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Author Topic:   Wright et al. on the Process of Mutation
Taq
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Message 16 of 296 (627881)
08-04-2011 10:03 PM


It's been a couple of days, so this first figure will be a bit of a refresher.

caption:

quote:
Figure 3
Ribonuclease protection assay of E. coli CP78 (relA+, ppGpp+) and CP79 (relA−, ppGpp−. Total RNA was recovered, and the levels of specific transcripts were determined by nuclease protection assay and densitometry (see Materials and Methods). Total RNA [5 μg (lanes 1–4) and 10 μg (lanes 5–8)] was hybridized simultaneously with probes for leuB, pyrD, and glpK mRNA. E. coli CP78 and CP79 cells were grown to log phase (L) and then were washed and starved for leucine for 60 min (S). RNA markers 200–500 nt in length were run in the last lane. The leuB hybridized fragment is 436 nt, pyrD is 313 nt, and glpK is 244 nt.

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 1
Correlation 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
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Posts: 4149
From: Edinburgh, Scotland
Joined: 04-09-2003


Message 17 of 296 (627900)
08-05-2011 4:41 AM
Reply to: Message 16 by Taq
08-04-2011 10:03 PM


Fig.3
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
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Message 18 of 296 (627927)
08-05-2011 11:44 AM
Reply to: Message 17 by Wounded King
08-05-2011 4:41 AM


Re: Fig.3
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
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Posts: 706
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Message 19 of 296 (627956)
08-05-2011 4:27 PM
Reply to: Message 16 by Taq
08-04-2011 10:03 PM


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
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Message 20 of 296 (627961)
08-05-2011 5:23 PM
Reply to: Message 19 by shadow71
08-05-2011 4:27 PM


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
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Message 21 of 296 (627986)
08-06-2011 12:25 AM


Hasn't the fundamental study been done yet?
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
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Message 22 of 296 (627987)
08-06-2011 12:31 AM
Reply to: Message 21 by Doctor Witch
08-06-2011 12:25 AM


Natural Law of Dynamic Equilibrium
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
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Message 23 of 296 (627997)
08-06-2011 1:26 AM
Reply to: Message 22 by Theodoric
08-06-2011 12:31 AM


Re: Natural Law of Dynamic Equilibrium
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
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Message 24 of 296 (628017)
08-06-2011 7:07 AM
Reply to: Message 1 by Taq
08-01-2011 6:23 PM


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
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Message 25 of 296 (628022)
08-06-2011 8:26 AM
Reply to: Message 24 by John Jones
08-06-2011 7:07 AM


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
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Message 26 of 296 (628023)
08-06-2011 8:42 AM
Reply to: Message 22 by Theodoric
08-06-2011 12:31 AM


Re: Natural Law of Dynamic Equilibrium
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
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Message 27 of 296 (628024)
08-06-2011 8:45 AM
Reply to: Message 21 by Doctor Witch
08-06-2011 12:25 AM


Re: Hasn't the fundamental study been done yet?
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
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Posts: 7051
From: Northwest, WI, USA
Joined: 08-15-2005


(1)
Message 28 of 296 (628026)
08-06-2011 8:55 AM
Reply to: Message 26 by Dr Adequate
08-06-2011 8:42 AM


Re: Natural Law of Dynamic Equilibrium
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
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Message 29 of 296 (628027)
08-06-2011 9:00 AM
Reply to: Message 24 by John Jones
08-06-2011 7:07 AM


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 3530 days)
Posts: 21
Joined: 08-05-2011


(2)
Message 30 of 296 (628029)
08-06-2011 9:07 AM
Reply to: Message 25 by Dr Adequate
08-06-2011 8:26 AM


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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