1. The Discovery And Characterization Of MPF -------Experimental Pathways
陈汪洋
吴 娅

2. Chief Contents The discovery of MPF The discovery of cyclin
The link between cyclin and MPF

3. The discovery of MPF Experiment 1: the discovery of MPF Experiment 2:
MPF activity

4. As an amphibian oocyte nears the end of oogenesis, the large nucleus( called a germinal vesicle ) moves toward the periphery of the cell. In subsequent steps, the nuclear envelope disassembles, the compacted chromosomes become aligned along a metaphase plate near one end (the animal pole )of the oocyte, and the cell undergoes the first meiotic division to produce a large secondary oocyte and a small polar body.

5. The processes of germinal vesicle breakdown and first meiotic are referred to as maturation and can be induced in fully grown oocytes by treatment with the steroid hormone progesterone.
Progesterone induces maturation only if it is applied to the external medium surrounding the oocytes.

6. Experiment 1: The discovery of MPF
Purpose ：to learn more about the nature of
thecytoplasmic change that
was responsible for triggering maturation
Material ： frog oocytes
Researchers ：Yoshio Masui & Clement Markert
Methods ：removed cytoplasm from isolated frog
oocytes at various stages following progester one treatment and injected 40—60 nl of the donor cytoplasm into fully grown, immature oocytes that had not been treated with the hormone

7. (2)After 12 hours the cytoplasm gained the ability to induce maturation in the recipient oocyte
（1）cytoplasm taken from oocytes during the first 12 hours following progesterone treatment had little or no effect on recipient oocytes
(4)Its effectiveness decline by 40 hours
(3)Maximally effective about 20 hours after progesterone treatment

8. Conclusion ：
Masui and Markert referred to the cytoplasm substance that induce maturation in recipient oocytes as “maturation promoting factor”, which became known as MPF.

9. Experiment 2: MPF activity
Purpose ：to learn more about the activity of
MPF
Material ： cleaving frog eggs
Researchers ：Wasserman & Smith
Methods ：to see the MPF activity in that cytoplasm taken from cleaving frog eggs
at different times
after fertilization, as
assayed by injection into immature oocytes

10. Test phenomenon ：
（1）within 30—60 minutes after fertilization: the cytoplasm contains little or no detectable MPF activity
(6)At 225 minutes after fertilization: MPF activity reach a peak, and then declines again to a very low level
(5)At 180 minutes after fertilization: the eggs undergo their first cytokinesis, no activity is detect
(3)At 120 minutes after fertilization: MPF activity reaches a peak
(2)At 90 minutes after fertilization: MPF activity can again be demonstrated
(4) At 150 minutes after fertilization: MPF activity decline again

11. Conclusion ：
　(1)MPF activity disappears and reappears on a time scale that correlates with the length of the cell cycle
(2)the peak of MPF activity corresponds to the time of nuclear membrane breakdown and the entry of the cells into mitosis
Inference ： MPF may play
a key role in regulating
the cell cycle of dividing cells

12. The discovery of cyclin
MPF activity is present in a wide variety of organisms.
Mammalian cells growing in culture also possess MPF activity as assayed by the ability of mammalian cell extracts to induce germinal vesical breakdown when injected into amphibian oocytes.

13. The discovery of cyclin
MPF activity of mammalian cells fluctuates with the cycle.
Extracts from cultured HeLa cells perpared from early G1-, late G1-, or S- phase cells lack MPF activity.
MPF appears in early G2, rises dramatically in late G2, and reaches a peak in mitosis.

14. The discovery of cyclin
Another element of the machinery that regulates the cell cycle was discovered in studies on sea urchin embryos.
If sea urchin eggs are fertilized in sea water containing an inhibitor of protein synthesis, the eggs fail to undergo the first mitotic division, arrest at a stage prior to chromosome compaction and breakdown of the nuclear envelope.

15. The discovery of cyclin
Similarly, each of the subsequent mitotic divisions can also be blocked if an inhibitor of protein synthesis is added to the medium at a time well before the division would normally occur.
This finding can suggested that one or more proteins must be synthesized during each of the early cell cycles if the ensuing mitotic division is to occur.
Early studies on cleaving sea urchin eggs failed to reveal the appearance of new species of proteins during this period.

