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Author | Topic: Wright et al. on the Process of Mutation | |||||||||||||||||||||||||||||||||||||||||||
Taq Member Posts: 10073 Joined: Member Rating: 5.2
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It's been a couple of days, so this first figure will be a bit of a refresher.
caption:
quote: It would take a few posts to explain how this blot was produced. Thankfully, the actual method is not that important, but the results are. The authors picked 3 different genes that should respond differently to ppGpp levels. As has been discussed, leuB is derepressed when ppGpp is elevated. pyrD is just the opposite. It is repressed by ppGpp. glpK is only controlled by the growth rate of the organism and is not sensitive to ppGpp. L stands for log phase (unstarved) and S stands for starvation. They tested both the relA- and relA+ strains. The results are just as they should be, so nothing too surprising here. Now we get to the meat of the paper. Table 1Correlation between leuB mRNA levels determined as described for Fig. Fig.22 and leuB− reversion rates (too lazy to transcribe the data here so click on this link) They measured two things. The amount of leuB mRNA compared to the total amount of mRNA for all genes. If this ratio increases then leuB has been derepressed. They also measured the number of leuB- clones that regained their ability to make leucine which are called leuB- reversions. The leuB- gene had a very specific mutation, "a C-to-T transition resulting in a serine-to-leucine substitution at amino acid residue 286 of the LeuB protein" according to the paper. The rate at which that T was reverted back to a C is the reversion rate. Also keep in mind the units that the reversion rates are given in, which is 10-9. So for the first number given in that column, 0.15, that is 0.15 reversions per billion bacteria. Yes, billion with a B. The table demonstrates that the reversion rate does correlate with the amount of leuB mRNA. It also correlates with conditions where ppGpp are elevated (in the relA+ and spoT strains with leucine starvation conditions). The highest reversion rate in this table is 2.5 reversions per billion bacteria. This is the almighty guided mutation, 2 reversions per billion bacteria. Powerful, isn't it?
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Wounded King Member Posts: 4149 From: Cincinnati, Ohio, USA Joined: |
Technically an RNase protection assay (RPA) is not a blot since there is no transfer of the sample material, instead the gel the sample is run out on is dried down straight onto filter paper to make it more stable. I spent practically my whole PhD running those damn things!
I have to say that looking at that RPA it doesn't really seem consistent with the data from figure 2 or Table 1. In Fig. 3 it looks like there is a definite induction of leuB in the starved CP79 relA- sample. The induction is certainly massively higher in the relA+ strain, but it just seems strange that the only actual raw data they show doesn't really look consistent with the other derived figures. It is a bit strange that they present exactly the same sort of data, RPAs, in two different ways in this paper. Although all of the data figures are from RPAs only Fig. 3 shows the actual bands on gel. If I were cynical I'd say that it suggests that the gels were pretty messy and possibly hard to interpret, though going by the derived graphs the results should have been pretty clear. TTFN, WK
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Taq Member Posts: 10073 Joined: Member Rating: 5.2 |
Technically an RNase protection assay (RPA) is not a blot since there is no transfer of the sample material, instead the gel the sample is run out on is dried down straight onto filter paper to make it more stable. I spent practically my whole PhD running those damn things! I have done a million Western's, so I tend to use the word "blot" where it isn't appropriate. I am much more of a protein guy than a gene jockey. I thought it would be confusing to describe how the RNase protection assay worked when the results are pretty straightforward. But thanks for the clarification anyway. Most appreciated.
In Fig. 3 it looks like there is a definite induction of leuB in the starved CP79 relA- sample.
I don't know if it was discussed in this paper or a related paper, but it is known that the relA2 (i.e. relA-) genotype is leaky. relA2 is not an allelic replacement knockout, but relA with a specific mutation. I think this shows up more in the spoT knockouts. For the purposes of this discussion, I am more than willing to conceed that lueB derepression is tied to leucine starvation and is partly dependent on ppGpp, leaky alleles or not.
If I were cynical I'd say that it suggests that the gels were pretty messy and possibly hard to interpret, though going by the derived graphs the results should have been pretty clear.
I would agree with your cynicism. Many of the papers I read do not show entire blots, only a slice of the blot that includes the band of interest. I have always hated that. Sometimes there is important data elsewhere on the blot. What one author calls "non-specific" binding/bands I call important information.
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shadow71 Member (Idle past 2960 days) Posts: 706 From: Joliet, il, USA Joined: |
As I told Aristotle when I visited Athens, this is pretty much all Greek to me.
I will keep reading and learning.
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Taq Member Posts: 10073 Joined: Member Rating: 5.2 |
As I told Aristotle when I visited Athens, this is pretty much all Greek to me. I will keep reading and learning. Now would be the time to ask questions. There are many knowledgable people participating in this thread that would be happy to answer them.
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Doctor Witch Junior Member (Idle past 4643 days) Posts: 27 From: Both Sides Joined: |
This paper appears to be yet another fragment of evidence for the non-random evolution of protein codes that carry biological advantage at the cellular level which pretty much started in the study of bacterial resistance to antibiotics.
