I survived my thesis defense! w00t!
Shemeska
Adventurer
Fenris said:
Yes, singly expressed Z localized to the mitotic chromosomes in Daudi, D98, NIH3T3, and HeLa cells. It did not in Ramos cells.
Can you speculate what features of Ramos cells would cause the non-binding?
And if this gives you flashbacks, feel free to ignore them
And this was one of the things that they picked on me for, and in the end I can't think of any obvious reasons. Ramos cells are EBV negative, and I'd already established in the earlier cell lines that EBV infection status didn't matter for Z localization, so Ramos lacking the full EBV genome isn't a reason here. I'd have to do more work to really have any worthwhile speculation.
Shemeska
Adventurer
Fenris said:Were you doing gel-shift assays or footprinting? Footprinting might tell you a bit why if the sequences are off in the binding area.
But hey, that can all be saved for your Ph.D.
Congrats again.
More flashbacks
Well we don't know where Z is binding, just that of its functional domains, only the DNA binding domain is required for localization to the mitotic chromosomes. I have some suspiscions for what it's doing there, but we don't have much evidence for where it's binding actually.
I raised the prospect of footprinting, but that got shot down during the proposal as being a bit beyond the scope of the project. If the department had a PhD program (we only go up to MS at the moment) I'd have wanted to look much closer at where it was specifically binding though.
And we did some gel shift assays, but only with respect to determining an interaction between Z and a few cellular proteins, not Z and the host chromatin. That work was done before I started my project, but it did form the basis for some of stuff I ended up doing.
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