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Two questions, mobility shift, primer extension analysis

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Two questions, mobility shift, primer extension analysis

Postby operon » Sun Apr 08, 2007 4:40 pm

Hello again duders! I have two questions here (they are attached as JPEGS), and I was wondering if some of you could help me. For the first question I got 6/10, for the second I got 7/10. So I was hoping you guys could help clarify what is missing!

Okay, question 1 (see img002.jpg).

a) What can you conclude about the expression of your favorite gene in the rat?

ANS.

AH is not expressed at all.
AL is expressed a little
EH is expressed a little more
EB is expressed some more
AH is expressed the most

QUES. Why does EB shift? I think it may have something to do with there being reverse primers or something (from what I remember hearing in class) - but I cannot really see this. I said that it must be a modified or different mRNA altogther. I got 6/10 for this question so I'm obviously missing something important.


Question 2 (see img001.jpg)

a) There a three molecular species that resolve from each other. What are the three species?

ANS. I labelled these on img001.jpg Number 1 is the labelled promoter DNA that is unbound to protein; 2 is the labelled promoter DNA bound to protein B; 3 is the labelled promoter DNA bound to protein B.

b) What do the results presented in the experiment tell us about the interaction of protein A and protein B with the probe DNA. Justify your answer.

ANS. I think this is where I lost all my marks. I wrote a lot but I'll summarize. 2 shifts because it interacts with protein A. In lane 3 we can see that promoter DNA can compete with binding sites for protein A and as such we see no mobility shift. But in lane 4 we see that non promoter DNA cannot compete for binding sites so we still get a shift. In lane 5 we see that protein B isn't as bulky as protein A so the DNA is more mobile. In 6 and 7 we see that promoter and nonpromoter DNA can successfulkly compete with protein B for binding sites and the labelled DNA can still migrate all the way.

QUES. What am I missing from this?


That's about it guys. I really appreciate any help anyone can give. Thanks a lot.
Attachments
img002.JPG
Question 1 (image is above, not below)
img001.JPG
Question 2
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Postby blcr11 » Mon Apr 09, 2007 9:21 am

1. Primer extension is used to map the 5' ends of mRNAs, as used here, anyway. The intensity of the band is a measure of the amount of mRNA expressed in the tissue. There is one form of the gene (the one where the primer is 95 bases downstream of the 5' end of the transcript) that is strongly expressed in embryonic liver (EL), moderately expressed in embryonic heart (EH), marginally expressed in adult liver (AL) and not expressed (to the limit of detection) in adult heart (AH). There is a second form of the gene--I would guess an alternatively spliced transcript--being expressed in embryonic brain (EB). The primer is now 126 bases downstream of the 5' end of this transcript.

2. Both A and B show specific binding to the same promoter sequence. The interactions must be specific because cold promoter DNA competes while cold non-promoter DNA does not compete for binding. Band 1 is P32-labeled promoter; Band 2 is A-promoter complex; Band 3 is B-promoter complex.
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Postby operon » Thu Apr 12, 2007 12:32 am

For question 1. Would it also be possible that another promoter lies just upstream and the primer extends to this one as well? In my question I foolishly mentioned a reverse primer. What I meant what a reverse promoter (I'm not sure I would understand why this might give some insight, I just remember my teacher mentioning it).
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Postby blcr11 » Fri Apr 13, 2007 3:04 pm

I suppose it could be an alternative upstream promoter that is activated in embryonic brain (or supressed in all the other tissues). I guess you'd have to presume the existence of tissue-specific cellular factors of some sort or alternative chromatin environments acting to supress or activate transcription from one or the other promoter. I don't know what a reverse promoter is.
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Postby blcr11 » Sat Apr 14, 2007 4:46 pm

I think I misspoke about the binding of B being specific. It is non-specific since cold promoter as well as non-promoter DNA competes for binding. My mistake. I must have mis-read the figure.
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Postby operon » Sun Apr 15, 2007 5:37 am

UGH. I did it again. I meant an OVERLAPPING promoter. Not a reverse promoter, not a reverse primer. ANYWAYS...I think the alternative splicing is the best answer. Thanks a lot!
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