1. 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
2. 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
4. 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.
5. 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…
6. 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.
7. 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
8. Why Xenopus? Containing Enzymes which help in chromatin assembly in vitro conditions. Competent in many physiological condtions
21. ATP-depleted Extracts Assemble Chromatin Onto DNA in Two Phases Below 1.2pN Exponential Condensation Phase Slow Condensation
22. Extension Reduction Occur at Same Rate, No Matter What is Size of Molecule… Graph Shows Same Pattern For Different Molecules
23. 4pN- A Dead-Line for Assembly… Disassembly Starts If force increase beyond this limit, chromatin molecule disassemble and DNA molecule show disassembly
24. Bit Confused Here… What is the Thing , I am Searching For? What is Key to Understand Chromatin Dynamics?
56. 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…
57. Experiment Containing ATP ATP less Extract But Still ATP containing extract show linear attribute… Not exponential as shown in ATP less Extract graph…
60. 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…
61. 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.
62. “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
