The Discovery And Characterization Of MPF

-------Experimental Pathways

池维丹吕丛伊谢芳

on P616

Chief Contents

• The discovery of MPF ( 池维丹 )

• The discovery of cyclin ( 吕丛伊 )

• The link between cyclin and MPF

( 谢芳 )

The discovery of MPF

• Experiment 1: the discovery of MPF

• Experiment 2: MPF activity

卵母细胞卵子发生末期

大的细胞核移向细胞周边（生发泡， germinal vesicle ）

核被膜崩解，压缩的染色体排列在卵母细胞靠近动物极的中期板上

进行第一次减数分裂，产生次级卵母细胞和极核

• The processes of germinal vesicle （生发泡） breakdown and first meiotic are referred to as maturation and can be induced in fully grown oocytes （卵母细胞） by treatment with the steroid hormone progesterone.

• Progesterone （黄体酮） induces maturation only if it is applied to the external medium surrounding the oocytes

Experiment 1: The discovery of MPF

• 实验目的： to learn more about the nature of thecytoplasmic （胞质） change that was responsible for triggering maturation

• 实验材料： frog oocytes• 实验人员： Yoshio Masui & Clement Markert• 实验方法： 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

（ 1 ） cytoplasm taken from oocytes during the first 12 hours following progesterone treatment had little or no effect on recipient oocytes

(2)After 12 hours the cytoplasm gained the ability to induce maturation in the recipient oocyte

(3)Maximally effective about 20 hours after progesterone treatment(4)Its effectiveness decline by 40 hours

• 实验结论： Masui and Markert referred to the c

ytoplasm substance that induce maturation in recipient oocytes as “maturation promoting factor”, which became known as MPF.

Experiment 2: MPF activity

• 实验目的： to learn more about the activity of MPF

• 实验材料： cleaving frog eggs• 实验人员： Wasserman & Smith• 实验方法： to see the MPF activity in that cytoplasm take

n from cleaving frog eggs at different times after fertilization, as assayed by injection into immature oocytes

实验现象：（ 1 ） within 30—60 minutes after fertilization: the cytoplasm contains little or no detectable MPF activity

(2)At 90 minutes after fertilization: MPF activity can again be demonstrated(3)At 120 minutes after fertilization: MPF activity reaches a peak(4) At 150 minutes after fertilization: MPF activity decline again

(5)At 180 minutes after fertilization: the eggs undergo their first cytokinesis, no activity is detect

(6)At 225 minutes after fertilization: MPF activity reach a peak, and then declines again to a very low level

• 实验结论：　 (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

推论： MPF may play a key role in regulating the cell cycle of dividing cells 　

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.

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.

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.

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.

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 Huntborn February 19, 1943 The Nobel Prize in Physiology or Medicine 2001

Tim Hunt receiving his Nobel Prize from His Majesty the King at the Stockholm Concert Hall.

The Prize Award Ceremony at the Stockholm Concert Hall. Tim Hunt is first from left, front row.

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.

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.

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.

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.

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

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.

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

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 （爪蟾）

oocytes,the cell underwent germinal vesicle breakdown and chromosome compaction over a time course alike that induced by progesterone( 黄体酮 )treatment

• 黄体酮和 cyclin A 的 mRNA 分别激活爪蟾卵母细胞的动力学

• GVBD( 生发泡破裂 )

What is the relationship between cyclins and MPF ?

• Purify and characterize the substance reponsible for MPF activity

• Use the fission yeast

The purification of MPF

• 1980 年，利用硫酸铵沉淀蛋白，再溶解后柱层析，将 MPF 纯化 20~30 倍

• 纯化物样品与 [32P] 体外温育，该样品中蛋白中掺入了 32P ，说明 MPF 将蛋白磷酸化—— MPF 可能作为蛋白激酶诱导成熟

The purification of MPF

• 1988 年，通过六次连续层析后完全纯化• 纯化制品的活性与两个亚基有关，相对分

子大小分别为 32000 ， 45000

• 纯化制品具高水平蛋白激酶活性，当其与[32P]ATP 一起温育时，分子量为 45000 的多肽被标记

background

• 蛙卵：蛙卵中存在失活的 MPF 前体分子，进入减数分裂后被激活

• 海蛤：海蛤的卵母细胞中完全没有 cyclin ，但在受精不久后出现

The development

• 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 ——MPF32000 亚基的同源物（抗体试验证明）

The development

• 脊椎动物中的 cdc2 同源蛋白不随细胞周期而波动（抗体试验） ——脊椎动物细胞中32000 蛋白激酶的活性依赖另一蛋白调节—— cyclin

• 人们把多种生物的 MPF 纯化，并分析其多肽成分后，证明了该假说

The conclution

• 相对分子质量为 34000 的亚基：含蛋白激酶活性位点，并与酵母 cdc2 蛋白激酶同源

• 相对分子质量为 45000 的亚基：即 cyclin