Has nobody thought of the general mechanism of this and investigated it? That mechanism would appear to be a Natural Law of Dynamic Equilibrium. According to this Natural Law, the reactions that take us from DNA to RNA to protein goes both ways. All of the conditions for the reverse reaction occur in the cytoplasm. PROTEIN CODES DNA! It does so in the cytoplasm. Protein fragments are coded into DNA (occasionally but enough to mechanically influence randomness) The codes for fragments of proteins are biologically useful to the cell, especially in terms of toxins where fragments and variants are capable of blocking the binding site of the toxin, or in the production of mirror images of the toxin that can bind to the toxin, even stressing it to breaking point. And we should expect a tendency towards coding DNA for any protein that enters a cell. To prove this general principle would be quite simple to test as a general rule that solves all of the statistical issues that are quoted against modern Evolutionary Theory.
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Theodoric Member Posts: 9197 From: Northwest, WI, USA Joined: Member Rating: 3.2
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Natural Law of Dynamic Equilibrium Not familiar with this. Please provide references for this. Edited by Theodoric, : No reason given.Facts don't lie or have an agenda. Facts are just facts
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Doctor Witch Junior Member (Idle past 4643 days) Posts: 27 From: Both Sides Joined: |
Any school chemistry book. You may remember the words that were indelibly indoctrinated into most of us in the classroom....
Every reaction proceeds in both directions unless on of the substrates is removed from that reaction.
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John Jones Junior Member (Idle past 4615 days) Posts: 21 Joined: |
quote 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. end quote
You might want to look at your phrase "fitness of offspring". As offspring are, by definition "fit" simply by being there, then "fitness" is not a quality "of" offspring. If, instead, you argue that success of reproduction of the offspring makes them fit, then i) success of reproduction doesn't necessarily incur advantage to the species, and ii) clearly, if the offspring do not reproduce then the parents reproductive success cannot be regarded as "fitness" or success. The best option I think is to drop the word "fitness". Edited by John Jones, : no quote option
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Dr Adequate Member (Idle past 310 days) Posts: 16113 Joined: |
You might want to look at your phrase "fitness of offspring". As offspring are, by definition "fit" simply by being there, then "fitness" is not a quality "of" offspring. If, instead, you argue that success of reproduction of the offspring makes them fit, then i) success of reproduction doesn't necessarily incur advantage to the species, and ii) clearly, if the offspring do not reproduce then the parents reproductive success cannot be regarded as "fitness" or success. The best option I think is to drop the word "fitness". If you don't understand the concept of fitness, that is no reason why biologists should abandon it; that's a sign that you should try harder to understand it. What is it about this concept that is giving you trouble?
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Dr Adequate Member (Idle past 310 days) Posts: 16113 Joined: |
Not familiar with this. Please provide references for this. I think he's trying to be wrong about the Law of Mass Action.
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Dr Adequate Member (Idle past 310 days) Posts: 16113 Joined: |
That mechanism would appear to be a Natural Law of Dynamic Equilibrium. According to this Natural Law, the reactions that take us from DNA to RNA to protein goes both ways. All of the conditions for the reverse reaction occur in the cytoplasm. PROTEIN CODES DNA! Since the process of transcription and translation does not destroy DNA, reversing it would not create it even if this reversal actually happened.
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Theodoric Member Posts: 9197 From: Northwest, WI, USA Joined: Member Rating: 3.2
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The phrase ''natural law", usually pegs my bullshit meter. I was wondering if he could even explain this"law". Per usual, it doesn't look like he knows what he is talking about.
I grant that dynamic equilibrium is a valid scientific principle, but "Law" is a stretch. Facts don't lie or have an agenda. Facts are just facts
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Percy Member Posts: 22490 From: New Hampshire Joined: Member Rating: 5.0 |
John Jones writes: You might want to look at your phrase "fitness of offspring". As offspring are, by definition "fit" simply by being there, then "fitness" is not a quality "of" offspring. As Dr Adequate implies, fitness has a well-defined meaning in biology. I can see that you're defining a "fit" offspring as one that has survived birth, but in biology fitness is measured by an organisms ability to generate offspring, or in genetic terms, to propagate its genes. Wikipedia has a good article on biological fitness. It actually calls it, and correctly in my view, "a central idea in evolutionary theory," so the odds are small that you're going to persuade evolutionary biologists to abandon the concept. --Percy
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John Jones Junior Member (Idle past 4615 days) Posts: 21 Joined:
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quote If you don't understand the concept of fitness, that is no reason why biologists should abandon it; that's a sign that you should try harder to understand it.
What is it about this concept that is giving you trouble? unquote Many disciplines have flags. That the word "fitness" should be meaningless yet purposeful appears to present us with a contradiction. We can begin to break the contradiction by noting that flags are like mandala's, invocational devices whose meaning is not derived from the context in which they are delivered. If the flag "fitness" is assumed to be a semantic/syntactical device, that is, with context dependent meaning, the assumption quickly unravels on inspection. So my advice is to use the flag "fitness" only as a flag - as an inspirational, promotional metaphor aimed at those not familiar with the subject. I don't think biologists should court favour with the "term" fitness. That would be very risky, as my analysis showed.
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