When did nylon-eating proteins actually evolve the ability to eat nylon?

It has been widely advertised that nylon eating genes evolved after 1940. I have no problem with that claim in principle since new antibiotic and malaria resistances have evolved since 1940. Even though I can easily accept the possibility of post-1940 nylon-eating evolution in principle, where is the slam dunk evidence that this is actually the case? Did a significant portion of the ability for bacteria to digest nylon take place after 1940 (or 1935 when nylon was first created)?

In an NCSE article New Proteins Without God’s Help we read:

Since nylon first came into commercial production in 1940, we know that the new enzymes have formed since that time.

A similar line of thinking was argued at BioLogos in Biological Information and Intelligent Design, De Novo or Ex Nihilo.

Three nylon eating genes NylA, NylB, NylC were discovered on the Flavobacteria plasmid pOAD2 from 1977-1992, but the researchers concede none of the three have significant sequence homology. Worse, in papers published in 2007, they reported other bacteria contain those same genes in their chromosomes. Unless the researchers have access to pre-1935 bacteria sitting in lab refrigerators, the claim that the genes actually evolved new proteins via mutation is dubious since we have no pre-1935 bacterial samples to actually do a comparison with, especially in the case of NylC. The claim that NylB’s nylon eating ability evolved by gene duplication from a non-functional NylB-prime gene could just as well be interpreted that a functionless NylB-prime gene is a defective copy of a functioning NylB gene!

What’s the proof new nylon eating genes actually evolved after 1940, or is it just speculation? Slam dunk proof would entail having strains of pre-1935 bacteria and then comparing it with the strains after 1935 that supposedly evolved new genes. Is that the case? No. Just speculation which began in 1977 but got less defensible over the next 40 years as more bacteria and non-sequence-homologous genes were discovered to have nylon eating capability.

At least we can credit Richard Lenski who can back up his claims of evolution because he has samples of bacteria in his lab before and after his creatures evolved, whereas the guys promoting the claim the nylon-eaters are new don’t have pre-1935 physical samples of the bacteria. The only pre-1935 samples of the bacteria they have are samples from their imagination.

Incidentally, it is worth pointing out the bacteria that is the centerpiece of the controversy has been mis-classified or misidentified, and thus the name of the bacteria has itself gone through some sort of macro evolution. It was first called Acromobacter gattatus, then renamed as Flavobacterium sp., but then renamed Arthrobacter sp. after it was realized it had been put in the wrong taxonomic category all along.

But anyway, here is the 1977 speculation that became uncritically accepted and exaggerated as fact for the last 40 years:

6-Aminohexanoic Acid Cyclic Dimer Hydrolase. A New Cyclic Amide Hydrolase Produced by Acromobacter guttatus KI 72

There are two possible mechanisms for an enzyme to be active towards unnatural synthetic substance such as 6-aminohexanoic acid cyclic dimer; one is that an unnatural compound is hydrolyzed as an analogue of the physiological substrate, and the other is that the compound is hydrolyzed by an evolved enzyme which originally had an activity on a physiological substrate but lost it by the evolutionary mutation. The data obtained in this experiment indicate that this enzyme did not hydrolyze any physiological substrates tested including peptides, cyclic amides, and amides. In addition to the lack of activity on natural compounds, its low turnover number (8 SKcI) compared to other cyclic amide hydrolases (35 – 260 s ~ for penicillinase [12]) supports the possibility that the enzyme has evolved by adaptation to a new synthetic substance which is a waste product of nylon-6 production.

I have no problem if they say “the enzyme has evolved by adaptation to a new synthetic substance which is a waste product of nylon-6 production” when they mean an existing enzyme has changed its pre-existing function to a different one. That is to say, it acquires a new function while also losing its previous one. However, I would have serious issue with the NCSE’s insinuation that this was such a spectacular change that it illustrates how to solve the origin things on the order of the spliceosome or ribosome.

They don’t have strains sitting in a refrigerator from which to compare the “newly evolved” genes with do they? They just guessed, and then 30 years later one of the same team (Negoro) found nylon eating genes on other bacteria, plus reasonable nylon-eating homologues on other bacteria to boot.

Worse, the claim that these are post-1935 genes is weakened by some facts that require a little elaboration. First there is the basic nylon monomer that forms the components of nylon oligomers. NylA, NylB, NylC have different specialties of which nylon oligomer they can degrade. These three enzymes provide what looks like a digestive cascade where N-oligomer substrates are degraded by NylC to dimers which can be further degraded by NylB (and even NylC but to lesser extent). The cascade is described in: Biodegradation of nylon oligomers.

But important to note: NylA, NylB, NylC are not sequence homologous. In A New Nylon Oligomer Degradation Gene (nylC) on Plasmid pOAD2 from a Flavobactenium sp we read:

Sequence alignment by the method of Wirbur and Lipman (21) showed no significant homology among the
nylA, nylB, and nylC genes. These results suggest that the three nylon oligomer-degradative enzymes evolved independently.

There is no pre-1935 bacteria from which to compare with to establish NylC’s true history, much less anything beyond speculation about the nature of NylC’s ancestor. So how can anyone claim it is new (as in post-1935-new)? Just guesses. They’d have to argue these the genes evolved independently since 1935, and in the case of NylC, the ancestor is almost totally without detailed description.

They however argue, a functional NylB arose from a non-functioning NylB-prime gene via gene duplication in a 1991 paper entitled: Evolutionary adaptation of plasmid-encoded enzymes for degrading nylon oligomers.

But for all we know NylB’ is a defective copy of a functioning NylB! Where is a Ken-Miller-pseudo-gene-like argument when you need it?

In fairness, another bacteria called Pseudomonas was shown in lab conditions to acquire enzymatic nylon-eating function after a few months. The gene however in Pseudomonas, according to BLASTN, has no sequence homology with Flavobacteria. However, the protein according to BLASTP can align 91% at 37% homology. But even the researchers were astonishingly vague about what actually changed in the bacteria that enabled nylon degredation. The two relevant papers are: Characterization of the 6-aminohexanoate-dimer hydrolase from Pseudomonas sp. NK87 and Emergence of Nylon Oligomer Degradation Enzymes in Pseudomonas aeruginosa PAO through Experimental Evolution.

Unless we have strains sitting in a refrigerator of pre-1935 bacteria, how do we know that the genes actually evolved to digest bacteria? For all we know, it had that capability already or very close to it. And unless we have genes from all existing bacteria in 1935 and can prove all the bacteria on the globe do not have horizontally-transferrable nylon eating genes which could end up on the plasmids of Flavobacteria, we can’t unequivocally argue the three nylon eating genes (NylA, NylB, NylC) in Flavobacteria didn’t already exist pre-1935.

Even if the capability of nylon eating evolved, it is also not necessarily because of a new gene, but new regulation. A mini-example of this possibility had to be raised because even in their lab, they had bacteria with identical nylon eating genes but which could not digest nylon.

it is conceivable that expression of the nylC gene is enhanced in these strains and the elevated enzyme activities made the cells Nom+. However, the following possibilities could not be ignored: (i) KI725R strains may possess an additional nylon oligomer-degrading enzyme which is active
toward a substrate included in Noml, but K1725 has no degradative ability toward the substrate; (ii) nylon oligomer transport proteins are activated in K1725R strains; or (iii) the EIII proteins were altered by mutations in the coding region of the nylC gene by which the specific activities and/or
substrate specificity of the enzyme were changed. The last possibility should be negligible,

So my question is, if we ignore the NCSE hype, does the scientific literature really even prove unequivocally a significant part of nylon eating genes evolved after 1935 and absolutely rule out these genes pre-existed? Maybe yes, maybe no, but the NCSE can’t now brag that any significant evolution (like say compared to evolution of antibiotic resistance) bacteria definitely evolved after 1940 without God’s help. We simply don’t know.

NOTES:

Here are some accession numbers:

NylB (protein) in Flavobacteria: WP_012476894.1

which generates a BLASTP hit on Agromyces accession number: BAE97621.1

NylC (protein) in Flavobacteria: BAA01528

which generates a BLASTP hit also on Agromyces accession number: BAE97629.1

in Pseudomonas, the nylon eating gene accession numbers are : BAA01524 (protein), D10678.1 (DNA)

which generates BLAST hits on a variety of organisms as well

240 thoughts on “When did nylon-eating proteins actually evolve the ability to eat nylon?

  1. John Harshman:

    Or perhaps NylB’ is the duplicate. You need to separate the duplication process from the evolution of nylon-digesting activity. Either copy of the gene could have evolved that activity. I would suggest that it’s more likely that NylB is the original copy

    I concur. I somewhat (not exactly) suggested the possibility in the OP:

    The claim that NylB’s nylon eating ability evolved by gene duplication from a non-functional NylB-prime gene could just as well be interpreted that a functionless NylB-prime gene is a defective copy of a functioning NylB gene!

    You’ve refined, corrected, and improve my original speculation. You’re improvement however seems to suggest NylB’ came from NylB, but then NylB evolved, whereas I suggested NylB was already functional before it made a copy of NylB’, and then NylB’ de-evolved to something functionless. I can live with your alternate version, however.

    Thanks.

  2. True, but Rubisco has two active sites. I think I read that NylB doesn’t (seem to?) in one of the papers. Don’t take my word for it, I have to go back and read it again.

