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Author Topic:   Wright et al. on the Process of Mutation
Taq
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(1)
Message 1 of 296 (627265)
08-01-2011 6:23 PM


In this thread I would like to explore a specific paper written by Wright et al.

quote:
Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):5089-94.
Hypermutation in derepressed operons of Escherichia coli K12.
Wright BE, Longacre A, Reimers JM.
Source

Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA.

Abstract

This article presents evidence that starvation for leucine in an Escherichia coli auxotroph triggers metabolic activities that specifically target the leu operon for derepression, increased rates of transcription, and mutation. Derepression of the leu operon was a prerequisite for its activation by the signal nucleotide, guanosine tetraphosphate, which accumulates in response to nutritional stress (the stringent response). A quantitative correlation was established between leuB mRNA abundance and leuB- reversion rates. To further demonstrate that derepression increased mutation rates, the chromosomal leu operon was placed under the control of the inducible tac promoter. When the leu operon was induced by isopropyl-D-thiogalactoside, both leuB mRNA abundance and leuB- reversion rates increased. These investigations suggest that guanosine tetraphosphate may contribute as much as attenuation in regulating leu operon expression and that higher rates of mutation are specifically associated with the derepressed leu operon.


The full text (in .html format) can be found here.

Once the topic is promoted I will describe the methods used and the results in a way that a layperson can relate to. After this, we will determine if these findings demonstrate a process of random or guided mutation.

Just to get it out of the way, I define random mutations as changes in the DNA sequence that are blind to the needs of the organism. IOW, mutations are random with respect to fitness. I am not saying that mutation rates are constant through time, nor am I saying that each base has an equal chance of being substituted, inserted, or deleted. With respect to the paper, I will attempt to demonstrate that the same mechanisms that produce reversions in leuB- organisms will also cause deleterious mutations in very important and vital genes as well as mutations which do not change the fitness of offspring.


Replies to this message:
 Message 5 by Dr Jack, posted 08-02-2011 6:18 AM Taq has responded
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 Message 225 by herebedragons, posted 12-22-2011 8:12 PM Taq has responded

  
AdminModulous
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Message 2 of 296 (627267)
08-01-2011 7:28 PM


Thread Copied from Proposed New Topics Forum
Thread copied here from the Wright et al. on the Process of Mutation thread in the Proposed New Topics forum.
    
Taq
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Posts: 7034
Joined: 03-06-2009
Member Rating: 3.8


(2)
Message 3 of 296 (627294)
08-01-2011 11:14 PM


Background on strains, genes, and biochemistry
I am assuming all of you understand the relationships between replication (DNA to DNA), trascription (DNA to RNA), and translation (RNA to protein). This is often called the Central Dogma. If you don't understand these concepts then use google for a quick refresher course. This is basic biology stuff, so it shouldn't be hard to find or to understand.

What I would like to do first is describe the different E. coli strains, genes, and the biochemistry that affects gene expression. This is vital for understanding the data in some of the figures and tables.

Strains and gene knockouts:

CP78: this strain lacks functional genes to produce the amino acids arginine (argH-), histidine (his-), threonine (thr-), and most importantly leucine (leuB-).

CP79: Same as CP78 but with one difference. It has a deleterious mutation in the relA gene. This relA gene product (i.e. relA protein) is responsible for producing guanosine tetraphosphate (ppGpp).

78AL: Same as CP78 but with a new promoter region for the leuB gene. This will be very important in my future posts, so pay careful attention to this. The replaced the portion of the leuB gene that controls its expression with a promoter that responds to another chemical, IPTG. This allows them to control the expression of the gene independently of leucine concentrations in the surrounding media. If they want more leuB gene products all they need to do is add IPTG. For those who are interested, they are replacing the native promoter with a modified lac promoter. A google for "lac promoter" should find hundreds of thousands of hits describing how this promoter works. For those who do not know much about promoters now would be a good time to do a google for that lac promoter. I would suggest this site, especially the animation tab which has narration describing how the lac system works.

So how do all of these genes interact with one another and the environment? Since the paper focuses on the leuB gene and derepression of the leuB gene we will focus on that.

First, what is derepression? This is where a gene is normally kept in the off position until a derepressor removes whatever is stopping the gene from being transcribed or creates a situation which turns the gene on. In the case of leuB, the gene is repressed when there is leucine present in the growth media. This prevents the bacteria from using energy to produce its own leucine. When the cell senses that its amino acid stores are low (be it arg, his, thr, or leu) it increases the expression of relA which then increases the intracellular levels of ppGpp. This is called the stringent response. In addition to ppGpp increases, there is also another system that senses leucine stores specifically (the paper doesn't mention this second level of control, but I can try and find it if someone is interested). In order to remove the repression on the leuB gene (i.e. derepression) you have to have both an increase in ppGpp and the lack of leucine. The result is an increase in leucine through "de novo" pathways instead of scavenging from the environment.

To sum up this section, when you have low leucine levels it kicks in two systems to derepress the leuB gene: the stringent response and the leucine specific system.

