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Generation of DNA nucleotides

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Postby kolean » Mon Jan 04, 2010 2:52 pm

Think of the DNA as a terabyte. And instead of 1 and 0 s, it is A, T, C, and G s. Basically a code to program by. Then think of all the coding you would need to do, just to do the basic metabolism of a complex system such as a human.

And please do not say "junk DNA". There is no such thing. It just shows our ignorance at our DNA knowledge that we don't know what that sequence of DNA does yet. We have just recently found new 'genes' that are really just produce the RNA (made from the 'junk' DNA) that is used for regulation. And yes, some of the DNA gets inserted into our genome from outside sources (not just the usual doubling of a DNA from the sliding model), but I figure this is raw material of DNA for the organism to use and develop new systems/programs.

Again, there is no short supply of nucleotides. They are in high concentration in the cell. Humans ingest cells all day long. And inside cells is DNA. You would only have to worry if you were malnourished/starving, and thus no cells to digest and salvage the nts, and if your ATP (and possibly GTP) weren't available because they were being used by the metabolic system as energy.
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Re:

Postby JackBean » Mon Jan 04, 2010 3:59 pm

kolean wrote:Again, there is no short supply of nucleotides. They are in high concentration in the cell. Humans ingest cells all day long. And inside cells is DNA. You would only have to worry if you were malnourished/starving, and thus no cells to digest and salvage the nts, and if your ATP (and possibly GTP) weren't available because they were being used by the metabolic system as energy.

Well, I think that in ióne of Biochemistry books was, that actually any of nt from food is actually used in the body, but these are all build de novo
http://www.biolib.cz/en/main/

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Re: Generation of DNA nucleotides

Postby gs99 » Mon Jan 04, 2010 8:14 pm

Kolean,
Thanks for the link to HHMI, and thanks to the people who produce the various media.
[edited 1/6/10. This link does not show the nucleotides. Please see correction in next post]
http://www.hhmi.org/biointeractive/dna/ ... n_vo2.html
They almost seem real! I would not have imagined the action of DNA replication being that fast.

The video reminded me of a sewing machine operation, when attaching two layers of material together.
In each cycle,
>The needle with thread "n" is pushed through the material layers.
>The bobbin with thread "b" (underneath the material) causes a connection to be made between the two threads.
>A small tool moves the materials exactly the same short distance for the next stitch.
In reality it goes Very fast, but this shows it in slow motion:
http://materialmama.wordpress.com/2007/ ... ern-ideas/
With a sewing machine, there is simply one needle thread and one bobbin thread.
The needle is not concerned about matching things, as polymerase is.

In the HHMI video, (If I understand correctly) the nts (all four types) appear "floating around" and get attracted somehow at the right time to where the polymerase is making attachments to the DNA strand.
The nts float in the nucleoplasm, a highly viscous liquid that evidently provides support for all the components.
But objects normally move slower through liquids.

My basic question remains: How does one of the needed nt move into position when and where it's needed?

----
I agree with your "junk DNA" thought. That's why I put it in parenthesis.

----
I disagree slightly about the 1s and 0s, which are the basic units of information in a typical computer.
The natural basic units are element atoms (not counting the smaller things being discovered).
Each nucleotide type is made in a separate way of certain atoms.
To a non-chemist, these are complicated in themselves.
But yes, each molecule type could be abbreviated with its letter.

----
"Then think of all the coding you would need to do, just to do the basic metabolism of a complex system such as a human."
But I try to understand the mechanics of "small" parts, like gene expression and DNA replication.

When I explain scientific things to others, I try to talk in terms people know.
When talking about the solar system, I find it useful to envision smaller models.
Like, if the earth is an inch in diameter, how big is the moon and sun. How far away?
Students may get a different feeling about the force of gravitation, seeing the objects together.

To explain small things such as DNA, we need to envision larger.
If DNA were produced in a factory, typical questions may be:
How are orders received; from where?
What does the Bill of Materials look like? How will the raw materials arrive?
What machines and tools are needed?
How much energy will be needed?
How quickly must the job be done?
What provisions for quality assurance?
I would like to see a web site dedicated to this subject.
Where the latest scientific discoveries about DNA replication are explained in language all can understand.
Videos that can be run in slow motion, and including text.

----
"Again, there is no short supply of nucleotides."
And they evidently are available in the ratio that is needed, in the nucleus.

I have found a way to order the book Biochemistry by Voit & Voit, through a state-wide library search facility.
I'm anxious to see if it answers any of my questions.

