logging in or signing up Milap Thaker - Harvard biology powerpoint - molecular milapthaker Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 149 Category: Science & Tech.. License: All Rights Reserved Like it (0) Dislike it (0) Added: February 09, 2011 This Presentation is Public Favorites: 0 Presentation Description Harvard Molecular and Cellular Biology powerpoint presentation by Milap Thaker, presenter. Topic is DNA Damage checkpoints and regulation via the mitotic exit network versus cell cycle regulation via CDC14 EAR Comments Posting comment... By: umaskumar (10 month(s) ago) it is good work Saving..... Post Reply Close Saving..... Edit Comment Close Premium member Presentation Transcript Fengshan Liang and Yanchang Wang: MOLECULAR AND CELLULAR BIOLOGY , July 2007, p. 5067–5078 Presenter: Milap Thaker Proseminar in Molecular and Cellular Biology Harvard University Date 11/29/2007 DNA Damage Checkpoints Inhibit Mitotic Exit by Two Different Mechanisms Fengshan Liang and Yanchang WangKey Point 1: Two Different Mechanisms: Key Point 1: Two Different Mechanisms 2 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology: Key Point 1: Two Different Mechanisms 3 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology: 4 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyRelevant context: How to activate Mitotic Exit Network?: Relevant context: How to activate Mitotic Exit Network? 5 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMitotic Exit Network: Take 2: Mitotic Exit Network: Take 2 6 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyAlternate Mechanism: The FEAR Pathway: Alternate Mechanism: The FEAR Pathway 7 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMEN VS. FEAR: Specific Temporal Differences: MEN VS. FEAR: Specific Temporal Differences 8 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMaterials and Methods: Materials and Methods Yeast Strains grown in YPD (standard yeast-extract-peptone-dextrose) medium Arrested in G1 by 5 µg/ml α -factor After 2 hours, washed 2x with water Cytology: Cells fixed with 3.7% formaldehyde for 5 min at RT, then washed twice with PBS buffer (phosphate buffered saline) and resuspended in PBS 9 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyDAPI Staining Technique: DAPI Staining Technique Cells fixed with 100% MeOH at -20 Celsius for 30 mins. Cells pelleted, resuspended in DAPI at a final concentration of 2.5µg/ml for 1 min at RT Cells finally resuspended in 1X PBS Fluorescence microscopy used to examine GFP and DNA 10 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyProtein Techniques: Protein Techniques For Immunoprecipitation, cells harvested (50 ml) and protein extracts are prepared with RIPA RIPA = Radioimmunoprecipitation Assay Incubated with Anti-HA (hemagglutinin) antibody Washed in SDS (sodium dodecyl sulfate) protein-loading buffer Boiled 5 mins, resolved with 8% SDS gelResults 1a: Results 1a KEY ELEMENTS TO NOTE: WILD TYPE HIGH COLONIZATION LANE 2 NO COLONIZATION ALSO, NOTE LANE 6 HIGH COLONIZATION (rad53) 12 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyResults 2 – short spindling: Results 2 – short spindling Fig 1b 13 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyResults 3 – Rebudding: Results 3 – Rebudding Fig 1c 14 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyRe-Incubated Cells: Re-Incubated Cells Fig 1c 15 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyResults 4 – cdc 14 release: Results 4 – cdc 14 release Fig 2a 16 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyCdc 14 Localization - Images: Cdc 14 Localization - Images Fig 2a(2) 17 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyCdc 14 Release: Cdc 14 Release Fig 2B 18 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyRebudding of Cells (2): Rebudding of Cells (2) Fig 3a 19 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyRebudding of Cells (3): Rebudding of Cells (3) Fig 3a(2) 20 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyCdc 14 Release (2): Cdc 14 Release (2) Fig 3B 21 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, CDC 14 Release (3): Budding, CDC 14 Release (3) Fig 4a,b 22 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMicrocolonies (2): Microcolonies (2) Fig 4C 23 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, CDC 14 (3): Budding, CDC 14 (3) Fig 5a 24 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, Cdc 14 Release (4): Budding, Cdc 14 Release (4) Fig 5B 25 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMicrocolonies (3): Microcolonies (3) Fig 5C 26 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, Cdc14 Release (5): Budding, Cdc14 Release (5) Fig 6a 27 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, Cdc 14 Release (6): Budding, Cdc 14 Release (6) Fig 6B 28 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyColonization across states: Colonization across states Fig 6C 29 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyNuclear Localization, Segregation: Nuclear Localization, Segregation Fig 7a 30 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, cdc 14 Localization (6): Budding, cdc 14 Localization (6) Fig 7b 31 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyCdc 5-1 mutation vs. Cdc5-1RAD53-21: Cdc 5-1 mutation vs. Cdc5-1RAD53-21 Fig 7c 32 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyWestern Blot, States: Western Blot, States Figs 8a, 8b 33 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMaster Conclusion Set (1): Master Conclusion Set (1) Fig 8c 34 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMen & Fear: Proposed Mechanism: Men & Fear: Proposed Mechanism 35 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology: 36 Master Conclusion Set (2) Rad53 key player in mitotic exit prevention This is because cdc13-1, rad53021 mutants exhibited cdc14 release Rad 53 kinase known to phosphorylate Dun1 directly, known activator of Dun1 Dun1 plays a more direct role in regulating MEN than Rad53 (downstream) Authors contend possibility that Dun1 kinase phosphorylates Bfa1 and prevents Bfa1/Tem1 dissociation. Support: Less Bfa1-Tem1 complex in Rad53-21 when DNA damaged Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMaster Conclusion Set (3): Master Conclusion Set (3) 37 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.
Milap Thaker - Harvard biology powerpoint - molecular milapthaker Download Post to : URL : Related Presentations : Share Add to Flag Embed Email Send to Blogs and Networks Add to Channel Uploaded from authorPOINT lite Insert YouTube videos in PowerPont slides with aS Desktop Copy embed code: (To copy code, click on the text box) Embed: URL: Thumbnail: WordPress Embed Customize Embed The presentation is successfully added In Your Favorites. Views: 149 Category: Science & Tech.. License: All Rights Reserved Like it (0) Dislike it (0) Added: February 09, 2011 This Presentation is Public Favorites: 0 Presentation Description Harvard Molecular and Cellular Biology powerpoint presentation by Milap Thaker, presenter. Topic is DNA Damage checkpoints and regulation via the mitotic exit network versus cell cycle regulation via CDC14 EAR Comments Posting comment... By: umaskumar (10 month(s) ago) it is good work Saving..... Post Reply Close Saving..... Edit Comment Close Premium member Presentation Transcript Fengshan Liang and Yanchang Wang: MOLECULAR AND CELLULAR BIOLOGY , July 2007, p. 5067–5078 Presenter: Milap Thaker Proseminar in Molecular and Cellular Biology Harvard University Date 11/29/2007 DNA Damage Checkpoints Inhibit Mitotic Exit by Two Different Mechanisms Fengshan Liang and Yanchang WangKey Point 1: Two Different Mechanisms: Key Point 1: Two Different Mechanisms 2 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology: Key Point 1: Two Different Mechanisms 3 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology: 4 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyRelevant context: How to activate Mitotic Exit Network?: Relevant context: How to activate Mitotic Exit Network? 