第二节 细胞周期的调控

2. 第二节 细胞周期的调控 • 一、MPF的发现和作用Rao和Johnson(1970、1972、1974)将Hela细胞同步于不同阶段，然后与M期细胞混合，在灭活仙台病毒介导下，诱导细胞融合，发现与M期细胞融合的间期细胞产生了形态各异的早熟凝集染色体(prematurely condensed chromosome，PCC)，这种现象叫做早熟染色体凝集(premature chromosome condensation)。

3. ** MPF, the mitosis-promoting factor Rao,70’

5. PCC早熟染色体凝集 G1+M S+M G2+M PCC现象说明M期细胞中有可能存在一种诱导染色体凝集的因子。

7. S期激活因子简称SPF 的细胞融合实验 1,S+G1结果是G1早进入S期（和S期激活因子有关） 2, G2+S结果 S期细胞继续合成DNA，说明G2期并未产生DNA合成抑制因子。但G2也不受S期诱导，G2不合成DNA，说明在一个细胞周期中DNA只合成一次。 3 ，G2+G1结果G2不能诱导G1期合成DNA。

8. 有人做如下提取液 G1提取液 G2提取液 S提取液 M提取液

9. 将这些提取液分别注射的蛙卵母细胞中，发现G1和S期不具MPA（促成熟活性）将这些提取液分别注射的蛙卵母细胞中，发现G1和S期不具MPA（促成熟活性） MPA在G2-M过度期达到高峰，这时正是染色体形成时期 有人将这种诱导减数分裂的物质称为有丝分裂因子（MF）后来称做MPF（成熟促进因子）

13. * MPF, the maturation-promoting factor Y. Masui and C. L. Markert. 1971. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes J. Exp. Zool. 177: 129-145.

14. 不仅同类M期细胞可以诱导PCC，不同类的M期细胞也可以诱导PCC产生，如人和蟾蜍的细胞融合时同样有这种效果，这就意味着M期细胞具有某种促进间期细胞进行分裂的因子，即成熟促进因子(maturation promoting factor，MPF)。

15. 早在1960s，Yoshio Masui发现成熟蛙卵的提取物能促进未成熟卵的胚胞破裂(Germinal Vesicle Breakdown，GVBD)，后来Sunkara将不同时期Hela细胞的提取液注射到蛙卵母细胞中，发现G1和S期的抽取物不能诱导GVBD，而G2和M期的则具有促进胚胞破裂的功能，它将这种诱导物质称为有丝分裂因子(MF)。后来在CHO细胞，酵母和粘菌中也提取出相同性质的MF。这类物质被统称为MPF。

16. 1960s Leland Hartwell以芽殖酵母（图13-16）为实验材料，利用阻断在不同细胞周期阶段的温度敏感突变株（在适宜的温度下和野生型一样），分离出了几十个与细胞分裂有关的基因(cell division cycle gene，CDC)。如芽殖酵母的cdc28基因，在G2/M转换点发挥重要的功能。Hartwell还通过研究酵母菌细胞对放射线的感受性，提出了checkpoint（细胞周期检验点）的概念，意指当DNA受到损伤时，细胞周期会停下来。

17. 1970s Paul Nurse等人以裂殖酵母（图 ）为实验材料，同样发现了许多细胞周期调控基因，如：裂殖酵母cdc2、cdc25的突变型和在限制的温度下无法分裂；wee1突变型则提早分裂，而cdc25和wee1都发生突变的个体却会正常地分裂（图）。

18. 进一步的研究发现cdc2和cdc28都编码一个34KD的蛋白激酶，促进细胞周期的进行。而weel和cdc25分别表现为抑制和促进CDC2的活性。这也解释了为何cdc25和wee1双重突变的个体可以恢复野生型的表型。进一步的研究发现cdc2和cdc28都编码一个34KD的蛋白激酶，促进细胞周期的进行。而weel和cdc25分别表现为抑制和促进CDC2的活性。这也解释了为何cdc25和wee1双重突变的个体可以恢复野生型的表型。

19. 二、 P34CDC2的发现和细胞周期调控因子MPF 的关系 MPF的主要成分就是P34CDC2和P56CDC13的同源物。 细胞周期蛋白为调节亚基 P34CDC2为催化亚基 正是由于不同的细胞周期蛋白和不同的CDC2基因产物的结合、分离、磷酸化和去磷酸化，激活催化亚基的活性，而成为驱动细胞周期运转的引擎。

20. The story of the work in identifying the key regulators of the cell division cycle and the 2001Nobel Prize for Physiology or Medicine

21. A, Nurse found cdc2 Nurse is Director General of the Cancer Research UK ( was the ICRF) and has his research group. In the mid-1970s, Nurse was working in the Scottish city of Edinburgh as a postdoc studying the fission yeast S. pombe. He found some mutant cells that were constantly small even though they multiplied at the same rate as other cells in the population.

22. The difference was that they appeared to divide before they had reached full size. He went on to isolate 50 of these mutants which he called wee， meaning small in the Scottish dialect; 49 had mutations in a gene he called wee1, and one was mutated in a different gene, wee2.