This is why they are comparing CP78 and CP79. They want to see how changes in ppGpp affect the overall mutation rate in leuB. By using a non-functional mutant of relA they are able to remove ppGpp from the picture and see what affects derepression has. 78AL was also used to move to a completely different repressor system to further verify that single stranded DNA is what is being hypermutated.

There are other mutants and rescued strains, but they are not central to the main argument. These are more of a check to verify certain findings. If need be, I will include those other strains once we discuss the data.

If I have any of this information wrong PLEASE correct me.

Edited by Taq, : confused stringent response with leucine specific system

Edited by Taq, : No reason given.

Edited by Taq, : edited strain names


Replies to this message:
 Message 4 by Wounded King, posted 08-02-2011 5:01 AM Taq has responded

  
Wounded King
Member (Idle past 1595 days)
Posts: 4149
From: Edinburgh, Scotland
Joined: 04-09-2003


(1)
Message 4 of 296 (627336)
08-02-2011 5:01 AM
Reply to: Message 3 by Taq
08-01-2011 11:14 PM


Re: Background on strains, genes, and biochemistry
That seems like a very concise introduction but there are a few points.

The strains are called CP78 and CP79 throughout the paper.

I would also suggest that the 'modified' Lac promoter, tac, is more accurately described as a fusion of two different promoters, the trp and lac uv5 promoters. I'd usually consider a modified promoter to be one that has been subjected to targeted mutations to alter its sequence rather than a chimera/fusion as in this case.

TTFN,

WK


This message is a reply to:
 Message 3 by Taq, posted 08-01-2011 11:14 PM Taq has responded

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Dr Jack
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Posts: 3506
From: Leicester, England
Joined: 07-14-2003


Message 5 of 296 (627346)
08-02-2011 6:18 AM
Reply to: Message 1 by Taq
08-01-2011 6:23 PM


Their claim is not well supported.
quote:
This article presents evidence that starvation for leucine in an Escherichia coli auxotroph triggers metabolic activities that specifically target the leu operon for derepression, increased rates of transcription, and mutation.

That seems a monumentally unsupported claim. It seems to me that their results are better explained in two ways, one of which is discussed in their own discussion section and one of which isn't.

1. As discussed in their discussion and supported by other papers: transcription increases the chances of mutation. This should surprise no-one: DNA is usually packaged in a manner to ensure its protection and only unravelled when copied or expressed.

2. As not discussed in the paper. A mutant that is already derepressed will experience a significant selective advantage when it is put into the starvation conditions as it will already have the required enzymes in place; this means a sample taken post-starvation will have a vastly increased chance of finding a bacterium that is descended from a pre-starvation mutant. I see nothing in their method that would distinguish between a pre-starvation mutant and one that occurred during starvation.


This message is a reply to:
 Message 1 by Taq, posted 08-01-2011 6:23 PM Taq has responded

Replies to this message:
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Taq
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Posts: 7034
Joined: 03-06-2009
Member Rating: 3.8


Message 6 of 296 (627400)
08-02-2011 11:27 AM
Reply to: Message 4 by Wounded King
08-02-2011 5:01 AM


Re: Background on strains, genes, and biochemistry
The strains are called CP78 and CP79 throughout the paper.

Good catch. It is now fixed.

I would also suggest that the 'modified' Lac promoter, tac, is more accurately described as a fusion of two different promoters, the trp and lac uv5 promoters. I'd usually consider a modified promoter to be one that has been subjected to targeted mutations to alter its sequence rather than a chimera/fusion as in this case.

That is a very fair criticism. For those who are interested, this paper outlines the features of the tac promoter.

I decided to leave the details out because I am trying to make this accessible to the scientific layperson. From my reading of the paper, the important bit is that the gene can now be controlled with IPTG instead of ppGpp. That is what I want to stress in further posts.

Edited by Taq, : No reason given.


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Taq
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Posts: 7034
Joined: 03-06-2009
Member Rating: 3.8


Message 7 of 296 (627403)
08-02-2011 11:32 AM
Reply to: Message 5 by Dr Jack
08-02-2011 6:18 AM


Re: Their claim is not well supported.
I see nothing in their method that would distinguish between a pre-starvation mutant and one that occurred during starvation.

It wouldn't distinguish between the two, but they do control for this by measuring the mutation rate in non-starvation conditions. They did not see any mutants in these controls, at least not at the concentrations they were plating at.


This message is a reply to:
 Message 5 by Dr Jack, posted 08-02-2011 6:18 AM Dr Jack has responded

Replies to this message:
 Message 8 by Dr Jack, posted 08-02-2011 12:16 PM Taq has responded

  
Dr Jack
Member
Posts: 3506
From: Leicester, England
Joined: 07-14-2003


Message 8 of 296 (627414)
08-02-2011 12:16 PM
Reply to: Message 7 by Taq
08-02-2011 11:32 AM


Re: Their claim is not well supported.
I'm remain unconvinced. The mutation rate is never going to be zero.
This message is a reply to:
 Message 7 by Taq, posted 08-02-2011 11:32 AM Taq has responded

Replies to this message:
 Message 9 by Taq, posted 08-02-2011 9:35 PM Dr Jack has responded

  
Taq
Member
Posts: 7034
Joined: 03-06-2009
Member Rating: 3.8


Message 9 of 296 (627581)
08-02-2011 9:35 PM
Reply to: Message 8 by Dr Jack
08-02-2011 12:16 PM


Re: Their claim is not well supported.
I'm remain unconvinced. The mutation rate is never going to be zero.

