Invited responses to my nylonase research and the question of “New Proteins Without God’s Help”

Susumo Ohno (who coined the term “junkDNA”) published a paper in 1984 through the National Academy of Sciences that was used by the NCSE, Ken Miller and Dennis Venema to claim “proteins can evolve without God’s help”. At the request of John Sanford, a courtesy associate research professor at Cornell, I was recruited to write a paper to refute Ohno’s evolutionary hypothesis on nylonases. I wrote it under John’s guidance based on his intuitions about genetics, his life-long specialty of 40 years and for which he became famous as attested by the fact he is one of the few geneticists who had their work featured in the Smithsonian National Museum of American History.

The actual paper is now in review, but it is not intended to be published in any journal, but will be released in a variety of channels shortly. It is hoped the material can be used by others to actually create papers that enter peer review. The motivation for releasing the paper in this way is to counter Venema’s book while it is still hot off the press.

The paper is being also published in this way so as to invite discussion since it isn’t intended to be considered a completely vetted product but one that welcomes improvement. That said, the sentiment among IDists and creationists who’ve seen the drafts is that paper has utterly discredited Ohno’s claims and thus the claims of the NCSE, Ken Miller and Dennis Venema connected to Ohno’s hypothesis of nylonase evolution.

Because the draft paper is a massive VJTorley-sized paper (15 pages in the main section and almost 80 pages of supplemental material) I’m establishing this thread at TSZ to invite review of the some of the themes from the paper that I’m releasing on the nylonase.XYZ website piece by piece in a format adapted for a website.

As I release each webpage, I’ll post in the comment section at TSZ a link to the newly constructed page so as to invite commentary on that page. Thank you in advance to all those willing to participate in this public review of my research on nylonase evolution.

NOTES:

1. “proteins can evolve without God’s help” is a paraphrase of the title of an NCSE article New Proteins Without God’s Help. Thwaites at the NCSE basically framed the debate over nylonase evolution in this way:

We’ve been trying to explain all this to the protein “experts” at ICR for the last seven years. We have told them that new proteins could indeed form from the random ordering of amino acids. We have warned them that their calculations were based on faulty assumptions and soon someone would document the natural formation of a new protein from the random association of amino acids.

Now it has happened! Not one, but two, new proteins have been discovered. In all probability new proteins are forming by this process all the time, but this seems to be the first documentation of this phenomenon. The newly discovered proteins are enzymes that break down some of the byproducts produced during nylon manufacture. Since nylon first came into commercial production in 1940, we know that the new enzymes have formed since that time.

2. The nylonase.XYZ website is under construction, so don’t click around the website too much yet. In the comment section at TSZ I will link to individual pages of the website that can be reviewed individually.

3. The first comments by me at TSZ will be more technical, not for the beginners regarding Ohno’s work. The beginner and introductory stuff will be added later to the website.

174 thoughts on “Invited responses to my nylonase research and the question of “New Proteins Without God’s Help”

  1. Here are two pages I just constructed. They are meaningful to those of you who are familiar with the technical details of Ohno’s theory.

    For those not familiar with the theory yet, just hang on as those web pages are forthcoming.

    I claim Ohno’s made typographical errors including errors that changed the genes! You all can be the judge of how severe this error is, but it just looks sloppy for a PNAS paper. Since this is a rather serious charge of a basic error on his part, I’m putting this item first on the table.

    Thus the first link in this thread is to my technical description of Ohno’s typo. This is just perhaps one minor flaw in his theory, but since it is a particularly biting charge (one of carelessness on his part), I put it on the table first. If I’m the one in error, feel free to correct me. That’s why I’m putting it on the table here at TSZ.

    The link describing Ohno’s typo is here:
    https://nylonase.wordpress.com/description-analysis-and-problems-with-ohnos-pr-c-seqeunce/

    A more mundane appendix is also provided for those already familiar with the nylonase papers. The appendix contains accession numbers and names of organisms and plasmids related to Ohno’s hypothesis.

    Of note in the appendix is that there are at least 4 nylonase genes, not 1. Ohno only attempted to explained the nylB and nylB’ genes. His ommission of an explanation for nylA and nylC makes his theory look especially ad hoc and thus dubious.

    The link to the nomenclature appendix is here:
    https://nylonase.wordpress.com/nomenclature-relevant-to-okada-and-ohnos-hypotheses/

  2. I wrote it under John’s guidance based on his intuitions about genetics, his life-long specialty of 40 years and for which he became famous as attested by the fact he is one of the few geneticists who had their work featured in the Smithsonian National Museum of American History.

    I find your constant attempts to puff up the credentials of creationists (and only creationists, too) to be annoying, as well as unprofessional. Hope that helps.

    it is not intended to be published in any journal

    Also unprofessional. Of course it makes sense if you’re a propagandist rather than a scientist.

    I claim Ohno’s made typographical errors including errors that changed the genes! You all can be the judge of how severe this error is, but it just looks sloppy for a PNAS paper. Since this is a rather serious charge of a basic error on his part, I’m putting this item first on the table.

    What makes you think it’s a serious charge? Does it invalidate his claims? If not, it’s hardly worth mentioning and merits a correction, nothing more. And you seem to be attempting an ad hominem argument: if Ohno made a mistake, you shouldn’t believe anything he said.

    His ommission of an explanation for nylA and nylC makes his theory look especially ad hoc and thus dubious.

    Why must a theory about one protein also explain all unrelated proteins of similar function? Doesn’t sound like a problem to me.

    Now as I see it, the big problem with Ohno’s paper is that he thinks, apparently (I haven’t read it recently so can’t be sure what it says), that the frame shift was recent and was the mutation that gave rise to nylonase, while the evidence shows that it was ancient and the resulting protein was not nylonase but an ancestor with unknown function. That wouldn’t affect the point that it shows a new protein arising by frame shift.

