Debate Help Required

Hi,I'm new here but a committed evolutionist having had (to date) three pro-evolution (anti-creation) websites to my name (used to go by UK Atheist but I've calmed down a bit).I am debating a young creationist on a UK board who is somewhat more qualified than I am (she's a PhD undergrad ... don't know how with her mentality but she is) in molecular biology and she posted the following in a thread about evolution being taught in schools and, though I will eventually get an answer together, I could really do with some help.To cut a long story short she has been posting a lot of the usual creationist bilge and I have been countering fairly easily whilst I am fairly critical of her ability to research (her trust in creationist sites) so finally she has done some and though I know there WILL BE flaws in her stuff (that being the creationist way) I do not yet know what they are and she is (obviously I suppose) vastly more qualified (and more recently so) than an aging degree level ex-biologist.I need help and searching for "Negoro journal microbiology 2000 nylon three" (for papers published by Negoro in some kind of Journal of Microbiology in 2000 citing three enzymes of 'the nylon bug'") I came across this forum ... evidently someone here knows something about this subject so ... help is required!!!She posted the following:

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So, about the nylon bug - I’m sorry, but this is going to be long and complicated. It has to be to get my point across. Again, being honest and upfront, I would like to point out that the basis of my argument comes from TJ Volume 17(3), which just happened to plop through my door Friday morning (by complete coincidence I’m sure). I’m guessing that you would not be willing to accept TJ as a reputable scientific journal, so I have cited mainstream peer-reviewed papers to back up my argument.

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The nmsr nylon bug link, supplied by Vampyre on the previous page, talks about evidence based largely on papers by Ohno in 1984 and Thwaites in 1985. However, more recent research has shown that the picture is not quite a simple as it is being painted here. The nmsr site claims that a new enzyme arose in Flavobacterium through a frame-shift mutation, but there are in fact three altered enzymes involved, not one (Negoro, Appl. Microbiol. Biotechnol, Vol. 54; 2000).

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All three types of nylon degrading genes appear on plasmids, not on the main bacterial chromosome. This does not look like some random origin of these genes - the chance of this happening is very low. Prijambada et. al. (Appl. & Env. Microbiol. Vol. 61; 1995) demonstrated that nylon degrading ability can be obtained in laboratory cultures of Pseudomonas aeruginosa, which initially had no enzymes capable of degrading nylon oligomers. This was achieved in a mere 9 days! The rapidity of this adaptation suggests a special mechanism for such adaptation, not something as haphazard as random mutations and selection.

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P. aeruginosa is renowned for its ability to adapt to unusual food sources, and all of these abilities reside on plasmids - they do not reside in the chromosome. Plasmids seem to be adaptive elements designed to make bacteria capable of adaptation to new situations while maintaining the integrity of the main chromosome.

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P. aeruginosa was first named in 1872 and it still has the same features that identify it as such. So, in spite of being so ubiquitous, so prolific and so rapidly adaptable, this bacterium has not evolved into a different type of bacterium. Note that the number of bacterial generations possible in over 130 years is huge — equivalent to tens of millions of years of human generations. And yet the bacterium shows no evidence of directional change — stasis rules, not progressive evolution. Flavobacterium was first named in 1889 and it likewise still has the same characteristics as originally described.

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It seems to be that plasmids are designed features of bacteria that enable adaptation to new food sources or the degradation of toxins. This mechanism might be analogous to the way that vertebrates rapidly generate novel effective antibodies with hypermutation in B-cell maturation.

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Some evidence to support the idea that these nylon-degrading mutations are not random:

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1. There are five transposable elements on the Flavobacterium plasmid. When activated transposable enzymes cause genetic recombination. Stress, such as high temperature, exposure to a poison, or starvation can activate transposases. The presence of the transposases in such numbers on the plasmid suggests that the plasmid is designed to adapt when the bacterium is under stress.

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2. All five transposable elements are identical, with 764 base pairs each (over 8% of the plasmid). How could random mutations produce three new catalytic genes (coding for the three new enzymes) without at least some changes being made to the transposable elements?

