Comparisons of Neandertal mtDNA with modern humans and modern chimpanzees

Thanks. I missed that.Mea Culpa.

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Aslan is not a Tame Lion

Actually, fragment size is not really a good indicator of authenticity as depending on the sample, some fairly large fragments of DNA may still remainComp Biochem Physiol B. 1985;81(4):1045-51. Related Articles, LinksIsolation and characterization of deoxyribonucleic acid from tissue of the woolly mammoth, Mammuthus primigenius.Johnson PH, Olson CB, Goodman M.DNA was isolated from tissue samples of several mammoth specimens, radiocarbon dated between 10,000 and 53,000 years old. The DNA was purified by chromatography on hydroxyapatite at 60 degrees C and was characterized as a heterogeneous population of fragments ranging in size from 3000 to 200 base pairs. Thermal denaturation analysis demonstrated that approximately 25% of the DNA had a base composition similar to Asian elephant DNA calculated as 36% G + C. Preliminary analysis by nucleic acid hybridization indicated that only a small fraction of DNA isolated from mammoth tissue (2-5%) was homologous to DNA of Asian elephant, a close living relative of the mammoth. Our results provide the first definitive isolation and characterization of DNA from ancient tissue and suggest a purification strategy that will lead to preparations of DNA from mammoth tissue significantly enriched in elephant-related DNA sequences.In addition, since it is usually assumed beforehand that the DNA is degraded, most researchers amplify short (100-300 bp) overlapping fragments and "walk out" to generate a larger sequence. The problem is if you land on a Numt and then build your sequence based on the Numt i.e. design primers that are sure to bind to the first sequence you retrieved, you will end up walking along the Numt and not the mtDNA. In any case, if you only get 100 bp fragments from a Cro-Magnon, no contamination, and can reproduce it in another lab, and it looks just like human, the question is, should it be considered authentic or should one have doubts? If it does not look human i.e. like the neandertal sequences, should one have doubts? Is it more convincing just because it looks different? I think this is the crucial problem facing those using aDNA to test human evolution hypotheses. Glad I work on megafauna

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Preliminary analysis by nucleic acid hybridization indicated that only a small fraction of DNA isolated from mammoth tissue (2-5%) was homologous to DNA of Asian elephant, a close living relative of the mammoth.

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I was somewhat surprised when I read this. I am assuming that you are using high stringency temperatures, and the low G+C content probably also contributes. Of course, I could just be talking out of my ass since I work on the protein side of things. My mol bio knowledge mostly comes from working with the gene jockies in my lab. Anyway, enough off topic tech talk for now.

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In addition, since it is usually assumed beforehand that the DNA is degraded, most researchers amplify short (100-300 bp) overlapping fragments and "walk out" to generate a larger sequence. The problem is if you land on a Numt and then build your sequence based on the Numt i.e. design primers that are sure to bind to the first sequence you retrieved, you will end up walking along the Numt and not the mtDNA.

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I can definitely picture this. Nested primers would create overlapping sequence, but the source would be difficult to discern unless the "walk" ran into genomic, non-Numt sequence. I seem to remember that the mutation rate for Numt is higher than mitDNA, so this could pose a huge problem. If you hypothesize that neandertal mitDNA is identical to Cro-Magnon mitDNA, you would have to show how the neandertal mitDNA is actually Numt while the Cro-Magnon mitDNA is truly mitDNA.I can definitely understand your preference for megafauna, that is unless you use elephants as lab techs.

I would find this very surprising. Mitochondria have much higher mutation rates than nuclei do -- their DNA polymerase lacks proofreading capability that the nuclear polymerases have.

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I would find this very surprising. Mitochondria have much higher mutation rates than nuclei do -- their DNA polymerase lacks proofreading capability that the nuclear polymerases have.