  3. I have to wonder how much of Salvador’s ‘research’ is original. Ann Gauger of ENV and Biologic did a recent series on the subject.

  4. Mung:
    Sal, be sure topuff up the credentials of John Harshman. He’s feeling left out.

    What are John Harshman’s real credentials? PhD in evolutionary biology and studies in zoology? If someone like him can’t explain the fundamentals, not to mention experimentally proving them (like the evolution of prokaryotic cell into eukaryotic for example) that his credentials stand on, what’s left? A birdwatcher credentials?

  5. J-Mac: what’s left? A birdwatcher credential?

    I’m sure he has that as well. Sal, don’t forget to add bird watching to John’s credentials. Wouldn’t want them to be under-inflated!

  6. Mung: I’m sure he has that as well. Sal, don’t forget to add bird watching to John’s credentials. Wouldn’t want them to be under-inflated!

    Lol

    No wonder people like him defend evolution to death…

    What would they be or mean to anybody if evolution were to be declared a fraud worldwide?

    What would happen to Harshman, Coyne, Moran and the like if they were no longer be able to bully people into accepting their beliefs in godless evolution as science, so that they could feel good about their over-inflated egos..?

  7. By the way, you really should provide a complete citation to the paper you are criticizing. And preferably a link too.

  8. Rereading Ohno, I discover that he did not claim that the frame shift is what caused the evolution of nylonase activity, but agrees that it happened very long ago. So your major criticism is not of Ohno but of some people who may have misread him. I say “may” because I don’t know what those folks wrote.

  9. I thought I did! You’d find the title and links if you follow the link to the nylonase.xyz website. I said I’d shell out the pieces of the research in bits in the comment section. Did you not read this comment?

    http://theskepticalzone.com/wp/invited-responses-to-my-nylonase-research-and-the-question-of-new-proteins-without-gods-help/#comment-185274

    And I specifically said:

    The link describing Ohno’s typo is here:

    https://nylonase.wordpress.com/description-analysis-and-problems-with-ohnos-pr-c-seqeunce/

    Let me know if that doesn’t do the job. Thanks for reading. I am interested to see if you concur that Ohno goofed the way I said he did.

  10. Rereading Ohno, I discover that he did not claim that the frame shift is what caused the evolution of nylonase activity, but agrees that it happened very long ago. So your major criticism is not of Ohno but of some people who may have misread him. I say “may” because I don’t know what those folks wrote

    Thanks for offering your opinion. I think you’re the one misreading his work.

    So how do you interpret the phrase in the title: “Birth of a unique enzyme.” What was unique about it? Could it be the ability to, ahem, degrade nylons?

    As far as other guys, I’ll post on that at nylonase.xyz.

    Thanks you for your participation.

  11. What makes you think it’s a serious charge? Does it invalidate his claims? If not, it’s hardly worth mentioning and merits a correction, nothing more. And you seem to be attempting an ad hominem argument: if Ohno made a mistake, you shouldn’t believe anything he said.

    Well, I’d like to know if I was right and if he made the errors I pointed out:

    1. a typo of “472” when he meant “427” for the length of the open reading frame

    2. accounting for why he deleted a guanine base at the end of PR.C sequence which would cause the stop codons to disappear. Given he’s arguing for the powerful effects of frame shift, his should be careful with such mutation-by-typo errors that create even more frame shifts. For all we know, hundreds of down stream residues got chopped off because of his mistake.

    What makes you think it’s a serious charge? Does it invalidate his claims? If not, it’s hardly worth mentioning and merits a correction, nothing more. And you seem to be attempting an ad hominem argument: if Ohno made a mistake, you shouldn’t believe anything he said.

    Hardly worth mentioning? How about all those poor evolutionary biologists in the future who might be searching for the missing link PR.C sequence in the databases for decades upon decades only to find out it had a typo and thus screwed up their searches. You could show little more gratitude for my service to the scientific enterprise for trying to set the record straight! Sheesh. No good deed goes unpunished.

  12. John said:

    Rereading Ohno, I discover that he did not claim that the frame shift is what caused the evolution of nylonase activity, but agrees that it happened very long ago. So your major criticism is not of Ohno but of some people who may have misread him. I say “may” because I don’t know what those folks wrote.

    Seems in conflict with this statement:

    From Ohno’s paper:

    I assume that the longer open reading frame identified as PR.C. was the original coding sequence of this stretch of plasmid DNA until several decades ago.

  13. Yes after recently re-reading that original paper by Ohno I discovered he actually inferred two independent frameshift mutations, recently, to explain NylB and NylB’.

    After our previous discussion I went back and read Ohno again (it’s remarkable how much one can miss, it seems we all missed this one). Ohno writes:

    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 being ancestral to another, for so extensive an amino acid sequence divergence is not expected to occur in so short a 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 preexisted gene of a related function, I suggest that each of these unique genes for degradation of nylon by-products arose de novo independently from an alternative reading frame of the preexisted, internally repetitious coding sequence. In particular, I suggest that the RS-IIa base sequence was originally a coding sequence for an arginine-rich polypeptide chain 427 or so residues long in it’s length and that the coding sequence for one of the two isozymic forms of 6-ALA LOH arose from its alternative open reading frame.

    My bolds. (From Ohno 1984 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC345072/pdf/pnas00609-0153.pdf).

    Remarkably Ohno is here suggesting that there were two independent frameshift mutations which created the NylB and NylB-prime genes. Also, that little “or so” thing where he estimates the length of the putative original reading frame suggests he knows he’s created a stop codon not found in the actual sequence?

    I think given what we now know today, Ohno’s original hypothesis doesn’t stand up to scrutiny as there are just too many strange discrepancies. At the time he made the inference it was reasonable, but I think he biggest obstacle that undermines his inference is that a very distantly related NylB sequence (P-NylB) with only about 37% amino acid sequence similarity to F-NylB, was found on a Pseudomonas plasmid. Annoyingly I could never find the full nucleotide sequence for the Pseudomonas plasmid, it doesn’t seem to exist in any database that I can find.