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3. The antisense DNA strand of the four nylon genes investigated in Flavobacterium and Pseudomonas lacks any stop codons (Yomo et. al. Proc. Nat. Acad. Sci. USA. Vol 89;1992). This is most remarkable in a total of 1535 bases (probability by chance is about 1 in 1,000,000,000,000). Some statements by Yomo et. al. express their surprise:
‘These results imply that there may be some unknown mechanism behind the evolution of these genes for nylon oligomers-degrading enzymes.
‘The presence of a long NSF (non-stop frame) in the antisense strand seems to be a rare case, but it may be due to the unusual characteristics of the genes or plasmids for nylon oligomers degradation.
‘Accordingly, the actual existence of these NSFs leads us to speculate that some special mechanism exists in the regions of these genes.’

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Interestingly, Yomo et. al. also show that it is highly unlikely that any of these genes arose through a frame-shift mutation, because such mutations would have generated lots of stop codons. This nullifies the claim of Thwaites and the nmsr site that a function gene arose from a purely random process.

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Any tips or hints would be most helpful.------------------
Vampyre UK
Editor: UK Tech Portal and UK Freethought

Plasmids are just small circular segments of DNA. They replicate in the same manner as the main bacterial chromosome. The main key is that they reproduce and mutate faster (they tend to be of things that you want faster mutation and reproduction of; you don't want all genes mutating at the same rate, as it is evolutionarily advantageous for certain genes to mutate more quickly). The key is: So? If random alterations to a plasmid can create novel abilities, then random alterations to the main chromosome can as well, just at a slower rate. Thus, her argument needs to be that the plasmid mutations are not random.1) It is evolutionarily advantageous for genes that need to mutate quickly (such as those for antibiotic resistance and food sources) to exist as plasmids.2) It is evolutionarily advantageous for stress to increase the rate of mutation there - no magic necessary!3) It is unrealistic to expect that the same transposon will always make the same mutation. If that were the case, the capability for the plasmids to adapt would be almost nil.4) She is further arguing via "God of The Gaps" concerning stop codons. She doesn't know why there aren't any, so her assumption is "God designed it, and it doesn't apply to other genes". A scientific approach would be to test try and learn why, instead of assuming. "God of the Gaps" has been assumed countless times throughout history, and each time humans have discovered not a God in the gap, but science. You should ask her if she *really* wants to stick a deity in this testable gap.------------------
"Illuminant light,
illuminate me."[This message has been edited by Rei, 12-02-2003][This message has been edited by Rei, 12-02-2003]

... for your help. I've composed a basic reply using yours and some other stuff here and based on my own knowledge but I want to do some more reading before I post to make sure I've got it right.I appreciate the help :-)

If it helps, the Yomo paper and the Prijambada paper are both available in their entirety in .pdf format. Yomo paperPrijambada paperI am actually looking at the plasmid sequence itself now. What is interesting is that the nylB' gene seems to be a duplication but lacks the long complementary sequence on the anti-sense strand. I'll have to read some more, but I think the nylB' gene product is more active than its counterpart (nylB). Anyway, the complete sequence for the pOAD2 plasmid be found here. The only problem I have with the listed sequence is that I can't find the nylA gene anywhere. It lists its location and aa sequence but the listed position isn't turning out the corresponding open reading frame. Maybe Mammuthus could take a look, he's probably had more experience with sequence data than I have. Give me another day and I might be able to pull together a more complete picture of the whole mess (NSF's and sudden nylonase production).PS: You can do a search for the Nogoro paper (and the others as well) at http://www.pubmed.com. Just use "nogoro nylb" as a search criteria, for example. I'm not sure which one you are looking for, but it should be there somewhere.

Can you link to the discussion on the UK site?thnaks

My reply:

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The nmsr nylon bug link, supplied by Vampyre on the previous page, talks about evidence based largely on papers by Ohno in 1984 and Thwaites in 1985. However, more recent research has shown that the picture is not quite a simple as it is being painted here. The nmsr site claims that a new enzyme arose in Flavobacterium through a frame-shift mutation, but there are in fact three altered enzymes involved, not one (Negoro, Appl. Microbiol. Biotechnol, Vol. 54; 2000).

