Lecture 30 Flashcards
Cancer is a disease of genetic stability, T or F
F – genetic instability
Controlling cellular proliferation involves control of cell death, T or F
T
What is the goal of the cell cycle
To produce 2 daughter cells that are accurate copies of the parent
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
Roughly what percentage of cells are in S phase at any one time
0.35
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
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
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
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
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
Schizsaccharomyces pombe spend very little time in M phase, T or F
F – they spend a long time in M phase
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
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
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
What is the name of the gene involved in the G2 to M phase transition during the cell cycle of S.cerevisiae
Cdc28/2
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
How often does each cell division in the early Xenopus embryo occur
Every 13 minutes
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
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
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
Which two proteins are responsible for mediating the stages of the cell cycle
Cyclin-dependant kinases and cyclins
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
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)
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
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
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
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
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
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
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
Regulation of cdk activity occurs only at the level of cyclin binding, T or F
F – additional regulation is going on
What are the two functions of cyclins
Activate catalytic subunits are target it within the cell to specific substrates
Explain the role of the pre-replicative complex in limiting S phase of the cell cycle
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
Describe and explain the results of the fairy liquid experiment that investigated S phase
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
Explain what is meant by the licencing factor hypothesis
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
What was licencing factor later identified to be
Licencing factor turned out to be components of the pre-replication complex
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
What are mcms
Mcms are helicases that unwind the DNA allowing S phase to begin
What is the significance of the pre-replication complex and the cell cycle in cancer testing
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