    As we went over in the other thread, this would imply that Ohno has to now infer either THREE independent frameshift mutations, or a single very very ancient one.

    But I think then some of the facts that lead to him inferring the frameshift mutation become stretched and difficult to assess the plausibility of.

    For example, what makes only little sense to me now is the remarkable degree of conservation of sequence, of the upstream region of the F-NylB and F-NylB’ genes that would have constituted the original reading frame. Remember that small piece of sequence 33 amino acids long that gave hits with 97% identity on BLAST searches? Why would that be there and sit at the c-terminal of the F-NylBs but N-terminal of pubmed proteins?

    This looks to me like the NylB genes on the pOAD2 plasmid are insertions horizontally transferred (possibly related to transposons?) and the upstream region has nothing to do with any previous reading frame.

    I’m still very much unsure about the qualities of the BLAST hits I got for longer stretches of amino acids in the putative older 427 amino acid proteins reading frame.

    Regardless of this, what we CAN say is that the NylB gene family must be old. Very old. With ~37% sequence similarity between distant homologues (F-NylB and P-NylB) of a 392 amino acid protein, we are talking hundreds of millions of years at least. I think the facts we have now today can not be used to infer a frameshift mutation with any appreciable level of confidence. But that’s just my own amateur opinion.

  14. stcordova: For all we know, hundreds of down stream residues got chopped off because of his mistake.
    (snipped comment)
    Hardly worth mentioning? How about all those poor evolutionary biologists in the future who might be searching for the missing link PR.C sequence in the databases for decades upon decades only to find out it had a typo and thus screwed up their searches. You could show little more gratitude for my service to the scientific enterprise for trying to set the record straight! Sheesh. No good deed goes unpunished.

    Don’t be silly. We already argued about that deletion in the other thread. The nucleotide sequences for the F-NylB and F-NylB’ strongly diverge immediately following the stop codons for both, so even if both genes have a shifted reading frame that continues for a while after the NylB stop codons, we can’t meaningfully infer an ancestral sequence when the downstream nucleotide sequences diverge as much as they do.

    Why didn’t you get this point the last five goddamn times I informed you of it? Look at this nice picture again and see it with your own eyes (from Okata H et al 1983 where the F-NylB and F-NylB’ nucleotide sequences are reported). The gene sequences immediately following the NylB stop codons are very much different:

  15. stcordova:
    John said:

    Seems in conflict with this statement:

    Yeah, you’re right. That paper could have used a rewrite for clarity. That’s my excuse. My initial objection came when I saw that he knew about the two genes on the plasmid, but I failed to see that he was positing two independent, nearly simultaneous, identical frame shifts. Now that’s carrying things a bit far.

    I will agree that the idea of one, or even two, recent frameshifts is untenable, and that the latter is what he was claiming. The evidence is clear that if there was a frameshift, it was many millions of years ago. (Of course you can’t put it that way, since you don’t believe in “millions of years”.)

  16. I wrote:

    it is not intended to be published in any journal

    and

    John wrote:

    Also unprofessional. Of course it makes sense if you’re a propagandist rather than a scientist.

    I no longer hold the title of scientist (my title was junior scientist while working at the Army Night Vision Laboratories in Ft. Belvoir).

    I’m just a trouble maker. 🙂

    But more seriously, I strongly suggested to John Sanford we don’t go the route of traditional peer-review, but instead go public and have our errors discussed and cleaned up, and then we can decide if we want to invest time shopping around the world (like say in China) to have our stuff published in some journal. We originally thought of some poster session or small symposium.

    I cautioned that we might have 90% of a good paper accepted by a reviewer but only the condition we drop the Ohno stuff on the grounds it was irrelevant to the major thesis of the paper which was actually the widespread conservation of nylonases, not Ohno’s frameshift hypothesis.

    I suggested the public review would help us decide if it was really worth our time to cross every “T” and dot every “I” (so to speak), and format the paper to death when one of our goals was to point out Venema’s error which is really related to the general discussion of the difficulty of “evolving a new protein without God’s help”.

    We’re not going to resolve the question of whether new proteins can evolve without God’s help, but refuting Ohno’s hypothesis is kind of important to that question. I suggested rather than us trying to write a paper and then trying to defend it after it gets in a peer-reviewed, let’s just put it on the table and see how good our idea is first. The journal editors also put themselves at risk when they publish stuff from a creationist. John manages to keep publishing in secular journals even though he is known to be a creationist, but we know the editors stick their neck out every time they do so. John’s last paper was on the waiting time problem (which is over my head).

    I’ll provide evidence of the nylonase conservation in this thread shortly. I alluded to it earlier in the other thread when I said proteases like trypsin (which are also found in vertebrates) were discovered as nylonases. Same with Papain (another protease). There are biochemical reasons for this.

    So really, as much as you and Rumraket and others have sharp disagreements with me, I say sincerely how much I appreciate the time you guys scrutinizing elements and themes from our paper.

    When John called contacted me and suggested the project, I initially thought John Sanford would be the lead author and I was just a research grunt, but then he listed me as principal author and him as last author (which sometimes indicates the head of the lab or group or department).

    So I’m releasing parts of this now just to double check for my own peace of mind whether I’m making a claim that isn’t appropriate for a formal paper. I make all sorts of outrageous claims on the net, but formal papers with someone else’s reputation on the line is another story. So that’s the reason we’re doing business this way.

    Really, sincere thanks for taking the time to engage and consider our work.

  17. Thanks Rumraket for the earlier comments, they were so good I don’t have much of a response.

    The last draft of the paper which I submitted for review had this in it. I expect it might get re-worded by the editor. But anyway:

    It is possible Ohno realized that since nylB’ is 88% sequence homologous in DNA to nylB this led to a dilemma of explaining how the corresponding NylB and NylB’ proteins could also maintain amino acid homology if a frame shift happened only in the open reading frame containing nylB but not the open reading frame containing nylB’.

