Lecture 6 - the cell cycle Flashcards Preview

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Flashcards in Lecture 6 - the cell cycle Deck (52):

What checkpoint is present in G1 

At the end of G1 - entrance to S is blocked if the genome is damaged. 


What checkpoint is present in S phase?

DNA damage checkpoint - DNA replication halted if genome is damaged


What checkpoint is present in G2?

At the end of G2 - entrance into M is blocked if DNA replication is not complete.


What checkpoint is present in M phase?

Anaphase is blocked if all of the chromatids are not assembled on mitotic spindle. 


How is confocal microscopy used to detect cells in S phase. 

Look for the incorporation of halogenated derivatives of deoxyridine which is incorporated into DNA instead of thymidine. Different antibodies can detect different halogenated forms of deoxyridine. If cells incorporate the iodo/chlorodeoxyridine then they are visualised by confocal microscopy and are shown to be in S phase. 


How are morphological markers used to detect cells undergoing mitosis. 

Cells are stained with a fluorophore for DNA and for mitotic spindle (tubulin). The proportion of cells at each stage of mitosis can be detected and gives an idea of how long each phase takes (mitosis = around one hour)


What model system was used to understand how the cell cycle works?

the early embryo was used as it only contains S and M phases. 

Embryos are stained with a dye and tubulin is replaced withGFP tubulin to allow investigstors to see how tubulin behaves. 

It takes 15 minutes to complete the cell cycle in the early drosophila embryo. 


What does analysis of DNA content tell us?

How many cells are in a particular phase of the cell cycle and how long that phase lasts in a particular cell type. 


What is flow cytometry?

It allows cells to flow through a thin narrow capillary tube. Light is then shone at this tubing and how much light reflected back is determined. 


How is flow cytometry used in determining the phase of the cell cycle. 

The area under the curve is proportional to the number of cells in that phase. Most cells in this example are in G1. 


What is univariate fluorescent activated cell sorting and why is it not as reliable as bivariate FACS?

It only measures one variable

Quite a lot of cells are in S phase are hidden as being in either G1 or G2. 


What is Bivariate FACS?

The DNA is measured, but the y-axis is different as the cells have been incubated with BrdU for 15 mins. This means S phase cells will have rapidly incorporated BrdU. The graph shows the intensity of hallogonation - if there is high intensity then it is in S phase, if low then it is in G1/G2.


Why is bivariate FACS more useful?

If there is a single set of cells that appear to be stuck in G1 phase we would assume they are in G1 arrest. If however you perform bivariate FACS on these cells it may reveal that in fact a set of the cells are beginning to incorporate BrdU so are in fact just beginning to start S phase. Helps us work out which part of a phase a cell is in. 


What advantages do studies of fission and budding yeats have 

Both are easily tractible (Easy to make mutants)

Both are cheap

No sophiticated equiptment is needed to find out which stage of the cell cycle they are in - just a light microsope to work out the length of the cell which only grows longitudinally. 


What are the differences between fission and budding yeast cells?

Budding yeast undergo an assymetric cell division and spend a lot of time in G1/ mitosis.

Fission yeast spends a lot of time in G2 phase. 


How are cdc temp sensitive mutant yeast used

At the permissive temperature there will be cells at different stages of the cell cycle

At the restrictive temperature, Cdc mutant cells will pause at the execution point. The number of mutants found is proportional to the length of time spent in the specific cell cycle stage. 


How do we know about cyclins?


They took cells in different stages of the cell cycle and fused them together using Sendai virus. They wanted to find out if one phase was biochimcally dominant then another. No matter where on the cell cycle cells are - mitotic cells always induced other phase cells into mitosis. i.e there is a biochemical entity which drives mitosis. 


What other experiment gave an understanding of cyclins?

Using the xenopus oocyte system. If the cytoplasm from an egg was injected into an oocyte arrested in G2, it caused mitotic entry. Conclusion is that the biochemical entity that drives mitosis is present in the cytoplasm and was named maturation promoting factor (MPF) 


What did purification of MPF discover?

Two bands seen on fractionating experiments. One of which appeared to be a kinase. 


What did experiments in fission yeast schizosccharomyces pombe show?

Cdc2 identified - at a restrictive temp, Cdc mutants fail to enter into mitosis. 

To genotype this - take a library of known genes from the yeast and reintroduce them into the mutant - look for the gene that rescues the phenotype, then this gene is sequenced. 


What did the simple 2 phase cell cycle reveal about the factors controlling mitotic entry. 

Transitions into mitosis are driven by activated protein kinase based machinery. These turned out to be Cdks. Once in mitosis it is made irreversible by the destruction of cyclin. 

Peak MPF activity also coincides with peak cyclin activity


What studies were performed after discovering Cdc2 and cyclin B in yeast?

Hybridisation studies between yeast and human cDNA to find similar genes. 


What are the two functions of cyclins 

Activate the catalytic subunit of the relevent kinase

Target the catalytic subunit so the kinase phosphorylates the appropriate target protein at the correct time to ensure the correct phase transition can occur. 