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The NMSR site claimed that a new nylonase gene, separate from the nylA, nylB, and nylB' gene arose because of a frame shift mutation in the first codon of nylC. From the paper you cited, there are not three nylonase enzymes that were altered, but rather three involved, one of which came about due to a frame shift mutation.

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All three types of nylon degrading genes appear on plasmids, not on the main bacterial chromosome. This does not look like some random origin of these genes - the chance of this happening is very low. Prijambada et. al. (Appl. & Env. Microbiol. Vol. 61; 1995) demonstrated that nylon degrading ability can be obtained in laboratory cultures of Pseudomonas aeruginosa, which initially had no enzymes capable of degrading nylon oligomers. This was achieved in a mere 9 days! The rapidity of this adaptation suggests a special mechanism for such adaptation, not something as haphazard as random mutations and selection.

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The fact that genes are on a plasmid or in the chromosome doesn't change the fact that random mutations can happen at both sites. In fact, it seems that it is argued later on that hypermutation on plasmids is important, so perhaps newer genes are produced in plasmids and later are integrated (perhaps through a transposable element) into the genome. Second, it doesn't look like anyone compared the two papers (Yomo and Prijambada) to closely. In the Yomo paper, curing the Flavobacterium of plasmid pOAD2 did not inhibit the bacterias growth on media when the sole carbon and nitrogen source was 6-aminohexanoate (abbrev. Ahx from now on). Therefore, the nylonase genes are not needed in Flavobacterium to digest Ahx. In the Prijambada paper, the Pseudomonas strains could not grow on Ahx as the sole source of C and N. However, 9 days later they could. They now possess the same abilities as pOAD2 negative Flavobacterium. No big woop. The purpose of the nylonase genes is to chop up linear polymers of Ahx (abbrev Ald as per the Prijambada paper). When Ahx positive cultures of Psuedo. were made, they were subjected to selection on polymerized Ahx. It took three months for a 1% innoculum to reach an Abs600nm of 1.0. Normal Psuedo on glucose only takes about 20 hours to do the same thing. Also, it took three weeks before any appreciable growth was seen (A600 0.02-0.05). This is a little different than the 9 days touted earlier. Thirdly, nine or 90 days, it doesn't matter. You still have to provide evidence as to the nature of the mutational mechanism that makes it "special". I am assuming that by special, we are talking a front loaded system that acts in very specific ways to specific stimuli (ala Intelligent Design). I don't see how rapidity speaks to this.

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P. aeruginosa is renowned for its ability to adapt to unusual food sources, and all of these abilities reside on plasmids - they do not reside in the chromosome. Plasmids seem to be adaptive elements designed to make bacteria capable of adaptation to new situations while maintaining the integrity of the main chromosome.

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Chitinase (which attacks chitin, what makes of the outershell of many invertebrates) is produced by some strains of P. aeruginosa. This does seem like a strange food source to attack, and the gene responsible also resides in the chromosome (ref. Characterization of Pseudomonas aeruginosa Chitinase, a Gradually Secreted Protein,
Jindra Folders, et al, Journal of Bacteriology, December 2001, p. 7044-7052, Vol. 183, No. 24). So, this argument seems to fall apart quite quickly. BTW, this was just the first example I could find, there may be quite a few more.

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P. aeruginosa was first named in 1872 and it still has the same features that identify it as such. So, in spite of being so ubiquitous, so prolific and so rapidly adaptable, this bacterium has not evolved into a different type of bacterium. Note that the number of bacterial generations possible in over 130 years is huge — equivalent to tens of millions of years of human generations. And yet the bacterium shows no evidence of directional change — stasis rules, not progressive evolution. Flavobacterium was first named in 1889 and it likewise still has the same characteristics as originally described.

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Yeah, nothing has changed except: the ability to grow in man made liquid soap, grow in derivatives of nylon, antibiotic resistance, ability to eat chitin, etc. Secondly, how do you know that the original P. aeruginosa is still here but sister species have not split off since the discovery of P. aeruginosa? Thirdly, how do you know that it hasn't speciated in the past? As to Flavobacterium, bacteria are often characterized by their ability or non-ability to survive in certain conditions. The loss of the ability to grow on glucose and total reliance on nylon deriviatives could be enough to call the strain of Flavobacterium a new species.

