New to this forum have a question

[rockind78]

Hello everyone,

As you can probably tell, I am new to this forum. I have been following some threads for quite some time and I have to say I am quite impressed! Anyway, I DID have a question amongst all this fluff. I am attempting to read a paper that was submitted and published back in 2002 for JMB. In this article, he refers to hydroxyl radical cleavage as a technique that is used. Does anyone know what this is or where I can do some reading on this particular technique? Thank you.

 

[Monique]

Are you talking about this publication??

Frazee RW, Taylor JA, Tullius TD.
Interchange of DNA-binding modes in the deformed and ultrabithorax
homeodomains: a structural role for the N-terminal arm.
J Mol Biol. 2002 Nov 1;323(4):665-83.
PMID: 12419257

 

[Monique]

This is what I found on it, it is basically a footprinting technique where a DNA binding protein is hybridized to the DNA, and thus protects that region from chemical interactions. The rest of the DNA is broken down, in this case by hydroxyl radical cleavage (which is high resolution, a low resolution method would be to use a frequent cutter enzyme), the part with the protein on it is not broken down.

The way the reaction is done is that every strand of DNA is ONLY cut once, every strand in a different position. One end is labeled with a fluorescent marker (for capillary gel electrophoresis) or with a radioactive marker (for regular electrophoresis and autoradiography). What will show up is a ladder, every time a different DNA molecule which was cut one base further from the previous one. The location of the protein is revealed by the lack of signal :D

Hydroxyl Radical Footprinting

Several methods have been employed to determine the general positioning of DNA binding proteins on DNA. One of the highest resolution methods takes advantage of Fenton1 chemistry in generating hydroxyl radicals in solution. This is achieved by complexing ferrous iron with ethylenediamine tetraacetic acid (EDTA) and adding ascorbate, as a reducing agent, together with hydrogen peroxide. Hydroxyl radicals diffuse to the DNA surface where strand breaks occur via radical cleavage of the sugar backbone2,3 . A limited reaction with DNA will cause on average less than one random break per molecule resulting in a ladder of DNA fragments differing by one nucleotide base in length. These fragments can be resolved by denaturing native polyacrylamide gel electrophoresis (PAGE) and visualized by autoradiography if the original DNA is end labeled with 32P. If a specific protein/DNA complex is allowed to form prior to hydroxyl radical treatment, then those regions of the DNA no longer exposed to solvent due to protein binding will not be cleaved. Thus, a comparison of bound and unbound samples will show a loss of some fragments in the ladder of the former relative to the latter. This region constitutes the "footprint" of the protein on DNA.

1 Fenton, H. J. H. (1894) J. Chem. Soc., 65: 899.
2 Tullius, T. D. and Dombroski, B. A. (1986) Proc. Natl. Acad. Sci. USA, 83: 5469-5473.
3 Dixon, W. J., Hayes, J. J., Levin, J. R., Weidner, M. F., Dombroski, B. A., and Tullius, T. D. (1991) Methods Enzymol., 208: 380-413.

taken from: http://www.rowan.edu/mars/chemphys/faculty/Frazee/research_proj/

 

[rockind78]

lol...thank you VERY much Monique. He did not provide that information at his old website, which I believe was hosted by U of M Flint, and I was unware that he had put up a new one. Thank you very much for the info.

 

[Monique]

lol, now I also just noticed that the first author of the JMB paper and the website are the same person that was actually a coincedence!

So was that actually the JMB paper you were looking at?

 

[rockind78]

Yeah, it was. That man is brilliant! He is a really good teacher too. Did you actually read it?

 

[Monique]

Originally posted by rockind78
Yeah, it was. That man is brilliant! He is a really good teacher too. Did you actually read it?

that was a lucky guess then

Unfortunately I am not able to get to the full text version at home, should I read it? You've been a student of Dr. Frazee?, and what makes him so good?

 

[rockind78]

I can send it to you if you would like. Is there a specific email address you would like it sent to? I don't know if you SHOULD read it, but at least I would have someone else to discuss it with...lol I don't even know how much I will be able to follow. I am in the last year of a molecular biology degree at UofM Flint, soI am thinking it won't be too bad.

What made him a good teacher is the way he ran his classes. I had him for 2 semesters of Biochemistry lab, and each week he would hold a discussion forum just dedicated to the concepts and techniques we would use in the lab. He would focus on the chemical concepts, but he would NOT lecture. He would ask us how we thought it worked and why we thought it worked like that. If we would ever ask him a question in the lab, he would answer with a series of questions, basically trying to lead you to the right answer without giving it to you. IF you were able to answer his questions, and you still could not answer your own question, he would give you a little help.

The labs were a two part series designed by him. Its been a little while so I can't remember all the details, but the first half of the year was a protein chemistry lab. The object of the lab was to purify E. coli alkaline phosphatase in its active form, and assess the activity. The second half of the lab entailed making a site directed mutation within the PhoA gene for alkaline phosphatase, DNA sequencing to confirm suspected mutation, and kinetics studies as to how that affected the enzymatic properties of wild type alkaline phosphatase.

Amazing series of labs, and I was sorry to see him go.

 

[Monique]

That DOES sound like a good teacher :) you are finishing up your Bachelor? If you don't mind I would like it if you'd send a copy of the article to my hotmail account: - :)

What will you do when you get your degree? Continue to study or are you planning to work?

 

[rockind78]

The file is over 3MB, so I am assuming you have more space than that at your homtail account? The reason I ask is because there is a 2MB limit on the free hotmail accounts.

You are correct in assuming I am finishing up my bachelor's. I will be applying to a number of graduate schools this coming year. As far as research goes, I would like to find a good microbial genetics program that is molecular intensive. I will also be applying to a few veterinary schools, and those would be my first choice.

 

[Monique]

THAT much?? Usually PDF files aren't that big...?

But I have got an extra storage account with 10 MB of space, and I just recently deleted some large files so I have about 4.6 MB of free space :)

I am still waiting for my University account with 25 MB storage capacity

 

[Another God]

Hey Rockind78. Welcome aboard. Its great to see another Molecular Biology Student in here (we're at exactly the same stage too. I have 4 weeks left in my UG degree (Although I won't graduate yet because I plan on doing honours... Not sure if things work the same over in the US...)

Anyway, that teacher does sound great. Not enough education is conducted in a 'thinking' way anymore. Its all rote learning and fact feeding: Which, in the end, doesn't teach much.

I might have to check out that paper...sounds interesting

 

[rockind78]

Hello Another God,

Thank you for the warm welcome. I fully agree with your philosophy on modern education and in fact Dr. Frazee referred his dislike for rote memorization and such when I asked him about my chemistry labs. I would be more than happy to send you a copy of the paper as well if you would like. Just give me an email address to send it to.

Monique,

You were right, the file size was only about 2.5 MB, so although smaller than I thought, I guess my concern was still valid. It is on the way as I am typing this, so you should have it in the next hour or two.

 

[Monique]

AG: the paper counts 19 pages, so be aware of the task that lays ahead of you I skimmed through it and it looks really complete with different techniques used, looks interesting.

 

[Another God]

It's OK, I just went and downloaded it myself. (I have access to just about every journal through my library =) You got to love electronic access from home...