    His wording on the matter might be subject to various interpretations, but it does appear he thought the frame shift that created nylB was mirrored by a corresponding frame shift that created nylB’. He said:

    “It is of interest to note here that this stretch of base sequence is duplicated elsewhere within the pOAD2 genome roughly 90˚ away and that the coding sequence for the second isozymic form of 6-AHA LOH is found in this duplicated stretch. Thus, a pair of isozymic preexisted coding sequences might have given rise independently to the coding sequences for two isozymic forms of 6-AHA LOH.”.

    Thwaites of the NCSE interpreted this passage by Ohno as evidence two frame shifts happened independently on the same plasmid and independently created two nylonases.

  18. Addendum to my immediately previous comment, I also said this in the paper:

    Furthermore, unless one invokes horizontal gene transfer, because the nylB and nylB’ are also found in Agromyces KYR5 and are also each 1179 bases in length, Ohno’s frame shift would have to happen independently in four separate reading frames spread across two organisms.

  19. Just for clarity, micro biologists use a different convention than those studying primates for naming of genes and corresponding proteins.

    The gene name has first three letters in lower case: nylB,

    the protein name has the first letter upper case and no italics: NylB

    http://www.biosciencewriters.com/Guidelines-for-Formatting-Gene-and-Protein-Names.aspx

    “Bacteria: Gene symbols are typically composed of three lower-case, italicized letters that serve as an abbreviation of the process or pathway in which the gene product is involved (e.g., rpo genes encode RNA polymerase). To distinguish among different alleles, the abbreviation is followed by an upper-case letter (e.g., the rpoB gene encodes the β subunit of RNA polymerase). Protein symbols are not italicized, and the first letter is upper-case (e.g., RpoB). ”

  20. stcordova: “Bacteria: Gene symbols are typically composed of three lower-case, italicized letters that serve as an abbreviation of the process or pathway in which the gene product is involved (e.g., rpo genes encode RNA polymerase). To distinguish among different alleles, the abbreviation is followed by an upper-case letter (e.g., the rpoB gene encodes the β subunit of RNA polymerase). Protein symbols are not italicized, and the first letter is upper-case (e.g., RpoB). ”

    Sal,
    If you are going to quote some text as an authority, you should probably check to make sure it is error-free. Can you spot the mistake in this passage?

  21. stcordova: Addendum to my immediately previous comment, I also said this in the paper:

    Furthermore, unless one invokes horizontal gene transfer, because the nylB and nylB’ are also found in Agromyces KYR5 and are also each 1179 bases in length, Ohno’s frame shift would have to happen independently in four separate reading frames spread across two organisms.

    I take it Agromyces KYR5 is a strain of Agromyces. If the nylB and nylB’ genes are also found in this species, are they also on plasmids?
    If they are, I think we couldn’t really say we’d have to claim four independent frameshifts to explain all four genes, as we could just be talking about a plasmid transfer of the pOAD2 plasmid from one Acromyces strain to another. Or even that the pOAD2 plasmid can be transferred and recombined with plasmids in other strains.

    There are multiple possible interpretations and we don’t have to go straight to four independent frameshifts if the nylB(‘) genes are found in other bacteria, in order to not buy into the frameshift scenario.

  22. DNA_Jock: If you are going to quote some text as an authority, you should probably check to make sure it is error-free. Can you spot the mistake in this passage?

    Looks fine to me?

    Reproducing the formatting directly from the source without putting it in quote tags gives this:
    Bacteria: Gene symbols are typically composed of three lower-case, italicized letters that serve as an abbreviation of the process or pathway in which the gene product is involved (e.g., rpo genes encode RNA polymerase). To distinguish among different alleles, the abbreviation is followed by an upper-case letter (e.g., the rpoB gene encodes the β subunit of RNA polymerase). Protein symbols are not italicized, and the first letter is upper-case (e.g., RpoB).

    I don’t see an error. At best you can say that they forgot to say that the uppercase letter is also italicized in the gene name. Is that what you are thinking of?

  23. Rumraket,

    KYR5 has its nylB in a reverse complement orientation on a chromosome. The Flavobacteria (Arthrobacteria) KI72 has it’s nylB in a forward orientation on the plasmid pOAD2.

    I have no strong opinion on horizontal gene transfer from KI72 to KYR5. My speculation nylB in KI72 and KYR5 descended from a common ancestor that was horizontally transferred into both. But that isn’t anything I’d officially defend. That’s for microbiologists and evolutionary biologists to figure out.

    BLASTN shows 99% sequence similarity between KYR5 and KI72.

    I certainly don’t think 4 frame shifts and 4 typos (ha!) created nylB in those species.

  24. stcordova: Rumraket,

    KYR5 has its nylB in a reverse complement orientation on a chromosome. The Flavobacteria (Arthrobacteria) KI72 has it’s nylB in a forward orientation on the plasmid pOAD2.

    I have no strong opinion on horizontal gene transfer from KI72 to KYR5.

    With respect to direction (from KI72 to KYR5 or reverse), neither have I. Either of them could have got it from the other as far as I’m concerned. As you suggest possibly even from a 3rd source, but then we’d have to have some evidence of that. Someone would have to do the work of finding all the different species with nylB homologues and build a phylogeny to make sense of it.

    BLASTN shows 99% sequence similarity between KYR5 and KI72.

    I assume this is for the nylB genes in particular.

    I certainly don’t think 4 frame shifts and 4 typos (ha!) created nylB in those species.

    We agree, that’s preposterous.

  25. At the time Ohno wrote his 1984 paper, he said “birth of a unique enzyme” believing NylB was only in bacteria KI72. But after 4 decades we find NylB to be wide spread. I provide evidence of 193 NylB homologs with genes actually labeled nylB in creatures different bacteria in the Indian Ocean far away from Japanese nylon factories. These bacteria are in the Indian Ocean. This suggests to me the NylB wasn’t an evolutionary response to the presence of nylon, but is conserved in nature.