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It seems to be that plasmids are designed features of bacteria that enable adaptation to new food sources or the degradation of toxins. This mechanism might be analogous to the way that vertebrates rapidly generate novel effective antibodies with hypermutation in B-cell maturation.

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Show us the design. Can you look at a plasmid and a complete genome and point to the "design" differences that differentiate the two? I think not. Perhaps it is the inter-species transmittability of plasmids that make them so important. That is, since plasmids are rapidly passed between species of bacteria they are loaded with muted sequence that was once selected for. Therefore, slight alterations in recently acquired plasmids can cause sudden changes in phenotype. This is more my theory than anyone elses, but at least its testable.

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Some evidence to support the idea that these nylon-degrading mutations are not random:

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1. There are five transposable elements on the Flavobacterium plasmid. When activated transposable enzymes cause genetic recombination. Stress, such as high temperature, exposure to a poison, or starvation can activate transposases. The presence of the transposases in such numbers on the plasmid suggests that the plasmid is designed to adapt when the bacterium is under stress.

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Genetic recombination could explain the nylB gene and its 88% homologous copy nylB'. However, it is shown quite distinctly that nylC arose because of a frame shift mutation and not because of transposition. There is not a high level of homology between nylC and any other protein, including the other 2 nyl genes. So the question is, does adaption occur by chance or by design? Show me how a bacteria adds in a frame shift mutation at a specific site in a plasmid when it comes into contact with nylon derivitaves and I might agree that it is by design (as in ID). Otherwise, it looks like selection among strains who differ genotypically because of faulty DNA replication.

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2. All five transposable elements are identical, with 764 base pairs each (over 8% of the plasmid). How could random mutations produce three new catalytic genes (coding for the three new enzymes) without at least some changes being made to the transposable elements?

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I see repeats of 859 bases, but not 764. I think this is what is being referred to. Anyway, there is no way of knowing when these repeats occurred, it could be very recently. The fact that it only took one base to get nylC speaks to the fact that mutations are rare but do happen. Is there a mechanism that specifically mutates the nylC gene and not the repeats, haven't seen one yet.

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3. The antisense DNA strand of the four nylon genes investigated in Flavobacterium and Pseudomonas lacks any stop codons (Yomo et. al. Proc. Nat. Acad. Sci. USA. Vol 89;1992). This is most remarkable in a total of 1535 bases (probability by chance is about 1 in 1,000,000,000,000). Some statements by Yomo et. al. express their surprise:
‘These results imply that there may be some unknown mechanism behind the evolution of these genes for nylon oligomers-degrading enzymes.
‘The presence of a long NSF (non-stop frame) in the antisense strand seems to be a rare case, but it may be due to the unusual characteristics of the genes or plasmids for nylon oligomers degradation.
‘Accordingly, the actual existence of these NSFs leads us to speculate that some special mechanism exists in the regions of these genes.’

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I actually found this part somewhate interesting. Open reading frames are quite popular among viruses who try and pack in genes as closely as possible. The repeats could be due to bacteriophages and the NSF's are a relic of past infection. However, the pOAD2 sequence shows that the nylB' gene (nylB duplicate) does not have the antisense open reading frame. Being that the nylB' gene is 100 times less effective as the nylB gene, the opposing open reading frame could also affect selection for a more active enzyme.

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Interestingly, Yomo et. al. also show that it is highly unlikely that any of these genes arose through a frame-shift mutation, because such mutations would have generated lots of stop codons. This nullifies the claim of Thwaites and the nmsr site that a function gene arose from a purely random process.

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Not true for the nylC gene. The nylC gene does have a complimentary open reading frame, as does the non-active unmutated gene. The active nylC gene has an NSF of 1.229 kilobases and the non-active unmutated nylC NSF is 1.346 kb, both of which are larger than the gene and extend past both ends. So, in the case of nylC, the gene in contention it seems, frame shift mutation did not cause stop codons to appear in the NSF.The pOAD2 sequence can be found here. I also used the ORF Finder on the NCBI site for open reading frame visualization (found here).

... than I thought!Thanks for the replies and links everyone.

