Topic 1 A Genetic Switch Flashcards
Slide 4 a bunch of numbers
Ask prof if need to remeber
Why are sex chromosomes important
They are structurally different (y is smaller)
Subject to diff forms of gene regulation
Have their own inheritance patterns (mom only give 1 x but dad gives x and y, causes imbalance)
Females have. ____ more x linked gene expression then male
2 times more since two x chromosomes
What is dosage compensation
Process where the amount of the gene product in two X chromosomes in women
becomes equal to a single dose of the one x in males
(By inactivation one of the X chromosome)
What is chromosome inactivation theory
In the somatic cells of females, one of the X chromosomes get inactivated RANDOMLY early in development
This equalizes the expression of the x link genes in both sexes
When and where does the inactivated X chromosome get deactivated
In oogenesis in the germ line
How was X chromosome inactivation discovered
The inactive chromosome came in the form of a Barr body (tight packed dna) in interphase cells
The ones that lit up were bar bodies
If two lit, two bar bodies and xxx
How does the inactivation of x linked genes actually happen
The X chromosome is decorated with XIST RNA at the XIC (x inactivating centre)
This rna changes the chromosome to be more tight and not allow its translation and expression
What percent of x linked genes are inactivation
85
How can you tell that it is the XIC/XIST that inactivates the X chromosome
Can use directed mutagenesis to mutate the XIST gene
Then to see if this had an affect on the rna you can use qPCR or a northern blot
What is an example of the effect of having more than one X chromosome
Klinefelter syndrome (XXY)
Only one x chromosome is inactivated
This causes decreased muscle tone, infertile, low testosterone, etc.
What happens as you have more and more X chromosomes but only one is inactivated
Ex. XXXXY
This causes more severe changes in phenotype because there is always leakage which makes more accumulation of the X chromosomes
What is a bacteriophage
What is the lambda phage
A virus that infects bacteria
A dna virus that infects bacterial species like e. Coli
What is a way to capture a bacteriophage on camera
Transmission EM
Scanner EM
How long is the lambda chromosome of a bacteriophage
How many genes does it encode
Where it it’s chromosome located
48kb bp
Encoded 50 genes
Located in the protien core in the phages head
What is in the protien coat of the bacteriophage
15 protiens
What are the two bacteriophage cycles and how do they work
Lytic: the bacteriophage inserts its chromosome into the bacteria. more bacteriophages form inside the cell because the host expressed the phage genes. they then lyses the cell and leaves
Lysogenic: the bacteriophage inserts its chromosome into the bacteria. Viral dna integrates into the bacterial plasmid and stay dormant. Enters lytic after uv induction.
What is prophage
What is lysogen
The bacteriophages dna in its dormant state where it’s integrated with the bacterial chromosome
The bacterial cell that carries the prophage
What makes it go from lysogenic to lytic
Uv light
What is a plaque assay
This is where you can see how many bacterial cells were in the lytic cycle
You mix the ecoli with the phage and see if plaques form
More plaques mean more phage because more cells were lysed
What does the clarity of the plaques in the plaque assay tell us
If the plaque is more clear, the virus is more likely to kill the host, so more likely the cell was in lytic phase.
If the plaque is turbid or cloudy, this means that the cell had the virus but is in the lysogenic phase and they virus doesn’t kill all the cells right away
What do repressor do to bacterial cells
They keep the host cell in lysogenic (dormant) state
They prevent further infection (from more bacteriophages) and lysis
What does the cl/lambda repressor do
It keeps the cell in lysogenic phase
It does this by the help of c2
What does the cro protien do
(Control of repressor)
Keeps the cell in the lytic cycle
What is on and off in
Lysogenic cycle
Lyric cycle
Lysogenic: cro gene off, cl repressor on
Lytic: cro gene on, cl repressor off
What is an operator
Promotor
A dna sequence that is recognized and bound by a repressor protien to block transcription of the gene
DNA sequence that is reconfnized and bound by RNA POL to initiate transcription of the gene
What is the OR
What is OR1 2 and 3
The oR is the operator between the lambda and cro genes
These are the 3 operator sites that repressor can bind to within the OR
What is the PRM and PR
The prm is the promoter of repressor maintence (keeping cl repressor on) and it’s on the or3 and 2
Pr is the right promoter which makes the cro protien and it’s on the OR 1 and 2
What type of promoter is pRM and what does this mean
What type of promoter is pR
Weak promoter, it needs a activator protien to start transcription
Strong promoter, doesn’t need an activator protien
What is the composition of the lambda repressor
236 AA long
Two domains (N and C) linked by 40 AA linker
On the c domain (top) it has a tetramerization domain and a dimerization domain
On the N domain it has the transactivating domain and the DNA binding domain where the operator binds
Why is the linker in the lambda repressor important
It’s helps with protien integrity
Why is the linker in the lambda repressor important
It’s helps with protien integrity
How does the lambda repressor become functional
They bind to each other to form a homo dimer (can’t work by itself)
Then they can bind COOPERATIVELY to the operators through the N domain
What is the composition of the cro protein
What do they bind to
66 AA
Have high affinity for each other, so they form dimers
Bind to the same operators as the lambda repressor but have the opposite effect (make cro not lambda)
What is a way to study the interaction of the repressor or cro with the dna
EMSA
Electrophoretic mobility shift assay
Use native PA gel , the dna blot shifts up if bound by protein.