    I believe Rubisco has one active site that can enable Carboxylase and Oxygenase activity. I got this from my Biochem textbook, Lehningher Principles of Biochem:

    Rubisco is not absolutely specific for CO2 as a substrate. Molecular oxygen (O2) competes with CO2 at the active site, and about once in every three or four turnovers, rubisco catalyzes the condensation of O2 with ribulose 1,5-bisphosphate to form 3-phosphoglycerate and 2-phosphoglycolate

    Nelson, David L.; Cox, Michael M.. Lehninger Principles of Biochemistry (Page 812). W.H. Freeman. Kindle Edition.

    Not that Rubisco has any necessary relevance, but it’s the only enzyme I can think of that can catalyze two fairly different reactions from the same active site. If you have a better enzyme to get the point across that nylonase activity possibly can coexist with other enzymatic functions, then I’ll happily use that example over Rubisco.

    If a NylB-like gene had been around millions of years, I have to imagine it was doing something before it got co-opted to eat nylon. How much it had to change before getting co-opted, we don’t know. There is a chance it didn’t change at all post-1935 but was used to catalyze a different reaction than nylon degredation pre-1935.

    Just for completeness:

    The enzyme that catalyzes incorporation of CO2 into an organic form is ribulose 1,5-bisphosphate carboxylase/oxygenase, a name mercifully shortened to rubisco. As a carboxylase, rubisco catalyzes the covalent attachment of CO2 to the five-carbon sugar ribulose 1,5-bisphosphate and cleavage of the unstable six-carbon intermediate to form two molecules of 3-phosphoglycerate, one of which bears the carbon introduced as CO2 in its carboxyl group (Fig. 20–4). The enzyme’s oxygenase activity is discussed in Section 20.2.

    Nelson, David L.; Cox, Michael M.. Lehninger Principles of Biochemistry (Page 802). W.H. Freeman. Kindle Edition.

  3. stcordova,

    Sal, let me clarify. I don’t think that NylB’ is nonfunctional or that the precursor of NylB (the gene before it gained the nylon-eating adaptations) was nonfunctional. I suspect they were both functional, just not as nylon-eaters. At least there is no particular reason to hypothesize non-functionality for either of the genes, now or in the past.

  4. John,

    Thanks for the clarification and thanks so much for taking the time to participate. The hypothesis that NylB came from NylB’ was put on the table before the discovery of NylC on the same plasmid. It may be that the technology wasn’t as good back then as it is now, so that’s probably why NylC was missed for almost a decade.

    The next thing on my list of things to do is to look more carefully at Ohno’s frameshift hypothesis and then the NylA gene. It may be a few days before I get back on this thread since there is so much to read and read carefully.

    I didn’t intend this thread to be contentious, just a review and discussion of what was actually published over the last 40 years and trying to understand the claims and looking at which claims may now be obsolete.

    Thanks for participating. I hope to drop in again after I’ve read some more.

  5. From the paper on Agromyces,

    NylA has not been found in Agromyces sp. KY5R by either enzyme assays of cell extracts or by Western blot analysis (9) using antiserum for p2-NylA (pOAD2-encoded NylA). Moreover, Southern blot analysis (9) using the p2-nylA probe exhibited no hybridization signal against that of the total DNA fragments from KY5R (data not shown). These results suggest that the absence of NylA activity in KY5R cells is not the result of the repression of gene expression, but rather the result of the absence of the responsible genes. Owing to the lack of NylA activity, strain KY5R does not degrade the Ahx-cyclic dimer, but degrades Ahx-linear oligomers and Ahx-cyclic oligomers (degree of polymerization >3) through the cooperative action of NylB and NylC (8). Therefore, we focused further analysis on the nylB/nylC regions

    So, I take that as evidence the pOAD2 plasmid did not originate from Agromyces. It would appear, the genes in Agromyces were integrated from some other source, possibly a plasmid like pOAD2.

  6. This comment comes after looking at the issue further.

    It turns out in 1959 a protease (protein degrading) enzyme could degrade nylon. Recently it was found also a lipase (fat degrading) enzyme could also degrade nylon.

    A UNIPROT search on yielded 193 organisms with a predicted nylB and 125 with a nylB’. The organisms included and experimentally confirmed nylon eating bacteria found in the Indian Ocean. Arctic bacteria discovered in 2011 have nylonase genes.

    There are no relics of Ohno’s supposed ancestral sequence in the databases. It’s absurd to think there are no relics floating around. So it seems the only place the frame-shift happened was in Ohno’s imagination, certainly not in any actual data available.

    The fact Kinoshita could not get nylonase to catalyze biological reactions suggest loss of function in a pre-existing gene that had biological function!

    One can mean de Novo genes implies actual new loci rather than new alleles.

    When nylonase evolution happens, if at all, I think it is the evolution by a few point mutations (as Kato demonstrated in 1991 with a mere 2-residue change) of pre existing gene. This is well within the adaptive capacity predicted by Luria and Delbruck principles for things like anti-biotic resistance.

    A search for 6-aminohexanoate hydrolases (a specific nylonase) yielded over 1800 organisms in the NIH-funded UNIPROT databases. Given that proteases and lipases can act as nylonases could put the number of organisms in the tens of thousands with nylonase enzymes.

    NOTE!!! Having a nylonase enzyme doesn’t imply an organism can live of nylon exclusively. There is a difference between an organism that can live off nylon and an organism that possesses a nylonase enzyme (like say trypsin).

  7. So to answer the question I posed:

    When did nylon-eating proteins actually evolve the ability to eat nylon?

    Given we know that “conserved” proteases and lipases can degrade nylons, it appeared they acquired the ability to degrade nylons when God made the world 6,000 years ago. 🙂

  8. stcordova,

    When did nylon-eating proteins actually evolve the ability to eat nylon?

    Ouch. This story is one of the primary foundations supporting the Bio Logos website.

  9. stcordova: This comment comes after looking at the issue further.

    It turns out in 1959 a protease (protein degrading) enzyme could degrade nylon. Recently it was found also a lipase (fat degrading) enzyme could also degrade nylon.

    Do you have references for these?

    A UNIPROT search on yielded 193 organisms with a predicted nylB and 125 with a nylB’. The organisms included and experimentally confirmed nylon eating bacteria found in the Indian Ocean. Arctic bacteria discovered in 2011 have nylonase genes.

    I don’t see why that should be a surprise now, given how plasmids are very prone to HGT, and nylon waste products and countless other forms of microplastics have totally invaded all of Earth’s ecosystems.

    There are no relics of Ohno’s supposed ancestral sequence in the databases.

    What’s a “relic” here ?

    It’s absurd to think there are no relics floating around. So it seems the only place the frame-shift happened was in Ohno’s imagination, certainly not in any actual data available.

    No, it doesn’t seem like that at all. You pulled this conclusion straight out of your ass.

    Are you saying Ohno simply made up a DNA sequence?

    While it may have been originally thought that the frameshift mutation was relatively recent, I think we established given the deep divergence of the P-NylB and F-NylB gene-family detected between the two different species of bacteria (Agromyces(formerly Flavobacterium) and Pseudomonas), that in so far as there was a frameshift mutation, this must have pre-dated the divergence into two separate lineages and their subsequent duplication.

    Having thought about it a couple of times since we first discussed this, I’ve now realized there are several different possible ways we can end up with the genes we see today. It might be something worth discussing because ultimately we’d want to find the most parsimonious hypothesis that best explains the data. But I’m not the man to do that.

    The fact Kinoshita could not get nylonase to catalyze biological reactions

    Where did you get this fact? I’ve read the NylB enzymes with the highest activity towards nylon oligomers are actually just duplications of naturally occuring beta-lactamases. As in, the NylB’ gene is probably the ancestral sequence, and this one is a bona fide beta-lactamase.

    suggest loss of function in a pre-existing gene that had biological function!

    That just sounds to me like an attempt to dismiss nylonase evolution with rhetoric. One could just as well say that nylonase evolved from pre-existing genes the same way whales lost the ability to walk on land when their legs became flippers.

    One can mean de Novo genes implies actual new loci rather than new alleles.

    Yes, I agree with that. A duplication followed by subfunctionalization of one copy wouldn’t constitute an example of de novo gene origination.

    When nylonase evolution happens, if at all, I think it is the evolution by a few point mutations (as Kato demonstrated in 1991 with a mere 2-residue change) of pre existing gene.

    Yes I think so too. We’ve been over this. The putative frameshift mutation is probably very old, possibly over 140 million years. Only somewhat recently did a gene-duplication of the NylB’ gene that we now call NylB, suffer point mutations that increased it’s specificity towards nylon waste oligomers (not sure if I got those two switched around).

    You seem to insinuate that there is no evidence for the frameshift mutation that Ohno reported, because you’ve failed to find the DNA sequence in some database.
    What have you specifically looked for? I get the impression you’re looking for an expressed protein, as in an amino acid sequence, rather than looking at the DNA sequence of the pOAD2 plasmid. The latter part is what Ohno did. He did actually identify a flanking coding region in a different reading frame, and an associated putative Shine-Delgarno-like (but obviously degraded) sequence. Those are among the lines of evidence he used to infer a frameshift mutation.

    Do you have the full DNA sequence of the pOAD2 plasmid? That should be easy to find, and with that you could simply seach for the DNA sequence that Ohno shows in his paper. That is after all the sequence from which he infers a frameshift mutation happened. It should be pretty easy to check if the sequence he reports is in fact the sequence one finds somewhere on the pOAD2 plasmid. Then you can see for yourself if there is, in fact, a flanking coding region, and whether there really is a Shine-Delgarno-like initiation sequence near the start of it.