16. The discovery of cyclin
In 1983, Tim Hunt and his colleagues reported on several proteins that are synthesized in fertilized sea urchin eggs but not unfertilized eggs.
Tim Hunt
born February 19, 1943
The Nobel Prize in Physiology or Medicine 2001

17. The discovery of cyclin
To study these proteins further
They incubated fertilized eggs in containing [35S]methionine and withdrew samples at 10-minute intervals beginning at 16 minutes after fertilization.
Crude protein extracts were prepared from the samples and subjected to polyacrylamide gel electrophoresis, and the labeled proteins were locates autoradiographically.

18. The discovery of cyclin
Several prominent bands were labeled in gels from fertilized egg extracts that were not evident in comparable extracts made from unfertilized eggs.
One of the bands that appeared strongly labeled at early stages after fertilization virtually disappeared from the gel by 85 minutes after fertilization, suggesting that the protein had been selectively degraded.

19. The discovery of cyclin
This same band then reappeared in gels from eggs sampled at later times and disappeared once again in a sample taken at 127 minutes after fertilization.

20. The discovery of cyclin
Correlation of the level of cyclin with the cell division cycle
The degradation of the protein occurs at about the same time that the cells undergo the first and the second division.

21. The experiment
An mRNA encoding cyclin A was transcribed in vitro from a cloned DNA fragment that contained the entire cyclin A coding sequence.
When the synthetic cyclin mRNA was injected into Xenopus oocytes, the cell underwent germinal vesicle breakdown
and chromosome compaction over a time course alike that induced by progesterone treatment

22. The discovery of cyclin
A similar protein was found in the eggs of the surfclam.
Hunt and colleagues named the protein “cyclin” and noted he striking parallel in behavior between the fluctuations in cyclin levels in their investigation and MPF activity in the earlier studies

23. The discovery of cyclin
Subsequent studies showed that there were two distinct cycles, A and B, which are degraded at different times during the cell cycle.
Cyclin A is degraded during a 5-6 minute period beginning just before the metaphase-anaphase transition, and cyclin B is degraded a few minutes after this transition.

24. The relationship between cyclin and MPF
The first clear link between cyclin and MPF was demonstrated by Joan Ruderman and her colleagues at the Woods Hole Marine Biological Laboratory

25. Conclusion:
The results suggested that the rise in cyclin A, which occurs normally during meiosis and mitosis, has a direct role in promoting entry into M phase. The amount of cyclin A normally drops rapidly and must be resynthesized prior to the next division or the cells cannot reenter the M phase.

26. Purify and characterize the substance responsible for MPF activity
Use the fission yeast

27. The relationship between cyclin and MPF
Difficulty: use of different organisms.
MPF in amphibians.
Cyclin in sea urchins and clams.

28. The relationship between cyclin and MPF
Evidence indicated that frog oocytes contain a pool of in active pre-MPF molecules, which are converted to active MPFs during meiosisⅠ. Cyclin, on the other hand, is totally absent from clam oocytes but appears soon after fertilization. Ruderman considered the possibility that cyclin A is an activator of MPF.

29. Characterization of MPF
The purification of MPF

30. The purification of MPF
In 1988, MPF was finally purified by a series of six successive chromatographic steps.

31. The purification of MPF
In 1980,reserchers accomplished a 20- to 30-fold purification of MPF by precipitating the protein in ammonium sulfate and subjecting the redissolved material to column chromatography.
When partially purified MPF preparations were incubated with [32p]ATP in vitro, proteins present within the sample became phosphorylated, suggesting that MPF induced maturation by acting as a protein kinase.

32. Characterization of MPF
Two polypeptides:
32kDa and 45kDa polypeptides.
A high level of protein kinase activity.
When partially purified MPF preparations were incubated with [32P]ATP in vitro, proteins present within the sample became phosphorylated.

33. The 32 kDa polypeptide of MPF
Paul Nurse and his colleagues used fission yeast to show that yeast produced a protein kinase with a molecular weight of 34 kDa whose activity was required for those cell to enter M phage ——cdc2 that is similar with 32kDa of MPF.

34. What is the relationship between 32kDa of MPF and 34kDa in yeast?

35. Antibody experiment
Antibodies formed against cdc2 from fission yeast were showm to react specifically with the 32kDa component of MPF isolated from Xenopus eggs. These findings indicate that this component of MPF is a homologue of the 34kDa yeast kinase .

36. The conclusion MPF consisting of two types of subunits:
(1)A 32-kDa subunit that contains the protein kinase active site and is homologousto the yeast cdc2 protein kinase.

37. (2)A larger subunit identified as a cyclin whose presence is required for kinase activity.

38. Thank you !