    To be fair, I’ve gone on record as saying nylonase homologs had a few residue mutations to get them to adapt to nylon, and further “nylonase” is a misnomer as we don’t know the pre-1935 role of these enzymes yet. It is suspected it is some sort of amidase or beta lactamase. I haven’t seen reports that actually went into what NylB’s role is in biological world.

    Furthermore, BLASTN comparisons among the homologs show almost no sequence homology among groups, thus the homology is structural as predicted by UNIPROT. A protein researcher named Rost pointed out structurally similar proteins often have only 12% sequence similarity.

    I provided the list of organisms, materials and methods and some references to the papers here. If I can find links to the papers I’ll insert them shortly.

    https://nylonase.wordpress.com/widespread-nylb-homology-of-form-not-of-sequence-among-193-nylb-homologs/

    Also, for those just joining the conversation, the table of contents will reflect the latest updates to the Nylonase.XYZ website:

    https://nylonase.wordpress.com/table-of-contents/

  26. I collected a list of over 1800 organisms with actual or predicted nylonases and found reasons to suspect the number organisms with nylonases is in the tens of thousands.

    I should be careful to point out, having a nylonase capable enzyme does not necessarily translate into having a metabolism that can actual digest nylon. There is more to nylon digestion than having a single nylonase enzyme!

    The fact there are so many organisms with nylonases spread out from the Arctic to Indian Ocean to human pathogenic bacteria suggests it is implausible to argue the emergence of nylonase homologs around the world emerged as an post-1935 evolutionary response to nylon being created by man.

    Here is the latest webpage on the nylonase.XYZ website. I will revise it to insert references as I have time, but one can easily get the list of organisms independently through the description of materials and methods on the webpage.

    https://nylonase.wordpress.com/stampede-of-organisms-with-nylonases-1800-with-6-ah-nylonases-tens-of-thousands-with-other-kinds-of-nylonases/

  27. I think the fact of the widespread existence of enzymes now known with activity on nylon oligomer manufacture waste products, is a much stronger argument against the frameshift scenario for the origin of the nylB genes. As you said, Ohno worked with the information available to him at the time and now we know a lot more.

    stcordova: Furthermore, BLASTN comparisons among the homologs show almost no sequence homology among groups, thus the homology is structural as predicted by UNIPROT.

    I find this a bit confusing. UNIPROT doesn’t do structural prediction as far as I’m aware so why would it predict structural homology with so low sequence homology? I’m assuming it is because these enzymes have been tested on linear oligomer and found to have similar activity to the first discovered NylB enzymes?

    I have some questions out of general interest.
    How many of all these reported nylonase homologoues have resolved structures as far as you are aware?
    Are they all active on the linear oligomer like the NylB enzymes are? Or are we talking about all the different types of enzymes active on all sorts of nylon manufacture waste (or nylon pollution degradation) byproducts?

    Regardless I would totally agree that the nylB gene family existed long before the nylon waste byproducts came to exist on Earth, and they definitely had other functions. And with so many distant homologoues (12% sequence identity) this totally undermines Ohno’s frameshift hypothesis.

  28. stcordova: I provided the list of organisms, materials and methods and some references to the papers here. If I can find links to the papers I’ll insert them shortly.

    https://nylonase.wordpress.com/widespread-nylb-homology-of-form-not-of-sequence-among-193-nylb-homologs/

    Also, for those just joining the conversation, the table of contents will reflect the latest updates to the Nylonase.XYZ website:

    https://nylonase.wordpress.com/table-of-contents/

    Thank you, this is interesting information. Just for you information, the screenshots are very small and it’s very difficult to read what they show.

  29. stcordova: We’re not going to resolve the question of whether new proteins can evolve without God’s help, but refuting Ohno’s hypothesis is kind of important to that question.

    I don’t believe that’s true. That isn’t a question that can be answered, because no empirical data can conceivably tell you whether a new protein evolved with or without god’s help. Nylonase is no exception. If it had evolved by frame shift, god might have caused the frame shift. If it had evolved by point mutation, god might have caused the point mutation. Phylogenetic evidence from paralogous proteins shows nothing either, since god could have caused both the gene duplication and subsequent changes. God can do anything. How can you detect his presence or absence in any event or process?

    stcordova: Furthermore, unless one invokes horizontal gene transfer…

    Hello, it’s on a plasmid. What do you think happens to plasmids, quite frequently?

  30. John Harshman:

    Hello, it’s on a plasmid. What do you think happens to plasmids, quite frequently?

    I was referring to post 1935 HGT.

    The problem is a bit more subtle than supposed.

    KYR5 not only has NylB but also NylC and NylB’ on it. The NylC on KYR5 is only 95-98.6% similar to NylC on KI72.

    Reference:
    http://aem.asm.org/content/73/21/7099.full

    On the KI72 plasmid nylC and nylB share a promoter. Doesn’t that make the nylB/nylC a polycistron?

    So not only did the frame shift have to happen, but also HGT early enough to effect enough residue changes on nylC. You’re the one whose in the best position to say how many residues you think can evolve post-1935. They are 355 residue in length in each organism. 4.2% of 355 = 15 residues. I guess 15 residues can evolve in 60 years. If not, then the HGT happened pre-1935.

    So I was criticizing post-1935 HGT. I could have worded it better. So your criticism is certainly valid in that respect. Thank you.

    But all this is moot if you agree with me and Rumraket about the fact widespread conservation of nylB is evidence against the frame-shift origin of nylB.

  31. stcordova: I provide evidence of 193 NylB homologs with genes actually labeled nylB in creatures different bacteria in the Indian Ocean far away from Japanese nylon factories.

    Oh, good. So you accept common descent.

  32. John Harshman: That isn’t a question that can be answered, because no empirical data can conceivably tell you whether a new protein evolved with or without god’s help.