    A search for 6-aminohexanoate hydrolases (a specific nylonase) yielded over 1800 organisms in the NIH-funded UNIPROT databases. Given that proteases and lipases can act as nylonases could put the number of organisms in the tens of thousands with nylonase enzymes.

    It is entirely possible that many enzymes have now adapted to degrade nylon waste products besides the originally reported NylA, NylB, and NylC proteins.

    What would be interesting to see is how much of the spread of the adaptation towards nylon metabolism owes to HGT of those three enzymes, and how much of it owes to point mutations in other genes that already had very low activity towards the nylon waste products.

    NOTE!!! Having a nylonase enzyme doesn’t imply an organism can live of nylon exclusively. There is a difference between an organism that can live off nylon and an organism that possesses a nylonase enzyme (like say trypsin).

    Sure. The products generated by the enzyme have to fit into other parts of carbon and possibly nitrogen metabolism. There’s no use degrading nylon oligomers if the products can not in turn be used by other metabolic pathways.

  10. Rumraket:

    You pulled this conclusion straight out of your ass.

    Pot calling the kettle black. Where did Ohno get his ancestral sequence?

    Anyone can cut and paste letters of a known gene from the gene banks, use the delete key on one of the letters and create a frame-shift effect then freaking proclaim this as evidence of how a brand spanking new gene emerged from after 1935.

    Did Ohno have a pre-1935 refrigerated sample of bacteria to work from? Nope, it was pure speculation. Amazing no one, absolutely no one is willing to call a spade a spade!

  11. Do you have references for these?

    Yeah, baby! Praise God that the papers are available!

    Ohno 1984 referenced Okada 1983 who referenced Kinoshita 1975 who referenced Fukumura 1966 who referenced Ebata’s 1959 trypsin paper.

    Commercially viable nylons are 100-mer or more, the “nylonases” can’t digest commercially viable 100-mer nylons, only short-mers (aka oligomers) as small as dimers.

    The nylon monomer C6H11NO is a dehydrated 6-aminohexanoic acid C6H13NO2 and aminohexanoic acid is a modified lysine! 6-Aminohexanoate is the conjugate base of 6-aminohexanoic acid.

    6-aminohexanoic acid can be called 6-aminocaproic acid or epsilon-aminocaproic acid. When the aminocaproic acid it is is called aminocaproyl.

    Thus a nylon dimer can be called epsilon-aminocaproyl-epsilon-aminocaproic acid. A nylon oligomer can be called aminocaproamide. All this to say, you won’t find searches for early literature calling nylonase a nylonase! It will be called something like 6-aminohexanoate hydrolase or some sort of aminocaproic acid hydrolase, etc. The ambiguity is dispelled with the molecular diagrams are provided.

    In fact if you go to UNIPROT you won’t get any hits if you type in “nylonase”. Instead type in “nylB” or “6-aminohexanoate hydrolase” in the search box. It’s easily done, see for yourself:

    http://www.uniprot.org/

    UNIPROT, by the way, is yet another NIH-funded project like ENCODE. 🙂

    Regarding trypsin, start here:

    https://www.jstage.jst.go.jp/article/biochemistry1922/59/6/59_6_531/_pdf

    The only report on the biochemistry of cyclic and linear oligomers of 6-aminocaproic acid is that of Ebata and Morita….

    Now go here for more about trypsin:

    Ok Ebata and Morita’s paper:

    https://www.jstage.jst.go.jp/article/biochemistry1922/46/4/46_4_407/_pdf

    The item of interest is episilon-aminocaproyl-epsilon-aminocaproic acid and poly-epsilon-aminocaproic acid.

    As I mentioned last month, I was sent down to Lipcomb university to meet with a pro-ID biochemist on a variety of matters:

    http://theskepticalzone.com/wp/my-presentation-at-lipscomb-university-in-front-of-faculty-and-deans-of-several-universities-available-for-free-online-expense-for-live-attendance-is-390/

    One of the followup issues is that of nylonases. We may actually try to duplicate the experiment, and if someone beats us to it, all the better as we have reproducibility.

    Trypsin is protease, but then there are lipases. Here is the experiment using lipases in 2009!

    http://onlinelibrary.wiley.com/doi/10.1002/app.31756/abstract

    They used lipex50T which is no longer available but it is derived from the LIP gene of Thermomyces lanuginosus (formerly Humicola lanuginosa) via directed evolution in the laboratory.

    Now, I’m being public about this because what’s the point of being secret, not to mention I get all this free-of-charge editorial review at TSZ should I choose to write about nylonases!

    I should note something interesting. The smaller the proportion of the nylon that has the lysine-like aminohexanoic acid terminus, the less the enzyme can effect it. Beyond hexamers, classic nylonases stop working. That gives a clue what that enzyme or it’s homolog was involved with. Trypsin is a serine protease that acts on lysine, btw.

    Kato in 1991 in trying to understand the mechanism of nylonases noticed serines having an important role and specifically mentioned trypsin.

  12. Ouch. This story is one of the primary foundations supporting the Bio Logos website.

    No kidding. Someone’s gonna be gettin’ a whuppin if I’m right. 🙂

  13. Do you have the full DNA sequence of the pOAD2 plasmid? That should be easy to find, and with that you could simply seach for the DNA sequence that Ohno shows in his paper.

    I did the searches, Ohno’s sequence doesn’t exist. The only sequence that comes up in BLAST is the real nylB gene!

    Of course if you just take any random gene from gene bank and pull a single letter to create a new sequence, you’ll get similarity hits to the original gene. No surprise.

    Just for grins I did not only BLASTN but BLASTP just to see if any organism actually had the relic of the supposed ancestor protein Ohno said existed. No hits. That means Ohno just concocted a story. He has no direct empirical evidence.

    Ohno also mad a serious typo, I think and added a termination codon where it shouldn’t be.

    Any how, here is Ohno’s sequence:

    ATGGGCTACATCGATCTCTCCGCCCCCGTCGCGATGATCGTCAGC
    GGTGGCCTCTACTATCTCTTCACCCGCCGCGGCTACACCTTCGGAGACACT
    CG agaacgcacgttccacc
    ggccagcaccccgccaggtatcccggagccgcggccggggagccgacactcgacagctgg
    caggaggccccgcacaaccgctgggccttcgcccgcctgggcgagctgctgcccacggcg
    gcggtctcccggcgcgacccggcgacgcccgcggagcccgtcgtgcggctcgacgcgctc
    gcgacgcggctccccgatctcgagcagcggctcgaggagacctgcaccgacgcattcctc
    gtgctgcgcggctccgaggtcctcgccgagtactaccgggcgggtttcgcacccgacgac
    cgtcacctgctgatgagcgtctcgaagtcgctgtgcggcacggtcgtcggcgcgctgatc
    gacgaggggcgcatcgatcccgcgcagcccgtcaccgagtatgtacccgagctcgcgggc
    tccgtctacgacgggccctccgtgctgcaggtgctcgacatgcagatctcgatcgactac
    aacgaggactacgtcgatccggcctcggaggtgcagacccacgatcgctccgccggctgg
    cgcacgcggcgagacggggaccccgccgacacctacgagttcctcaccaccctccgcggc
    gacggcggcaccggcgagttccagtactgctcggcgaacaccgacgtgctcgcctggatc
    gtcgagcgggtcaccggtctgcgctacgtcgaagcgctctccacgtacctgtgggcgaag
    ctcgacgccgatcgggatgcgaccatcacggtcgaccagaccggcttcggcttcgcgaac
    gggggcgtctcctgcaccgcgcgggatctcgcacgcgtgggccgcatgatgctcgacggc
    ggcgtcgctcccggcggacgggtcgtatcgcagggctgggtggaaagcgtgctggccggc
    ggctcccgcgaagccatgaccgacgagggtttcacctccgcattccccgagggcagctac
    acgcgccagtggtggtgcacgggcaacgagcgcggcaacgtgagcggcatcggcatccac
    ggccagaacctctggctcgatccgcgcaccgactcggtgatcgtcaagctctcgtcgtgg
    cccgatcccgacacccggcactggcacgggctgcagagcgggatcctgctcgacgtcagc
    cgtgccctcgacgcggtgtag GCGGCTGA

    When you do a BLASTN guess what, you get a 99% similarity hit on the real sequence. So what about that missing 1%? That’s the 1% Ohno claims was the ancestor, but it’s nowhere to be found.

    This may sound kind of mean, but anyone can pull this trick with any gene with cut, paste, and delete and say: “this was the ancestor prior to 1935”. How the heck can this be tested if someone will go to any length to try to explain the erasure of ancestral relics that should still be floating around. That would entail changing all the strains of the bacteria around the globe to erase the relic, assuming our databases are a representative sample of all the global bacteria.

    All this to say, the only place so far the frame-shift happened is in Ohno’s imagination.

    And this is the same guy who coined the term “junk DNA”. So where is the junkDNA floating around to help his case on nylonase? Only in his imagination.

  14. Here is a summary of my findings so far:

    ABSTRACT

    Nylon comprises a family of a man-made substances that originated in 1935. Nylonases are biological enzymes that can break down nylon oligomers. Although the most prominent nylonases are within the family of enzymes classified as 6-aminohexanoate hydrolases, some enzymes not formally classified as 6-aminohexanoate hydrolases also have enough ability to breakdown nylons and can properly be also called nylonases.