Hi Loudmouth ...You gave me a link to pubmed above and I've used it but it doesn't seem to allow me to get at the whole article only the abstract. Is there something I'm missing here?

As a rule, only abstracts are listed on Pubmed. However, you will notice that next to each search result is a yellow piece of paper. If that logo has a green or orange (I think) heading on it then you may be able to access the entire article for free. Otherwise you will have to set up an account with Pubmed which usually requires you to pay for each article, about 5-15 American. Most libraries will can send a .pdf file of a scanned image of the entire paper (strongly suggested) but it will still cost you. Free articles are few and far between, that's why I was surprised both the Yomo and Prijambada papers were both available at no charge.You might also look at local libraries, especially Universities with a strong biology department. They should have subscriptions to quite a few journals and you can probably photocopy those for cheap. Anyway, happy reading.

{Somehow, 25 minutes later, a duplicate posting - Contents blanked out - Adminnemooseus}[This message has been edited by Adminnemooseus, 12-03-2003]

Hi,I thought I'd post this here for review first (I plan to post it in the forum tomorrow) to see what you guys/girls thought and, particularly (since I freely admit it's been a long, long time since I read things of this kind of complexity and I'm very, very rusty) there are likely to be things I have misunderstood or got plain wrong.I also have to concede that, whilst I have tried to prcis and reword some of your stuff into something I am fairly sure means something to me, there are times I think I've simply plagiarised ... I apologise for this and hopefully you'll forgive that in the light of the fact that some passages confused the hell out of me. If you fancy trying to explain the passages I did simply quote I'd appreciate it (LM: I opened with a quote from you and it is a quote because it said what it said and I couldn't think of a better way to say it, it doesn't need to be explained).Once again my thanks for the advice you have all given ... it is truly appreciated.******************************************

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I accept that the enzymes are coded for on plasmids and indeed the link supplied also mentioned that so I am a little confused as to why you seem to think that is such a big thing?

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Some Corrections
"The NMSR site claimed that a new nylonase gene, separate from the nylA, nylB, and nylB' gene arose because of a frame shift mutation in the first codon of nylC. From the paper you cited, there are not three nylonase enzymes that were altered, but rather three involved, one of which came about due to a frame shift mutation" Loudmouth, EvC Forum.

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You appear to have missed that Negoro also contributed to the Prijambada et al paper (just a curiosity but ... well ... creationists!), that the title ("Emergence of nylon oligomer degradation enzymes in Pseudomonas aeruginosa PAO through experimental evolution") and abstract, "Through selective cultivation with 6-aminohexanoate linear dimer, a by- product of nylon-6 manufacture, as the sole source of carbon and nitrogen, Pseudomonas aeruginosa PAO, which initially has no enzyme activity to degrade this xenobiotic compound, was successfully expanded in its metabolic ability. Two new enzyme activities, 6-aminohexanoate cyclic dimer hydrolase and 6-aminohexanoate dimer hydrolase, were detected in the adapted strains." imply very, very strongly that the authors of the paper very much consider this to be an evolutionary advancement.

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It also seems to me somewhat irrelevant that the process can be achieved under laboratory conditions in a mere 9 days since I have little doubt the entire experiment was set up to provide observational evidence that it can occur.

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The Detail
A comparison of the two papers by Yomo and Prijambada reveals that curing the Flavobacterium of plasmid pOAD2 did nothing to stop that bacteria's growth on media where the only carbon and nitrogen sources derived from 6-aminohexanoate so we can conclude that the nylonase genes are not needed to digest 6-aminohexanoate. Yet Prijambada's paper, despite confirming that the Pseudomonas strain was unable to grow on 6-aminohexanoate, reveals that nine days later they could and that they now carry the same functionality as the pOAD2 negative Flavobacterium. The rate of bacterial growth in culture appears to have been ignored by you or your source: in 6-aminohexanoate positive cultures of Pseudomonas nearly three months was required for the innoculum to reach Abs600nm of 1.0 (indeed it took three weeks before any appreciable growth was noted) whereas only 20 hours was required for normal Pseudomonas to achieve this optical density on glucose substrate. So, regardless of whether we are talking 9 days or 90, you still have to provide evidence of the mechanism that makes this mutation "special" in other words evidence of a system that is pre-designed to act in very specific ways in response to very specific environments. It is hard to see how the rate of genetic change reflects on this.

