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Author
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Topic: Data, interpretation and false positive inferences
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charlie d.
Member
Member # 159
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posted 29. September 2003 19:47
Nice use of ellipsis, Nelson. How about the rest:
"...ask yourself whether you know how many new substitutions can the anti-cephalosporin enzyme now tolerate, before its function is lost and yet another function is found."
Care to answer? How many mutations can you introduce into the Wang cephalosporin-active mutants before you lose 99% of that activity and gain a new one? Don't just guess, give a real answer.
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Nel
Member
Member # 614
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posted 29. September 2003 19:52
Charlie,
Modification of key residues in the active site of the cephalosporin-active mutants would probably produce mutants that are better poised for evolving a new function. This is probably because they would retain a stable folded structure.
[ 29. September 2003, 19:53: Message edited by: Nelson-Alonso ]
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charlie d.
Member
Member # 159
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posted 29. September 2003 20:13
That's not the answer to the question I was asking. We are talking about ther Axe assay of mutating "neutral" sites.
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Nel
Member
Member # 614
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posted 29. September 2003 20:15
In that case I would think that the cephalosporin activity would cease.
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rafe gutman
Member
Member # 134
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posted 29. September 2003 22:03
nelson, you never answered my question about how many mutations can be considered "slight". axe never uses the term "slight", at least as far as i can tell. so how many is slight? 1? 2? 5? 10? 20?
also, looking at table 1 of the axe paper, i see that one of the double-group mutants, blaMY, has a relative activity of 0.023 compared to wild-type TEM, which according to the paper is still considered enough for penicilin resistance. according to figure 1, there are 10 mutations in group M, and 10 mutations in group Y, yielding a total of 20 mutations of TEM in blaMY.
so given that 20 mutations are not enough to abolish TEM's penicilin resistance function, do you consider this summary of the axe paper by dembski to be accurate?
"But there is now mounting evidence of biological systems for which any slight modification does not merely destroy the system's existing function, but also destroys the possibility of any function of the system whatsoever (Axe, 2000)."
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charlie d.
Member
Member # 159
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posted 30. September 2003 07:24
Still not answered my question. Go back and re-read it. Also, remember you can't "guess".
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Nel
Member
Member # 614
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posted 30. September 2003 13:21
Rafe writes:
quote:
nelson, you never answered my question about how many mutations can be considered "slight". axe never uses the term "slight", at least as far as i can tell. so how many is slight? 1? 2? 5? 10? 20?
I did actually. As I showed with the Douglas Axe quote, combining replacements until one in five are changed causes inactivation.
Rafe writes:
quote:
also, looking at table 1 of the axe paper, i see that one of the double-group mutants, blaMY, has a relative activity of 0.023 compared to wild-type TEM, which according to the paper is still considered enough for penicilin resistance.
Actually, the paper doesn't say whether this is enough to confer resistance to pen. Whether it can afford to lose this much, I am not sure. The paper actually says that when combined (double and triple groups) there is severe inactivation (see below).
As for the usage of the word "slight", Douglas Axe states this:
quote:
The single-group substitutions in blaM, blaY,
and blaG affect function only mildly, yet these
substitutions result in >99% inactivation when
combined.
quote:
The fact that conservative replacement
of only fifth of the exterior residues causes near total loss of activity in both enzymes shows that function actually places very tight constraints on amino acid identities at exterior positions (a quantitative estimate follows).
I think this is what Dembski is referring to when he talks about slight modifications, he gets a lot more specific in his book No Free Lunch:
quote:
Preliminary indications are that proteins permit a pertubation tolerance factor of no more than 10 percent (thermodynamic considerations seem to preclude proer protein folding for more than this percentage of random substitutions.