    Experimentally confirmed nylonase ability appears in soil bacteria such as Arthrobacter KI72, marine bacteria strains such as Bacilus cereus from the Indian Ocean, and thermophilic bacteria such as Anoxybacillus rupiensis Ir3 (JQ912241) from the soils of Iraq. Computationally predicted (and thus provisional) 6-aminohexanoate hydrolase genes appear in pathogenic bacteria such as Streptoccous pneumoniae from a refugee camp in Thailand and Cryobacteria articum from the Arctic to name a few of the over 1800 organisms with predicted 6-aminohexanoate hydrolase genes deposited in the NIH-funded UNIPROT knowledge base. Even if these 1800 predicted nylonases are not yet experimentally confirmed to digest nylons, the predicted nylonases arguably have significant homology to experimentally confirmed nylonases.

    In addition to 6-aminohexanoate hydrolases, proteases like trypsin and certain lipases have been experimentally demonstrated to be nylonases. The inclusion of certain proteases and lipases as nylonases possibly increases the potential catalog of species specific biological nylonases into the tens of thousands.

    Ironically, this present review regarding the conservation of nylonases was initially framed as a response to speculative ideas by Kinoshita, Ohno and Okada in the 1980s that claimed nylonases only emerged through post-1935 evolution of completely de Nove genes. These ideas hinged on Kinoshita’s 1977 claim that nylonases were so rare that they must have only emerged after 1935. One might speculate that the persistence of Kinoshita’s ideas and the accompanying post-1935 evolutionary theories result from the apparently common misunderstanding that an organism having a nylonase enzyme must necessarily be able to live solely off nylon. But this notion would be clearly erroneous for organisms which cannot solely live off nylon but still possess trypisin nylonases for biological processes other than degrading nylon. Whatever the reasons for the persistence of Kinoshita’s claim of the exceeding rarity of nylonases , the data presented here argues for the widespread presence of nylonase enzymes in the biological world rather than rarity and also strongly argues against the viability of Ohno and Okada’s post-1935 de Novo nylonase gene emergence theories.

  15. Are you saying Ohno simply made up a DNA sequence?

    Yes. Take an existing gene from the gene bank, and pull a letter and create a frame shift and say that’s the ancestor. When the world can’t actually find directed evidence of the ancestor (a relic) or that the ancestor ever existed, then hope your friends will come to your defense and make up stories as to why the evidence was erased.

    Ohno’s claim is just an urban legend. It’s nothing based on actually having a pre-1935 refrigerated sample of bacteria that was later sequenced prior to Ohno’s paper.

    I’ve already said, if there was adaptation at all, I’d go with Kato’s 2-residue change — point mutations vs. a frame shift insertion. That is believable and credible.

    The reason the Biologos people wanted Ohno’s claim to be true is that is would show a random amino acid sequence can suddenly become functional. Well, yes sort of. Take any functional gene sequence and delete a single letter and create a frame-shifted sequence which looks random and then proclaim it as the ancestor without any empirical proof. That’s essentially what Ohno did. It’s mean to say that, but that essentially is the substance of his claim.

    I should note the Pseudomonas evolution of nylon digestion over 9 days doesn’t necessarily have to be a change in the nylonase gene. No one knows what actually changed to enable pseudomonas to evolve the ability to digest nylon. Having a nylonase enzyme is only a necessary, but not sufficient condition to digest nylons.

    If human trypsins are indeed nylonases, then this is evidence having a nylonase enzyme doesn’t necessarily imply the organism has the ability to actually metabolize nylon as a food source. Something else in Pseudomonas could have changed than it’s nylonase gene. I’m just pointing that out.

    I don’t see why that should be a surprise now, given how plasmids are very prone to HGT,

    That’s a reasonable claim, however, UNIPROT returns genes of structurally homologous proteins not genetically homologous nylB genes. As we discussed in your “Beating a Dead Horse” thread regarding lysyl oxidase, there doesn’t have to be sequence homology for the folds and functions to be homologous. In fact many similar proteins only have 12% sequence homology. A fact I just reminded TSZ of:

    http://theskepticalzone.com/wp/sandbox-3/comment-page-18/#comment-180712

    UNIPROT classified the nylB that were non-gene homologous, but protein structure and function homologous. I confirmed the lack of sequence similarity with BLASTP on a few of the nylB predicted proteins….(like say the Flavobacteria nylB against the Streptococcus pneumonia nylB).

    The other problem with the adaptationist story toward nylon is trypsin, which is a nylonase, can be found in humans and other vertebrates! The adaptationist story in the case of human or vertebrate nylonases makes little sense.

    Unfortunately Ebata didn’t specify where he got the trypsin for his experiments, but I suspect given that protein degrading enzymes like trypsin and fat degrading enzymes like lipex50T/lipolase can break down nylon, it seems to me lots of things in the biological world are candidates for breaking down nylon.

    NOTE: when I say nylon, I mean the nylon oligomers, which isn’t commercially viable nylons. Bacteria can’t live off of nylon stockings and parachutes since the polymers are too long. Hence, “nylonase” is a bit of a misnomer since a lot of them can’t digest beyond hexamer forms of nylon, whereas commercial nylons are 100-mer of more in length.

  16. stcordova: Take an existing gene from the gene bank, and pull a letter and create a frame shift and say that’s the ancestor.

    That wouldn’t work with most genes, since it wouldn’t leave a long open reading frame. Presumably that’s one reason Ohno made the proposal.

    Anyway, I think you still misunderstand the scenario, despite many people having explained it to you. The proposed frameshift would have been millions of years ago and would have nothing to do with the evolution of nylonase. It would have happened before the duplication that led to nylB and nylB’, itself fairly old. What results in nylonase is a couple of mutations to the nylB ancestor, long post-duplication, long post frameshift (if any).

    Have you searched for the predicted pre-frameshift protein sequence? It isn’t clear from what you say.

  17. Anyway, I think you still misunderstand the scenario, despite many people having explained it to you. The proposed frameshift would have been millions of years ago and would have nothing to do with the evolution of nylonase.

    Ohno’s paper suggests he’s claiming it for post-1935. If that’s the case no one “explained’ it to me several time and that’s not how it’s represented by those promoting it as an example of de Novo gene evolution like the Bio Logos people or the NCSE. Rather, that’s how it was mis-read to me.

  18. It appears Ohno made a typo, on minor, one fairly serious. I highlight the serious one first as it modifies a gene sequence!

    The actual end sequence by Okada from which Ohno made his construction in genebank is “GCGGCGTGA” not “GCGGCTGA” as reported by Ohno. Did he make a mistake???? Ohno effectively added a stop codon “TGA” where there shouldn’t be one by deleting a “G” in the original sequence! Ohno’s last sequence does not agree with Okada’s actual reported sequences as far as I can tell.

    Does his abstract also has a typo of “472” instead of “427” as well as one of his footnotes?

  19. Have you searched for the predicted pre-frameshift protein sequence?

    Yes.

    Here is the predicted protein from Ohno’s hypothetical PR.C ancestral which I derived using Ex-Pasy:


    MGYIDLSAPVAMIVSGGLYYLFTRRGYTFGDTRERTFHRPAPRQVSRSRGRGADTRQLAG
    GPAQPLGLRPPGRAAAHGGGLPARPGDARGARRAARRARDAAPRSRAAARGDLHRRIPRA
    ARLRGPRRVLPGGFRTRRPSPADERLEVAVRHGRRRADRRGAHRSRAARHRVCTRARGLR
    LRRALRAAGARHADLDRLQRGLRRSGLGGADPRSLRRLAHAARRGPRRHLRVPHHPPRRR
    RHRRVPVLLGEHRRARLDRRAGHRSALRRSALHVPVGEARRRSGCDHHGRPDRLRLRERG
    RLLHRAGSRTRGPHDARRRRRSRRTGRIAGLGGKRAGRRLPRSHDRRGFHLRIPRGQLHA
    PVVVHGQRARQRERHRHPRPEPLARSAHRLRDRQALVVARSRHPALARAAERDPARRQPC
    PRRGVGG

    As a double check that I collected and converted the gene sequence correctly it reads 427 letters. I also double checked by making an audio recording of what Ohno printed and then seeing that it agreed with what I type in. Then I wised up and actually used the gene bank sequences!

    The result was:

    No significant similarity found.

    One might argue the frame-shift happened millions of years ago, but then on what basis empirical relative to a more parsimonious explanation involving no frame shift?

    If it was not functional and not conserved over the years, then the problem is how did stop codons not pop up in the junk sequences by random mutation.

    If it was conserved, then is BLASTP not picking it up because of a sampling issue, that somehow the sequence is floating around but we haven’t found it even though we’ve found homologs of the real sequence all over the place.

    Not to mention millions of years in the past not how his paper is represented, it is represented as happening post 1935.

    If there was enzyme adaptation, I’d accept Kato’s 2-residue mutation as more likely. But gain of nylonase function could also imply loss of function of something else. Kato’s scenario is well within expected changes that happen with periodically re-occurring “alleles” in bacterial populations according to models by Luria and Delbruck.

  20. The biologos account of nylonase by Dennis Venema:

    http://biologos.org/blogs/dennis-venema-letters-to-the-duchess/intelligent-design-and-nylon-eating-bacteria

    In the 1970s, scientists made a surprising discovery: a bacterium that can digest nylon, a synthetic chemical not found in nature. These bacteria were living in the wastewater ponds of chemical factories, and they were able to use nylon as their only source of food.

    Wrong. The bacteria were gathered from soil. They were then tested on waste nylon waste products.

    how had these bacteria adapted to this novel chemical in their environment so quickly? Intrigued, the scientists investigated. What they discovered was that the bacteria had an enzyme (which they called “nylonase”) that effectively digested the chemical. **This enzyme, interestingly, arose from scratch as an insertion mutation into the coding sequence of another gene. This insertion simultaneously formed a “stop” codon early in the original gene (a codon that tells the ribosome to stop adding amino acids to a protein) and formed a brand new “start” codon in a different reading frame. The new reading frame ran for 392 amino acids before the first “stop” codon, producing a large, novel protein.** As in our example above, this new protein was based on different codons due to the frameshift. It was truly “de novo” – a new sequence.

