2. Recovery and analysis of old/ancient DNA: molecular archaeology/anthropology Why study ancient DNA (aDNA)? Obtaining aDNA Early studies of aDNA Guidelines for studying aDNA Reconstructing extinct gene sequence Hofreiter et al.(2001) Nature Reviews Genetics 2, p. 353 Thornton (2004), Nature Reviews Genetics 5, p. 366.

3. What is aDNA, and why the interest? aDNA is isolated from archaeological, paleontological remains, museum specimens, etc. aDNA potentially a rich source of information for molecular evolution studies –Compare DNA sequences of modern organisms to ancestral organisms –trace speciation at the molecular level –great interest in human evolution/speciation aDNA can be used to define animal diets

4. DNA sequences from extinct animals-- a time line

5. What happens to DNA following death? Typically decays quickly: nucleases, microbial decomposition Occasionally DNA is spared this fate: –Rapid dessication –Low temperatures –High salinity Slow decay: –Depurination (loss of A and G bases) –Oxidative damage –Hydrolytic damage

6. Sites in DNA affected by long-term chemical processes

7. Effects of DNA damage Backbone breakage--fragmentation C and T residues converted to hydantoins, blocking DNA polymerases (PCR) Deamination of C causes wrong base to be added during PCR--false mutations Increasing time, increasing degradation, decreasing utility 100,000 to 1,000,000 years considered the age limit for DNA to yield useful sequences

8. SB Carroll (2003) Nature 422 p. 849

9. First retrievals of old DNA Quagga (extinct relative of the zebra) DNA cloned from museum specimen (Higuchi et al. 1984) 2430 year-old Mummy DNA cloned (Paabo 1985) 1) Isolate DNA (20 micrograms/gram mummy tissue 2) Treat with Klenow enzyme (DNA polymerase) to make DNA fragments blunt ended 3) Cloned into alkaline phosphatase treated pUC8 (pMUM plasmids) ***But cloning presents problems, eg. repair of DNA following transformation (leading to false mutations)

10. Revolution in ancient DNA isolation: PCR PCR--get a lot from a little, sequence PCR products directly (no cloning artifacts to worry about) Permits targeted studies of specific genes or DNA regions –Mitochondrial DNA is typical target in aDNA PCR isolations –Copy number of mitochondria is high relative to nuclear DNA

11. Identifying ancient remains that are likely to yield good PCR Amino acid racemization: conversion of L-amino acids to D-amino acids Rate depends on water, temperature, chelated metal ions (things that also affect rate of DNA depurination) The higher the D/L ratio, the less likely that DNA can be isolated: >0.08, no DNA will be isolated Calculation of D/L ratios is easy, rapid

12. Cautions for PCR of ancient DNA (particularly human remains)

14. Fossilized poo (coprolites): a rich source of information To recover DNA that can by amplified by PCR: treat with a reagent that breaks sugar cross-links

15. Two coprolite DNA studies From ground sloth coprolites: pine forests present in south Nevada 28,500 years ago, but by 20,000 years ago pine forests likely gone Analysis of diet of ancient humans: one study found 8 different plants (looking at mitochondrial DNA) and meat from several types of large animals

16. New advances in ancient DNA sequencing Mammoth sequencing Complete mitochondrial sequence obtained: “multiplex” PCR (simultaneous amplification of many targets at once-- good for minimal DNA situations) 28 million base pairs of mammoth “metagenome” sequence obtained, using very short sequences and the pyrosequencing technique (massively parallel short sequencing runs) “the high percentage of endogenous DNA recoverable from this single mammoth would allow for completion of its genome, unleashing the field of paleogenomics”

17. Previous sequencing techniques: one DNA molecule at a time New: many DNA molecules at a time -- arrays One example: “pyrosequencing” Cut a genome to DNA fragments 300 - 500 bases long Immobilize single strands on a very small plastic bead (one piece of DNA per bead) Amplify the DNA on each bead to cover each bead to boost the signal Separate each bead on a plate with up to 1.6 million wells

18. Sequence by DNA polymerase -dependent chain extension, one base at a time in the presence of a reporter (luciferase) Luciferase is an enzyme that will emit a photon of light in response to the pyrophosphate (PPi) released upon nucleotide addition by DNA polymerase Flashes of light and their intensity are recorded