    John says something I can agree with.

  33. stcordova: KYR5 not only has NylB but also NylC and NylB’ on it. The NylC on KYR5 is only 95-98.6% similar to NylC on KI72.

    Reference:
    http://aem.asm.org/content/73/21/7099.full

    On the KI72 plasmid nylC and nylB share a promoter. Doesn’t that make the nylB/nylC a polycistron?

    Sal, KI72 and KYR5 are bacterial strains, not plasmids.

    There can’t be a NylC “on” KI72 (or KYR5) unless you mean on the actual chromosome of the bacterium, instead of on the plasmid it might carry around.

  34. stcordova: So not only did the frame shift have to happen, but also HGT early enough to effect enough residue changes on nylC. You’re the one whose in the best position to say how many residues you think can evolve post-1935. They are 355 residue in length in each organism. 4.2% of 355 = 15 residues. I guess 15 residues can evolve in 60 years. If not, then the HGT happened pre-1935.

    I think the HGT of the NylB gene family would have had to happen waaaay before 1935 to see it in as many places as we do (assuming here all the reported versions of it truly are homologous).
    At a 12% sequence similarity score but with a retained 2ndary/tertiary protein structure and function, we are looking at several billion year divergence times. At those ages we might not even be talking about HGT through plasmids as part of an explanation for their divergence any more for some of them, as they could be different (and in different species) simply because the different bacterial species (not just strains of the same species) that carry them on their chromosomes, split off from each other that long ago. As in we could be talking about a gene family that goes back to the common ancestor of some of these bacteria.

  35. stcordova: But all this is moot if you agree with me and Rumraket about the fact widespread conservation of nylB is evidence against the frame-shift origin of nylB.

    You mean widespread existence but great divergence, not widespread conservation. It isn’t highly conserved other than in the sense it’s existence is conserved, but it’s sequence seems to be pretty much as divergent as purported homologous proteins can get.

  36. What we can agree on is that the evolution of NylB did not result from a recent frame shift and that the wide distribution of NylB homologs did not result from a recent horizontal transfer.

  37. Sal, KI72 and KYR5 are bacterial strains, not plasmids.

    Agreed, that was sloppy language on my part. I did point this out in the nomenclature page on nylonase.XYZ.

    But, a rather dumb question, when I see something like

    Flavobacterium sp KI72

    I presume, “sp” means species? So if I see the phrase “a Flavobacterium sp.” that means species. Stupid as it sounds, it’s hard to google “meaning of sp” and expect to get an explanation!

    There can’t be a NylC “on” KI72 (or KYR5) unless you mean on the actual chromosome of the bacterium, instead of on the plasmid it might carry around.

    Actually, I’m glad you’re forcing me to double check this. Yikes!

    NylC was on the same promoter. In fact, I just realized John and I didn’t provide reference for the claim of the shared promoter.

    The paper is:
    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC182563/

    Flavobacterium sp. strain KI725 harbors plasmid pOAD21, a derivative of nylon oligomer-degradative plasmid pOAD2, ….

    This suggests that the nylB and nylC genes are expressed from a common promoter.

    Yikes. I can’t believe we didn’t affix a reference to Negoro 1993 for this in the paper. We owe you!!!

    Regarding Agromyces KYR5:
    http://www.sciencedirect.com/science/article/pii/S1389172308700148?via%3Dihub

    If you don’t have institutional access I can arrange a copy for you since you are helping John and I with our paper.

    These results suggest taht at least two nylB-related sequences are located on the chromosome of KYR5

    Dumb question #2. I presume there is a distinction between a plasmid and a chromosome as far as bacteria are concerned? Aren’t they both circular? Bacteria isn’t my gig….

    Here is a link to the photo below which is from the paper just reference by Yasuhira. I provide the link so you can zoom in:
    https://nylonase.files.wordpress.com/2017/08/yasuhira_agromyces_2007.png

    It says

    B Agromyces sp. KYR6 chromosome

    C Arthrobacter sp. KI7 plasmid pOAD2

  38. As is so often the case, you ignored the main content of my comment a

    Ok so I can’t prove proteins can or cannot evolve without God’s help, I just don’t find it believable, but we don’t say that in our paper.

    “Proteins without God’s help” is only referenced in our paper on the section where we talk about the role of the nylonase question in popular culture, namely, the NCSE using Ohno’s paper to say, “New Proteins Without God’s Help”. We were essentially refuting the premise of Thwaites “New Proteins Without God’s Help” which was Ohno.

    Now that we’ve refuted post 1935 frame-shift origin of nylB, it still doesn’t mean we’ve formally proven or disproven proteins can or can’t evolve with or without God’s help in that paper, or any paper in the future. But thanks anyway for this:

    What we can agree on is that the evolution of NylB did not result from a recent frame shift and that the wide distribution of NylB homologs did not result from a recent horizontal transfer.

    Maybe that’s about all we can agree on, all things considered.

    FWIW, John, unlike me, feels some remorse having to discredit some of the work of a hard working scientist like Ohno. I suppose he views Ohno like a comrade or venerable soldier on an opposing side. But he does feel strongly the record has to be set straight.

    Our paper will not go into theological type issues except to acknolwedge that Thwaites, Venema, and Ken Miller have referenced Ohno’s work in their writings.

    I wasn’t trying to diss you, it just wasn’t the focus of our paper, which I’m trying to clean up. And along those lines, I may ditch the paragraph on Agromyces for brevity in light of your criticism.

  39. Rumraket:

    I think the fact of the widespread existence of enzymes now known with activity on nylon oligomer manufacture waste products, is a much stronger argument against the frameshift scenario for the origin of the nylB genes. As you said, Ohno worked with the information available to him at the time and now we know a lot more.

    Thanks. Some of the other stuff like that about the typos was a bit petty, on the other hand, we included a solitary paragraph pointing it out in our paper as it is a data point worth being cognizant of.