    And here’s the kicker: this brand-new protein folded into a stable shape, and acted as a weak nylonase. Later duplications, mutations and selection would make a very efficient nylonase from this starting point.

    I got a present for Dennis Venema. This is Earth Wind and Fire’s song FANTASY:

    https://youtu.be/L0CVoFsUhC4

    Every man has a place
    In his heart there’s a space
    And the world can’t erase his fantasies
    Take a ride in the sky
    On our ship, fantasize
    All your dreams will come true right away

    And we will live together
    Until the twelfth of never
    Our voices will ring forever, as one

    Every thought is a dream
    Rushing by in a stream
    Bringing life to the kingdom of doing
    Take a ride in the sky
    On our ship, fantasize
    All your dreams will come true miles away

    Our voices will ring together
    Until the twelfth of never
    We all will live love forever, as one

    Come to see victory
    In a land called fantasy
    Loving life, a new degree
    Bring your mind to everlasting liberty

    As one, come to see victory
    In a land called fantasy
    Loving life for you and me
    To behold, to your soul is ecstasy

    You will find other kind
    That has been in search of you
    Many lives has brought you to
    Recognize, it’s your life now in review

    And as you stay for the play
    Fantasy has in store for you
    A glowing light will see you through
    It’s your day, shining day
    All your dreams come true

    As you glide in your stride
    With the wind as you fly away
    Give a smile from your lips and say
    I’m free, yes I’m free, now I’m on my way

    Come to see victory
    In a land called fantasy
    Loving life for you and me
    To behold, to your soul is ecstasy

    You will find other kind
    That has been in search of you
    Many lives has brought you to
    Recognize, it’s your life now in review

    🙂

  21. stcordova: Pot calling the kettle black.Where did Ohno get his ancestral sequence?

    The DNA sequence is right there in the pOAD2 plasmid for fucks sake. Ohno wasn’t working with the amino acid sequence of the ancestral protein, he had the DNA sequence from the plasmid. This DNA sequence also has regions flanking the coding regions of NylB and NylB’. It is from the flanking regions, and the particular sequence of NylB and NylB’, that Ohno infers a bigger coding region once existed.

    Anyone can cut and paste letters of a known gene from the gene banks, use the delete key on one of the letters and create a frame-shift effect then freaking proclaim this as evidence of how a brand spanking new gene emerged from after 1935.

    This doesn’t make sense at all, you are conflating DNA sequence with amino acid sequence from protein.

    If you already have a coding region for the NylBs, you can’t just “delete” something and expect to find a bigger reading frame encompassing the previous one.

    Did Ohno have a pre-1935 refrigerated sample of bacteria to work from? Nope, it was pure speculation. Amazing no one, absolutely no one is willing to call a spade a spade!

    No what is amazing is that in your desperation to reach your creationist conclusion, you’re too worked up to take a dispassionate look at the arguments Ohno originally raised.

    We already agreed, given the evidence for the deep divergence of F-NylB and P-NylB, that the frameshift mutation, supposing it did happen, could not have happened somewhere around 1935. It would have, then, to predate divergence of F-NylB and P-NylB. Given that these two 392 amino acid proteins are only about 37% similar in sequence, they would have probably diverged many millions of years ago.

    So if the NylB gene-family really did emerge from a framshift mutation, it would have to be ancient. The question is, what kind of evidence is there that such a mutation happened? This is what Ohno argues in his paper with several lines of evidence. First of all, as you yourself can see there does appear to be a bracketing reading frame encompassing a larger 427 amino acid protein. Was this really a functional protein once upon a time, or just some statistical fluke? Well, if there really was a larger protein there once upon a time, you’d expect there to be other things normally associated with functional protein coding genes, like upstream enhancers and promoters of their various kinds. Ohno did do that at least in part.

    One of these things that you can look for is called a Shine-Dalgarno sequence. This is not to say that initiation sequences have to be exact matches to the consensus sequences, they can be sloppy.

    Ohno argues there a remnant of one in the proper location. Another thing to look for is if the bracketing reading frame really does contain a proper start and stop codon so in addition to there being a continous uninterrupted sequence of triplets coding for amino acids, it also has what a proper coding sequence should have: Stop and Start codons.

    Now, if the frameshift mutation really did happen those approximately 140 million years ago, you’d also expect these flanking pieces associated with the original gene to have degraded, since they’re no longer used. It would be strange if they were all fully intact after 140 million years.

    I submit that these lines of evidence that Ohno really did find, are consistent with what you’d expect from an ancient frameshift mutation.

    It seems to me the mistake Ohno made is not in inferring there was a frameshift mutation, but when.

  22. stcordova:
    It appears Ohno made a typo, on minor, one fairly serious.I highlight the serious one first as it modifies a gene sequence!

    The actual end sequence by Okada from which Ohno made his construction in genebank is “GCGGCGTGA” not “GCGGCTGA” as reported by Ohno.Did he make a mistake????Ohno effectively added astop codon “TGA” where there shouldn’t be one by deleting a “G” in the original sequence!Ohno’s last sequence does not agree with Okada’s actual reported sequences as far as I can tell.

    Having now checked the two references you are correct, it does appear Ohno’s end sequence lacks a G. With the G back in, the putative ancestral coding region seems to go on for a while uninterrupted by any stops, if going by Okata’s reported sequence. But I don’t think this means anything, because there’s a serious lack of sequence similarity between the P-NylB and F-NylB regions following the NylBs stop codon. So whatever evidence there could have been there is now pretty much erased. As such this is definitely a counter-argument to one of the evidences Ohno uses for his inference.

    This factoid is to me more damning than anything else you’ve said about the lack of a protein sequence for the ancestral sequence in UNIPROT. Again, I wouldn’t expect it to be expressed anywhere if it has not been a functional gene for over a hundred million years. In contrast, as reported by Okata et al, the upstream region before the start codon and Shine-Delgarno sequences are still highly similar indicating they’re under purifying selection. My guess is it has to do with transcription.

    Does his abstract also has a typo of “472” instead of “427” as well as one of his footnotes?

    It appears he only makes that typo in the abstract.

  23. stcordova: One might argue the frame-shift happened millions of years ago, but then on what basis empirical relative to a more parsimonious explanation involving no frame shift?

    If it was not functional and not conserved over the years, then the problem is how did stop codons not pop up in the junk sequences by random mutation.

    I think it would go something like this: The original frameshift supposing it happened, resulted in some sort of proto-NylB gene, which was already then a functional gene (of the beta-lactamase fold, therefore possibly related to antibiotic resistance) and so was subsequently retained by selection.

    Even disregarding the frameshift mutation, the NylB gene family must have had some other function prior to 1935, than their chance ability to act on nylon waste oligomers, which would explain why they exist as protein coding genes in the first place. As has been found out, they are beta-lactamases, so they’re probably genes involved in antibiotic resistance.

    I would agree that the evidence for the frameshift mutation is not that strong, though Ohno does give some other interesting arguments for why we can infer it happened, such as the sequence of the putative ancestral sequence having this remarkable ability to result in such a long coding region uninterrupted by stop codons.

    But besides this there isn’t any good reason to think that frameshift mutations cannot produce functional genes, other than some sort of a priori committment to the idea that functional genes are extremely rare in amino acid sequence space. A committment only found among religious fundamentalists, and contradicted by experiments.

    Looking at Okata H et al’s reported sequences for F-NylB, and P-NylB, it appears relatively few nucleotide substitutions have resulted in the rather significant changes in amino acid sequence for the NylBs, which in turn indicates that the putative primordial gene that suffered the frameshift mutation might still be possible to reconstruct in the lab.
    It would be interesting to see if this could be done, and if it shows any structural or functional relationships to other known protein coding genes. I get that the amino acid sequence does not return any hits on blast searches, but that doesn’t actually allow us to rule out that it could have been a functional protein coding gene of some sort, with structural similarity to other known proteins.

    I wonder if it is possible to use some sort of fold prediction software to see if it gives any hints about what kind of structure, if any, that sequence might adopt.

  24. Actually I found that a fragment of the putative “ancestral” protein (the beginning part of the sequence) produces an alignment to known protein:
    nitrate reductase [Leucobacter celer]
    NCBI Reference Sequence: WP_083451415.1

    https://www.ncbi.nlm.nih.gov/protein/1179929060?report=genbank&log$=protalign&blast_rank=2&RID=PBWUESGX014

    This is the part of the 427 aa protein that aligns to Nitrate Reductase:
    MGYIDLSAPVAMIVSGGLYYLFTRRGYTFGDTR

    This is the portion of the Nitrate Reductase sequence it aligns to:
    MGYIDLSAPVAMIVSGGLYYLFTRRGYTFGDAR

    Oddly enough this piece is at the c-terminal of Nitrate Reductase, while it corresponds to the n-terminal of Ohno’s hypothetical protein.

    There is a single amino acid difference for this alignment, as one ends in DTR and the other DAR. That’s 32 of 33 amino acids straight.

    The thing is that this is the beginning part of the putative ancestral sequence which spans upstream to where NylB begins, which pretty substantially confirms that Ohno is right, there really was another protein there before. Strangely enough this fragment stops where NylB begins.

