Lecture 1&2 - The cell cycle and cell cycle control

Question 1

What are the four phases of the cell cycle and what happens in each?

Answer 1

G1: Cell grows and carries out normal metabolism, cell receives external signals to enter the cell cycle and the organelles duplicate
S: Dna replication and synthesis
G2: Cell grows and prepares for mitosis, resolves problems
M: Mitosis and Cytokinesis

Question 2

What is Quiescence?

Answer 2

When a cell is in an inactive state and not actively dividing, a cycling of G1 (G0)

• can last for years
• must receive signals to exit G0

Question 3

How did Walther Flemming illustrate the major stages of the cell cycle?

Answer 3

Stained cells genetic material

-Observed that in each cell cycle the interphase chromatin is condensed into transmissible chromosomes then decondensed

Question 4

What are the major componants of DNA condensation?

Answer 4

• Nucleosomes (DNA wrapped around histone protein cores)
• Solenoid (Winding of the nucleosomes on top of each other)
• Mitotic chromosome scaffold

Question 5

What occurs in the first stage of DNA condensation? (Nucleosome)

Answer 5

• 146bp length of DNA is wrapped around an octamer of histone subunits, made up of pairs of: H2A, H2B, H3, H4, forming a Nucleosome
• H1 tightens the connections between DNA wrapped around the histone octamer
• Creates a ‘beads on a string’ structure
• results in a 6-fold reduction in length

Question 6

What is the function of the H1 molecule in the nucleosome?

Answer 6

Tightens the connections between DNA wrapped around the octamer by binding to linker DNA 20-80bp long
-stabilises chromatin structure

Question 7

What occurs in the second stage of DNA condensation? (Solenoid)

Answer 7

Nucleosomes wind up on top of each others, forming chromatin fibres 30nm thick = Solenoid
-results in a 40 fold reduction in length

Question 8

What occurs in the third stage of DNA condensation?

Answer 8

After coiling and supercoiling, chromosome fibres attach to the mitotic chromosome scaffold

• results into 46 chromosomes with total length ~200µm
• 10,000 fold reduction in length

Question 9

For what percentage of time is a cell in M phase?

Answer 9

5%

Question 10

What is the structure of microtubules in Interphase and the processes in the 5 stages of Mitosis?

Answer 10

Interphase: Microtubules are long and diffuse, not attached to centromere
Prophase: Chromosomes condense
Prometaphase: Nuclear envelope breaks down
Metaphase: Chromosomes align at the spindle equator
Anaphase: Sister chromatids separate along the spindle as the astral microtubules grow
Telophase: Cleavage furrow constricts around the central microtubules and central spindle as nuclei reassemble

Question 11

What is the cleavage furrow?

Answer 11

An indentation of a cells surface which begins the progression of cleavage leading to cytokinesis

Question 12

What are the three types of astrotubules formed when the spindle apparatus forms, and what are they?

Answer 12

• Kinetochore microtubules: attaches to kinetochores of condensed chromosomes and moves towards centrosomes
• Polar microtubules: emanates from 2 centrosomes and overlap with each other. Push against each other to force centrosomes towards cell plates
• Astral microtubules: extend from centrosome to cell membrane and anchor to the periphery of the cell. Pull centrosome towards one of the poles

Question 13

What is the kinetochore?

Answer 13

Sequences of DNA in the centromere where microtubules bind

Question 14

What experimental benefits can be gained from the natural syncrony in fly embryos?

Answer 14

can study many cells at the same stage at the same time

Question 15

Give two examples of chromosome segregation gone wrong

Answer 15

• Anaphase bridges: when telomeres of sister chromatids fuse together and fail to completely segregate into their respective daughter cells
• Aneuploidy: inaccurate segregation of chromosomes

Question 16

What is the chromosome complement at each of the four stages of the cell cycle?

Answer 16

G1: 2N
S: 2N/4N
G2: 4N
gametes: N

Question 17

How can multiple cell cycle synchrony be achieved?

