DNA

Another attempt for a direct radiocarbon date on the Tarkio Valley sloths failed.  Bob Feranec reports that the National Ocean Sciences AMS (Accelerator Mass Spectrometry) Facility at the Woods Hole Oceanographic Institution could not obtain a sufficient quantity of CO2 from the 2.5 milligram collagen sample that he collected via seven extractions from a molariform.

Extraction on the tooth proved to be a labor intensive process to even recover a milligram. The sample, NOSAMS # 81815 labeled 10 RSF C14 005, which was adequate by weight, yielded only 1.7 micromoles of C02. When asked if sacrifice of a whole tooth would produce a date, Bob replied that we needed to recover at least 50 micromoles. This would require at least 150 extractions, would be a few months work and still probably would not generate sufficient CO2 for our purposes. He concluded,  “I think that it is not going to be a datable specimen.”

Earlier, we had submitted a bone sample and a dental sample from the adult to the Keck Carbon Cycle Accelerator Mass Spectrometry facility at the University of California-Irvine. They could not recover enough collagen to date the specimen either. After attempts by two world class facilities, we are convinced that it is not possible to directly date sloth remains with current technology. Alternatives for direct dating are under consideration.  Pollen and seeds are in the matrix and offer radiocarbon alternatives. There is also the potential to date the sediments above and below the sloth-bearing matrix. 

 Exciting as they are, the Tarkio Valley sloths continue to be analytically evasive. 

Sloth on. Holmes

This news from Andy about our DNA tests:

Holmes,

I got a mitochondrial amp from one of the extracts, but I haven’t cloned it yet to check for contamination.  I wasn’t able to get nuclear amplifications using our current stock of primers.  It wouldn’t surprise me if we get endogenous DNA from this thing, but it may not be in high enough copy numbers to do much more than generate a few scraps.  If we can determine that there is endogenous DNA via old school PCR and cloning(what I’m trying to do now), then the next step might be large scale extracts.  We’d then use the concentrate to build libraries to go on GS (Genome Sequencer) FLX plates and try to shotgun generate at least a significant portion of the mitochondrial genome.

Beth Shapiro at Penn State has her sample. . . hope to hear from her soon.

(GS-FLX) is actually the way to go. . . . Old school targeted PCR characterization is plagued by a lot of issues, including low endogenous copy number, PCR inhibitors in the extract, contamination (from a bunch of possible sources)  and primer specificity. . . .

We would initially generate say ~100,000 reads of ~100bp apiece and then sort through those to determine which represent environmental contamination (bacteria, fungus, etc), human contamination and actual endogenous sloth DNA (if any). . . .  When you do it on that scale you can get a much better idea of what’s going on in the specimen/extract. You can then use that info to design species specific primers of appropriate base composition and target length.

Best

AC

Andy’s results aren’t unexpected. Finding ancient DNA is like winning the lottery–its almost always highly degraded and finding a specific segment still intact with the PCR process that he’s using is a long shot. That’s especially true for nuclear DNA (nDNA). A cell has only one nucleus, and one set of nDNA, but it has 1,000 or more mitochondria and thus 1,000X as many copies of the mDNA genes. We’ll keep our fingers crossed about the mDNA signal, but as we learned before, chances are good it’s contamination. Winning this lottery is hard even when you are holding 1,000 extra tickets. No one has ever succeeded in the Megalonyx DNA sequencing game.

Even if you are lucky enough to have your ancient DNA (aDNA) survive 12,000 years of oxidation, heat and humidity, the fundamental limitation of the traditional Polymerase Chain Reaction (PCR) process is the focus on specific genes.  Old school (i.e. targeted) PCR is like going fishing—there may be a lot of DNA swimming around but you’ll never know it if you are using the wrong “bait,” or primer.   Primers are synthesized strings of nucleotides–just 15-25 base pairs usually–designed to complement a specific DNA segment (the target).  Drop the primer in the test tube and if a DNA fragment with the complementary sequence is in there, chances are  they’ll link up  and you can reel them in–hopefully with a long piece of the ancient  genome  attached.  Andy’s primers are derived from tree sloths and have proven successful with other ground sloths, but millions of years of separate evolution increases the likelihood that a mutation will inhibit primer binding. 

Andy’s recommendation takes advantage of new technology that avoids many of the problems that come from working in a heterogeous soup of contaminants, inhibitors and aDNA fragments.  The rapid genome sequencer GS-FLX from Roche moves each DNA fragment in the extraction to an individual microscopic compartment before  PCR amplification, and then  sequences everything.   It’s a formidable task made easier by a fast computer and massively parallel lab-on-a-chip technology.  DNA scientists have assembled a huge database  of known DNA sequences from every kind of life-form; workers worldwide add new species to the GenBank library every day. Basic Local Alignment Search Tool (BLAST) software provides a quick means of searching the database and identifying unknown sequences.   The vast majority of the DNA Andy finds will be contamination, of course—from the vast array of life that moves in after death, browsing on the different organic molecules that remain, and each other.  Many of the sequences aren’t identifiable yet, but the success rate improves daily.