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I am also disappointed (though not particularly surprised) at your comment that mutation is random with it's implication that evolution is a theory of chance when, as Dawkins puts it, "it is grindingly, creakingly, crashingly obvious that if Darwinism was really a theory of chance, it could not work." It seems to me that your understanding of what evolution really is, is woefully lacking.

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To say that "all of these abilities reside on plasmids" implying that the ability was due to the plasmids alone is a frankly ludicrous claim (akin to saying that the heart pumps blood because of chromosomes) ... true, it does but the link is so tenuous, so vague as to be meaningless and I can only assume that you, through inability or sheer unwillingness to do so, do not understand the issues you are prating upon. Your remarks infer that the genes have arrived from other bacteria, presumably via inter-special recombination (known to occur) however the important point to note is that the new nylon gene did not simply arrive, it had been sequenced into the plasmid genome just like the original carbohydrate by the addition of a single thymine nucleotide (1 of 400 nucleotides). What's, more the entire experiment was shown to be repeatable.

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Have you ever given thought to what a plasmid is? It is a small cellular inclusion consisting of a ring of DNA that is not in a central chromosome but is capable of autonomous replication ... did you get that? They are capable of autonomous replication! Indeed they replicate in exactly the same way as the main bacterial chromosome replicates, yet your entire post attempts to infer that they are not ... I wonder why that is?

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Plasmids tend to host the genes of those things that one can envisage are required to mutate faster, to respond in genetic terms quicker than main bacterial genome so it is unsurprising that changes might be found in these yet not in the main genome especially since it is not necessarily desirable that all genetic components mutate at the same rate ... it is, in fact, easy to make a case for variable mutation rates. But the key point is this ... if it is observed that random changes to the plasmid genome can create novel abilities then it is not unreasonable to expect that over a sufficiently long period random changes to the main bacterial genome can do the same. It is likely that the same process is happening in both main and plasmid genomes simply at differing rates. It is also possible that the more rapidly occurring mutations of the plasmid later become integrated into the core genome.

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In evolutionary terms it is advantageous to have those genes that can mutate quickly (for instance those involved in resistance and digestion) in plasmids. It is also advantageous for adverse conditions (stress) to increase the rate of mutation. It is not realistic to expect the same transposon to unfailingly make the same mutation as that would reduce plasmid adaptability to almost none.

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Chitinase (an enzyme that attacks the chitin armour of insects) might, at first glance appear to be a somewhat strange enzyme and yet is produced by some (not all) varieties of Pseudomonas aeruginosa, it is located in the central genome so you are wrong to infer that all such abilities lie in the plasmids.

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You claim that both Flavobacterium and Pseudomonas have not evolved in 110 and 130 years respectively ... firstly this is wrong since both have been noted to develop novel abilities such as the ability to digest nylon, grow in soap and resist antibiotics. Secondly how do you know there hasn't been a split-off derivative species since then?

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According to my sources bacteria are often defined by their ability or lack of same to survive in given environments so the loss of an ability to grow on glucose is every bit enough to demonstrate evolution and speciation.

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You could explain the nylA and nylB (an 88% homologous copy of nylA) genes through genetic recombination but not nylC as it is apparent that that arose through a frame-shift mutation rather than transposition. The similarity between nylC and any other protein (including both nylA & nylB) is relatively low and though it is possible, one supposes, that this might have arisen through design (see later) the evidence has been interpreted as being the product of faulty DNA replication.

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One of my sources reckons that the repeats consists of 859 bases rather than 764 ... regardless there's no way of ascertaining exactly when these repeats occurred but it could well be recently and that it took only one base change to create nylC hints that mutations, though rare, do occur.

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The following is a direct quote from one of my sources since I am not absolutely sure of what he/she means: "I actually found this part somewhat interesting. Open reading frames are quite popular among viruses who try and pack in genes as closely as possible. The repeats could be due to bacteriophages and the NSF's are a relic of past infection. However, the pOAD2 sequence shows that the nylB' gene (nylB duplicate) does not have the antisense open reading frame. Being that the nylB' gene is 100 times less effective as the nylB gene, the opposing open reading frame could also affect selection for a more active enzyme."

