L13: Eukaryotic Cell Cycle control

Question 1

Cell fusion experiments

Answer 1

1. Fusing M phase cell w/ G2 cell results in both sets of chromosomes being condense
   -> mitosis promoting factor in M phase cells
2. Fusing M phase w/ G1 or G2 or S has same result
   -> response is not unique to G2 cells, MPF is biochemically dominant
3. S fused w. G1 -> SPF
4. S fused w/ G2 -> don’t enter S phase; only G1 cells respond to SPF

Question 2

Experimental systems for elucidation of proteins req. for G2/M transition (4 benefits)

Answer 2

• Eggs and early embryos of Xenopus (african tree frogs)
• Eggs and early embryos of marine invertebrates
• Relatively large cells, high protein content, readily available
• Naturally synchronized
  -> can obtain extracts from specific points in cycle

Question 3

Egg arresting, investigating MPF activity

Answer 3

• Eggs are arrested in G2 phase
• Progesterone promotes maturation to Meiosis 1
  -> meiotic division, 2nd division (meiotic interphase)
  -> egg arrested in meiosis 2 until fertilised, can assay for MPF…
• Transfer cytoplasm to G2 arrested oocyte
  -> -> Driven through metaphase

Question 4

MPF activity

Answer 4

• Rises and falls in parallel w/ mitosis
• High MPF arrests the oocyte in meiosis until its abundance drops

Question 5

Tim Hunt experiment

Answer 5

• Identified cyclin as a component of MPF
• Added 35S methionine to fertilised sea urchin oocytes; only incorporated into freshly made protein
• Took extracts at different points to identify a protein made and degraded in sync w/ mitotic divisions
• Visualised w/ autoradiography, analysed by SDS-PAGE

Question 6

Further experimental system development (cyclin levels in oocytes)

Answer 6

• Identifying gene encoding cyclin
• Used frog oocytes, added sperm chromatin to extracts enabling mitosis in test tube
• Visualisable by microscope
• Showed that Cyclin B levels parallel MPF
• RNase treated extracts no longer undergo mitosis (but addition of wild type mRNA for cyclin B restore cycle progress)
• Adding mutant mRNA containing undegradable cyclin B stimulates entry into M, A but not T as cyclin B must be degraded to exit mitosis