Lecture 30 Flashcards

1
Q

Cancer is a disease of genetic stability, T or F

A

F – genetic instability

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2
Q

Controlling cellular proliferation involves control of cell death, T or F

A

T

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3
Q

What is the goal of the cell cycle

A

To produce 2 daughter cells that are accurate copies of the parent

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4
Q

How can you determine that amount of time a cell is in S phase

A

To determine the amount of time a cell spends in the synthesis phase of the cell cycle you first need to establish the number of cells in S phase and synthesising DNA within a colony. To do this 32P containing phosphate is included in the medium on which the cells are cultured. The cells actively synthesising DNA and are hence replicating will incorporate this radioactive phosphate into the genome. These cells can then be visualised by exposing a film to the colony and the radiation emitted from the cells undergoing S phase will leave black spots in the film. These can then be counted

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5
Q

Roughly what percentage of cells are in S phase at any one time

A

0.35

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6
Q

Given that the average S phase lasts 7 hours and this accounts for 35% of the cell cycle, how long is the average cell cycle

A

21.42 hours – 7.5/0.35

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7
Q

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

A

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

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8
Q

If the cells spend roughly 1 hour in M phase, what percentage of cells in a colony would show tubulin staining indicative of mitosis

A

5% - 1/20 x 100

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9
Q

Why are Drosophila embyros ideal models for S and M phase

A

The Drosophila embryo spends 15 minutes in S phase to replicate its genome and then another 15mins in mitosis

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10
Q

Explain the usefulness of schizosaccharomyces pombe in studying the cell cycle

A

S. pombe only grows in one direction and so the length of the organism can tell you which phase of the cell cycle it is in

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11
Q

Schizsaccharomyces pombe spend very little time in M phase, T or F

A

F – they spend a long time in M phase

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12
Q

How can mutations that affect the cell cycle be easily studied in S. pombe

A

Mutations in genes that cause changes in the length of the yeast are affecting the various stages of the cell cycle

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13
Q

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

A

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. Instead temperature sensitive mutations are used where permissive temperatures can be changed to restrictive ones to screen for specific cell cycle genes

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14
Q

What is meant by the execution point of a particular gene

A

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

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15
Q

What is the name of the gene involved in the G2 to M phase transition during the cell cycle of S.cerevisiae

A

Cdc28/2

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16
Q

Xenopus laevis ooctyes grow without dividing for months before they are laid and fertilised, what stage of the cell cycle are these cells arrested in

A

G2

17
Q

How often does each cell division in the early Xenopus embryo occur

A

Every 13 minutes

18
Q

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

A

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

19
Q

What phenomena is seen during the first 8 cell cycles of the developing Xenopus embryo

A

There is no change in the size of the embryo as the cells aren’t undergoing transcription. The cells themselves get smaller but increase in number so no overall change is seen

20
Q

How have Xenopus eggs been used to observe replication and mitosis

A

Centrifugation of Xenopus eggs creates a pellet containing the nuclei and a cellular extract. 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

21
Q

Which two proteins are responsible for mediating the stages of the cell cycle

A

Cyclin-dependant kinases and cyclins

22
Q

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

A

Rao and Johnson took cells that were in interphase and metaphase and fused them together by infecting both with a 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

23
Q

What was the effect of injecting cytoplasm from a Xenopus egg into an arrested oocyte, why is this

A

Injection of the cytoplasm into an oocyte led to that cell maturing and induced early entry into M phase. This is due to the action of what was deemed maturation promoting factor (MPF)

24
Q

How was it determined that kinase activity was involved in the progression of a cell in the cell cycle

A

Fractions were isolated from a cell containing the maturing promoting factor activity. These were then incubated with histone H1 and radiolabelled ATP. The histone H1 proteins were then observed for radioactivity which revealed that indeed the radioactive phosphate had been transferred from ATP to the protein. This indicates the action of a kinase

25
Q

How did Tim Hunt discover cyclins

A

Bathed sea urchin eggs in radioactive methionine and observed the radioactive proteins produced. He observed that a particular protein made in sea urchin eggs 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. These were later found to be cyclins, whose levels fluctuate during the cell cycle

26
Q

Explain the phenotype of cdc2 temperature sensitive mutant S.pombe relative to the cell cycle

A

Cdc2 mutants at restrictive temperature are elongated because they aren’t dividing

27
Q

Explain the phenotype of cdc25 temperature sensitive mutant S.pombe relative to the cell cycle

A

Cdc25 mutants at restrictive temperature are elongated too because they aren’t dividing – hence have the same phenotype as cdc2

28
Q

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

A

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

29
Q

Explain the interaction between cdc2, cdc25 and wee1

A

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

30
Q

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

A

MPF was later determined to be almost identical to cdc2. It was found to consist of cdc2 and its corresponding cyclin B

31
Q

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

A

F – additional regulation is going on

32
Q

What are the two functions of cyclins

A

Activate catalytic subunits are target it within the cell to specific substrates

33
Q

Explain the role of the pre-replicative complex in limiting S phase of the cell cycle

A

The pre-replicative complex marks where the origins of replication are. During S phase, the pre-replicative complex is knocked off and is degraded which means that it needs to be re-made before the next cell cycle. This acts to limit the cycle to a single S phase per cycle

34
Q

Describe and explain the results of the fairy liquid experiment that investigated S phase

A

In this experiment researchers isolated the nuclei from an extract that had already undergone replication (S phase). These were exposed to a detergent before introducing them into another extract. It was observed that these nuclei underwent another round of replication seen (double S phase). This showed that permeabilising the nuclear envelope was sufficient to get the nuclei to replicate again

35
Q

Explain what is meant by the licencing factor hypothesis

A

In every cell cycle DNA is licenced to undergo one round of replication only. During replication, the licence is destroyed/removed from DNA. Thus, DNA inside the nucleus cannot access licensing factor because it resides in the cytoplasm

36
Q

What was licencing factor later identified to be

A

Licencing factor turned out to be components of the pre-replication complex

37
Q

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

A

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

38
Q

What are mcms

A

Mcms are helicases that unwind the DNA allowing S phase to begin

39
Q

What is the significance of the pre-replication complex and the cell cycle in cancer testing

A

Antibodies that recognise the pre-replication complex (specifically Mcms) are profoundly useful for detecting malignant cells in cervical samples. It makes it far easier to identify malignant epithelial cells in smear tests