[ 30. September 2003, 13:35: Message edited by: Nelson-Alonso ]
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rafe gutman
Member
Member # 134
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posted 30. September 2003 15:08
quote:
nelson: As for the usage of the word "slight", Douglas Axe states this, "The single-group substitutions in blaM, blaY, and blaG affect function only mildly, yet these substitutions result in >99% inactivation when combined."
okay, so let me get this straight, changing 30 amino acids of this protein is considered a "slight" modification?
quote:
rafe: looking at table 1 of the axe paper, i see that one of the double-group mutants, blaMY, has a relative activity of 0.023 compared to wild-type TEM, which according to the paper is still considered enough for penicilin resistance.
nelson: Actually, the paper doesn't say whether this is enough to confer resistance to pen. Whether it can afford to lose this much, I am not sure.
so, in your opinion, this level of activity is good enough for penicilin resistance for the wang mutants, but not for the axe mutants? your hypocrisy is duly noted.
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Nel
Member
Member # 614
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posted 30. September 2003 15:38
Rafe writes:
quote:
okay, so let me get this straight, changing 30 amino acids of this protein is considered a "slight" modification?
Rafe, why do you think he calls this an extreme functional sensitivity? I think that when loss is seen when combining blaM, BlaY, etc I would call this slight modifications.
rafe writes:
quote:
so, in your opinion, this level of activity is good enough for penicilin resistance for the wang mutants, but not for the axe mutants? your hypocrisy is duly noted.
There is no hyprocisy here. Those were two compeltely different experiements, one does not contradict the other.
[ 30. September 2003, 15:55: Message edited by: Nelson-Alonso ]
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charlie d.
Member
Member # 159
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posted 30. September 2003 18:33
quote:
Those were two compeltely different experiements, one does not contradict the other.
On this I agree. Axe's experiments deal strictly with the potential for neutral evolution of a protein under a single selective constraint. Wang's deals with the possibility of proteins traversing fitness valleys when multiple selective contraints can be applied. Wang experiments are closer to the real evolutionary situation in which many selective forces can act simultaneously, or at different times, on any single trait or protein.
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rafe gutman
Member
Member # 134
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posted 01. October 2003 16:45
quote:
rafe: okay, so let me get this straight, changing 30 amino acids of this protein is considered a "slight" modification?
nelson: why do you think he calls this an extreme functional sensitivity? I think that when loss is seen when combining blaM, BlaY, etc I would call this slight modifications.
i think dembski calls it "extreme functional sensitivity" because he doesn't understand the paper. this is highlighted by his use of the word "slight" to describe changes of up to 30 amino acids. just out of curiosity, what adjective would you use to describe the 20 amino acid substitutions that still retained function?
quote:
rafe: so, in your opinion, this level of activity is good enough for penicilin resistance for the wang mutants, but not for the axe mutants? your hypocrisy is duly noted.
nelson: There is no hyprocisy here. Those were two compeltely different experiements, one does not contradict the other.
what does the fact that the experiments were different have to do with your hypocrisy? is a relative penicilin resistance activity of 0.023 for the mutant good enough or not?
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Nel
Member
Member # 614
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posted 01. October 2003 19:19
Rafe writes:
quote:
i think dembski calls it "extreme functional sensitivity" because he doesn't understand the paper.
Well this is ironic considering the fact that it was not Dembski that calls it "extreme functional sensitivity", but Douglas Axe, the author of the paper. I found this interesting in light of your comments on hypocrisy.
By the way Rafe, do you agree with this?
quote:
The structural stability of the new cephalosporin-active enzyme can then be recovered by an otherwise silent mutation, Met182-->Thr (of the kind that Axe would have said are ultimately detrimental).
Rafe writes:
quote:
this is highlighted by his use of the word "slight" to describe changes of up to 30 amino acids. just out of curiosity, what adjective would you use to describe the 20 amino acid substitutions that still retained function?
The problem is that I don't think that that much retained function such that the bacteria can resist the drug.
Rafe writes:
quote:
what does the fact that the experiments were different have to do with your hypocrisy? is a relative penicilin resistance activity of 0.023 for the mutant good enough or not?
Well for starters, the Wang experiment didn't seem to destabilize the folded structure (since the bacteria could still resist penicillin), whereas Axe was conscerned with the mutations at non-active sites, such that the folded structure was destabilized, and therefore the protein had no function.
With the Wang paper, there was still substantial pen activity. With the Axe paper, there was very little to nothing (inactivation). With the former, co-option had a functional leg to walk on. I don't think it did with the latter.
