BIOL211 - L14

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  • Created by: Katherine
  • Created on: 12-04-17 12:34
What is the yeast cell cycle?
Start in G1, move past start to see bud emergence. The bud gets larger and then mitosis takes place across the plane of the bud. DNA replication.
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In budding yeast, the major control point is at
The G1/S transition: Start
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How do you clone y complementation?
Isolate the mutations which are temp sensitive. At 35'C, the cells stop dividing (arrest in G1 - NO BUD). If you mutangenise your culture of yeast, changing the nucleotide sequence. You do this randomly, killing 95% of culture cells. I
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What happens if we arrest at restrictive temperature?
All the cells stop dividing at a specific point in the cell cycle. You do mutageneisis, shift the temp then look at morphology, throwing away all the ones with mixed morphology and keeping the specific ones.
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What do we do with the cells that can grow at restrictive temperature?
Separate this one and clone it
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Fission yeast looks like ...
A big bacterial cell
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How does fission yeast divide?
By fission.
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What is the difference between fission and budding yeast?
The major control point in fission yeast is G2/M not start.
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All the genes involved in the cell cycle in eukaryotes are...
Highly conserved
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Fission yeast grows as a large bacterial cell and...
Divides down the middles
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The cell always divides at the same..
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How do the control the G2/M transition?q
We isolate the temperature sensistive mutation and find that the cell continues to grow but doesn't divide.
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cdc2+ is both...
Required and rate limiting in mitosis
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The cdc2 gene encodes a
cdk1 Cyclin dependent kinase
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cdc2 is the second subunit of
MPF - It is only active with cyclin and provides catalytic activity.
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How many genes are central to the control of the G1/M transition?
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In the absence of cdc25 you can't do
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What do we see if we lack wee1?
Advanced mitosis - this shows that it's not absolutely required for mitosis.
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What is rate limiting and a negative regulator of mitosis?
Wee1 - when it's present, mitosis occurs normally, but when it's absent, you do it quicker
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What are activators of mitosis?
Cdc2 and cdc25 are needed to do mitosis. If you increase expression of cdc25, you advance mitosis
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What happens if you increase cdc25 expression?
You advance mitosis
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What is the fission yeast cyclin B?
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What makes up the fission yeast MPF?
Cdc13 and Cdc2
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What is the role of CDC25?
It is a protein phosphatse (remves phosphate groups) and therefore the activator of the MPF complex
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Wee1 is an... of the complex
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Wee1 is a
Protein kinase
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What is the process you have to go through to assemble an active MPF?
Cdc13 and cdc2 are complexed together. This is now the substrate for wee1. Wee1 phosphorylates it. It's still inactive. Cak phosphorylates the complex. cdc25 remobes the tyrosine 15 and makes it active. Cdc25 unblocks the binding surface
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What does phosphorylation do?
It changes the charge of the complex, which in turn changes the shape. This changes the behaviour of the protein within the cell.
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What creates rapid transition from CDK inactive to active state?
Positive and negative feedback loops.
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How do we activate mytotic cyclin?
By removing the phosphate groups
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What is the equivalent of the fission yeast cdc2, for budding yeast?
cdc28 - it's the same protein, they're highly conserved
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What is the SPF?
S phase promoting factor
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What does the amino acid do?
It changes the structure of the protein slightly
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When do the cells become arrested in g1?
When we have low affinity cdc28, rather than high affinity
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Why might any gene you encode in a gene be expressed more?
Because you have up to 30 copies of the plasmid per cell - any gene is expressed more than usual. We're producing excess of wildtype g1 which shifts the equilibrium.
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What shifts the equilibirum?
Producing excess of the wild type G1 cyclin, so you can form some active complexes to get you through.
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Having excess of the G1 wild type cyclins drives...
Colony formation
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Are cyclin dependent kinases all active act the same time of the cell cycle?
No they are not, they activate at different points of the cycle
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Is cdk1 conserved?
Highly throughout evolution - it is structurally and functionally conserved amongst eukaryotes
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Having got the amino acid sequence of multiple organisms, you can redict...
the potential DNA sequence of any organism and attempt to clone it.
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Amino acid sequence means you can predict...
potential DNA sequence of coding region
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You can synthesize oligonucleotides corresponding to the DNA sequence coding...
For regions 1 and 2
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You can use oligonucleotides to amplify DNA between regions 1 and 2 and then
The whole open reading frame from cDNA source
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You can clone...
Open reading frame into yeast expression plasmid.
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How can you test conservation?
By complementation
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What makes up MPF?
Cyclin andcdc2
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The MPF is also know as
Cyclin dependent kinase 1 complex (CDK1)
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Is there just one CDK activity funcitioning at different points in the cell cycle?
No, there are multiple CDK activities
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The whole process of mitosis is regulated by...
Cycles of phosphorylation and dephosphorylation.
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Card 2


In budding yeast, the major control point is at


The G1/S transition: Start

Card 3


How do you clone y complementation?


Preview of the front of card 3

Card 4


What happens if we arrest at restrictive temperature?


Preview of the front of card 4

Card 5


What do we do with the cells that can grow at restrictive temperature?


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