    I should add that when you do BLAST searches for similar sequences using amino acids, the default settings of BLAST are actually quite restrictive, so you have to play around with the algorithm settings if you wish to find any alignable fragment parts of the larger protein, or pieces with lower similarity scores.

    Another stretch of Ohno’s hypothetical protein, an 247 amino acid stretch at the c-terminal end, aligns to an uncharacterized protein from the fungus Pneumocystis murina (strain B123), with a sequence-similarity of about 29%.

  25. stcordova: I got a present for Dennis Venema. This is Earth Wind and Fire’s song FANTASY:

    As an aside, this overweening triumphalism is not one of your more attractive features. Venema was wrong. Leave it at that. If you just remember that you are much more profoundly wrong about a great many things, perhaps your Christian humility and charity will kick in.

  26. John Harshman: As an aside, this overweening triumphalism is not one of your more attractive features. Venema was wrong. Leave it at that. If you just remember that you are much more profoundly wrong about a great many things, perhaps your Christian humility and charity will kick in.

    Judge not, that ye be not judged.
    For with what judgment ye judge, ye shall be judged: and with what measure ye mete, it shall be measured to you again.
    And why beholdest thou the mote that is in thy brother’s eye, but considerest not the beam that is in thine own eye?
    Or how wilt thou say to thy brother, Let me pull out the mote out of thine eye; and, behold, a beam is in thine own eye?
    Thou hypocrite, first cast out the beam out of thine own eye; and then shalt thou see clearly to cast out the mote out of thy brother’s eye.

  27. Rumraket:

    The DNA sequence is right there in the pOAD2 plasmid for fucks sake

    It most certainly isn’t.

    Immediately below is the actual sequence where nylB resides (with some spaces to identify the start codon of nylB and where Ohno eventually made a typo. The lowercase is where nylB is within the relevant part of the R-IIA transcript:


    ATGGGCTACATCGATCTCTCCGCCCCCGTCGCGATGATCGTCAGC
    GGTGGCCTCTACTATCTCTTCACCCGCCGCGGCTACACCTTCGGAGACACT
    CG atgaacgcacgttccacc
    ggccagcaccccgccaggtatcccggagccgcggccggggagccgacactcgacagctgg
    caggaggccccgcacaaccgctgggccttcgcccgcctgggcgagctgctgcccacggcg
    gcggtctcccggcgcgacccggcgacgcccgcggagcccgtcgtgcggctcgacgcgctc
    gcgacgcggctccccgatctcgagcagcggctcgaggagacctgcaccgacgcattcctc
    gtgctgcgcggctccgaggtcctcgccgagtactaccgggcgggtttcgcacccgacgac
    cgtcacctgctgatgagcgtctcgaagtcgctgtgcggcacggtcgtcggcgcgctgatc
    gacgaggggcgcatcgatcccgcgcagcccgtcaccgagtatgtacccgagctcgcgggc
    tccgtctacgacgggccctccgtgctgcaggtgctcgacatgcagatctcgatcgactac
    aacgaggactacgtcgatccggcctcggaggtgcagacccacgatcgctccgccggctgg
    cgcacgcggcgagacggggaccccgccgacacctacgagttcctcaccaccctccgcggc
    gacggcggcaccggcgagttccagtactgctcggcgaacaccgacgtgctcgcctggatc
    gtcgagcgggtcaccggtctgcgctacgtcgaagcgctctccacgtacctgtgggcgaag
    ctcgacgccgatcgggatgcgaccatcacggtcgaccagaccggcttcggcttcgcgaac
    gggggcgtctcctgcaccgcgcgggatctcgcacgcgtgggccgcatgatgctcgacggc
    ggcgtcgctcccggcggacgggtcgtatcgcagggctgggtggaaagcgtgctggccggc
    ggctcccgcgaagccatgaccgacgagggtttcacctccgcattccccgagggcagctac
    acgcgccagtggtggtgcacgggcaacgagcgcggcaacgtgagcggcatcggcatccac
    ggccagaacctctggctcgatccgcgcaccgactcggtgatcgtcaagctctcgtcgtgg
    cccgatcccgacacccggcactggcacgggctgcagagcgggatcctgctcgacgtcagc
    cgtgccctcgacgcggtgtag GCGGCGTGA

    But any clown can delete a single letter because he thinks it would be cool to see what happens, and then delete another one because he made a typo and not realize it and then let it go through peer review at the National Academy of Science and then get evolutionists like Venema to proclaim what a great experimental discovery it was of the ancestor of nylB.

    What do you get when you do this, tada, Ohno’s hypothetical sequence which looks so much like the original, only frame-shifted with a stop codon thrown in due to a typo. Heck, let’s do that to every gene in our genebanks and proclaim our willy nilly removals constitute actual experimentally proven ancestors. Amazing crap like this persists for 4 decades and is uncritically accepted by both evolutionists and IDists as if it were an actual experimental discovery.

    Below is ohno’s FANTASY sequence which he calls PR.C:


    ATGGGCTACATCGATCTCTCCGCCCCCGTCGCGATGATCGTCAGC
    GGTGGCCTCTACTATCTCTTCACCCGCCGCGGCTACACCTTCGGAGACACT
    CG agaacgcacgttccacc
    ggccagcaccccgccaggtatcccggagccgcggccggggagccgacactcgacagctgg
    caggaggccccgcacaaccgctgggccttcgcccgcctgggcgagctgctgcccacggcg
    gcggtctcccggcgcgacccggcgacgcccgcggagcccgtcgtgcggctcgacgcgctc
    gcgacgcggctccccgatctcgagcagcggctcgaggagacctgcaccgacgcattcctc
    gtgctgcgcggctccgaggtcctcgccgagtactaccgggcgggtttcgcacccgacgac
    cgtcacctgctgatgagcgtctcgaagtcgctgtgcggcacggtcgtcggcgcgctgatc
    gacgaggggcgcatcgatcccgcgcagcccgtcaccgagtatgtacccgagctcgcgggc
    tccgtctacgacgggccctccgtgctgcaggtgctcgacatgcagatctcgatcgactac
    aacgaggactacgtcgatccggcctcggaggtgcagacccacgatcgctccgccggctgg
    cgcacgcggcgagacggggaccccgccgacacctacgagttcctcaccaccctccgcggc
    gacggcggcaccggcgagttccagtactgctcggcgaacaccgacgtgctcgcctggatc
    gtcgagcgggtcaccggtctgcgctacgtcgaagcgctctccacgtacctgtgggcgaag
    ctcgacgccgatcgggatgcgaccatcacggtcgaccagaccggcttcggcttcgcgaac
    gggggcgtctcctgcaccgcgcgggatctcgcacgcgtgggccgcatgatgctcgacggc
    ggcgtcgctcccggcggacgggtcgtatcgcagggctgggtggaaagcgtgctggccggc
    ggctcccgcgaagccatgaccgacgagggtttcacctccgcattccccgagggcagctac
    acgcgccagtggtggtgcacgggcaacgagcgcggcaacgtgagcggcatcggcatccac
    ggccagaacctctggctcgatccgcgcaccgactcggtgatcgtcaagctctcgtcgtgg
    cccgatcccgacacccggcactggcacgggctgcagagcgggatcctgctcgacgtcagc
    cgtgccctcgacgcggtgtag GCGGCTGA

  28. John Harshman:

    That wouldn’t work with most genes, since it wouldn’t leave a long open reading frame.

    Actually it would work even better because it doesn’t leave long open reading frames. It shows that a “random” sequence chock full of stop codons every 20 triplets or so can suddenly lose all of them except the one that counts and then create a brand spanking new protein. That would even be a more Earth shattering discovery than Ohno’s!

  29. stcordova: Rumraket:

    The DNA sequence is right there in the pOAD2 plasmid for fucks sake

    It most certainly isn’t.

    Oh for fucks sake, you make it sound like he made up over a thousand new nucleotides de novo. They’re all already there, he simply inferred the ancestral sequence from the observation that a T insertion would create a new, smaller reading frame inside it.

    The lack of the stop codon due to his “mistake” is still only one aspect of a greater argument for why that alternative reading frame can be inferred.

    But any clown can delete a single letter because he thinks it would be cool to see what happens

    Yes, but in the majority of proteins this is expected to produce premature stop codons, rather than several hundred contigous amino acid reading frames. The purported ancestral sequence is from that perspective exactly of the sort that could suffer a frameshift somewhere and result in another long protein.

    What do you get when you do this, tada, Ohno’s hypothetical sequence which looks so much like the original, only frame-shifted with a stop codon thrown in due to a typo. Heck, let’s do that to every gene in our genebanks and proclaim our willy nilly removals constitute actual experimentally proven ancestors.

    Nobody claims they’re “experimentally proven”, Salvador “rhetoric” Cordova. It was always an inference about an event nobody saw actually happen. Nobody has claimed otherwise. It is true there has been a failure of scholarship in missing Ohno’s mistake regarding the stop codon, and pointing out the extreme implausibility of the frameshift and subsequent HGT of P-NylB and F-NylB happening in the 20th century.

    The most substantial mistake is in when this frameshift mutation is thought to have occurred. But that doesn’t actually matter from your perspective, because regardless of whether it happened somewhere in the 1900’s, or a hundred million years ago, nobody actually saw it happen and nobody has ever claimed to see it happen, and nobody claims to have a specimen with the ancestral sequence, nor was the inference ever based on such a thing and it was never implied. And this isn’t a requirement to be able to infer a frameshift mutation.