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Very good point. Still, there are scenarios that could cause problems if numt instead of mitDNA is sequenced. I think we can all agree that mitDNA genomic insertions will experience different selective pressures than the mitDNA pool. Also, the two sequences will accrue different mutations. I would assume that mutations in numt's would be considered neutral for the most part. However, if this was a problem it should have shown up in the modern human vs Cro-Magnon pairwise comparison.A question for the experts. If there were a mix of numt and mitDNA in the PCR, what would the sequencing results look like. From my limited experience, it would seem that the predominant sequence at the start of the PCR would overpower the less predominant sequence. That is, the peaks coming off of the sequencer would reflect the predominant base at each PCR termination. The argument then is that the ratio of mitDNA to numt is high enough to preclude contamination from genomic sequences.But then again: From:Thalmann O, Hebler J, Poinar HN, Paabo S, Vigilant L. Unreliable mtDNA data due to nuclear insertions: a cautionary tale from analysis of humans and other great apes. Mol Ecol. 2004 Feb;13(2):321-35.I guess I am straddling the mitDNA reliability fence. It would seem that even Mammuthus would have to control for numt PCR amplification, but luckily avoids the possibility of modern DNA contamination. For now, I will stick with the theory of neandertal mitDNA divergence from H. sapien, but tentatively so. Perhaps further studies will demonstrate non mitDNA flanking sequence, and therefore evidence numt amplification. Given the fragmented nature of ancient DNA a shotgun library would be impractical, but perhaps narrowing down possible insertion sites through phylogenetic analysis could result in canidates for genomic flanking sequence [1].[1] Bensasson D, Feldman MW, Petrov DA. Rates of DNA duplication and mitochondrial DNA insertion in the human genome. J Mol Evol. 2003 Sep;57(3):343-54.

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I think we can all agree that mitDNA genomic insertions will experience different selective pressures than the mitDNA pool. Also, the two sequences will accrue different mutations. I would assume that mutations in numt's would be considered neutral for the most part. However, if this was a problem it should have shown up in the modern human vs Cro-Magnon pairwise comparison.

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This is true. However, Numt generation is an ongoing process and a newly inserted Numt could be identical or only slightly different from bona fide mtDNA. Here is an experiment that shows that really minor mutations that appeared to correlate with occurrence of Alzheimer's were in fact all Numts..they are very very similar to mtDNA and would fit nicely in the middle of the pairwise distributionBiochem Biophys Res Commun. 1998 Mar 27;244(3):877-83.
Evidence that two reports of mtDNA cytochrome c oxidase "mutations" in Alzheimer's disease are based on nDNA pseudogenes of recent evolutionary origin.Davis JN 2nd, Parker WD Jr.Center for the Study of Neurodegenerative Diseases, University of Virginia Health Sciences Center, Charlottesville 22908, USA.Recently, two reports [R. E. Davis et al. (1997) Proc. Natl. Acad. Sci. USA 94, 4564-4569 and E. Fahy et al. (1997) Nucleic Acids Res. 25, 3102-3109] described a series of heteroplasmic mitochondrial DNA (mtDNA) mutations in the genes encoding two cytochrome c oxidase subunits (CO1 and CO2) which segregated in higher abundance with Alzheimer's disease subjects than controls. Using mtDNA-depleted NT2 cells, we provide further evidence that these two reports are erroneously based on a PCR artifact arising from the amplification of nuclear DNA encoded mtDNA pseudogenes (mtDNA psi s). Our findings are similar, but not identical, to other recent studies of these putative mtDNA psi sequences. This sequence variability may indicate that multiple mtDNA psi s, all of comparatively recent evolutionary origin are involved. While such pseudogenes are interesting in that they provide a molecular evolutionary "snapshot" of human ancestral mtDNA, it is unlikely that they play any role in the etiology of Alzheimer's disease.

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A question for the experts. If there were a mix of numt and mitDNA in the PCR, what would the sequencing results look like. From my limited experience, it would seem that the predominant sequence at the start of the PCR would overpower the less predominant sequence. That is, the peaks coming off of the sequencer would reflect the predominant base at each PCR termination. The argument then is that the ratio of mitDNA to numt is high enough to preclude contamination from genomic sequences.

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If you look at the sequenceing figure in this paper, you can see what a mess Numts can cause...it was not possible from DNA alone to determine the correct sequence...in fact, direct sequencing produced exclusively Numt sequenceGreenwood AD, Paabo S.
Nuclear insertion sequences of mitochondrial DNA predominate in hair but not in blood of elephants.
Mol Ecol. 1999 Jan;8(1):133-7.these guys wrote a nice review of the Numt problem for different speciesBensasson D, Zhang D, Hartl DL, Hewitt GM. Related Articles, Links
Mitochondrial pseudogenes: evolution's misplaced witnesses.
Trends Ecol Evol. 2001 Jun 1;16(6):314-321.

