Lecture 13

Question 1

What happens to complex signalling pathways when cells divide

Answer 1

They integrate

Question 1

How do cells control cell growth?

Answer 1

Responses to intra and extracellular signals/stimuli which utilize systems

Question 2

What do Cyclin-CDKs ensure?

Answer 2

They ensure only one round of DNA replication occurs per each turn of the cell cycle, and ensure chromosomes equally separate into the daughter cells

Question 3

Why study cell cycle contro?

Answer 3

• Fundamental property of cellular life
• Organelles segregated to daughter cells
• Defects cause disease such as cancer

Question 4

Different stages of the cell cycle

Answer 4

G1, S, G2, M

Question 5

What does cell cycle link?

Answer 5

-Microtubule cytoskeleton formation
-genome dynamics

Question 6

What are major experimental questions in understanding cell cycle control?

Answer 6

• Factors controlling cell cycle transitions
• Why does S-phase always occur before mitosis

Question 7

Cell fusion experiments by Rao and Johnson

Answer 7

M-phase + G2 -> M-phase
M-phase cells have a mitosis promoting factor (MPF)

M-phase + G1 or G2 or S-phase -> M-phase
Cells at any stage of the cycle are forced to enter mitosis (M-phase) by the MPF in M-phase cells

S-phase + G1 -> S-phase
S-phase promoting factor in S-phase cells

S-phase + G2 -> No cells entering S-phase
Caused by SPF being responsive by only G1 cells, not G2.

Question 8

Identification of proteins required for G2/M transition

Answer 8

• Used eggs or early embryos from African tree frogs or marine invertebrates such as sea urchins
• Used as they’re large, high protein content, and many of them
• Possible to obtain biochemical extracts as naturally synchronised and undergo rapid cell divisions

Oocyte maturation in vitro
G2-arrested oocyte -> (Progesterone) -> Meiosis I (metaphase) -> Meiotic interphase -> Egg arrested in meiosis II (metaphase) -> (Sperm added) -> Fertilized egg -> First cleveage -> Second cleaveage

Assay for MPF:
Egg arrested in meiosis II (metaphase) -> (Cytoplasm transfer) -> G2-arrested oocyte -> Meiosis I (metaphase) -> Meiotic interphase -> Meiosis II metaphase

Question 9

What stages was MPF high?

Answer 9

• Meiosis I (metaphase)
• Meiosis II
• First embryonic mitosis
• Second embryonic mitosis

High MPF arrests the oocyte in meiosis until its abundance drops

Question 10

How was cyclin identified as component of MPF

Answer 10

• Autoradiograph showed newly synthesised protein
• Cyclin rises and falls after fertilisation

Question 11

How were sea urchin oocytes used to locate presence of cyclin

Answer 11

Oocytes fertilized and 35S methionine added
Methionine only incorporated into newly synthesized proteins
Protein extracts taken at different time points postfertilization and analysed by SDS-PAGE
Autoradiography used

Question 12

Why was sperm chromatin added?

Answer 12

• Frog oocytes good for identification of gene encoding cyclin
• Addition of sperm chromatin to extracts enabled mitosis to occur in test tube
• Therefore, formation of mitotic spindles, chromosomes condense, generate metaphase plate, subsequently proceed through mitosis in vitro

Question 13

Cyclin B is a constituent of MPF

Answer 13

Cyclin B parallels with MPF activity

Protein synthesis required

Rnase treated extracts don’t undergo mitosis

Question 14

Cyclin B mRNA

Answer 14

Has ubiquitin ligase binding site on Deg

Question 15

Degradation of cyclin B protein required for mitotic exit

Answer 15

• Normal mitotic program caused by addition of cyclin B mRNA
• Cyclin B required for mitotic entry
• Deletion of cyclin B 5’ sequences forms a catalytically active truncated cyclin B protein
• Shortened cyclin B construct stable - Can’t be degraded by proteosome
• Activity prevents mitotic exit

-Cyclin B destruction required for mitotic exit