    Regardless, I have now given you actual evidence that substantial parts of the alternative reading frame suggested by Ohno, in fact yields amino acid sequences that can be aligned to proteins known to exist. Some of which have known functions. So Ohno wasn’t actually wrong, even though he seems to have made up a stop codon somehow. You could actually check to see how much further the reading frame continues, it’s going to run into a stop codon eventually. This could simply imply the original reading frame was even larger than Ohno mistakenly identified it to be. Though it should be said, if you look at Okata H et al where Ohno got the sequence from, the similarity between F-NylB and P-NylB drops off pretty significantly immediately following the NylB stop codons, implying that even if the original reading frame once did extend into this zone, it has not been retained by purifying selection so whatever evidence there could be found of an extended reading frame terminating later on should probable not be taken at face value.

  30. Rumraket:

    Nobody claims they’re “experimentally proven”, Salvador “rhetoric” Cordova. It was always an inference about an event nobody saw actually happen. Nobody has claimed otherwise. It is true there has been a failure of scholarship in missing Ohno’s mistake regarding the stop codon, and pointing out the extreme implausibility of the frameshift and subsequent HGT of P-NylB and F-NylB happening in the 20th century.

    I know what I say irritates you, but I value what you just said. Thank you.

  31. stcordova: I know what I say irritates you, but I value what you just said.Thank you.

    Would it be possible to have layman’s version of what you exposed?

  32. Rumraket:

    I have now given you actual evidence that substantial parts of the alternative reading frame suggested by Ohno, in fact yields amino acid sequences that can be aligned to proteins known to exist.

    Nope because you aligned proteins not DNA sequences.

    You have to go to the original CDS here:

    https://www.ncbi.nlm.nih.gov/nuccore/922788014

    and here

    https://www.ncbi.nlm.nih.gov/nuccore/NZ_JHEI01000132.1?from=343711&to=345216&sat=19&sat_key=63278035

    Then you have to take the gene sequence and reverse-complement it since the gene is in the complementary strand. I used this:

    http://www.bioinformatics.org/sms/rev_comp.html

    Then when you extract the relevant fragment you get this DNA sequence:


    atgggctacatcgatctctccgctcccgttgcgatgatcgtcagcggcggactgtactacctcttcacccgtcgcggctacaccttcggagacgctcgatga

    which you can confirm is the sequence you claim aligns to Ohno’s by using Ex-PASy:

    http://web.expasy.org/translate/

    Now use a two sequence blast comparison of:


    atgggctacatcgatctctccgctcccgttgcgatgatcgtcagcggcggactgtactacctcttcacccgtcgcggctacaccttcggagacgctcgatga

    to Ohno’s sequence

    ATGGGCTACATCGATCTCTCCGCCCCCGTCGCGATGATCGTCAGC
    GGTGGCCTCTACTATCTCTTCACCCGCCGCGGCTACACCTTCGGAGACACT
    CG agaacgcacgttccacc
    ggccagcaccccgccaggtatcccggagccgcggccggggagccgacactcgacagctgg
    caggaggccccgcacaaccgctgggccttcgcccgcctgggcgagctgctgcccacggcg
    gcggtctcccggcgcgacccggcgacgcccgcggagcccgtcgtgcggctcgacgcgctc
    gcgacgcggctccccgatctcgagcagcggctcgaggagacctgcaccgacgcattcctc
    gtgctgcgcggctccgaggtcctcgccgagtactaccgggcgggtttcgcacccgacgac
    cgtcacctgctgatgagcgtctcgaagtcgctgtgcggcacggtcgtcggcgcgctgatc
    gacgaggggcgcatcgatcccgcgcagcccgtcaccgagtatgtacccgagctcgcgggc
    tccgtctacgacgggccctccgtgctgcaggtgctcgacatgcagatctcgatcgactac
    aacgaggactacgtcgatccggcctcggaggtgcagacccacgatcgctccgccggctgg
    cgcacgcggcgagacggggaccccgccgacacctacgagttcctcaccaccctccgcggc
    gacggcggcaccggcgagttccagtactgctcggcgaacaccgacgtgctcgcctggatc
    gtcgagcgggtcaccggtctgcgctacgtcgaagcgctctccacgtacctgtgggcgaag
    ctcgacgccgatcgggatgcgaccatcacggtcgaccagaccggcttcggcttcgcgaac
    gggggcgtctcctgcaccgcgcgggatctcgcacgcgtgggccgcatgatgctcgacggc
    ggcgtcgctcccggcggacgggtcgtatcgcagggctgggtggaaagcgtgctggccggc
    ggctcccgcgaagccatgaccgacgagggtttcacctccgcattccccgagggcagctac
    acgcgccagtggtggtgcacgggcaacgagcgcggcaacgtgagcggcatcggcatccac
    ggccagaacctctggctcgatccgcgcaccgactcggtgatcgtcaagctctcgtcgtgg
    cccgatcccgacacccggcactggcacgggctgcagagcgggatcctgctcgacgtcagc
    cgtgccctcgacgcggtgtag GCGGCTGA

    And what do you really get? TADA!

    No significant similarity found. For reasons why,click here

    Conclusion: you didn’t find the missing link needed to vindicate Ohno.

    However in gratitude for your efforts at defending Ohno’s fantasy here is a version of Philip Baily hitting some high notes. Man that guy can sing!

    https://www.youtube.com/watch?v=gL2MsEbrtgI

  33. Sal, just look at the two DNA sequences. Just look at them. You can do the alignment by hand. I can see straight up the first 23 nucletides align. The rest aligns too with a few point mutations. LOL

    So now we know both the amino acid and the DNA aligns. You really should try to resist the triumphalism.

  34. LOL! Rumraket, those weren’t the sequences that were shifted.

    NOTE where OHNO put the frigging frame shift, it was after that sequence.

    . You really should try to resist the triumphalism.

    Yeah, but I’m right and you’re shown wrong. Twice. HAHAHA!

    I even freaking put the sequence before the shift in capital leters and the sequence on the “atg” which Ohno made “ag” to induce the frame shift in lower case, and you still don’t get it.

    ATGGGCTACATCGATCTCTCCGCCCCCGTCGCGATGATCGTCAGC
    GGTGGCCTCTACTATCTCTTCACCCGCCGCGGCTACACCTTCGGAGACACT
    CG agaacgcacgttccacc...

    HAHAHA!

  35. Sal,

    Yeah, but I’m right and you’re shown wrong. Twice. HAHAHA!

    And how do you suppose we think about you given the number of times you’ve been shown to be wrong and just ignored it?

  36. stcordova: Yes. What Ohno’s “scientific” paper represents can be described by this:

    This from someone who makes money lying about science. When will you be publishing a scientific paper Sal, instead of lying to rubes about what the science says about biology?

  37. stcordova: LOL! Rumraket, those weren’t the sequences that were shifted.

    That’s irrelevant, it show that the beginning sequence of Ohno’s inferred protein exists and codes for proteins in other organisms.

    NOTE where OHNO put the frigging frame shift, it was after that sequence.

    I know, I myself informed you of this in my large post above when I wrote
    “The thing is that this is the beginning part of the putative ancestral sequence which spans upstream to where NylB begins, which pretty substantially confirms that Ohno is right, there really was another protein there before. Strangely enough this fragment stops where NylB begins.”

    I also found a second, 247 long alignment IN THE FRAMESHIFTED portion, to a protein with unknown function from a fungus, though with a much lower similarity score of about 30%.

    Yeah, but I’m right and you’re shown wrong. Twice. HAHAHA!

    But you aren’t right you moron. And with every post you make yourself look more stupid.

    I even freaking put the sequence before the shift in capital leters and the sequence on the “atg” which Ohno made “ag” to induce the frame shift in lower case, and you still don’t get it.

    ATGGGCTACATCGATCTCTCCGCCCCCGTCGCGATGATCGTCAGC
    GGTGGCCTCTACTATCTCTTCACCCGCCGCGGCTACACCTTCGGAGACACT
    CG agaacgcacgttccacc…

    HAHAHA!

    Are we laughing at your moronity? That must be it, becuase you have no other reason to laugh than at your own persistent incompetence.

    The 33 amino acid sequence I informed you I had found an alignment for, above, is OUT OF FRAME from the NylB sequence. Meaning if that small portion was part of a bigger sequence, that sequence would have to extend into the NylB gene, but in a reading frame shifted by a single nucleotide.

    The last of the first 33 amino acids in the sequence I showed you is coded for by the codon CGA. The A nucleotide in that codon, is the first nucleotide in the NylB start-codon in both Okata H et al 1983, and in Ohno 1984. It really is out of frame and the nucleotide sequence for the beginning portion REALLY IS accurately reported by Ohno.

    Ohno has not altered the nucleotide sequence for the beginning of the NylB gene (he has only messed up the stop-codon near the end), he has simply inferred that the T in the ATG start-codon for NylB originally got there by a frameshift. He didn’t DELETE a nucleotide there as you seem to think. Rather, he simply shifted his view and looked at the same sequence, but with a reading frame shifted by a single nucleotide. That’s when he saw that there’s a continous unbroken reading frame overlapping the NylB gene. If you look at the reading frame for Ohno’s putative 427 residue protein following the NylB start codon, you will see it is actually shifted by a single nucleotide compared to the beginning portion. But it is identical to the sequence from Okata H.

    Given that I have also found an alignment for a stretch of 247 amino acids with a similarity of ~30% in the later portion of Ohno’s 427 residue putative ancestral sequence, this substantially confirms there used to exist a protein in that reading frame.

