is very efficient
only very small amounts of dsRNA are needed to induce a shutdown of target genes
Two reason why it is so efficient
-RNA-dependent RNA polymerase (RdRP) makes more dsRNA off of the RNA template

- RISC complex can cleave several mRNAs
How can small RNAs silence genes?
Condensing the chromatin (low acetalyation, high methlyation
Since centromeres are usually silenced, they have
low histone acetylation and high methlyation
The loss of dicer or Argonaute leads to the moss of
methlyation and the centromeres are no longer silenced
When centromeric DNA get transcribed what is produced
dsRNA
where does this dsRNA come from
centromere repeats
Once the dsRNA has been formed, what happens
Dicer cuts the dsRNA into small RNA
What does this new small RNA do
Bind the RITS complex
what does this small RNA-RITS complex do next
binds the transcripts coming from the centromeric repeats
What does the binding of the small RNA-RITS complex to the transcripts coming from the centromeric repeats do
recruits histone modifiers Swi6 and Clr4
what do the histone modifiers Swi6 and Clr4 do
they methlyate and silence the chromatin
Lin-14 is responsible for
stage 1 larval development
What does the worm need to process to stage 2 larval development
Lin-14 needs to be absent
How does this happen
Lin-4 target Lin-14 and decreases the levels of Lin-14
When does Lin-4 start to rise
stage 1
and what does it do
targets Lin-14 and decreases its levels
Lin-4 is responsible for
Stage 2 larval development
Between Lin-4 and Lin-14, which one is the repressor
Lin-4
what does Lin-4 encode for
small RNAs
How does Lin-4 repress Lin-14
it binds its Lin-14 3' UTR site
how Lin-4 has bound Lin-14 3' UTR site
with imperfect complementarity
what does this mean
it doesn't have to be an exact match
The better Lin-4 can Lin-14 means that
it has more binding sites
The more binding site,
the greater transcriptional repression of Lin-14