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It would seem that even Mammuthus would have to control for numt PCR amplification, but luckily avoids the possibility of modern DNA contamination. For now, I will stick with the theory of neandertal mitDNA divergence from H. sapien, but tentatively so

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Actually, I don't control for it..I completely switched over to nuclear sequences for elephantid work. I could not get around the problem. There is even some reasonably convincing evidence that several of the published mammoth mtDNA sequences are Numts. But I do not think the neandertal sequence is a Numt. Krings did a huge number of controls with modern DNA samples to demonstrate that the sequence he obtained is not present in the modern human nuclear genome. Adcock et al. did not do this with the Mungo Lake sample which is a problem since it looks almost exactly like a known human Numt. My real problem with the nea/Cro-magnon work is that it is fairly widely assumed (in the aDNA community) that a sequence is only authentic if it looks different and thus testing the hypothesis of intermixing is not possible or falsifiable...that is a pretty big problem.By the way for both Sylas and yourself, there is a newly published nea study that anyone can access since it is in the free access journalsPLoS Biol. 2004 Mar;2(3):E57. Epub 2004 Mar 16. Related Articles, LinksNo Evidence of Neandertal mtDNA Contribution to Early Modern Humans.Serre D, Langaney A, Chech M, Teschler-Nicola M, Paunovic M, Mennecier P, Hofreiter M, Possnert G G, Paabo S.Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.The retrieval of mitochondrial DNA (mtDNA) sequences from four Neandertal fossils from Germany, Russia, and Croatia has demonstrated that these individuals carried closely related mtDNAs that are not found among current humans. However, these results do not definitively resolve the question of a possible Neandertal contribution to the gene pool of modern humans since such a contribution might have been erased by genetic drift or by the continuous influx of modern human DNA into the Neandertal gene pool. A further concern is that if some Neandertals carried mtDNA sequences similar to contemporaneous humans, such sequences may be erroneously regarded as modern contaminations when retrieved from fossils. Here we address these issues by the analysis of 24 Neandertal and 40 early modern human remains. The biomolecular preservation of four Neandertals and of five early modern humans was good enough to suggest the preservation of DNA. All four Neandertals yielded mtDNA sequences similar to those previously determined from Neandertal individuals, whereas none of the five early modern humans contained such mtDNA sequences. In combination with current mtDNA data, this excludes any large genetic contribution by Neandertals to early modern humans, but does not rule out the possibility of a smaller contribution.Cheers,
M

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This is true. However, Numt generation is an ongoing process and a newly inserted Numt could be identical or only slightly different from bona fide mtDNA.

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Yep, you got me there. Just from my recent readings, numt generation happens quite often, more than often enough to back your assertion (in primates at least).

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Biochem Biophys Res Commun. 1998 Mar 27;244(3):877-83.
Evidence that two reports of mtDNA cytochrome c oxidase "mutations" in Alzheimer's disease are based on nDNA pseudogenes of recent evolutionary origin.

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This is a great cautionary tale for all scientists. Researchers (myself included) can be tempted to latch onto an observation that fits our hypothesis without fully qualifying or testing the reliability of the observation. Mutated cytC would be a tempting path for further research, but all for not given that the mutation happens outside of the mitDNA.

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My real problem with the nea/Cro-magnon work is that it is fairly widely assumed (in the aDNA community) that a sequence is only authentic if it looks different and thus testing the hypothesis of intermixing is not possible or falsifiable...that is a pretty big problem.

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I hear ya brother. My response to the cytC mutations applies here as well. I think we can agree that scientists jumped on the nea mitDNA data right away, but are only now (ie last few years) calling the data into question. This should be a lesson for creationists, that trying to falsify theory can only strengthen it, or completely falsify it, whichever the case. I think the current research into hominid aDNA is a great example of what we always tell skeptical anti-science types, that science really is competitive and no theory goes unchallenged.