23. Nurse realised that wee2 is crucial for cell division when he noticed that cells can enter mitosis if this gene is mutated so that its protein kinase product loses its activity, whereas cells with hyperactive wee2 enter mitosis prematurely. The wee2 gene was later renamed cell division cycle 2 (CDC2).

24. C, thediscovery of cdc mutants of fission yeast p34cdc2, a serine/threonine protein kinase (Photographs by S.Moreno) In the cdc2- mutant, which has a recessive phenotype, the cdc2 gene is inactivated. At the restrictive condition, the cell fails to pass the mitotic entry checkpoint, continues growing, and so becomes enormously large. Conversely, the dominant cdc2 mutant, cdc2D, in which the cdc2 gene product is hyperactive, passes the checkpoint prematurely, at an abnormally small size.

25. Hartwell found cdc28 In 1982, Nurse and his colleagues found that CDC2 was identical in function to CDC28, one of the hundred or so cell cycle-related genes that, father of the field, Hartwell had previously isolated from bakers yeast, S. cerevisiae. This was fitting as Nurse‘s work was originally inspired by Hartwell’s four seminal papers published in 1970 and 1971 on genetic control of the cell division cycle in yeast. Hartwell went on to characterise many of these S. cerevisiae cdc genes and also, with Ted Weinert, to develop the concept of checkpoints in the cell cycle that verify that the cell‘s DNA is intact and complete before allowing the division to procede.

26. 三、细胞周期蛋白 1 P34CDC2+周期蛋白的引擎作用外还有若干个检验点的监视。 2 P34CDC2的量是相对恒定的，而细胞周期蛋白是随着细胞周期的变化而波动的。 周期蛋白和P34CDC2结合在一定条件下激活P34CDC2的活性

27. Tim Hunt found cyclin in 1983 Hunt was working on a totally unrelated project. His background was in protein synthesis and its control. "I was a rather odd creature of a sort of mixture of a biochemist and a molecular physiologist, and I was interested in seeing how systems worked," explained Hunt, who was looking at how sea urchin eggs turn on protein synthesis after they are fertilised.

28. One day, Hunt did an experiment to look at the pattern of protein synthesis in fertilised eggs when they start to divide. "I have no real idea why I did the experiment ?it just seemed a good idea at the time," he admits. "I decided to take time points. When the gel was developed on Friday 23 July 1982, I found that the strongest band early on suddenly got very much weaker at about the time when the eggs were about to divide in two. This was a stunning, startling discovery."

29. Immediately, Hunt realised the implications. Researchers were aware that some stages of the cell cycle required protein synthesis, but until then nobody in the field had even remotely suggested that something would be specifically degraded to allow a cell to progress from one stage of the cell cycle to another. The disappearing protein became known as cyclin. "Twenty years later people are still struggling to find out how this mechanism works," says Hunt.

31. # Behavior of the temperature-sensitive cell-division-cycle (cdc) mutants Most conditional cell-cycle mutants are temperature-sensitive mutants in which the mutant protein fails to function at high temperatures but functions well enough to allow cell division at low temperatures. A temperature-sensitive cdc mutant strain of cells can be grown at low temperature (the permissive condition) and then raised to a higher temperature (the restrictive condition) to switch off the function of the affected gene. All of the cells will continue their cycling until they reach the point where the function of the mutant gene is required for further progress, and at this point they will all stop

32. 蛙P32 + P45 • 裂殖酵母 P34CDC2 + P56CDC13 • 海胆卵细胞 cdc2蛋白 + 周期蛋白B

33. 6, the molecular features of Cyclins Cyclins CYCLIN BOX 42 A2 48 kDa R Destruction Box B1 48 kDa PEST C 33 kDa PEST D 32 kDa PEST E 44 kDa PEST Cln3 66 kDa PEST F 87 kDa 29 kDa G

35. 细胞周期蛋白 哺乳动物-M期有 周期蛋白B S期有周期蛋白A 酵母中G1期周期蛋白，有CLN1 CLN2，CLN3 脊椎动物中有周期蛋白C、D、E等。

37. 四、CDK激酶和它的抑制物401 Cyclin-dependent kinase(CDK) 表11-2 P401 可以看到CDK激酶的种类、可以结合的周期蛋白以及执行功能的可能时期。

39. 7, the molecular features of CDKs PSTAIRE 100% Cdc2 (CDK1) PSTAIRE CDK2 65% PSTAIRE CDK3 66% PV/STVRE CDK4 44% PSSALRE CDK5 57% PLSTIRE 47% CDK6 NRTAIRE 40% CDK7 (p40MO15)

40. 五、细胞周期运转调控402 （一）G2和M期转化与CDK1激酶的调控作用

42. 实际上CDK激酶和周期蛋白结合后仍不能激活激酶。还需要其他激酶如CAK激酶（ CDK1活性激酶）。

44. b, by phosphorylatinon/dephospholylation of CDK subunit Thr14 Tyr15 Cyclin B/A Cyclin B/A CDK1 Thr161 Inactive APC + + CAK + Thr14 Tyr15 P P Thr14 Tyr15 CDK1 CDK1 Thr161 P Thr161 P Cyclin B/A Cyclin B/A Inactive Active + Cdc25c

47. 21-10

48. （二）M期周期蛋白与分裂中期向后期转化

50. E1 泛素激活酶 E2 泛素结合酶 E3 泛素连接酶