    Stated another way, I found what you failed to find, and which you looked for because you correctly inferred these would substantiate Ohno’s inference of a frameshift, if a protein product that is similar to the one Ohno inferred, could be found. I have found that. Both for the part that overlaps the NylB reading frame, and the beginning portion that does not.

    You really really really need to take a step back and think before you post any more crap.

  38. newton: Would it be possible to have layman’s version of what you exposed?

    Since Sal can’t think when he’s high on the Jesus-juices, I’ll answer for him.

    He has discovered that in Ohno’s 1984 publication, near the last portion of a nucleotide sequence copied from Okata H et al 1983, Ohno appears to have deleted a single G nucleotide. This G deletion in turn makes it appear like there is a stop-codon in a shifted reading frame, when in fact there isn’t.

    That is it. That’s Sal’s big discovery.

    What we here in this thread have done (actually it was mostly John Harshman who pointed this out early on), is that a frameshift mutation Ohno inferred to have happened (not one caused by his presumably accidental G deletion) somewhere in the 1930’s, did not actually happen in the 1930’s, but probably happened over a hundred million years ago.

    Now, the problem is that many people have incorrectly believed that this frameshift mutation happened in the 1930s and resulted in the ability of bacteria to digest the waste products of nylon manufacture.

    What Sal is trying to do is at least threeo things.
    1. He wants to undermine the evidence for the frameshift mutation (he tries to do this by showing how he fails to find amino acid and DNA sequences that align to Ohno’s putative ancestral protein).

    We are supposed to take his personal failure to find these things, as convincing evidence that there are no such tings. And if there are no such things, then we are supposed to not believe there is any evidence for a frameshift mutation (there actually still is even if we could not align DNA or protein sequences to other genes, that’s what I’ve been saying several times).

    2. He wants to blow up Ohno’s G-deletion as some sort of colossal failure of scholarship (after all, the reviewers didn’t catch it, nor has anyone who has read these papers since, so in that sense it is true).
    This one is probably just purely about Sal’s pride. He wants to go to biologos and “own” the people over there about how they’ve all failed to check their references thoroughly and bla bla bla

    3. He wants to use the fact that the timing of the frameshift mutation has been incorrectly inferred to have happened in the 20th century, as another piece of evidence in his case for a failure of scholarship.

    Sal has systematically tried to undermine Ohno’s inference that there was a frameshift mutation, and when he has failed to find the things he thought there should exist if such a frameshift mutation occurred, he has declared victory.

    And now I have checked, and I found what Sal failed to find: homologous amino acid sequences to the protein that must have once existed before the frameshift mutation rendered it nonfunctional.

    What Sal is now trying to do is cast doubt on my findings, but he’s actually just making a lot of mistakes. For example, he seems to think Ohno has somehow deleted something from the a DNA sequence in Okata H et al 1983 in order to make it appear that there was a frameshift mutation. But Ohno has done no such thing. Ohno is accurately reproducing the DNA sequence, he hasn’t deleted anything other than that G near the very last portion of the sequence, which results in Ohno mistakenly inferring a stop-codon for his “ancestral” protein, which it does not appear there actually is.

    I looked at all the codons up to and including the ones coding for the first four amino acids in NylB, as reported in both Ohno and Okata (See pic). They match exactly. Ohno didn’t delete or insert anything, the T the black arrow points to in Ohno et al is the start codon for NylB (coding for Met – Methionine), that same T is also there in Okata H et al, also coding for Met. All the codons are identical. Ohno only points his arrow there because he infers that is the frameshift-causing T-insertion that must have happened. And when he makes this inference, he finds an alternative reading frame without premature stop codons.

    How the hell is that a problem for Ohno and his inference?

  39. OMagain: This from someone who makes money lying about science. When will you be publishing a scientific paper Sal, instead of lying to rubes about what the science says about biology?

    I gather this is actually what Sal was trying to do, write a paper and get it published about how there’s no evidence for Ohno’s ancestral sequence. Up in this post he seems to be giving us his “abstract”.

    But it appears he doesn’t have the giant knock-down argument he had hoped for.

  40. Rumraket,

    I gather this is actually what Sal was trying to do, write a paper and get it published about how there’s no evidence for Ohno’s ancestral sequence. Up in this post he seems to be giving us his “abstract”.

    Yes, and the language of the abstract is mostly un-Sal-like, suggesting that he has a collaborator or ghostwriter who is more literate than he is. However, there are still Salisms like “de Novo” and “de Nove” for “de novo”, and “breakdown” where “break down” should be used.

  41. So I went to uniprot to search for similar amino acid sequences to the putative ancestral protein Ohno inferred. I cut out the beginning portion of the sequence that does not overlap with NylB, and simply seached the uniprot database with much more relaxed search criteria.

    I got hundreds of hits. To pick an example, I got a 303 residue alignment to Ribonuclease E from streptomyces with a 36.6% identity and E-value of 2.9e-13

    Or how about this 394 residue alignment with a 36.3% identity and E-value of 1.1e-22.

  42. Rumraket:

    That’s irrelevant, it show that the beginning sequence of Ohno’s inferred protein exists and codes for proteins in other organisms

    You’d get those first 21 nucleotides even without Ohno’s shift. Get a clue.

  43. Rumraket:

    a second, 247 long alignment IN THE FRAMESHIFTED portion, to a protein with unknown function from a fungus,

    A fungus is a eukaryote. So how do you explain a fungus having Ohno’s prokaryotic sequence that has only 30% identity since 1935?

  44. stcordova: You’d get those first 21 nucleotides even without Ohno’s shift.Get a clue.

    There’d be no reason for the upstream region to be coding for protein if there didn’t used to be a protein there. The fact that a 97% similar match is found in another protein is a significant piece of evidence in Ohno’s favor.

    Sure, you can just rationalize it away with some other ad-hoc hypothesis, but the fact is it fits with Ohno’s prediction.

    stcordova: A fungus is a eukaryote. So how do you explain a fungus having Ohno’s prokaryotic sequence that has only 30% identity since 1935?

    Irrelevant since I’m not claiming the sequences that are similar to Ohno’s inferred one, came from the plasmid at any point in the 20th century. Rather, they are homologous because they both evolved from common ancestors and got horizontally transferred, probably through plasmids, many hundreds of millions of years ago.

  45. it show that the beginning sequence of Ohno’s inferred protein exists and codes for proteins in other organisms

    So does the unshifted one!

    The translated sequence from the actual RS-IIA transcript sequenced by Okada:

    MGYIDLSAPVAMIVSGGLYYLFTRRGYTFGDTR-

    Note the dash indicates the “tga” stop codon.

    Gee it looks so much like what Rumraket claims is created by Ohno:

    MGYIDLSAPVAMIVSGGLYYLFTRRGYTFGDTR

    So the relevant portion already exists without Ohno’s frameshift.

    So how does Rumraket propose this sequence got into the middle nitrate reductases since 1935?

  46. Rumraket:

    Rather, they are homologous because they both evolved from common ancestors and got horizontally transferred, probably through plasmids, many hundreds of millions of years ago.

    Well that’s problematic for Ohno’s claim because he’s claiming the frame-shift happened only after 1935!

    From Ohno’s paper:

    In this paper, I shal show that this mechanism can be invoked to explain the sudden birth of a plasmid-encoded bacterial enzyme, 6-aminohexanoic acid linear oligomer hydrolase…

    The swiftness with which these two enzymes evolved is truly remarkable, for several decades are but a flash in the evolutionary timescale.
    ….
    Furthermore, 11.35% amino acid sequence divergence observed between two isozymic forms of 6-ALA LOH suggests their independent origin from the same family of repeated sequences identified within pOAD2 plasmid, rather than one bein ancestral to another for
    so extensive an amino acid sequence divergence is not expected to occur in so short at time span–i.e., 40 years or
    thereabout.

    As an alternative to the customary process of the birth of a unique gene from a redundant copy of the pre-existed gene of a
    related function, I suggest that each of these unique genes fro degradation of nylon by-products arose de novo independently
    from an alternative reading frame of the preexisted, internally repetitious coding sequence.

    The two Isozymes he is referring to are NylB and NylB’ which have 11.35% similarity. They are both 1179 nucleotides long. He didn’t show the nylB-prime gene in his paper but you can find it in Okada’s paper. So he’s proposing simultaneous frame shift in 2 genes in the same place and one of them, nylB’, creates an enzyme (NylB’) that only has 1 / 1000 the nylonase ability as NylB.

    He argues for the frame shift because he complains a simple gene duplication does not cause a radical enough change

    The mechanism of gene duplication, however, is inherently self limiting in that a new protein arising by this mechanism invariably retains substantial amino acid sequence homology and, therefore, functional relatedness with its immediate ancestor. Thus, one wonders if this mechanism alone sufficed at the very beginning of life when a large variety of polypeptide chains with divergent function had to be created almost simultaneously. The same can be said of this 20th century when a variety of microorganisms suddenly found themselves facing an onslaught of man-made artificial compounds.

    Unfortunately, Kato showed in 1991, 7 years later, a mere 2 residue change in NylB’ makes it as capable as NylB on nylon, so Ohno didn’t need to invoke a frame-shift which changes practically all the residues!

    And Venema and other anti-IDists cite Ohno’s claim of a post-1935 as Darwin’s truth, science be praised!

  47. dazz:

    Rum owning Sal’s ass…
    will make for a good bike parking rack

    Hey Rumraket, congratulations, you managed to bamboozle someone who has no clue. Excellent work, even though I just pointed out you proved Ohno wrong since you invoked millions of years rather than 40 as Ohno claimed. Well done.

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