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transcriptional cascade

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transcriptional cascade

Postby Marielle » Sat Mar 31, 2007 4:25 pm

Hello, I'm a beginner and I really need help from you.
My problem is that I want to make a construct which I will transfect into an animal cell and I have to use a particular gene and a particular promoter which is very strong.
The problem is that I don't want this gene xx to be expressed so much.
Would it be possible to put an activator protein and promoter which would be activated by this activator protein between promotor S and gene xx?
So the construct would look like this:
promoter S-activator-promoter-gene xx
S stands for strong.
The next problem is that I don't know which activator and which promoter to use! I want that promoter 2 wouldn't be so strong which means that gene xx won't be expressed so much.
Please, if u have any idea - help me (as soon as possible)
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Postby blcr11 » Sat Mar 31, 2007 5:51 pm

Not sure I understand the problem, exactly. It sounds like you want to down-regulate a strong eukaryotic promoter. Yea verily—you want to completely shut it down. I’m aware of silencer sequences in yeast, but I’m not sure they work in just any old cell, and then how do you un-silence it when you actually want to express the gene, which I assume you want to do at some point, you just don’t want it constitutively expressed, or am I wrong? Why do you need to express xx off the strong promoter at all if what you want to do is control the timing of xx expression? Is the object to learn something about the promoter, or the gene, or is this an exercise in exogenous gene expression for, say, therapeutics? I think I must be missing something.
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Postby Marielle_ » Sat Mar 31, 2007 6:22 pm

What I would like to do is to make a strong inducible promoter less stronger.
So what I want to do is to put this promoter before a gene for activator of another promoter which would be less stronger than the first ...
I'm asking u whether it is possible or not ...
It sounds strange , I know :)
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Postby blcr11 » Sun Apr 01, 2007 12:33 pm

I don’t know that I have any great suggestions, and I still don’t understand why exactly you want to do it. I’m not sure how sequence-specific they are, but you could try inserting sequences associated with the upstream regions of genes known to be transcriptionally inactive in the cells you are thinking of using. These sequences may be signals for nucleosome accumulation and genes packed tightly into nucleosomes tend to be less active than genes in nucleosome-poor chromatin. That probably won’t work if you’re using a transient expression system, or if your gene is not actually incorporated in chromatin. And I don’t know if this applies to yeast systems, either. The only thing I recall from yeast are the silencer sequences that shut off transcription from the unused mating cassette—only one can be active at a time and yeast can spontaneously switch between them.

You might look at hormone-sensitive promoters. There is reported to be a sequence of the parathyroid hormone receptor promoter that is used by insulin-like growth factor to inhibit PTH transcription. Another place to look at might be the glucocorticoid receptor system. Lastly, maybe you could look at what’s known about viral early-, middle-, and late-promoters. Virus transcription is regulated in part by upstream sequences (promoters, enhancers) and viral and cellular transcription factors. When in the course of infection the virus has to switch from, say, early-gene to middle-gene expression, there must be a way to turn off an early gene’s and turn on a middle gene’s expression. SV40 and adenoviruses are probably the most widely studied—I don’t remember too many details of the mechanisms of regulation, but you might find something useful there.

I’m reluctant to say it can’t be done—it probably can—maybe it’s even as simple as inserting a GC-rich sequence or something like that. But neither do I think I know how to do it.
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