[ 01. October 2003, 22:02: Message edited by: Nelson-Alonso ]
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rafe gutman
Member
Member # 134
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posted 01. October 2003 22:15
quote:
nelson: why do you think he calls this an extreme functional sensitivity?
rafe: i think dembski calls it "extreme functional sensitivity" because he doesn't understand the paper.
nelson: Well this is ironic considering the fact that it was not Dembski that calls it "extreme functional sensitivity", but Douglas Axe, the author of the paper. I found this interesting in light of your comments on hipocrisy.
(boldface mine)
nelson, if you use pronouns like "he", how am i supposed to know whether you're referring to dembski or axe? if you want people to know who you're talking about, provide them with the source in your original quote. furthermore, contrary to your proclamation, dembski does use the term "extreme functional sensitivity", in his ID FAQ:
quote:
[axe's] work shows that certain enzymes are extremely sensitive to perturbation. Perturbation in this case does not simply diminish existing function or alter function, but removes all possibility of function. This implies that neo-Darwinian theory has no purchase on these systems. Moreover, the probabilities implicit in such extreme-functional-sensitivity analyses are precisely those needed for a design inference.
i skimmed the axe, paper, but i can't find where he used this term. could you please direct me?
(edited to add: ah, found it. i spent about 10 minutes rereading the conclusion, only to find it in the title!)
quote:
nelson: By the way Rafe, do you agree with this?
"The structural stability of the new cephalosporin-active enzyme can then be recovered by an otherwise silent mutation, Met182-->Thr (of the kind that Axe would have said are ultimately detrimental)."
(boldface nelson's, i think)
again, another unattributed quote. is the entire quote wang's, axe's, dembski's, or yours? from my reading of the wang paper, i would have to agree with the first part. i don't understand the boldfaced part though. is the silent mutation what axe would have considered detrimental? what are you referring to here?
quote:
rafe: this is highlighted by his use of the word "slight" to describe changes of up to 30 amino acids. just out of curiosity, what adjective would you use to describe the 20 amino acid substitutions that still retained function?
nelson: The problem is that I don't think that that much retained function such that the bacteria can resist the drug.
again with the hypocrisy. look in figure 1 of the axe paper, he considers the activity of the blaMY mutant to be above threshold. also, somewhere else he provides the value for what he considers to be the threshold level of relative activity required for penicilin resistance. i think it's 0.0025, but i don't have the paper in front of me right now. you'll notice that a relative activity of 0.023 is equivalent to about a 40-fold decrease in activity relative to wild-type TEM. in the wang paper, they generated mutants with 100-fold decreases in activity relative to wild-type TEM, yet they could still resist penicilin, which you were all too eager to point out at the time.
quote:
nelson: With the Wang paper, there was still substantial pen activity. With the Axe paper, there was very little to nothing (inactivation). With the former, co-option had a functional leg to walk on. I don't think it did with the latter.
(emphasis mine)
so a mutant with a 100-fold decrease in activity is considered to retain "substantial" activity, but a mutant with a 40-fold decrease has "little to nothing". just like dembski, you're playing fast and furious with the adjectives, and contradicting yourself in the process.
so if 30 substitutions are considered "slight", how would you characterize a single substitution? massive?
[ 01. October 2003, 22:50: Message edited by: rafe gutman ]
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Nel
Member
Member # 614
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posted 01. October 2003 22:39
Rafe writes:
quote:
nelson, if you use pronouns like "he", how am i supposed to know whether you're referring to dembski or axe? if you want people to know who you're talking about, provide them with the source in your original quote.
But this just illustrates my point. If Dembski says it, he misunderstood the paper, he's wrong. But if Axe says it, now what? What does that tell you?
Rafe writes:
quote:
i skimmed the paper looking for where axe used that term, but i can't find it. could you please direct me?
Umm, it's the very title of the paper. Also note this statement:
quote:
The fact that conservative replacement
of only a fifth of the exterior residues causes near total loss of activity in both enzymes shows that function actually places very tight constraints on amino acid identities at exterior positions (a quantitative estimate follows).