19. Extension with individual dNTPs gives a readout A B A B The readout is recorded by a detector that measures position of light flashes and intensity of light flashes

20. APS = Adenosine phosphosulfate From www.454.com 25 million bases in about 4 hours

21. Height of peak indicates the number of dNTPs added This sequence: TTTGGGGTTGCAGTT

22. Human evolution and DNA sequencing 1) Compare sequences among all living humans (gene flow, origins, the sum total of human variation) 2) Compare sequences between us and closest living ancestor -- Pan troglodytes, chimpanzee -- where are the differences, which genes were selected during this divergence? 3) Compare sequences between us and other extinct hominids -- Homo neanderthalensis

23. The way we were…. (?) (The genome(s) of Homo neanderthalensis) Mitochondrial genome: Paabo et al, 1997 Neanderthals split from modern human lineage ~500,000 years ago (Homo sapiens: about 200,000 years ago, Homo sapiens sapiens, about 45,000 years ago) Neanderthals and humans: coexisted until about 30 - 40,000 years ago Did interbreeding occur?

24. Model for the expansion of modern humans neanderthal Modern human N = generations

25. Hypothetically up to 25% interbreeding rate But there are no Neanderthal mitochondria in modern Europeans (thousands tested) This is consistent with a less than 0.1% interbreeding rate -or- A higher rate of interbreeding but sterility of interbreeding outcomes

26. Value of a complete Neanderthal genome? 1)Which changes in human relative to the chimpanzee genome are recent? 2) Where have “selective sweeps” occurred in the human genome since divergence of Neanderthals? (selective sweep: reduction of variation in genomic DNA adjacent to a mutation that is under powerful selective pressure) 3)What was Neanderthal biology like?

27. Neanderthal nuclear sequences -- what were Neanderthal genetics like? How did they compare to modern humans? “Amplification-independent direct cloning” (no PCR artifacts) “Metagenome” from 38,000 year old bone

28. Average fragment size ~ 50 bp. 65250 bp total (NE1) Pyrosequencing versus Sanger: 99.89% agreement How do we know it’s Neanderthal (not modern human contaminants)? 1)Modern human mito contamination: ~2% 2)Damage “signatures” of ancient DNA 3)Hominid sequences: human-specific changes (that differ from chimpanzee sequences) were often not present in the Neanderthal sequence

29. Vi80: Vindja Cave, Croatia 1 million bp of Vi-80 were sequenced

30. Majority of DNA from old samples: organisms that colonize after death…

31. Distribution of neanderthal DNA sequence by chromosome matches expected frequencies based on lengths of human chromosomes Also--the fossil was from a male

32. …of the way we were Memories… Proposed time line for divergence

33. FOXP2: a key gene in human evolution (language and speech)? Mutation of this gene leads to deficits in “linguistic processing” and “orofacial movements” Two specific mutations in FOX2P are “fixed” in humans compared to chimpanzees There was a relatively recent (200,000 years ago) “selective sweep” in the FOX2P region of the human genome (selective sweep: a region of the genome that stays relatively unchanged because of selective pressure on beneficial mutations within that region)

35. Implication: FOXP2 variants not a guarantee of survival Other mutations necessary as well? (brain structure/function?) Neanderthals perhaps could talk, but may have had little interesting to say…

36. Resurrecting extinct genes: the phylogenetic approach What is (was) the function of an ancestral/ intermediate form of today’s genes? Few molecular fossils exist, and they don’t go back very far in time BUT Methods exist for inferring ancestral gene sequence The inferred ancestral gene can be synthesized, cloned, the gene can be overexpressed, the protein can be purified and studied…

38. How to infer ancestral gene sequence? Maximum likelihood analysis: A phylogenetic tree is constructed At any internal node, each possible ancestral state is evaluated for its likelihood of yielding the present day sequences Highest likelihood gives the best guess for ancestral sequence

39. A success story: ancestral bacterial EF-Tu (>1 billion years ago) At which temperature does the ancestral EF- Tu function best (were early bacteria thermophiles?) The reconstructed ancestral EF-Tu binds to GTP best at 65°C, suggesting a thermophilic ancestor to bacteria

40. Caveats: 1) How good is your Max. Likelihood prediction? Perform many predictions and compare the results--do they give similar results? 2) Is the function of the ancestral protein being assayed under relevant conditions? What if the protein functions as part of a complex?