    Our case was built mostly on the widespread existence of nylonases which the paper focused ad nauseam on. I mean 90 pages (including the supplement) ad nauseam. One reviewer said he liked the ad nauseam lists included. I suppose it has value in as much as it will serve as a snapshot of UNIPROT in 2017 and we’ll see how the list changes over the years as data flows in.

    We didn’t not take the angle of other creationists which you can find on wiki and which, imho, are somewhat cringeworthy compared to what John and I wrote:

    https://en.wikipedia.org/wiki/Nylon-eating_bacteria_and_creationism

    It seems clear that plasmids are designed features of bacteria that enable adaptation to new food sources or the degradation of toxins. The details of just how they do this remains to be elucidated. The results so far clearly suggest that these adaptations did not come about by chance mutations, but by some designed mechanism.[8]

    –Creationist Don Batten

    ….
    MSNBC published an editorial from science writer Ker Than that stated that the evolution of the enzymes, known as nylonase, produced by nylon-eating bacteria was a compelling argument against the claim made by intelligent design proponents that specified complexity required an intelligent designer, since nylonase function was both specified and complex.[10] The intelligent design proponent William Dembski posted a response that questioned whether the genetic changes that produced nylonase were complex enough to be considered specified complexity.[11] Ken Miller said that intelligent design proponents claim that we can’t see either design or evolution taking place and that therefore intelligent design and evolution are both just matters of faith or world view. However, Miller said, the evolution of the enzyme nylonase, which scientists were able to repeat in the lab with another strain of bacteria, is one of a number of cases that show that evolution can be observed as it occurs.[12]

  40. Have you ever looked at the literature citing Ohno’s paper to see what others have said about it?

  41. stcordova: I presume, “sp” means species? So if I see the phrase “a Flavobacterium sp.” that means species. Stupid as it sounds, it’s hard to google “meaning of sp” and expect to get an explanation!

    I actually didn’t know this myself and had to google around too. I found this from wikipedia: https://en.wikipedia.org/wiki/Binomial_nomenclature

    The abbreviation “sp.” is used when the actual specific name cannot or need not be specified. The abbreviation “spp.” (plural) indicates “several species”. These abbreviations are not italicised (or underlined).[45] For example: “Canis sp.” means “an unspecified species of the genus Canis”, while “Canis spp.” means “two or more species of the genus Canis”. (The abbreviations “sp.” and “spp.” can easily be confused with the abbreviations “ssp.” (zoology) or “subsp.” (botany), plurals “sspp.” or “subspp.”, referring to one or more subspecies. See trinomen (zoology) and infraspecific name.)

  42. Rumraket:

    I have some questions out of general interest.
    How many of all these reported nylonase homologoues have resolved structures as far as you are aware?
    Are they all active on the linear oligomer like the NylB enzymes are? Or are we talking about all the different types of enzymes active on all sorts of nylon manufacture waste (or nylon pollution degradation) byproducts?

    These are great questions and since they are such good questions, I’ll try to construct pages on the nylonase.XYZ website to help me respond to them. I’ll get to work on it as soon as I can.

    But as far as resolved structures, UNIPROT affixes quality scores to the gene predictions with 1 star being the lowest, 5 stars being the highest. None of them get 5 stars including the genes from confirmed cases of nylon eating bacteria!

    I only saw a couple papers where X-ray crystallography was used and none with cryo-electron microscopy.

    99% of the UNIPROT gene predictions get 1 star ratings. We noted in our paper the gene predictions were provisional (which is pretty much the case for most genes in most databases) but pointed to circumstantial evidence confirming the gene or a least nylonase activity by pointing out cases like Bacillus cereus and a few other species that have been confirmed to digest nylon. Despite the circumstantial evidence, UNIPROT curators still mark the nylonase homologs in Bacillus cereus at 1 star. So 1 star doesn’t mean bad or unreliable, it means the level of direct experimental confirmation.

    Circumstantial evidence weighs on the side of the gene prediction being correct in those cases.

    I should point out something about the BLAST searches. The nylB genes cluster in groups. That is you can BLAST the Flavobacteria nylB and get only a few limited hits. When you BLAST the Bacillus cereus nylB gene you get a different set of limited hits.

    If I had to hazard a guess, it seems there were plasmids with discrete nylB forms that seem almost like distinct subfamilies. How UNIPROT can still recognize them as nylB genes despite lack of sequence homology is a mystery and a black box which I hope to find out when I visit the NIH once a week for 3 months in the Fall to study protein bioinformatics.

    I find this a bit confusing. UNIPROT doesn’t do structural prediction as far as I’m aware so why would it predict structural homology with so low sequence homology? I’m assuming it is because these enzymes have been tested on linear oligomer and found to have similar activity to the first discovered NylB enzymes?

    Thank you for the criticism. I’ll have to think on it as the right words are hard to come by. And I strive for clarity.

    My understanding is that secondary structures (alpha helices, beta sheets) can be inferred from primary sturctures (the actual amino acid sequences). I’ll be studying how this is done the Fall at the NIH (including some study of protein phylogeny).

    I don’t think anyone can predict native folds (which I presume is predicting tertiary and quatenary structure), but maybe guess at them.

    Since we can group amino acids into to polar, non-polar, aromatic, etc. we can see which residues can be readily substituted and still get the same secondary structure. I suspect UNIPROT does a lot of this. It also has huge databases of chemical reactions and does some sort of association of motifs and conserved domains.

    Somehow UNIPROT computed an organism like Bacillus cereus would have nylonase capability even though its nylonase genes have no sequence homology to experimentally confirmed nylonases. Pretty amazing. UNIPROT algorithms have some serious black magic in them! 🙂

  43. stcordova: Dumb question #2. I presume there is a distinction between a plasmid and a chromosome as far as bacteria are concerned? Aren’t they both circular? Bacteria isn’t my gig….