Thanks again Kolean and JackBean
Last edited by gs99 on Wed Jan 06, 2010 4:25 pm, edited 1 time in total.
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Re: Generation of DNA nucleotides

Postby gs99 » Tue Jan 05, 2010 3:44 pm

I apologize for providing the incorrect link to Howard Hughes Medical Institute.
The one that I mentioned does not show the nucleotides.
But this page has a video of "DNA transcription (basic detail)", a similar process to DNA replication.
http://www.hhmi.org/biointeractive/dna/ ... n_vo1.html

As I looked closer at this video, I noticed the nt "building blocks" appear as two colors, white and yellow. Perhaps to indicate a protein moving each nt through an "intake hole" of the polymerase.
And at the right time...

Here's a possibility:
All A's dock at one waiting line, the T's dock in an adjacent line etc..
Like people getting onto a roller coaster, each waiting line gets into different cars.
Only here, the roller coaster has only one "car", the next spot on the DNA string.
The DNA polymerase controls the "dispensing" of next appropriate nt waiting for its ride.
That would enable fast operation!

I've found a book "The Way of the Cell", by Franklin M. Harold (Oxford University Press 2001),
that describes the many ways that proteins function, and other things explained clearly.

With my background in computers, I noticed this:
"There are cascades of such "kinases", one switching on the next, and each potentially the target for a signal.
The common conception of proteins serving either as catalysts or as structural elements is incomplete:
for many purposes it is more useful to emphasize their roles as computational elements in information-processing networks."
(page 53, with reference to Bray, 1995)

Recently I've thought of proteins as being computer applications that do the work for an organism.
Is there an operating system that enables the apps?
Like the news reporter said, "This story has many moving parts."
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Postby JackBean » Tue Jan 05, 2010 10:06 pm

The polymerase has simply high affinity for the nts. If the nt fits to the parental strain, it is incorporated, if not, it is released, eventually hydrolysed away, if it was already incorporated.
http://www.biolib.cz/en/main/

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Re: Generation of DNA nucleotides

Postby kolean » Wed Jan 06, 2010 2:05 pm

gs99 wrote:Here's a possibility:
All A's dock at one waiting line, the T's dock in an adjacent line etc..
Like people getting onto a roller coaster, each waiting line gets into different cars.
Only here, the roller coaster has only one "car", the next spot on the DNA string.
The DNA polymerase controls the "dispensing" of next appropriate nt waiting for its ride.
That would enable fast operation!

Now, how would you go about proving this theory?
What kind of experiment would you perform to show that this is what happens?
And you could go about this with a model organism, or perhaps just dividing cells in culture (though there is a difference between in vivo and in vitro).

There are chaperone proteins that help in the proper folding of proteins as they are being translated. RNA also has some help in folding into the proper hairpin structure. Why not help in getting proper nucleotides for processing into the DNA? (You might like to learn about translation: mRNA into a protein structure. There is tRNA that holds onto the amino acid while the polymerase 'reads' the mRNA template codon sequence for translation).

Remember that the DNA polymerase is only one subunit of the whole replication complex (for the initiation and elongation of the DNA replication mechanism).
I am studying epigenetics and they have just theorized that there is another subunit to add to the replication complex that helps to maintain the methylation status of the DNA during DNA replication.
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Re: Generation of DNA nucleotides

Postby gs99 » Thu Jan 07, 2010 1:11 am

Kolean wrote:
Now, how would you go about proving this theory?


I don't have the education and experience to know that.
I'm just guessing based on how a robotic system might do it.

The more I learn about proteins, the awe increases.

I don't understand how scientists discover what happens within the nucleus, itself being within a cell.
Microscopes don't work at that level do they?
And I would think everything has the same general color, not like the nice pictures we see in the books or the HHMI videos!
How do they learn how things move from here to there?

I don't understand how DNA polymerase can:
# process each of the billions of nucleotides in sequence (the exact number still being worked out)
# determine what kind of nucleotide is on the original strand (Could a lab expert identify these molecules easily?)
# do what's needed to obtain a new complementary nucleotide (What is there about an A that says: "Give me a T"?)
# enable the new nt to be connnected to the original nt with hydrogen
# keep track of what segments are done in reverse
I'm sure this is not a complete list.
And it does this accurately and (according to HHMI) very very fast.

The answer evidently is in your comment about the replication complex; it is a complex mixture of organized things.
But aren't they all just a collection of molecules made of amino acids?
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Postby JackBean » Thu Jan 07, 2010 6:20 pm

The polemerase does not recognise actually anything, everything is done by complementary bonding of paires of nucleotides. If is this bonding strong enough, the polymerase goes on if not, it is released again and searched for new proper nucleotide

# keep track of what segments are done in reverse
what?

But aren't they all just a collection of molecules made of amino acids?

Sure, they are, like all enzymes. Aren't the machines just composed of other pieces? And can they produce them?
http://www.biolib.cz/en/main/

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Re: Generation of DNA nucleotides

Postby gs99 » Sun Jan 10, 2010 2:19 am

JackBean wrote:The polemerase does not recognise actually anything, everything is done by complementary bonding of paires of nucleotides.