I don't think the authors ever stated that it was. What they are trying to show is a major increase in mutations under specific conditions. I think their methodology and results can show and do show an increase in mutations for actively transcribed genes. I think the authors would agree that if you plated enough bacteria that you would find a leuB- reversion in the non-starved controls.


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Replies to this message:
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Taq
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Posts: 7034
Joined: 03-06-2009
Member Rating: 3.8


Message 10 of 296 (627584)
08-02-2011 9:44 PM


To help clarify the 3rd post in this thread, let's look at figure 2 from the paper:

Caption:

quote:
Figure 2
leuB mRNA abundance in E. coli CP78 (relA+, ppGpp+) and CP79 (relA−, ppGpp−. Cells were grown to log phase (log) in minimal medium or were grown to log phase and were washed and transferred to minimal medium without arginine (−arg), threonine (−thr), or leucine (−leu) for 15 min. Total RNA was recovered, and the level of leuB transcript was determined by nuclease protection assay and densitometry (see Materials and Methods).

This figure is straightforward. It demonstrates that in CP78 (relA+) the amount of leuB mRNA is tied to leucine, and only leucine, starvation. In media that lacks threonine or arginine there is no significant increase in leuB over control (which is the clear bar labelled "log"). However, there is no signficant increase in leuB in CP79 (relA-). This indicates that relA, and by extension ppGpp, is important for the leucine starvation specific derepression of leuB.

Any questions so far (I am talking to you zi ko and shadow71)?


Replies to this message:
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Dr Jack
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Posts: 3506
From: Leicester, England
Joined: 07-14-2003


Message 11 of 296 (627636)
08-03-2011 4:56 AM
Reply to: Message 9 by Taq
08-02-2011 9:35 PM


Re: Their claim is not well supported.
I don't see how what they've done rules out a selection effect, especially as they haven't worked out the prevalence of the mutation in the basal population.
This message is a reply to:
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Wounded King
Member (Idle past 1595 days)
Posts: 4149
From: Edinburgh, Scotland
Joined: 04-09-2003


Message 12 of 296 (627641)
08-03-2011 6:31 AM
Reply to: Message 11 by Dr Jack
08-03-2011 4:56 AM


Re: Their claim is not well supported.
they haven't worked out the prevalence of the mutation in the basal population.

Without doing a population wide sequencing screen there would be no way to detect such revertants in an unstarved population. The selective conditions that would reveal the mutation through a standard screen would themselves trigger the starvation response. What the authors do is to use starvation for another amino acid, threonine or arginine, as the control condition for culturing prior to plating.

It is definitely worth noting that in fact these levels are essentially the same as those for cultures raised in leu starved conditions as those raised in arg starved conditions. Unfortunately revertants arising before or after plating can't be ditinguished, again short of wide scale genetic screening, so there is no way to tell when the reversions occurred.

I think we might be kind of preempting Taq's progression through the paper a bit.

TTFN,

WK


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shadow71
Member (Idle past 434 days)
Posts: 706
From: Joliet, il, USA
Joined: 08-31-2010


Message 13 of 296 (627655)
08-03-2011 8:49 AM
Reply to: Message 10 by Taq
08-02-2011 9:44 PM


I assume in message 3, 1st sentence, you meant to type "transcripton" correct? Not being picky, just want to make sure I am not missing something.

So far so good in following your explanations.


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Replies to this message:
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Taq
Member
Posts: 7034
Joined: 03-06-2009
Member Rating: 3.8


Message 14 of 296 (627700)
08-03-2011 1:51 PM
Reply to: Message 13 by shadow71
08-03-2011 8:49 AM


I assume in message 3, 1st sentence, you meant to type "transcripton" correct? Not being picky, just want to make sure I am not missing something.

That is correct. It took me 3 or 4 tries to figure out what you were talking about, hehe.

If you want to work ahead I will be focusing on Figure 3 as well as Tables 1 and 2 for my next post. As a brief preview, pyrD and glpK have the opposite expression profile as leuB, and spoT is the gene responsible for ppGpp degradation. spoT knockouts will have higher ppGpp levels due to lack of degradation.


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Taq
Member
Posts: 7034
Joined: 03-06-2009
Member Rating: 3.8


Message 15 of 296 (627701)
08-03-2011 1:54 PM
Reply to: Message 12 by Wounded King
08-03-2011 6:31 AM


Re: Their claim is not well supported.
I think we might be kind of preempting Taq's progression through the paper a bit.

If you can keep from ruining the punch line it would be most appreciated.

Of course, this is a public thread so anyone can say whatever they want (as long as it is on topic). I am trying to go through the paper slowly so others have time to ask for clarification and ask questions. If it was presented all at once it would be too overwhelming, IMHO.


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