Holmes gave Andy the go-ahead. We have a great-looking bone and no one wants to give up yet.  It would have been nice if it had been buried in permafrost or protected inside a dry cave, but the thick clay blanket that we found it sealed in gives us as much hope as anyone has ever had with this species.  Stay tuned. . . . Dave

We received an urgent inquiry from Andy last week asking if we had used shellac on the ankle bone that we sent him. . . he was having problems dissolving the sample.  Standard procedure after cleaning and grinding up the bone  is soaking the fragments in ethylene diamine tetra acetic acid (EDTA) which dissolves the calcium and frees the DNA inside.   We assured Andy we hadn’t used shellac or anything else, anticipating his tests and not wanting to introduce a  foreign chemical that could interfere.   We suggested the mystery coating might be one of the most impregnable known to man—greasy fingerprints and peanut butter and jelly.  The bone is sturdy and it has been handled by at least 50,000 children!   He went back and remixed his solutions. From the email we received at the end of the week, it looks like we’re moving forward again:

Hey Holmes,

Acratocnus Ye was discovered in a sinkhole in Haiti in 1984, and subsequently at several other sites in the country.  “Ye,” pronounced “yeh,” is a Hatian Creole word meaning  “yesterday,” hence the common name: Yesterday’s Acratocnus. (MacPhee et al., 2000)  Radiocarbon dates show the Megalonychid sloths survived in Haiti until about 4,500 years ago when humans arrived on the island. (Steadman et al., 2005)

If Beth and Andy get DNA from the Acratocnus ye sample and fail with ours we can assume there’s probably no DNA in the ankle bone, but that doesn’t necessarily mean things are hopeless.  Conditions vary tremendously around the site and DNA preservation may have been favored in a different location.  We may have to explore trying some inexpensive bone screening tests to identify candidates for further analysis.

When you are doing a destructive test on an irreplaceable sample it’s comforting  to know  you have people like the staff at the McMaster Ancient DNA Centre  and the  Ancient DNA Lab at Penn State who are checking every step 3X before they proceed. . . . Dave

References

 MacPhee, R. D. E., White, Woods, C. A. 2000.  New Megalonychid sloths (Phyllophaga, Xenarthra) from the Quaternary of Hispaniola.  American Museum Novitates 3303:  1-32.

 Steadman, D. W., Martin, P. S., MacPhee, R. D. E., Jull, A. J. T., McDonald, H. G., Woods, C. A., Iturralde-Vinent, M., and Hodgins, G. W. L. 2005.  Asynchronous extinction of late Quaternary sloths on continents and islands.  Proceedings of the National Academy of Science 102: 11763-11768 

The experience Andy and his colleagues are getting with analyzing ancient bones, and using clean room facilities, body suits, facemasks, etc. to avoid contamination are the very skills that will be needed in twenty years when astronauts return from Mars  with samples to test for traces of ancient life.  [previous post: Ancient DNA Centre]

Then, as now, the challenge will be uncovering the biomolecular markers preserved inside and proving any positive findings derive from the sample and not mishandling.  So we study our sloths to better understand them and the effects of global climate change, and prepare for the future. . . and maybe to visit the stars. . . . Dave

We sent a bone sample for DNA testing to the McMaster Ancient DNA Centre, McMaster University, Hamilton, Ontario two weeks ago.   Andy Clack, a PhD student in the Centre, sent this encouraging reply:

Holmes,

I have the talus/ankle bone in the lab now… wow! That thing is like a rock.  I couldn’t ask for a better specimen!  I’m going to use a sterile dremel tool and a particular type of bit that generates less heat and produces shards of bone and not powder.

I’ll try to drill in about 1.5 inches, collect the powder, decalcify and then hit it with proK to kill any lingering proteins in there.  I’m going to make up fresh reagents this week.  I want everything to be right.  Will likely test for nuclear and mt DNA fragments next weekend.

It’s all about the specimens on your end, and you really came through with this one…. I won’t hurt it too bad, I promise.

Best,
AC

  Andy found all kinds of  DNA in the first samples we sent–horse, pig, cow, deer, human, etc.  No sloth DNA though.  The bones were small and broken–just fragments.  They had probably been soaking up rural Iowa DNA for years.  Now Andy has the thickest, densest, unbroken bone we have. The ICLIC people did a CT scan and it looks perfect inside too.

