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1971

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1971

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  1. 1971 j. of Exp. Zoology

  2. Criteria for CSF 1) It should appear in the oocyte cytoplasm after Progesterone treatment 2) It should cause nuclei to arrest in metaphase 3) It should be maintained until the oocyte is activated 4) It should become inactivated during fertilization or egg activation

  3. Assays used to investigate CSF A and B are gain of function experiments. C is a loss of function assay.

  4. Nature 1989

  5. Criteria 1) It should appear in the oocyte cytoplasm after Progesterone treatment Synthesis and Modification of Mos during oocyte maturation Metabolic label with [35S] Methionine Antigenic peptide: 35S labeling followed by IP with anti Mos Slight shift in MW Metabolic labeled with [32P] ATP In the three cases, the experiments showed autoradiographies after IP with anti Mos

  6. Criteria 4) It should become inactivated during fertilization or egg activation Selective proteolysis of Mos upon egg activation In vitro matured oocytes pre-labelled with [35S]Methionine were washed and chased for one hour without activation (none/chase), or after activation by either pricking with a glass nnedle (Prick/chase), or treatment with Ca2+ ionophore (A23187/chase). B. Time curve of Mos degradation.

  7. 1989 Criteria for CSF 1) It should appear in the oocyte cytoplasm after Progesterone treatment 2) It should cause nuclei to arrest in metaphase 3) It should be maintained until the oocyte is activated 4) It should become inactivated during fertilization or egg activation

  8. 3) It should be maintained until the oocyte is activated 4) It should become inactivated during fertilization or egg activation Absence of Mos protein in Xenopus fertilized eggs Autoradiography Western blot Oocytes are pre labeled with 35S Methionine and then immunoprecipitated with anti Mos. Immunoprecipitates are then analyzed by PAGE and Mos visualized either by autoradiography or by Western blot using anti Mos. Labeling is done after fertilization for 2 hours. Why is the WB an important experiment?

  9. Criteria 2) It should cause nuclei to arrest in metaphase Arrested embryo with capped Mos mRNA In these experiments mos RNA was injected in one of the cells of a two-cell embryo. a. Morphology. b. Chromosome staining. C. MPF activity from cytosol of mos-arrested embryos in immature oocytes. Morphology of embryos and chromosomes as well as MPF activity in mos RNA-arrested embryos MPF activity CN-Mos: capped normal Mos

  10. Cleavage arrest induced by injected RNA Criteria 2) It should cause nuclei to arrest in metaphase CN-mos: Capped mos mRNA CN-mos + sense: same + sense mRNA UN-mos: Uncapped mos mRNA CT-mos: Capped truncated (minus kinase domain) mos RNA UT-mos: Uncapped truncated mos RNA TMV: tobacco mosaic virus total mRNA (Amersham) Globin: b=globin mRNA tRNA: Yeast tRNA (Sigma) Xenopus oocyte: total mRNA from Immature Xenopus oocytes

  11. Additional criteria: Loss of function experiment Effect of Mos-specific antibodies on CSF activity B-Mos: Polyclonal antibody against Mos. It is able to block Mos kinase activity in vitro 5S: Monoclonal antibody against Mos. It does not block Mos kinase activity. C232: Another monoclonal antibody against Mos. Two types of experiments: 1) Neutralization with specific antibodies. 2) Immunodepletion with specific antibodies In all these cases CSF extracts were obtained from matured-unfertilized eggs. The treatment with the different antibodies was conducted before microinjection in 2-cell embryos.

  12. MPF and CSF levels during oocyte maturation and fertilization

  13. Mos has the same activity than Raf; it is a MAP kinase kinase kinase MEK1 is a Map kinase kinase P90Rsk is another kinase that mediates the CSF action

  14. Avram Hershko Aaron Ciechanover Irwin Rose The Nobel Prize in Chemistry 2004 "for the discovery of ubiquitin-mediated protein degradation"

  15. A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes Aharon Ciechanover, Yaacov Hod and Avram Hershko1Technion-Israel Institute of Technology, School of Medicine, Haifa, Israel Received 8 March 1978.  Available online 02 December 2004. Abstract The degradation of denatured globin in reticulocyte lysates is markedly stimulated by ATP. This system has now been resolved into two components, designated fractions I and II, in the order of their elution from DEAE-cellulose. Fraction II has a neutral protease activity but is stimulated only slightly by ATP, whereas fraction I has no proteolytic activity but restores ATP-dependent proteolysis when combined with fraction II. The active principle of fraction I is remarkably heat-stable, but it is non-dialysable, precipitable with ammonium sulfate and it is destroyed by treatment with proteolytic enzymes. In gel filtration on Sephadex-G-75, it behaves as a single component with a molecular weight of approximately 9,000.

  16. Biochem Biophys Res Commun. 1978 Apr 28;81(4):1100-5               • A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. • Ciechanover A, Hod Y, Hershko A.

  17. To examine the possible association of the heat stable polypeptide with other cellular proteins and the role of the ATP in this process, 125I-APF-1 was incubated with fraction II in the presence of ATP, and was analyzed on columns of Sephadex G-75. On such columns the low MW polypeptide is retained while most of the proteins of fraction II are excluded and eluted at the void volume (V0). 1. - ATP 2-6. + ATP Different conditions

  18. JBC 1981 JBC 1983

  19. JBC 1983

  20. Current model of Ubiquitin-induced proteasome-mediated degradation

  21. FERTILIZATION 1. Fusion of the sperm and egg plasma membranes. 2. Increase in Calcium (Ca2+) inside the egg. 3. Cortical granule exocytosis. 4. Block to Polyspermy. A) Plasma membrane block to polyspermy. B) Zona block to polyspermy 5. Cyclin Destruction and inactivation of cyclin-dependent kinase (cdk). 6. Sperm Chromosomes decondensation. 7. Male and Female Pronuclear formation. 8. Syngamy and first mitosis.

  22. Summary of Murray and Kirshner paper Tim Hunt group instead of degrading all the mRNA, they specifically target cyclin B using an antisense approach

  23. Nature 1991 Two approaches to analyze the sequence important for cyclin degradation: 1) cyclin deletion mutants; 2) addition of sequence to another protein. In this case Protein A.

  24. Autoradiography 125I proteins are added to Xenopus mitotic or interphase extracts. Degradation is evaluated after different time periods. Cyclin deletion mutants Protein A-cyclin fusion

  25. 125I ubiquitin form high Mr complexes with cyclin-Protein A fusion proteins. The autoradiography is developed after immunoprecipitation of Protein A with Ig-Sepaharose. 13-91cyclin-protein A fusion is degraded and it is ubiquitinated. However, 13-91prA-R42C mutant is not degraded and not ubiquitinated. Kinetic analysis of degradation of 13-91prA. A) extracts; B) Immunoprecipitated with Ig Sepharose