Lecture 30

Question 1

Cancer is a disease of genetic stability, T or F

Answer 1

F – genetic instability

Question 2

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

Answer 2

T

Question 3

What is the goal of the cell cycle

Answer 3

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

Question 4

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

Answer 4

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

Question 5

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

Answer 5

0.35

Question 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

Answer 6

21.42 hours – 7.5/0.35

Question 7

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

Answer 7

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

Question 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

Answer 8

5% - 1/20 x 100

Question 9

Why are Drosophila embyros ideal models for S and M phase

Answer 9

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

Question 10

Explain the usefulness of schizosaccharomyces pombe in studying the cell cycle

Answer 10

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

Question 11

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

Answer 11

F – they spend a long time in M phase

Question 12

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

Answer 12

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

Question 13

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

Answer 13

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

Question 14

What is meant by the execution point of a particular gene

Answer 14

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

Question 15

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

Answer 15

Cdc28/2

Question 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

Answer 16

G2

Question 17

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

Answer 17

Every 13 minutes

Question 18

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

Answer 18

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

Question 19

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

Answer 19

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

Question 20

How have Xenopus eggs been used to observe replication and mitosis

Answer 20

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

Question 21

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

Answer 21

Cyclin-dependant kinases and cyclins

Question 22

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

Answer 22

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

Question 23

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

Answer 23

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)

Question 24

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

Answer 24

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

Question 25

How did Tim Hunt discover cyclins

Answer 25

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

Question 26

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

Answer 26

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

Question 27

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

Answer 27

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

Question 28

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

Answer 28

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

Question 29

Explain the interaction between cdc2, cdc25 and wee1

Answer 29

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

Question 30

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

Answer 30

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

Question 31

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

Answer 31

F – additional regulation is going on

Question 32

What are the two functions of cyclins

Answer 32

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

Question 33

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

Answer 33

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

Question 34

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

Answer 34

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

Question 35

Explain what is meant by the licencing factor hypothesis

Answer 35

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

Question 36

What was licencing factor later identified to be

Answer 36

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

Question 37

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

Answer 37

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

Question 38

What are mcms

Answer 38

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

Question 39

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

Answer 39

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