Lecture 30 Flashcards Preview

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Flashcards in Lecture 30 Deck (39)
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1

Cancer is a disease of genetic stability, T or F

F – genetic instability

2

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

T

3

What is the goal of the cell cycle

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

4

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

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

5

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

0.35

6

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

21.42 hours – 7.5/0.35

7

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

8

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

5% - 1/20 x 100

9

Why are Drosophila embyros ideal models for S and M phase

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

10

Explain the usefulness of schizosaccharomyces pombe in studying the cell cycle

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

11

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

F – they spend a long time in M phase

12

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

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

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

14

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

15

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

Cdc28/2

16

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

G2

17

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

Every 13 minutes

18

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

19

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

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

How have Xenopus eggs been used to observe replication and mitosis

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

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

Cyclin-dependant kinases and cyclins

22

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 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

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

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

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

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

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 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

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

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

27

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

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

28

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

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

29

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

30

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

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