Answer 17

Chemical inhibition
-blocks cell cycle before mitosis or before S phase leading to aggregation of cells at a particular stage in the cell cycle
-check by flow cytometry - measures the DNA content of a cell (SS, labelling, fluorescent peak) Normally smallest amount of light scattered in G1, smear for S, largest in G2
Natural synchrony - use process of quiescence to synchronise

Question 18

What are the chemicals that can be used to attain multiple cell cycle synchrony?

Answer 18

Nacodazole (G2)
Cytochalasin B
Thymidine (G1) 
Aphidicolin 
Flow cytometry analysis - Nacodazole shows biggest fluorescent peak for larger cells (G2); Thymidine shows biggest fluorescent peak for smaller cells (G1)

Question 19

What are the benefits of Natural synchrony over chemical inhibition when achieving experimental cell cycle synchrony?

Answer 19

• adding chemical may prevent validity

- more natural and can stimulate cells with a chemical signal to enter cell cycle at the same time

Question 20

What are five classic experiments that identified the cell cycle?

Answer 20

Cell fusion experiments - Roa and Johnson
Discovery of cyclins - Hunt
Discovery of MPF - Lokha and Masui
Discovery of cell cycle genes - Hartwell
Discovery of Cdc2 - Nurse

Question 21

Describe the Cell Fusion Experiments done by Roa and Johnson

Answer 21

Aim - (1)discover what happens when take a cell in one stage of cell cycle and fuse with one in another; (2)what happens when cells in cell cycle phases are exposed to different diffusible factors

(1) combined:
Cells in S + G1 => all cells were in S phase. Hypothesised that it was by an S phase specific factor (SPF)

Cells in S + G2 => No change. Therefore, SPF factor not recognised by G2 nucleus

found there was a difference in G1 cells being able to respond and G2 not.

(2)MPF (mitosis promoting factor)

Combined G1 and G2 cells => neither produce any dominant chemical signal

M phase cells fusing with any other cell in a different phase the M phase is dominant, acts on all cells in any phase

Consequently M phase cells produce MPF

Question 22

What is MPF?

Answer 22

Mitosis promoting factor or Maturation promoting factor: diffusible factor that triggers mitosis

Question 23

Why use Xenopus eggs as an experimental organism?

Answer 23

• rapid cleavage divisions
• lay hundreds of eggs at the same time
• fertilisation and maturation is external and visable
• only get one gamete, all the nutrients go to one cell resulting in a robust cell with high volume of enzymes and nutrients

Question 24

What is the process of Oocyte maturation in frogs that Masui observed, and what did he conclude?

Answer 24

Oocyte is arrested at G2 phase, where an influx of progesterone stimulate progression into Meiosis I followed by meiotic interphase. The egg is released and the cell cycle is arrested again at metaphase meiosis II. Fertilisation then occurs and the cell cycle continues and undergoes the first cleavage. (post fertilisation colours segregate to show first division)
Concluded - there are 2 key points of regulation in meiotic cell cycle

Question 25

How did Lokha and Masui identify MPF?

Answer 25

1- Took cytoplasm from an egg in Metaphase of MEiosis II, injected it into a G2 arrested oocyte. => Initiated the maturation process and G2 arrested oocyte matured into a metaphase meiosis II arrested egg Concluded - must be a chemical in the cytoplasm of metaphase meiosis II arrested egg that promotes meiosis = MPF 2- Used serial cytoplasmic transfers to show that this was always the case, and that MPF was always present (Developed a robust assay) Used this to test for MPF activity over time

Question 26

What test did Lokha and Masui do to test MPF activity of a frog oocyte over time and what did they find?