 Cloning DNA has become routine–the stuff of 7^th grade science classes, but contamination remains the biggest challenge of ancient DNA study. DNA is everywhere—people are constantly shedding it in dry skin cells, etc., not to mention what they carry around belonging to family members, pets, etc.,  and the microbes that live everywhere.  Chemical changes, water and warm temps begin breaking down DNA after death, while a host of decay organisms feed on the organic molecules and each other.  Retrieving verifiable ancient sequences from this rich DNA stew  after thousands of years is a major achievement.  Many an announcement of an ancient DNA discovery has been withdrawn when later analysis showed it merely to be the result of contamination.  For that reason special labs like the Ancient DNA Centre have been created with personnel trained in techniques designed to reduce the risk of errors.   No one has ever sequenced any part of the Megalonyx genome, but if anyone can do it, Andy can. . . . Dave

What kind of evidence of a disease would survive after 12,000 years?  As a cause of death among animals, disease is probably a much more significant factor than predation, especially if they are under stress, though given the penchant of predators to pick out the weak and infirm, it may be difficult to distinguish (Shipman, 1981).  A disease that could kill 3 sloths at once would have to have been dangerous and fast-acting, but can you prove it from fossils?

A hypervirulent disease has been suggested as a possible cause of the Pleistocene mass-extinction (MacPhee and Marx, 1997). The theory is humans or their dogs (or their fleas) brought a pathogen with them when they arrived in the New World, and while they were immune, the New World fauna was not, and large mammals died in unprecedented numbers–a dress rehearsal, if you will, for the devastation wreaked by measles, small pox, etc. when those pathogens arrived with Europeans 12,000 years later. But small pox didn’t exterminate dozens of species.

As a rule, disease-causing microbes adapt to a limited number of hosts with a life-style and genes that make them susceptible. Diseases that can infect widely disparate species are rare. MacPhee and Marx offer rabies and Leptospira as models for the plague microbe, though neither has the requisite characteristics today (e.g. aerosol transmission, range of hosts, etc.). You also need to assume some different social systems among the infected animals, i.e. more direct contact to maintain the plague. Of course, you don’t need to assume a super bug killed the Tarkio Valley Trio–a bad sloth-cold could have had the same effect if stress had left them vulnerable.

Proving the sloths died of a fast-acting acute disease is a challenge. Such diseases generally act on soft tissues, rarely do they leave an imprint on bone (Manchester, 1987). A long-term chronic disease has a better chance of leaving fossil evidence, but arthritis, or another such disease couldn’t kill three sloths at once. Does that just leave us with an interesting theory and no way to prove it? Maybe not.

The ability to detect disease directly in fossils took a giant leap forward in 2005 when Hendrik Poinar, in the Ancient DNA Center at McMaster University in Hamilton, Ontario, and an international team, succeeded in sequencing 28 million base pairs (bp) of DNA from a well-preserved Siberian wooly mammoth jaw (Poinar et al., 2006).  In a field where getting a few hundred bp of DNA is viewed as a significant achievement, they recovered 13 million bp of the mammoth’s genome. The remaining sequences were traced to a wide range of internal viruses and bacteria, a number of soil organisms, and even the mammoth’s last meal. Poinar’s team used a database of DNA sequences on file at the National Center for Biotechnology Information (NCBI), and software they developed, to identify the DNA sequences down to the species level in many cases.  

Sequencing DNA from our fossils is a long shot. The mammoth was preserved under ideal conditions. Iowa’s wet temperate climate makes finding DNA extremely unlikely. However, the NSF wants us to try and Poinar has agreed to help. Who knows what we’ll discover along the way–hopefully proof we have a sloth family, and maybe entire internal and external ecosystems in those fragile strands . . . . Dave

References

Poinar, HN, Schwarz, C, Qi J, Shapiro, B, MacPhee, RDE, Buigues, B, Tikhonov, A, Huson, DH, Tomsho, LP, Auch, A, Rampp, M, Miller, W, Schuster, SC. 2006. Metagenomics to Paleogenomics: large scale sequencing of Mammoth DNA. Science 311(5759): 392-294.
http://www.sciencemag.org/cgi/content/full/1123360?DC1#REF14

Rowe, B. 2005. Re disease and mass extinction The sneeze heard ’round the world: disease and the great Pleistocene extinction. Abstracts, Society for American Archaeology 70th Annual Meeting, March 30-April 3, 2005, Salt Lake City. P. 254.

MacPhee, R.D.E. and Marx, P.A. 1997. The 40,000 year plague: humans, hyperdisease and first-contact extinctions. In Natural Change and Human Impact in Madagascar. S. Goodman and B. Patterson (eds.) Smithsonian Institution Press.

Manchester, K. 1987. Skeletal evidence for health and disease. In Death, decay and reconstruction: Approaches to archaeology and forensic science. A. Boddington, AN Garland and RC Janaway (eds.) Manchester University Press.

National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html

Shipman, P. 1981. Life History of a Fossil: An Introduction to Taphonomy and Paleoecology, Harvard University Press.