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Cmmb 411 > Topic 1 A Genetic Switch > Flashcards

Topic 1 A Genetic Switch Flashcards

1
Q

Slide 4 a bunch of numbers

A

Ask prof if need to remeber

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2
Q

Why are sex chromosomes important

A

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)

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3
Q

Females have. ____ more x linked gene expression then male

A

2 times more since two x chromosomes

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4
Q

What is dosage compensation

A

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)

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5
Q

What is chromosome inactivation theory

A

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

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6
Q

When and where does the inactivated X chromosome get deactivated

A

In oogenesis in the germ line

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7
Q

How was X chromosome inactivation discovered

A

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

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8
Q

How does the inactivation of x linked genes actually happen

A

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

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9
Q

What percent of x linked genes are inactivation

A

85

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10
Q

How can you tell that it is the XIC/XIST that inactivates the X chromosome

A

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

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11
Q

What is an example of the effect of having more than one X chromosome

A

Klinefelter syndrome (XXY)

Only one x chromosome is inactivated

This causes decreased muscle tone, infertile, low testosterone, etc.

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12
Q

What happens as you have more and more X chromosomes but only one is inactivated

A

Ex. XXXXY

This causes more severe changes in phenotype because there is always leakage which makes more accumulation of the X chromosomes

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13
Q

What is a bacteriophage

What is the lambda phage

A

A virus that infects bacteria

A dna virus that infects bacterial species like e. Coli

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14
Q

What is a way to capture a bacteriophage on camera

A

Transmission EM

Scanner EM

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15
Q

How long is the lambda chromosome of a bacteriophage

How many genes does it encode

Where it it’s chromosome located

A

48kb bp

Encoded 50 genes

Located in the protien core in the phages head

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16
Q

What is in the protien coat of the bacteriophage

A

15 protiens

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17
Q

What are the two bacteriophage cycles and how do they work

A

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.

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18
Q

What is prophage

What is lysogen

A

The bacteriophages dna in its dormant state where it’s integrated with the bacterial chromosome

The bacterial cell that carries the prophage

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19
Q

What makes it go from lysogenic to lytic

A

Uv light

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20
Q

What is a plaque assay

A

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

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21
Q

What does the clarity of the plaques in the plaque assay tell us

A

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

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22
Q

What do repressor do to bacterial cells

A

They keep the host cell in lysogenic (dormant) state

They prevent further infection (from more bacteriophages) and lysis

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23
Q

What does the cl/lambda repressor do

A

It keeps the cell in lysogenic phase

It does this by the help of c2

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24
Q

What does the cro protien do

A

(Control of repressor)

Keeps the cell in the lytic cycle

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25
Q

What is on and off in

Lysogenic cycle

Lyric cycle

A

Lysogenic: cro gene off, cl repressor on

Lytic: cro gene on, cl repressor off

26
Q

What is an operator

Promotor

A

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

27
Q

What is the OR

What is OR1 2 and 3

A

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

28
Q

What is the PRM and PR

A

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

29
Q

What type of promoter is pRM and what does this mean

What type of promoter is pR

A

Weak promoter, it needs a activator protien to start transcription

Strong promoter, doesn’t need an activator protien

30
Q

What is the composition of the lambda repressor

A

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

31
Q

Why is the linker in the lambda repressor important

A

It’s helps with protien integrity

32
Q

Why is the linker in the lambda repressor important

A

It’s helps with protien integrity

33
Q

How does the lambda repressor become functional

A

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

34
Q

What is the composition of the cro protein

What do they bind to

A

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)

35
Q

What is a way to study the interaction of the repressor or cro with the dna

A

EMSA

Electrophoretic mobility shift assay

Use native PA gel , the dna blot shifts up if bound by protein.

36
Q

What happens when the lambda repressor dimer binds the or2

A

It triggers binding of rna pol to or3

This physically blocks it from binding to the pR and stop it from making cro

37
Q

What types of regulation does the cI repressor do when bound to oR2

A

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

38
Q

What type of regulation does the cl repressor do when bound to the or3

A

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

39
Q

What type of regulation does the cl repressor do when bound to or1

A

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

40
Q

What percent of the time is both or1 and 2 occupied

What about all three

Why

A

90

10

Repressor has diff affinities for the sites

41
Q

What is the order of affinity of the cl repressor dimer

And how can it be explain

A

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

42
Q

What is cooperativity with the repressor dimers

A

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

43
Q

Why is bind of the repressor dimer at OR3 so weak

A

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

44
Q

Repressor molecules are continuously doing what

A

Dissociating and reaccositong between monomeric and dimeric forms

45
Q

If the repressor is binding to the promoter what does this mean for the dimer cro protien

A

It is not bound

46
Q

What is the mechanism of switching from lysogenic to lytic growth

A

The main cause is UV damage

The dna damage due to uv light activates the E. coli protein RecA

47
Q

What are the recA functions

A

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

48
Q

What are the recA functions

A

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

49
Q

What is the SOS response

A

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

50
Q

What happens after the SOS response

A

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

51
Q

what is special about translesion dna synthesis

A

This is the way for the r coli to survive but still have damaged dna rather than just dying

52
Q

In bacteriophages what is rec A role

A

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

53
Q

If the lamda repressor concentration is decreased, what happens

A

Less binding to the prm and or sites

So eventually the prm is off because no repressor synthesis

54
Q

What is different about the regulation of CRO vs lambda repressor

A

Lambda repressor can do postive regulate if it’s own expression or negative regulation

But cro can only do negative regulation (suppress gene expression)

55
Q

CRO binds ___

What does this mean

A

Independently to Or sites

Not cooperatively

Has a hyperbolic curve

56
Q

What is the affinity order of CRO to OR sites

A

OR3 > Or1=Or2

Highest is or3

57
Q

If the CRO dimer binds to or3 what happens

A

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

58
Q

What happens if CRO binds to Or1 or O2

A

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

59
Q

Why does cro suppress its own sysnthesis

A

Since already the lambda repressor synthesis is off (due to Rec A cleavage)

Only need to control how much it is sysnthesized

60
Q

How can we tell if the concentration of CRO is increased how much it actually binds to DNA

A

Electrophoretic mobility assay

61
Q

What does the pra promoter do

A

Used for the CII activator which turns on rna pol

Cmmb 411 (6 decks)

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