What is the Cdk for cyclin D and at what phase is this found 

Cdk4/6 - found in G1


What is the Cdk partner of cyclin E and what phase is this found in 

Cdk2 and found in G1/S checkpoint.


What is the Cdk partner of Cyclin A and which phase is it present in 

Cdk2/1 found in S phase


What is the Cdk partner of Cyclin B and which phase is it found in 

Cdk1 and in M phase. 


Why does S phase only happen once>

DNA replication starts from very specific origins of replication. Origin recognition complexes (ORC) bind to these sites very early in G1 phase. 
ORC binding allows recruitment of Cdc6 and Cdt1, these bind ORC and also recruit the Mcm complex. 
This completes the pre-replicative complex (Pre-RC). Mcm is the principle DNA helicase that unwinds DNA for replication. 
The cell remains in G1 until the presence of an S phase cyclin-Cdk trigger
This complex phosphorylates Cdc6 causing its destruction 
Cdt1 is released and inhibited by geminim
After this a preinitiation complex is recruited and opens up the two replication forks by phosphorylating ORC
replication is performed and the ORC remains phosphoryalted in G2 phase. This marks it as replicated and it wont occur again at this origin. 


What binds the specific origins of replication 

The origin replication complex



What does ORC binding cause 

Allows recruitment of Cdc6 and Cdt1, these recruit the Mcm complex


What can occur if Geminim is mutated

Multiple rounds of replication as Cdt1 is no longer released or inhibited. 


What is the use of antibodies that recognise the pre-replication complex (PRC) 

Very useful for detecting malignant cells. An example of this is the cervical smear (papinicolaou test) 


What are CKIs and where in the cell cycle do they operate?

cyclin dependent kinase inhibitors which operate mainly in G1 and S phase


How are Cdks regulated by phosphorylation?

They must be phosphorylated on a specific threonine residue in order to be activated


Which threonine is phosphorylated in Cdk2 and Cdk4

T160 and T127 respectively 


What carries out the phosphorylation required for Cdk activity 

The cdk7-cyclin H complex - a serine threonin kinase thats also involved in transcription and DNA repair.


What inhibitory phosphorylations can be performed on Cdks

Phosphorylation of adjacent threonine and tyrosine residues (T14 and Y15 in cdk1) which is performed by dual specific kinases such as WEE-1 and MYT1. 


How is inhibitory phosphorylation relieved 

When Cdc25 phosphatases (CDC25A/B/C) dephosphorylate these residues. 


What is responsible for the sudden burst of MPF concentration seen below?

The action of Cdc25 and Wee1 regulating the action of Cdc2 (Cdk1). This is also responsible for MPF activity not following the cyclin pattern of expression. 


Which CKIs are responsible for the inhibition of Cyclin D/Cdk4/6 complexes.?

The INK proteins


What are the roles of the other CKIs (not the INK proteins)

P21 clip1, P27 kip1 and P57 kip2 are more widely acting at later stages of the cell cycle


What effect does TGFbeta have on the cell cycle

When applied to epithelia, it increases the expression of P15 INK4B

This prevents cells progressing through G1 to the restriction point. 


Which CKI is induced following physiological stress eg from DNA damage. 

P21 clip1 - can act on E/A cdk2 and A/B cdc2 to stop a cell in its tracks. 


What effect does mitogen signalling have on CKIs and how is it achieved

Control the cell cycle clock by regulating the function of CKIs. Mitogen (Growth factor) binding of receptors induces PI3K activity - then AKT/PKB which inhibit the expression of CKIs P21 clip1 and P27 kip1


What does the misregulation of CKIs have in common with clinical consequences of cancer

If CKIs are misregulated and found in the cytoplasm of cancerous tumours then the outcome of the patients is worse, reducing survival. 


What is the restriction point?

Found in late G1 - the restriction point is where cells either commit to DNA replication or death. Growth factors are responsible for cells getting past this point. 


What is the restriction point controlled by?

The pRb tumour suppressor


What keeps S phase genes off in G1 

E2F - a TF is unable to function because it is bound to Rb. Rb also ensures the presence of histone deacetylase in that region which ensures DNA can't form compacted chromatin. 


What role does cyclin D have in the restriction point and what is the effect of mitogens on this role 

Mitogens upregulate the expression of Cyclin D. Cyclin D Cdk4/6 complexes are able to phosphorylate Rb to its hypophosphorylated state, which is a pre-requisite to the R point transition. 

Hypophosphorylated Rb is not as good at sitting at the E2F binding site so S phase gene transcription can occur. 

One of the genes transcribed is Cyclin E - Cyclin E Cdk2 complexes are capable of hyperphosphorylating Rb making it completely inactive. S phase proteins are transcribed.


What can be said of receptors that activate cell proliferation

All operate through the induction of Cyclin D synthesis and stability 


Is Rb function lost in only few cancers?

No, peturbed in most, if not all cancers. 


What Cdk4/6 inhibtor is used for the treatment of breast cancer

The CDK4/6 inhibitor palbociclib combined with aromatase inhibitors for the treatment of estrogen receptor-positive breast cancers. Multiple studies have demonstrated that the CDK-RB-E2F pathway is critical for the control of cell proliferation.