Answer 26

1- Took cytoplasm from different stages of frog egg maturation and tested for MPF activity by the ability to induce egg maturation when injected Found - High MPF activity: - After G2 arrest when cell stimulated with progesterone at Meiosis I - During Meiosis II Metaphase arrest, declines at fertilisation - Then continued ossilations of high MPF activity during embyronic mitosis cycles

Question 27

Why use sea urchins as an experimental organism for developmental studies?

Answer 27

- large eggs that develop externally | - visible early divisions

Question 28

How did Tom Hunt discover cyclins?

Answer 28

-SDS-PAGE analysis of proteins present at each stage of the cell cycle after the fertilisation of Sea urchin eggs. Identified that: -cyclin levels ossilate after fertilisation whilst other proteins remained constant -cyclin peaks precede cell division, correlating with the cell cycle (intensity of bands were tested by densitometry)

Question 29

How did Hunt show that oscillating levels of cyclin (falling during cell division) were achieved by periodic destruction?

Answer 29

1- labelled proteins either: a) continuously to visualise the total amount of protein at any one time (SDS-PAGE) b) in short pulses to visualise newly synthesised proteins (SDS-PAGE) Found - a) different level of cyclins compared to other proteins in the total amount of proteins b) but the same level of cyclins were being newly synthesised Conclusion - that cyclins were synthesised constantly but degraded at specific points during the cell cycle

Question 30

How did Hunt check that the oscillating levels of cyclin were directly related to the cell cycle?

Answer 30

blocked the cell cycle and analysed the total and contiually synthesised products by SDS-PAGE found - no destruction occurs if the cell cycle is blocked

Question 31

How did Hunt's cyclin observations match up to previous observations about MPF?

Answer 31

Cyclin disappears as anaphase begins, correlating with MPF activity at cyclin peaks

Question 32

Why use Sacromyces cerevisae as an experimental organism in developmental biology?

Answer 32

- Unicellular eukaryote, life cycle = cell cycle - can grow and visually mark the stages of the cell cycle - can analyse gametes - start of the cell cycle = restriction point in mammalian cells

Question 33

How did Lee Hartwell identify the cell cycle genes in Sacromyces cerevisae?

Answer 33

1-Created temperature sensitive mutants, identified their mutant phenotype and revealed a complex set of interconnecting pathways e.g. action of Cdc8, Cdc10 and Cdc 24 mutants 2- once identified, placed the point of action of mutants in order and identified the secondary mutations (supressors) that counteract them

Question 34

How do temperature sensitive mutants work?

Answer 34

- at permissive temperature the growth of all mutants is the same as the WT - restrictive temp cell arrests at a certain point in the cell cycle

Question 35

What temperature sensitive mutants did Hartwell use?

Answer 35

cdc (cell division cycle) mutants = arrest before phase in which its gene product is needed non-cdc mutants = arrest immediately irrespective of what cell cycle stage it is at

Question 36

What did Hartwell observe about Cdc8, 10, 24 mutants and what could he conclude about the involvement of their mutated genes in the cell cycle

Answer 36

Cdc8 mutants arrested before DNA replicated => gene product is involved after initation of DNA synthesis but before full synthesis could occur Cdc10 stops during cyctokinesis => gene product involved in cytokinesis Cdc24 arrests before fusion during yeast mating => involved in bud formation

Question 37

What were the supressor genes that Hartwell identified for some of the cell cycle genes?

Answer 37

Cdc8 - Cdc21 | Cdc10 - Cdc3, Cdc11

Question 38

How did Nurse discover Cdc2 (key cell cycle regulator) in Schizosaccharomyces pombe (fission yeast)?

Answer 38

- found temperature sensitive mutants that did not preogress through the cell cycle at the restrictive temperature - identified mutant gene through plasmid rescue - identified cyclin dependent protein kinase 2

Question 39

What is the process of plasmid rescue?

