Marcu Lecture 5

Marcu Lecture 5 - Marcu Lecture 5 Slide 70- 72 - HMGB1-...

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Marcu Lecture 5 Slide 70- 72 - HMGB1- protein, when in the nucleus that acts as a chromatin architectural protein that binds inbetween nucleosomes, in the linker DNA between nucleosomes - It binds to about the same site in DNA that Histone H1 binds - Also Helps assemble the transcriptional apparatus - By kinking the DNA, sometimes HMGB1 binding site finds itself very close to the binding site to the transcriptional activator - the glucocorticoid receptor, which functions as a dimer is a transcriptional activator, that is activated in by glucocorticoid hormones - its binds to its DNA target sequence and if there is an HMGB1 binding site nearby, and if they are both bound to DNA, they help each other bond better and they cooperate in bonding - there is no ovelap in their sequences, and they both bind independently - how do we know aobut this mutual bonding? - The original methods is called FRET (fluorescence resonant energy transfer) - Modified version is pbFRET (photobleaching FRET) - You want to see if two molecules are nearby enough to be interacting in REAL TIME - You covalently link a fluorophore to one molecule and take a different fluophore and connect it another molecule - You target the laser that gives you activation of the first molecule - If the distance between the two molecules are very close, the second molecule will absorb some of the engery emitted from the first molecule when it was hit with the laser and this will happen extremely fast - If no energy is received what will happene very quickly is because you hit the first molecule with a laser, it energizes the molecules so much, you will lose the emitted color very quickly because the excited state is too unstable - Destabalization results from bleaching - However if you do have an acceptor molecule nearby, it will protect the first guy from total bleach and you will only get a partial bleach - this means that the color will persist longer and you can measure the color longer in real-time, which helps you determine its half life - the only way that the color will remain for a long time is if a fluorophore is very close Slide 73 - CFP is the donor - covalently linked to the gluco-corticoid receptor transcriptional activator - fluorophore gene is expressed through an expression cassette which will become mRNA into a protein; the protein will go into the nucleus - you put an expression casette of this transcription factor fused with the fluorophore in the cell o expressed at a very high level over the endogenous gene - when you do this gene transfer experiment, instead you mix two different expression cassettes - one expression vector has just CFP and GR - the other expression cassete has HMGB1 covalently fused to aceptor fluorophore, YFP o the cyan is the one your following, your not following yellow - when you co-transfect and mix the two DNA’s together simulaenously, you see persistence of the
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Marcu Lecture 5 - Marcu Lecture 5 Slide 70- 72 - HMGB1-...

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