    Yes there is a distinction. The chromosome is the bacteriums “normal” genome, the plasmid is a sort of small extra chromosome it carries around and will some times readily eject.

    As far as I’m aware bacterial chromosomes are normally circular like plasmids generally are. My guess is, as with everything in biology, you can find an obscure exception.

    Interestingly when it comes to plasmids, bacteria are generally in the habit of ejecting them after relatively small number of cell divisions, as carrying them around if they aren’t needed slows down replication, in addition to incurring a metabolic cost of having them replicated at cell division and through expressing the genes they encode.

    But it is super easy to get bacteria to both eject and pick up plasmids. The bacterial tendency to horizontally transfer plasmids is literally exploited in molecular and cell biology. Remove the selective pressure that forces them to retain it, and they will quickly expel it. Put them through a single cycle of cooling on ice and heat them up again and they’ll take up plasmids readily. So simple environmental changes will make them exchange plasmids.

    I happen to work with molecular and cell biology, so quite a lot of my work involves transforming and transfecting both bacterial and human cells with plasmids (as it’s an easy way to get a gene into a cell and see the effect the gene has on the organism).

    As bacteria in particular have this tendency to eject the plasmids, a selective pressure to retain them are artificially constructed in the growth medium. To do that, the bacteria are grown in a medium containing an antibiotic that kills them, but the plasmid we transform (which is the term for getting a plasmid into a bacterium) into the bacteria is constructed to carry a gene that gives them resistance to this antibiotic. That way, if any of the bacteria eject the plasmid, they die shortly after as they can no longer synthesize the resistance gene. This ensures the bacteria that grow and divide in the flask all carry the plasmid with the genes of interest.

  44. stcordova: I should point out something about the BLAST searches. The nylB genes cluster in groups. That is you can BLAST the Flavobacteria nylB and get only a few limited hits. When you BLAST the Bacillus cereus nylB gene you get a different set of limited hits.

    If I had to hazard a guess, it seems there were plasmids with discrete nylB forms that seem almost like distinct subfamilies. How UNIPROT can still recognize them as nylB genes despite lack of sequence homology is a mystery and a black box which I hope to find out when I visit the NIH once a week for 3 months in the Fall to study protein bioinformatics.

    I think what it does is that it can bridge very distantly related genes through more closely related ones.

    So if A is 80% similar to B, and B is 80% similar to C, and C is 80% similar to D, and D is 80% similar to E, and so on, you can get to a point where A and K are 10% similar, for example. Yet because there is that bridge of highly similar genes between them, A->B->C->D->E->F->G->H->I->J->K, you can still get them detected as homologous even though A and K almost no sequence similarity.

    In the case you bring up, there are probably clusters of nylBs for bacterial species that are either of closely related species, or they occupy the same environments. One might hazard a guess that soil bacteria in general have a tendency to share plasmids as they live next to each other, so you’d get a cluster of soil-bacterial nylBs. And then this cluster might be sorta similar to another cluster that is removed from, but still related to soil bacteria.

    But this leads me to an important point. As I pointed out in the previous thread, BLAST searches have some common pitfalls. For example, just doing a naive BLAST search with the default settings might not turn up anything, and you’d think that means there isn’t anything similar in the databases.

    But in actually the algorithm has to make shortcuts, because there’s simply too much data for it to be able to literally go through every single sequence in the database and try to align(which itself also take CPU time), it is waaay too complutationally intensive. So something is done to try to filter things out and save time and resources. But that also means there’s a chance that one of the things you might be interested in, is filtered out.

    To prevent that, you have to alter the algorithm’s parameters, and there are several ways to do this. How should it evaluate alignments? When it starts to align a sequence, what should count as a “hit” that spurs it align more? Two, three, or six nucleotides? How about gaps? Any sequence can be made to align with another if there’s no gap penalty, so clearly gaps can’t just be allowed willy-nilly or everything would come back as a significant alignment.

    This is why I was able to find hits for Ohno’s putative ancestral sequence when you were not, as I played around with the algorithm parameters. But as you might suspect, this can be both a blessing and a curse, as when you relax some criteria, you also pollute your results with potentially lots of insignificant stuff. In truth, it really takes an expert to evaluate what you get out a BLAST search. It’s really not so simple as just putting in a sequence and pressing “BLAST”.

    I got both obviously crap hits, and some pretty good ones. Interestingly the best hit I got was for the upstream region of nylB on the pOAD2 plasmid from Flavobacterium KI72, where that strange 33 amino acid peptide with a 97% identity score to the c-terminal of a protein in a microfungus.

  45. Sal,
    When I asked you where you would be publishing this paper, you replied you’ll publish it here at TSZ. Can I remind you of something you previously said:

    The other side dreads being put under critical scrutiny. They know their theory will not be seen favorably once it is examined by the standards of real science.

    https://uncommondescent.com/evolution/the-vise-strategy-squeezing-the-truth-out-of-darwinists/#comment-216

    Do you consider posting a blog post here and thereby gaining the ability to avoid answering specific questions the standard of real science?

    John Harshman: As is so often the case, you ignored the main content of my comment and addressed a trivial point.

    You won’t have that option Sal if you are actually getting work peer reviewed to the standards of real science. If you don’t address a point you don’t pass review. So it’s clear why you are avoiding having your work examined by the standards of real science. You already know it’ll fail.

  46. Thus, in a crucial way, the Kansas hearings repeat the pattern set by the Scopes Trial, which has
    been repeated many times since, namely, evolutionists escaped critical scrutiny by not having to
    undergo cross-examination. In this case, they accomplished the feat by boycotting the hearings. I
    therefore await the day when the hearings are not voluntary but involve subpoenas that compel
    evolutionists to be deposed and interrogated at length on their views.

    https://billdembski.com/documents/2005.11.Vise_Strategy.pdf
    Seem to me you’ve become the Darwinists youself Sal. Your “boycott the hearings” is “avoid real peer review”.

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