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It may well be that it is unlikely that the nylA and nylB genes arose through frame-shift mutation but that is not true of the nylC gene. Whilst the active nylC gene does have a complimentary open reading frame (no different from the unmutated gene) it has a non-stop frame of 1229 bases (compared to 1346) which is larger than the gene extending, as it obviously does in the unmutated version, past both ends. Thus it appears that frame shift mutation did not cause stop codons to appear in the NSF.

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Conclusion
Let's review the data so far.

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* Nylonase genes are not needed to digest 6-aminohexanoate yet in a very short time period the Pseudomonas strain, unable to grow on 6-aminohexanoate, was able to do so.
* It took nearly three months for 6-aminohexanoate positive cultures of Pseudomonas to reach Abs600nm of 1.wheras the same growth for normal Pseudomonas on glucose substrate was 20 hours.
* The new nylon gene was repeatably demonstrated to have been sequenced into the plasmid genome just like the original carbohydrate by the addition of a single thymine nucleotide.
* Plasmids tend to host those genes that are required to mutate faster and are capable of autonomous replication.
* Rapid mutation equates to evolutionary advantage conferring greater potential to survive in adverse conditions.
* It is not possible from the available data to claim that either Flavobacterium or Pseudomonas have not evolved especially since both have been noted to gain additional functionality and to lose others ... characteristics upon which evidence of speciation is often based.
* The differences in nylC compared to nylA & nylB have been attributed to faulty DNA replication.
* It took only one base change to mutate nylC to nylC'
* It appears that the nylC gene arose through frame-shift mutation because it has a non-stop frame of 1229 bases larger than the bacterial main genome extending past both ends. Frame shift mutation did not cause stop codons to appear in the NSF.

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This leads us to conclude that the organism has evolved.

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Now to your final comments which are, quite frankly, astounding.

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You say that plasmids are designed? How can you possibly know this? Can you somehow point to a given bacterium's plasmid and its genome and say this is designed but that is not and supply evidence to support this assertion? I think not!

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Quite apart from the fact that your argument hints at a creative process as yet undiscovered (a "god of the gaps" style argument as it is commonly referred to) you have yet to provide evidence for this supposed god that you apparently feel can fit nicely in such a gap. Since the typical scientific approach is to test, observe and explain, since this gap appears to be relatively small and relatively testable one is forced to wonder if you really do want to stick your god in it?

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References
It would be unfair of me to claim that all the above work was my own ... it isn't.

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Due to the technical nature of the claims being made and my lack of specialism in the area concerned I did a search on the net and ultimately arrived at an Evolution versus Creation forum where this stuff had already been discussed, signed up and posted your piece along with an explanation of what I was trying to achieve.

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So my sincere thanks to Rei and Loudmouth (their handles on that forum) who helped me immensely and provided several links to aid my quest.

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Actually, nylB and nylB' are 88% homologous. I might also mention that the Yomo paper stated that there were no stop codons in the anti-sense nylB' sequence. However, I took the pOAD2 sequence from the NCBI site and ran it through their ORF Finder and there were stop codons. It might be that the Yomo paper used a different plasmid or sequence than I did, but the facts are still there for anyone to look at. For websites, reference my previous post.To admin: I might have hit the back button to the redirect page (the page that pops up briefly after you submit your reply). I didn't go all the way back to my original message. It may be a software glitch of somekind.

Can someone define what a stop codon is for me please?My genetics is seriously rusty but I think a stop codon is probably a code triplet ... IIRC (from my Applied Biology years) information is coded in triplets, yes? I also seem to recall that there are only a few valid stop codons, is that correct?Questions:Is the stop codon unique to a given g organisms genome?Does the position of the codon matter? In other words if the codon triplet was shifted one to the left or right would it still be a stop codon?

Could you please be more explicit as to your sources? This debate is being picked up all over the place; it now also appears in TheologyWeb. It would be useful to have good referencing in place.Thanks -- Sylas