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Micromanipulation
 

Micromanipulation

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    Micromanipulation Micromanipulation Presentation Transcript

    • Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics
      Presented To:
      Ms. NaziaJamil
      Presented By:
      MazharHussain
      MMG-07-37
      BS-6
      Microbiology & Molecular Genetics
    • Micromanipulation Studies of Chromatin Fibers in Xenopus Egg Extracts Reveal ATP-dependent Chromatin Assembly Dynamics
      Jie Yan
      Thomas J. Maresca
      DunjaSkoko
      Christian D. Adams
      Botao Xiao
      Morten O. Christensen
      Rebecca Heald
      John F. Marko
    • Introduction
    • What is Chromatin?
      Chromatin is DNA molecule which is associated with different types of proteins…
      Provides basic structure for chromosome formation
      Histone is one of those proteins.
    • Nucleosome
      Consist of 160-180 nucleotides wound up on octamer of histone proteins…
      H2A, H2B, H3, H4
      Nucleotides make 2 left hand turns on core proteins…
    • Advantages of Biophysical Experiments
      Chromatin dynamics can be visulised by this appreach under light microscope
      Dynamics include : Assembly and Disassembly reactions
      Reveal details about structures
      Details about procedures like chromatin remodeling, mitotic chromosome condensation.
    • ΔG for Nucleosome Assembly
      ΔG is free energy for nucleosome formation
      Its value measured to be 3.5pN
      This is the force which is required to stall assembly and disassembly for nucleosome formation.
      ΔG is counted as equilibrium point
    • Why Xenopus?
      Containing Enzymes which help in chromatin assembly in vitro conditions.
      Competent in many physiological condtions
    • Materials & Methods
    • Single DNA Tethering
      Paramagnetic Bead (Streptoviridin)
      Dioxygen Labeled Plastic Bead
      Glass Slide
      DNA
      Biotin Labeled
    • Interphase Xenopus Egg Extracts
      1
      2
      Activated Eggs
      55000rpm for 2h at 4⁰C
      Cyclohexamide 50 µg/ml
      10200rpm 15mins
      HB-6
      3. Middle Layer Extracted Using 18 Gauge Niddle
      4. Again Centrifuged at 55000rpm for 30mins at 4 ⁰C
      5. Aliquoted Immediately, Flash Frozen at -80 ⁰C.
    • Real Experiments
    • Micrococcal Nuclease Gel Assay for Nucleosome Assembly by Extracts
      • Assembly reactions were run in extracts diluted 1:4 in HEPES buffer
      • With energy mix 5µl
      • Without Energy Mix
      Performed to check Viability of Extracts to Perform Assembly in vitro conditions
    • Results
      • Digital Video Microscopy
      • Bead Tracking System
      • Focusing Microscope to Single DNA molecule
      MT Setups Allow Precise Observation and Control of Chromatin Assembly Under Force Control
    • Magnetic Tweezers Employed
    • ATP-depleted Extracts Assemble Chromatin Onto DNA in Two Phases Below 1.2pN
      Exponential Condensation Phase
      Slow Condensation
    • Extension Reduction Occur at Same Rate, No Matter What is Size of Molecule…
      Graph Shows Same Pattern For Different Molecules
    • 4pN- A Dead-Line for Assembly… Disassembly Starts
      If force increase beyond this limit, chromatin molecule disassemble and DNA molecule show disassembly
    • Bit Confused Here…
      What is the Thing , I am Searching For?
      What is Key to Understand Chromatin Dynamics?
    • Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly
      • 1pN…
      • 2.8pN…
      • 3.5pN…
      • 4.5pN…
      • 15pN…
      Cannot Be Assembly Reaction is There…
    • Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly
      • 1pN…
      • 2.8pN…
      • 3.5pN…
      • 4.5pN…
      • 15pN…
      Figure a showed that reaction is in equilibrium… But it was’nt… Because Using Fresh Extracts again showed Assembly…
    • Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly
      • 1pN…
      • 2.8pN…
      • 3.5pN…
      • 4.5pN…
      • 15pN…
      When Force Increased to 3.5pN… Ideal Equilibrium Maintained…
    • Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly
      • 1pN…
      • 2.8pN…
      • 3.5pN…
      • 4.5pN…
      • 15pN…
      Extension in DNA fiber started… Equilibrium Passed…
    • Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly
      • 1pN…
      • 2.8pN…
      • 3.5pN…
      • 4.5pN…
      • 15pN…
      Now Rapid Expension Starts… Very Sharp Plateau of Extension Observed where Physical Noise is Minimum…
    • Quest for Force at Which Rate of Assembly stand Equivalent to Rate of Disassembly
      • 1pN…
      • 2.8pN…
      • 3.5pN…
      • 4.5pN…
      • 15pN…
      3.5pN
      Was the equilibrium point for ATP less Extracts…
    • 180 Steps Observed
      50nm
      25nM
      75nm
    • Experiment Containing ATP
      In graph (a), pattern was quite linear and Gradual Contraction of DNA molecule was observed…
      Graph (a) was further resolved in graph (b), showing very dynamic ups and downs in the presence of ATP…
    • Experiment Containing ATP
      ATP less Extract
      But Still ATP containing extract show linear attribute… Not exponential as shown in ATP less Extract graph…
    • Addition of ATP Caused Large V and ^ Steps
    • DISCUSSION
    • 1. Chromatin Assembles onto Naked DNA in Cytoplasmic Extracts in the Absence of ATP
      Evidence:
      Every ATP depleted reaction tend to go for DNA assembly below 3.5pN force… Never Extension Appeared without force…
      2. Are Nucleosomes Present in the Assembled Fibers?
      Evidence:
      Nucleosomes were there because extension steps observed were exactly 50nm and total number of steps were equal to total number of Nucleosomes estimated…
    • 3. Initial Assembly of Nucleosomes onto DNA by ATP-depleted Reaction Is Not Processive
      Evidence:
      Assembly of Nucleosome occur in random manner. Assembly and disassembly procedures continue side by side.
      4. Stall Force of Assembly Reaction Indicates Nucleosome
      Assembly Free Energy
      5. Nucleosome Disassembly require slightly larger force than 3.5pN. This force is much less then previously reported force.
    • “It was not only a revenge from those who made me bore by their presentations, but also a humble return for the knowledge, they poured on me.”
      NeverAsk.com