What happens when the lambda repressor dimer binds the or2
It triggers binding of rna pol to or3
This physically blocks it from binding to the pR and stop it from making cro
What types of regulation does the cI repressor do when bound to oR2
It positively regulates expression of its own gene by enhancing the bind of rna pol to prm and make more cl
Negatively regulates cro transcription by blocking rna pol from pr and suppress transcription of the cro
What type of regulation does the cl repressor do when bound to the or3
Negatively regulates cl transcription by binding to or3 and making rna pol bind to pr and make cro not cl
And since the pr is strong promoter, he lytic cycle happens right away
What type of regulation does the cl repressor do when bound to or1
Negative regulation of both Cl and CRO
Block rna from binding to Pr to cro isn’t made
Prm is weakly activated by this but since it is a weak promoter and repressor is far , rna pol isn’t recruited and cl isn’t made
What percent of the time is both or1 and 2 occupied
What about all three
Why
90
10
Repressor has diff affinities for the sites
What is the order of affinity of the cl repressor dimer
And how can it be explain
1>2>3
Is giving the choice, the dimer will bind to or1.
As repressor concentration increases, it binds to OR2 as well and forms a tetramer with the dimer at or1
Weakest affinity is or3
What is cooperativity with the repressor dimers
The binding of the dimer at or1 increases the affinity of a dimer at or2
The concentration of repressor needed to bind to or2 is lower with coorperativirt than if it was alone
So this show Sigmoidal kinetics
Why is bind of the repressor dimer at OR3 so weak
There’s no cooperativity since it can’t form interaction with the other dimers.
so the concentration needed for it to bind to the or3 isn’t lowered.
So It binds more weakly to this site
Repressor molecules are continuously doing what
Dissociating and reaccositong between monomeric and dimeric forms
If the repressor is binding to the promoter what does this mean for the dimer cro protien
It is not bound
What is the mechanism of switching from lysogenic to lytic growth
The main cause is UV damage
The dna damage due to uv light activates the E. coli protein RecA
What are the recA functions
A recombinant:
It promotes DNA exchange during homologous recombination
Also triggers DNA repair mechanisms in the the E. coli dna by activating protien degradation so that the dna replication stops
What are the recA functions
A recombinant:
It promotes DNA exchange during homologous recombination
Also triggers DNA repair mechanisms in the the E. coli dna by activating protien degradation so that the dna replication stops
What is the SOS response
After rec a activates protein degredation, the sos response happens
In this: the three different genes in the dna: UVRA UMUC, UMUD
Lex a (transcription repressor of the three genes) gets degraded by Rec A
The promoter is recognized by the rna polymerase and the three genes are then transcribed
What happens after the SOS response
When the UVRA gene is on, nuclear excision repair occurs
And it tries to repair the damaged nucleotides
If NER doesn’t work, the UMUC AND UMUD GENES will turn on translesion DNA synthesis
This is where the replication keeps going but skips the damaged nucleotides
what is special about translesion dna synthesis
This is the way for the r coli to survive but still have damaged dna rather than just dying
In bacteriophages what is rec A role
It cleaves the lambda repressor monomer
So that now the split monomers can’t dimeriza and make more lambda repressor
Decreases concentration of monomeric lambda repressor, less chance of binding to or1 or2
If the lamda repressor concentration is decreased, what happens
Less binding to the prm and or sites
So eventually the prm is off because no repressor synthesis
What is different about the regulation of CRO vs lambda repressor
Lambda repressor can do postive regulate if it’s own expression or negative regulation
But cro can only do negative regulation (suppress gene expression)
CRO binds ___
What does this mean
Independently to Or sites
Not cooperatively
Has a hyperbolic curve
What is the affinity order of CRO to OR sites
OR3 > Or1=Or2
Highest is or3
If the CRO dimer binds to or3 what happens
RNA pol is blocked from binding to prm so the repressor expression is turned off
It’s opens up the pr side so that the rna pol can bind there and express the CRO gene
Indirectly allows CRO expression
What happens if CRO binds to Or1 or O2
In this case CRO is already at Or3
If at either or1 or or2, the pr promoter is blocked by CRO and it shuts down its own expression
Negative regulation
Why does cro suppress its own sysnthesis
Since already the lambda repressor synthesis is off (due to Rec A cleavage)
Only need to control how much it is sysnthesized
How can we tell if the concentration of CRO is increased how much it actually binds to DNA
Electrophoretic mobility assay
What does the pra promoter do
Used for the CII activator which turns on rna pol
