Lecture 6 Cell Cycle Flashcards Preview

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Flashcards in Lecture 6 Cell Cycle Deck (79)
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1

What are the two functions of cyclins

Activate catalytic subunits of the relevant Cdk and target these catalytic subunits within the cell to specific substrates

2

How are temperature-senstivite mutations in yeast used to study cell cycle genes

Temperature sensitive mutations are used to screen for specific cell cycle genes. These are mutations that only manifest at specific temperatures when the permissive temperature is changed to a restrictive temperature

3

What was the significance in the similarity between the protein sequence of MPF and the base sequence of cdc2

The sequence of the cdc2 gene identified by Paul Nurse was almost identical to that of MPF identified by Masui and Lokha. It was later determined that MPF actually consisted of cdc2 and cyclin B the protein found to oscillate in sea urchin eggs by Tim Hunt

4

What is useful about female frogs that allows the study of their eggs extremely easy

Injection of progesterone into a female frog will cause her to lay eggs

5

Which region of cdks becomes phosphorylated to increase catalytic activity

T loop

6

Explain the experiments carried out by Rao and Johnson and how this developed our understanding of the cell cycle

Rao and Johnson took cells that were in interphase and metaphase and fused them together by infecting both with Sendai virus that elicited fusion. Fusion of the interphase and mitotic cells caused the interphase cells to enter mitosis prematurely regardless of where they were in the cell cycle. These experiments demonstrated that mitosis was somehow the dominant program for a cell

7

What is meant by the execution point of a particular gene

The point in the cell cycle that a mutation in a gene causes arrest

8

What is significant about yeast cdks

Yeast possess only one cdk and this is capable of mediating the entire cell cycle

9

Explain the phenotype of wee1 temperature sensitive mutant S.pombe relative to the cell cycle

Wee1 mutants are shorter than normal because they are dividing prematurely and spending less time in G2

10

How can you morphologically determine where S. cerevisiae yeast are in the cell cycle

Depending on the size of the daughter bud you can determine where the cell is in the cell cycle

11

Describe an in vivo assay that can be use to determine whether cells are in S phase

Incubate cells with halogenated nucleotide derivatives such as bromo-deoxyuridine which will be incorporated into the DNA if the cells are in S phase. These halogenated bases can then be recognised with specific antibodies that recognise individual halogens

12

Below is a table containing a list of the stages of the cell cycle. Fill in the column with the average time that a cell spends in each phase?

See completed table below

13

Why can’t we investigate the effects of mutating genes involved in the cell cycle and how is this overcome

If you mutate a gene that controls the cell cycle in a yeast cell it is likely to kill that cell and hence there will be no cells to study.

14

How was cdc2 identified

Yeast cells were mutagenized and colonies with the elongated phenotype were identified. A library of yeast genes were then introduced into cells from these colonies to see which one rescued the wild type phenotype. This lead to the identification of cdc2

15

How did Tim Hunt discover cyclins

Bathed sea urchin eggs in radioactive methionine and observed the radioactive proteins produced. He observed that a particular protein accumulated for a time but then periodically disappeared just before the cells divided (mitosis). This implied that these proteins were being destroyed and this operated in parallel with egg division. This was later found to be cyclin B whose levels fluctuate during the cell cycle and peak before mitosis

16

How have Xenopus eggs been used to observe replication and mitosis

Centrifugation of Xenopus eggs creates a pellet containing the nuclei and on top of that a cellular extract. This concentrated cell extract retains all of the functionality of the egg containing the proteins and lipids as well as preserving the functional mRNA. If DNA or chromatin is added to this cell extract with will acquire its own nuclear envelope in vitro. This allows you to observe replication and mitosis easily

17

How does Eukaryotic replicator selection occur

Origin replication complex (ORC) binds to the replicator sequence. Helicase loading proteins Cdc6 and Cdt1 then bind to ORC to convert the single stranded replicator sequence into a pair of replication forks. Mcm2-8 then also binds to complete formation of the pre-RC

18

What is the effect on the cell cycle of introducing dominant negative Akt into cells

This increases the activity of p21Cip1 and p27Kip1 subsequently leading to a global inhibition of proliferation

19

Which component of the pre-replication complex was found to mediate the transition into S phase

Removal of cdt1 allows the functional recruitment of mcms and S phase to occur

20

Describe the role of phosphatases in regulating cdk activity

The cdc25 phosphatases antagonise wee1 and dephosphorylate cdks returning them to an active state

21

What kind of cell cycle arrest is indicated by the two FACS traces below how do they suggest different arrest points

The univariate FACS graph on the left shows that all the cells are arrested in G1 phase. However the bivariate FACS trace suggest that these cells are trying to enter S phase indicated by cells in the dashed box however these cells are unable to complete S phase and enter G2. Hence this is a DNA replication problem rather than a growth one.

22

Which proteins are responsible for the phosphorylation of Cdks that increase their catalytic activity

The CDK7–cyclin H complex consisting of the cdk-activating kinase (CAK)

23

Regulation of cdk activity occurs only at the level of cyclin binding T or F

F – additional regulation is going on

24

What kind of protein is S. pombe cdc2

Cyclin-dependent kinase (Cdk1)

25

Given that the average S phase lasts 7.5 hours and this accounts for 35% of the cell cycle how long is the average cell cycle

21.42 hours – 7.5/0.35

26

How can you determine the amount of time cells spend in M phase

Stain the cells for tubulin using fluorescently labelled antibodies for tubulin. Then count the number of cells that have formed the mitotic spindle/metaphase plate. The percentage of the cells can then be used to work out the percentage of the cell cycle this stage must account for

27

Explain the interaction between cdc2 cdc25 and wee1

Cdc25 and wee1 both act upstream of Cdc2. Cdc25 is a positive upstream regulator whereas wee1 is a negative regulator

28

How can phosphorylation of cdks be used to negatively regulate its activity

Phosphorylation of threonine 14 and tyrosine 15 residues by wee1 and myt1 act to inhibit cdk activity

29

What is the limitation of temperature-sensitive mutations

You will always miss some genes due to the permissive and restrictive temperatures you chose

30

Specifically how does the CKI p15INK4B act

p15INK4B binds cdks directly at the kinase active site blocking its activity by interfering with ATP binding