Marcu Lecture 7

Marcu Lecture 7 - Marcu Lecture 7 Slide 107 - You can use...

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Marcu Lecture 7 Slide 107 - You can use this methodology to look at dynamic exchange of DNA binding trasncriptional activators on and off DNA - Asks how long it stays on or off the DNA and what regulates this process - Does not tell you about protein-protein interaction where we look for transferance of energy - Over expressed p65-gfp protein compared to endogenous expression; its extremely difficult to obtain expression that is exactly physiologically normal - The only way to get normal expression is to do a gene knockout of the original gene, and place the exogenous gene in its normal location o Now the fusion gene is under the same control as the endogenous one o Physiological expression o You can look at dynamic exchange on target sites o But its sensitivity is far lower since the expression is far lower if you knock it into the endogenous location o If DNA is mutated that it cant bind to DNA, theyn the dynamic exhchange rate is even faster - Evidence that p65 is coming on and off o P65 is overexpressed o Similar techniques used to knock genes out - Take p65 fusion gene and replace the endogenous p65 - Now the p65 gene is subject to the same regulation as the endogenous one o Allows you to get physiological expression o However sensitivity is far lower since expression is far lower so this is why we overexpress the gene - Evidence of dynamic exchange - P65 GFP binds to DNA o Half life is faster if it cant bind to DNA (1-2 sec) o If bound to DNA half life is slower because it has some affinity, so you have to give it a chance to dissociate and get replaced by another molecule Slide 108 - This is proof that p65 binds to NFKB binding site? - Plasmid vector (A) of multiple nfkb binding sites o Tremendous landing pad for p65 o 384 tandem repeats of nfkb binding center o Randomly integrated into chromosomes o So where its really green is where a lot of p65-gfp is binding too - Now you ask how fast is this dynamic exchange? - Shine the laser on the bright spot and on a spot where its not so bright but its green, this is a chromosomal site - As expected, the curve for the multimerized sites is shifted because it takes a longer time for the p65-gfp to come off those sites - Takes about 6 seconds to be replaced - So dynamic exchange is specific and fast Slide 109 - Flip side of the previous experiment
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- The whole nucleus gets hit with the laser except for the blocked off section - Measure the time of fluorescence loss after photobleaching - You lose the fluorescence from the spot because it measn that they dissociate and they go come place off and nobody can replace them because everybody else is bleached out and you get the flip of the prior curve
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This note was uploaded on 09/14/2011 for the course BIO 362 taught by Professor Walikarzai during the Spring '10 term at SUNY Stony Brook.

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Marcu Lecture 7 - Marcu Lecture 7 Slide 107 - You can use...

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