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By the way for both Sylas and yourself, there is a newly published nea study that anyone can access since it is in the free access journals

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PLoS Biol. 2004 Mar;2(3):E57. Epub 2004 Mar 16. Related Articles, Links

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No Evidence of Neandertal mtDNA Contribution to Early Modern Humans.

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Serre D, Langaney A, Chech M, Teschler-Nicola M, Paunovic M, Mennecier P, Hofreiter M, Possnert G G, Paabo S.

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Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.

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Do you feel that this is a valid control, and that this study adds credibility to the nea and amh mitDNA data? I tend to think it does, but not being an expert in the field . . .And just out of curiosity, are the authors above colleagues of yours? You don't have to sacrifice your anonymity, just wondering due to both you and the above research group are located in Germany and you are both into aDNA.Added in edit:

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If you look at the sequenceing figure in this paper, you can see what a mess Numts can cause...it was not possible from DNA alone to determine the correct sequence...in fact, direct sequencing produced exclusively Numt sequence

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Greenwood AD, Paabo S.
Nuclear insertion sequences of mitochondrial DNA predominate in hair but not in blood of elephants.
Mol Ecol. 1999 Jan;8(1):133-7.

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I could order the paper, but that would be abusing our grant money And, I'm far to lazy to go to a local uni library. But, just from your description of "messed up" I can picture what the peaks coming off of the sequence would look like (if that is what you are talking about). Anyway, fully understand the technical problems numts create.This message has been edited by Loudmouth, 05-06-2004 12:25 PM

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Do you feel that this is a valid control, and that this study adds credibility to the nea and amh mitDNA data? I tend to think it does, but not being an expert in the field . . .And just out of curiosity, are the authors above colleagues of yours? You don't have to sacrifice your anonymity, just wondering due to both you and the above research group are located in Germany and you are both into aDNA.

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I think it strengthens neandertal studies to have more samples processed. I worry a great deal that if there are neandertals that yield more human like sequences that they are not reported because the labs dismiss them as contaminants and focus on the ones that look different....as to you second question, they were my colleagues several years ago..I arrived in the lab the very week the first neandertal yielded its first PCR product

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I could order the paper, but that would be abusing our grant money And, I'm far to lazy to go to a local uni library. But, just from your description of "messed up" I can picture what the peaks coming off of the sequence would look like (if that is what you are talking about). Anyway, fully understand the technical problems numts create.

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Do you know if I post the journal name volume number and date etc like a normal reference, if I post the figure here does that violate the copyright? If it does not I will scan it and post it. If it does..I'll re-do the alignment from the Genbank sequences...it is worth showing I think...wish all journals would switch to open access so I could just link to the data cheers,
M

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Do you know if I post the journal name volume number and date etc like a normal reference, if I post the figure here does that violate the copyright? If it does not I will scan it and post it.

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As far as I know, posting selected figures for no profit is considered "fair usage" under international copyright. Of course, you can't post the paper whole cloth, but selected quotes and figures sounds kosher to me.

Let's see if this works(had to edit out the figure..I could not resize it and it kept crashing my computer when I tried to open the page...sorry Loudmouth...I will either have to send you a reprint or email the figure to you via Percy)This message has been edited by Mammuthus, 05-11-2004 05:43 AM

can't you set the scanner to make a smaller copy?I resize pictures in an html editor (frontpage is microsoft version, there is also firstpage {HTML Editor, Website Builder & Web Design Software} and other freewares) then copy the html code to use.

MammuthusIf you have problems email it to me and I can resize it and provide you a link.

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Aslan is not a Tame Lion

Thanks to both you and RAZD for your suggestions. I am pressed for time and on my way to a series of meetings over the next two weeks so I will try to send the figure to Percy before I go and see if he can get it in appropriate format....this is a wonderful example of why academic publishing that is not free access is really crap ..all the BMC journals and PLoS are open access and you can access all figures and data...without paying..the author pays to publish instead.

Often not a good idea. What you are doing is instructing the browser to resize the picture. Browsers typically aren't optimized for that task.The best thing to do is resize it in animage editor.

I sent the figure off to the Percy/Admin to see if he can get it in at a size that is reasonable.