Rafe writes:
quote:
again, another unattributed quote. is the entire quote wang's, axe's, dembski's, or yours? from my reading of the wang paper, i would have to agree with the first part. i don't understand the boldfaced part though. is the silent mutation what axe would have considered detrimental? what are you referring to here?
Thats my question to you. It doesn't matter who said it, do you agree with it? Is that silent mutation what Axe would have considered deterimental?
Rafe writes:
quote:
in the wang paper, they generated mutants with 100-fold decreases in activity relative to wild-type TEM, yet they could still resist penicilin, which you were all too eager to point out at the time.
And will point out again and again. Along with the fact that these are two completely different experiments. I really don't think that a 0.0023 would confer resistance to penicillin in the Axe paper. As I have repeatedly stated, this is because of what Axe points out:
quote:
With exterior constraints being so severe, how is
it that mutant studies often and greater tolerance
at exterior positions? The catastrophic effect
referred to above suggests an answer. When TEM-1 substitution groups are combined (Table 1), the
resulting relative hydrolysis activities are always lower than the product of the relative activities for the various groups. One likely explanation for this co-operative functional disruption is that it results from progressive loss of stability of the native fold...If loss of function were due exclusively to destabilisation, however, we would
expect to and the same relative activities against
penicillin G that we ®nd for ampicillin. The fact
that relative activities show significant substrate dependence (Table 1) indicates that distortion of the active site is also occurring and may contribute to functional disruption. The important and unexpected finding, of course, is not that destabilisation or distortion can be inactivating, but that stability and active-site conformation place such extreme constraints on the acceptable residues at exterior sites.
As to whether Axe's mutants could still confer resistance to penicillin in a clinical setting as it did with the Wang mutants, as I also stated, I'm not quite sure. One would need to take a closer look at the activities.
[ 01. October 2003, 22:43: Message edited by: Nelson-Alonso ]
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rafe gutman
Member
Member # 134
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posted 15. October 2003 22:34
nelson, sorry for the delay in responding.
i admit i screwed up by not noticing the "extreme functional sensitivity" phrase in the title of axe's paper. my first exposure to the phrase was through dembski's summary of the paper, so subconsciously i always thought it was dembski's phrase.
if the author really wants to conclude that TEM has "extreme functional sensitivity", then he's entitled to do so, but as soon as the wang paper came out, axe's conclusion was falsified. my guess is that axe was basing this conclusion on his work with the TEM hybrids in the second part of his paper. however, i really don't think 20 hybrids with no function constitutes very convincing evidence, especially given the wang data (if you think i'm being unfairly skeptical, look below). additionally, techniques similar to axe's have been around for awhile now, and have produced functional proteins. for example, the biotech company diversa utilizes a method called gene reassembly, where they splice portions of a gene together from homologues in different species and strains.
here is a brief description of their technology:
quote:
In addition to altering single genes using the GSSM technique, Diversa uses proprietary GeneReassembly technologies for the reassembly of related or unrelated genes from two or more different species or strains. GeneReassembly technologies recombine multiple genes to create a large population of new gene variants. The new genes created by GeneReassembly are then screened for one or more desired characteristics. This evolutionary process can be repeated on reassembled genes until new genes expressing the desired properties are identified. GeneReassembly technologies can be used to evolve properties which are coded for by single genes, multiple genes and entire genomes. While Diversa has received a patent for one process of gene shuffling based on interrupted DNA synthesis and reassembly, Diversa’s suite of multiple, proprietary evolution technologies is not limited to traditional shuffling techniques. For instance, unlike widespread shuffling technologies that require highly related gene sequences to achieve successful recombination, Diversa’s proprietary GeneReassembly technology also allows unrelated genes to be combined to maximize evolved improvements.
here's another description:
quote:
In evolving specific genes or gene pathways of interest, Diversa's GeneReassembly methodology significantly expands the genetic diversity available to be screened, first by creating populations of DNA fragments of varying lengths and then reassembling them via computer-designed DNA ligation to generate new genetic variants. An important advantage of Diversa's GeneReassembly technology is that, unlike the Polymerase Chain Reaction (PCR) and other recombination-based methods, it does not require the use of starting genes having DNA sequence identity, or genetic similarity.
