|
Register | Sign In |
|
QuickSearch
Thread ▼ Details |
|
Thread Info
|
|
|
Author | Topic: Wright et al. on the Process of Mutation | |||||||||||||||||||||||||||||||||||||||||
Taq Member Posts: 10085 Joined: Member Rating: 5.6
|
In this thread I would like to explore a specific paper written by Wright et al.
quote: 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.
|
|||||||||||||||||||||||||||||||||||||||||
AdminModulous Administrator Posts: 897 Joined:
|
Thread copied here from the Wright et al. on the Process of Mutation thread in the Proposed New Topics forum.
|
|||||||||||||||||||||||||||||||||||||||||
Taq Member Posts: 10085 Joined: Member Rating: 5.6
|
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
|
|||||||||||||||||||||||||||||||||||||||||
Wounded King Member Posts: 4149 From: Cincinnati, Ohio, USA Joined:
|
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
|
|||||||||||||||||||||||||||||||||||||||||
Dr Jack Member Posts: 3514 From: Immigrant in the land of Deutsch Joined: Member Rating: 9.2 |
quote: 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.
|
|||||||||||||||||||||||||||||||||||||||||
Taq Member Posts: 10085 Joined: Member Rating: 5.6 |
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.
|
|||||||||||||||||||||||||||||||||||||||||
Taq Member Posts: 10085 Joined: Member Rating: 5.6 |
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.
|
|||||||||||||||||||||||||||||||||||||||||
Dr Jack Member Posts: 3514 From: Immigrant in the land of Deutsch Joined: Member Rating: 9.2 |
I'm remain unconvinced. The mutation rate is never going to be zero.
|
|||||||||||||||||||||||||||||||||||||||||
Taq Member Posts: 10085 Joined: Member Rating: 5.6 |
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.
|
|||||||||||||||||||||||||||||||||||||||||
Taq Member Posts: 10085 Joined: Member Rating: 5.6 |
To help clarify the 3rd post in this thread, let's look at figure 2 from the paper:
Caption:
quote: 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)?
|
|||||||||||||||||||||||||||||||||||||||||
Dr Jack Member Posts: 3514 From: Immigrant in the land of Deutsch Joined: Member Rating: 9.2 |
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.
|
|||||||||||||||||||||||||||||||||||||||||
Wounded King Member Posts: 4149 From: Cincinnati, Ohio, USA Joined: |
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
|
|||||||||||||||||||||||||||||||||||||||||
shadow71 Member (Idle past 2964 days) Posts: 706 From: Joliet, il, USA Joined: |
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.
|
|||||||||||||||||||||||||||||||||||||||||
Taq Member Posts: 10085 Joined: Member Rating: 5.6 |
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.
|
|||||||||||||||||||||||||||||||||||||||||
Taq Member Posts: 10085 Joined: Member Rating: 5.6 |
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.
|
|
|
Do Nothing Button
Copyright 2001-2023 by EvC Forum, All Rights Reserved
Version 4.2
Innovative software from Qwixotic © 2024