5 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMitotic Exit Network: Take 2: Mitotic Exit Network: Take 2 6 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyAlternate Mechanism: The FEAR Pathway: Alternate Mechanism: The FEAR Pathway 7 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMEN VS. FEAR: Specific Temporal Differences: MEN VS. FEAR: Specific Temporal Differences 8 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMaterials and Methods: Materials and Methods Yeast Strains grown in YPD (standard yeast-extract-peptone-dextrose) medium Arrested in G1 by 5 µg/ml α -factor After 2 hours, washed 2x with water Cytology: Cells fixed with 3.7% formaldehyde for 5 min at RT, then washed twice with PBS buffer (phosphate buffered saline) and resuspended in PBS 9 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyDAPI Staining Technique: DAPI Staining Technique Cells fixed with 100% MeOH at -20 Celsius for 30 mins. Cells pelleted, resuspended in DAPI at a final concentration of 2.5µg/ml for 1 min at RT Cells finally resuspended in 1X PBS Fluorescence microscopy used to examine GFP and DNA 10 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyProtein Techniques: Protein Techniques For Immunoprecipitation, cells harvested (50 ml) and protein extracts are prepared with RIPA RIPA = Radioimmunoprecipitation Assay Incubated with Anti-HA (hemagglutinin) antibody Washed in SDS (sodium dodecyl sulfate) protein-loading buffer Boiled 5 mins, resolved with 8% SDS gelResults 1a: Results 1a KEY ELEMENTS TO NOTE: WILD TYPE HIGH COLONIZATION LANE 2 NO COLONIZATION ALSO, NOTE LANE 6 HIGH COLONIZATION (rad53) 12 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyResults 2 – short spindling: Results 2 – short spindling Fig 1b 13 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyResults 3 – Rebudding: Results 3 – Rebudding Fig 1c 14 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyRe-Incubated Cells: Re-Incubated Cells Fig 1c 15 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyResults 4 – cdc 14 release: Results 4 – cdc 14 release Fig 2a 16 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyCdc 14 Localization - Images: Cdc 14 Localization - Images Fig 2a(2) 17 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyCdc 14 Release: Cdc 14 Release Fig 2B 18 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyRebudding of Cells (2): Rebudding of Cells (2) Fig 3a 19 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyRebudding of Cells (3): Rebudding of Cells (3) Fig 3a(2) 20 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyCdc 14 Release (2): Cdc 14 Release (2) Fig 3B 21 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, CDC 14 Release (3): Budding, CDC 14 Release (3) Fig 4a,b 22 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMicrocolonies (2): Microcolonies (2) Fig 4C 23 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, CDC 14 (3): Budding, CDC 14 (3) Fig 5a 24 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, Cdc 14 Release (4): Budding, Cdc 14 Release (4) Fig 5B 25 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMicrocolonies (3): Microcolonies (3) Fig 5C 26 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, Cdc14 Release (5): Budding, Cdc14 Release (5) Fig 6a 27 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, Cdc 14 Release (6): Budding, Cdc 14 Release (6) Fig 6B 28 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyColonization across states: Colonization across states Fig 6C 29 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyNuclear Localization, Segregation: Nuclear Localization, Segregation Fig 7a 30 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyBudding, cdc 14 Localization (6): Budding, cdc 14 Localization (6) Fig 7b 31 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyCdc 5-1 mutation vs. Cdc5-1RAD53-21: Cdc 5-1 mutation vs. Cdc5-1RAD53-21 Fig 7c 32 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyWestern Blot, States: Western Blot, States Figs 8a, 8b 33 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMaster Conclusion Set (1): Master Conclusion Set (1) Fig 8c 34 Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMen & Fear: Proposed Mechanism: Men & Fear: Proposed Mechanism 35 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology: 36 Master Conclusion Set (2) Rad53 key player in mitotic exit prevention This is because cdc13-1, rad53021 mutants exhibited cdc14 release Rad 53 kinase known to phosphorylate Dun1 directly, known activator of Dun1 Dun1 plays a more direct role in regulating MEN than Rad53 (downstream) Authors contend possibility that Dun1 kinase phosphorylates Bfa1 and prevents Bfa1/Tem1 dissociation. Support: Less Bfa1-Tem1 complex in Rad53-21 when DNA damaged Milap Thaker - Graduate Proseminar - Molecular and Cellular BiologyMaster Conclusion Set (3): Master Conclusion Set (3) 37 Milap Thaker - Graduate Proseminar - Molecular and Cellular Biology