Regarding the important role of new nucleotides, various web sites explain it in different ways:
>You mention "complementary bonding". I could not find anything about this term.
Could you please provide a web site that explains?

>The HHMI movie ignores them altogether.
http://www.hhmi.org/biointeractive/medi ... vo2-lg.mov

>Talking of transcription (similar process) this page says:
"As RNA polymerase reads each nt, it brings in the complementary nt and bonds them together forming the mRNA strand".
http://www-class.unl.edu/biochem/gp2/m_ ... ne_a2.html
Notice it says the polymerase READS each nt.

>Others say the nucleotides are "attracted".

DNA replication is done in a liquid, which normally impedes motion of objects.
How are these nucleotide molecules floating in the nucleoplasm pursuaded to move quickly at the right time to where they are needed?

JackBean wrote:keep track of what segments are done in reverse
what?

I probably used the wrong words. I was referring to the additional steps completed in the lagging strand, where synthesizing is done "backward".
"Two polymerase enzymes are required, one for each parental DNA strand.
Due to the antiparallel nature of the DNA strands, however, the polymerase enzymes on the two strands start to move in opposite directions."
http://www.wiley.com/college/pratt/0471 ... index.html

The Primase enzyme is utilized often in the lagging strand.
It has the ability to move itself "down the line" and generate short segments of RNA primer as needed.
Now how does this collection of atoms know when and how to do that?

The initial experience of chemistry is to learn the characteristics of elements and compounds.
Something happens when mixing four grams of this with a liter of that.
And matter normally has standard properties when in a solid, liquid, or gaseous state.

But in biochemistry, it seems to be different.
A single molecule comprised of atoms or other molecules (eg. DNA polymerase, Primase enzyme) has the ability to do things on its own!

One web site claims there may be thirty different tools (proteins, enzymes etc.) working together in DNA replication. Another uses the term "military precision".

There are various claims about multiple processing. Most web sites show only a single replication fork.
One claims there are hundreds of concurrent DNA replication activities in a cell.
Is the DNA separated into chromosomes when replication occurs?
There are also various claims about the speed.
I appreciate the internet, but sometimes it's difficult to know what's the latest information.
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Postby JackBean » Sun Jan 10, 2010 11:30 am

>Others say the nucleotides are "attracted".
This is the complementary bonding. Complementary bonding (or binding) means, that A binds always T and C binds always G and vice versa ;)
http://www.elmhurst.edu/~chm/vchembook/ ... sepair.gif
This is due to spatial restrictions and H-bonding restrictions (position of donors and acceptors).

You are still NOT understanding, that the solution in cell is actually very dense, containing many molecules.

As I mentioned above, in bacteria you have only one replication origin, so you have only one replication at a time (although after some time, new replication can start, although the previous did not finish yet), but in humans you have replication origin each 3-300 thousands of bps. These are fired several of them at a time in a coordinated manner.
The speed of replication varies in dependence of polymerase.
http://www.biolib.cz/en/main/

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Re: Generation of DNA nucleotides

Postby gs99 » Mon Jan 11, 2010 3:37 pm

Polymerase Chain Reaction (PCR) seems to provide some insight.
http://en.wikipedia.org/wiki/Polymerase_chain_reaction

Why Taq is utilized
http://en.wikipedia.org/wiki/Taq_polymerase

And a Virtual lab where anyone can do it:
http://learn.genetics.utah.edu/content/labs/pcr/
These contents are placed into the pcr tube:
# extracted DNA (to be copied)
# Primer 1
# Primer 2
# New nucleotides (ATCG building blocks)
# DNA polymerase
The pcr tube is then put into the DNA Thermal Cycler.
The Cycler automatically changes the temperature in cycles to simulate what happens in the cell nucleus.
Many copies of a specific DNA segment are thereby generated.

(1) I noticed that no additional proteins were needed to facilitate movement of the nucleotides.
That seems in opposition to my guess that "proteins" were utilized.
I still am curious how they move, as if by magnetic force.

(2) The new nucleotides are not attracted automatically to complementary nts on their own.
Otherwise, why is DNA Pol needed?

(3) The DNA Pol "read the DNA code and then attach matching nucleotides".
This seems in opposition to what JackBean said:
"The polemerase does not recognise actually anything"
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Postby JackBean » Mon Jan 11, 2010 4:06 pm

Well, I'm not master in these things, probably MrMistery could shed some light into this topic...

(1) that's, why there is temperature change, not to need other proteins...

(2) Pol is used for the same thing as other enzymes - to catalyse reaction, to speed it up
http://www.biolib.cz/en/main/

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