Answer 39

1. Make cDNA library of the whole genome of yeast against human 2. Transform mutant Cdc2^ts cells with plasmid cDNA library and grow at the restrictive temperature 3. Only cells that have been transformed with the WT copy of the mutated gene will grow 4. Re-isolate cDNA and clone from rescued cells 5. Sequence gene and predict protein 6. Identify orthologs in other organisms

Question 40

How was it concluded that MPF is a complex of cyclin B and Cdc2?

Answer 40

- Cdc13 and Cdc2 mutants have similar phenotype and therefore functions - the predicted amino acid sequence reveals that cdc2 is a protein kinase and cdc13 is a cyclin - cdc13 and cdc2 form a heterodimeric complex -the activity of Cdc2 kinase correlated with rising level of cdc13 (like MPF, shown in previous experiments: Hunt - in sea urchin eggs cyclin levels fluctuate with the cell cycle; Masui - Xenopus MPF induces cell division and activity fluctuates with cell cycle) Therefore MPF is made up of two subunits, a kinase and a cyclin

Question 41

The expression of what genes (and how) act as checkpoints in the cell cycle?

Answer 41

G1: CDK4/6 forms a complex with cyclin D -expression falls as S phase cyclins expression increases S: CDK2 complexes with either cyclin E or A -expression falls as M phase cyclins take over, CDK1 (Cdc2) complexes with either A or B (MPF) These molecules are expression sequentially and are dependent on what was previously expressed, regulating the cell cycle

Question 42

When is MPF involved in M-phase checkpoints (mainly in yeast and embryonic systems) and how is it regulated?

Answer 42

MPF is involved in G2 progressing to the M phase Regulation relies upon: Induction of MPF -blocked until the S phase is complete and damage is repaired (phosphorylation of CDK1/Cdc2) Destruction of MPG -blocked until chromosomes are aligned on metaphase plate (spindle assembly checkpoint)

Question 43

What is the destruction of MPF controlled by?

Answer 43

APC/C (anaphase promoting complex), followed by destruction by the proteosome

Question 44

During the cell cycle what is MPF destruction required for?

Answer 44

- for cells to separate | - phosphorylation only occurs when all chromosome kinetochores are occupied and chromosomes are aligned

Question 45

What is the main checkpoint in mammalian cells?

Answer 45

S phase checkpoint | -Roe and Johnson showed S phase cell contains a factor which acts upon G1 to progress to S phase

Question 46

Why can G1 cells respond to SPF and not G2 cells?

Answer 46

In mammalian cells, S phase cyclins (CDK2, cyclins E and A) confer specificity of the kinase -Cyclin E synthesised first but levels drop as cyclin A levels rise (dependent upon the stability of the proteins, E less stable)

Question 47

How does the cell ensure that it's genome is replicated only once in the cell cycle?

Answer 47

Tightly control the assembly and activation of proteins that initiate DNA replication Early G1: origin of replication bound by the Origin recognition complex Late G1: MCM complex forms (MCM with CDt1 and CDC6) [6 proteins in a ring structure] and binds to sites specified by ORC. MCM complex is a DNA helicase and opens up DNA to allow access. (Pre replication complex) S phase: Replication complex is assembled and activated, DNA synthesis occurs (assisted by polymerases and ligases) Movement from late G1 into S phase is under control of cyclins E and A - Cdk2 1. Cyclin E/Cdk2 complex controls the assembly step by promoting phosphorylation events to support assembly 2. As Cyclin A/Cdk2 levels rise they add different phosphorylation events to assembly proteins to prevent it assembling again 4. As cyclin A/cdk2 levels rise futher they promote phosphorylation event that activate replication complexes

Question 48

How does the ORC select its binding site in lower eukaryotes and higher eukaryotes?

Answer 48

- lower eukaryotes: ORC selects binding site based on well defined sequence of DNA - higher eukaryotes: ORC recognises binding site by DNA sequence, which nearby genes are active, whether packaged into chromatin

Question 49

What is Geminin?

Answer 49

Protein that regulates cell cycle, builds up during S phase to act as an inhibitor for assembly