so why do i think that axe's 20 hybrids is insufficient to make a conclusion? take a look at diversa's screening procedure (same link as above):
quote:
Improved product candidates can be identified quickly and efficiently using Diversa's patented and proprietary ultra high-throughput screening technologies, including Diversa's recently announced GigaMatrix(TM) 100,000-well plate screening system. Diversa is using these patented and proprietary technologies to discover and improve products such as environmentally friendly pesticides and herbicides, more efficient enzymes for synthesizing pharmaceutical intermediates, novel antibiotics, and more effective protein therapeutics.
these plates are about the size of a 3 X 5 inch index card. to give you a sense of the scale at which they can screen mutants, take a look at this:
quote:
Diversa’s DirectEvolution technologies include Gene Site Saturation Mutagenesis (GSSM) and GeneReassembly. Diversa’s GSSM technology is a patented method of creating a family of related genes that all differ from a parent gene by at least a single amino acid change at a defined position. By performing GSSM on a gene encoding a protein, all possible single, double, and/or triple amino acid codon substitutions within a protein are created, removing the need for prior knowledge about the protein structure and allowing all possibilities to be tested in an unbiased manner. The family of variant genes created using GSSM is then available to be screened for proteins with improved qualities, such as increased ability to work at high temperature, increased reaction rate, resistance to deactivating chemicals, or other properties important in a chemical process. Individual changes in the gene that cause improvements can then be combined to create a single highly improved version of the protein. Additionally, Diversa’s patented GSSM methodology employs a more cost-effective approach than other methods of site-directed mutation.
when i went to a talk given by the president/CEO of diversa, he said they can make every combination of double mutation of a protein in a matter of weeks.
if you're skeptical of news releases, here's a paper from JBC demonstrating the utility of diversa's technology (however, i don't think they used the full-scale gene reassembly technology):
quote:
J. Biol. Chem., Vol. 277, Issue 29, 26501-26507, July 19, 2002
A Novel, High Performance Enzyme for Starch Liquefaction
DISCOVERY AND OPTIMIZATION OF A LOW pH, THERMOSTABLE -AMYLASE*
Toby H. Richardson, Xuqiu Tan, Gerhard Frey, Walter Callen, Mark Cabell, David Lam, John Macomber, Jay M. Short, Dan E. Robertson, and Carl Miller
abstract:
High throughput screening of microbial DNA libraries was used to identify -amylases with phenotypic characteristics compatible with large scale corn wet milling process conditions. Single and multiorganism DNA libraries originating from various environments were targeted for activity and sequence-based screening approaches. After initial screening, 15 clones were designated as primary hits based upon activity at pH 4.5 or 95 °C without addition of endogenous Ca2+. After further characterization, three enzyme candidates were chosen each with an exceptional expression of one or more aspects of the necessary phenotype: temperature stability, pH optimum, lowered reliance on Ca2+ and/or enzyme rate. To combine the best aspects of the three phenotypes to optimize process compatibility, the natural gene homologues were used as a parental sequence set for gene reassembly. Approximately 21,000 chimeric daughter sequences were generated and subsets screened using a process-specific, high throughput activity assay. Gene reassembly resulted in numerous improved mutants with combined optimal phenotypes of expression, temperature stability, and pH optimum. After biochemical and process-specific characterization of these gene products, one -amylase with exceptional process compatibility and economics was identified. This paper describes the synergistic approach of combining environmental discovery and laboratory evolution for identification and optimization of industrially important biocatalysts.
so who are we to believe? axe, whose own conclusion (or more specifically, dembski's interpretation of it) is based on 20 mutants and has already been falsified by wang's paper, or companies like diversa, who can screen 100,000 clones per plate and are getting significant results using technologies that wouldn't even work if dembski's conclusions were true? maybe you'd like to make a prediction that diversa's technology will be fruitless, or better yet, claim that diversa's technology is an example of the utility of intelligent design?
[ 15. October 2003, 22:41: Message edited by: rafe gutman ]
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