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- 1. “Great” Grandma and You:Methods of Analyzing Human MtDNA Substitution Rate BY SETH NELSON THURSDAY, OCTOBER 8TH, 2015 1
- 2. Outline I. Mitochondria andDNA II. MtDNA anatomy and replication III. Methods of finding substitution rate IV. Improvement on current findings V. Using the rate 2
- 3. I. Mitochondria and DNA I.MITOCHONDRIA AND DNA II. MTDNA ANATOMY III. METHODS OF FINDING SUBSTITUTION RATE IV. IMPROVEMENT ON CURRENT FINDINGS V. USING THE RATE 3
- 4. Mitochondria Endosymbionts with proto-eukaryotes (Anderssonet al., 2003) Applications in forensics and evolutionary relatedness Need to know mutation rates for accurate judgment 4 Figures: Sadava et al. (2005)
- 5. Using DNA forphylogenetic inference Figure: García et al. (2011) 5
- 6. Why use mtDNA More mtDNA copies than nDNA (Robin and Wong, 1988) Mitochondria are inherited from mother (Schwartz and Vissing, 2003) High mutation rate, good for closely related individuals (Butler and Levin, 1998) Image from https://www.thermofisher.com/us/en/home/technical-resources/research-tools/image- gallery/image-gallery-detail.2643.html 6
- 7. II. MtDNA Anatomy and Replication I.MITOCHONDRIA AND DNA II. MTDNA ANATOMY AND REPLICATION III. METHODS OF FINDING SUBSTITUTION RATE IV. IMPROVEMENT ON CURRENT FINDINGS V. USING THE RATE 7
- 8. Anatomy of mtDNA Figure:Pakendorf and Stoneking (2005) Transfer RNAs NADH Dehydrogenase subunits Cytochrome c Oxidase subunits Cytochrome bRibosomal RNAs ATP Synthase subunits 8
- 9. Control region Controlsreplication No protein product Two hypervariable regions Figure: Pakendorf and Stoneking (2005) 9
- 10. Beginning of replication Initiatesat heavy strand origin Light strand synthesis follows Figure: Clayton (2000) 10
- 11. Mutations in replicationof DNA Insertion Deletion Frameshift Substitution Transition Transversion Figure: Sadava et al., 2011 11
- 12. Substitutions happen atspecific rates Substitutions per site per million years Numerator: Number of sequence differences, only counting substitutions That is, no insertions, deletions, etc. Denominator: Time since last common ancestor between sequences of comparison 12
- 13. III. Methods of Finding Substitution Rate I.MITOCHONDRIA AND DNA II. MTDNA ANATOMY III. METHODS OF FINDING SUBSTITUTION RATE I. Considerations II. Pedigree III. Phylogenetic IV. IMPROVEMENT ON CURRENT FINDINGS V. USING THE RATE 13
- 14. Secondary structure forms Light strand forms loop structure (Pereira et al., 2008) Selective pressure on control region Figure: Pereira et al. (2008) 14
- 15. MtDNA can recombine Mitochondria possess recombinase activity (Thyagarajan et al., 1996) Does not affect substitution rate (Kraytsberg et al., 2004) 15 Figure: Thyagarajan et al. (1996)
- 16. Some paternal inheritance Single case of paternal inheritance in man (Kraytsberg et al., 2004) Figure: Kraytsberg et al. (2004) 16
- 17. Pedigree analysis isdirect observation Analyze mtDNA from closely related individuals English family with Leber’s hereditary optic neuropathy Age of last common ancestor is known with certainty Figure: Howell et al. (2003) 17
- 18. Less time meansfewer mutations Pedigree analysis tends to count fast mutations Potentially overestimate substitution rate 18
- 19. Phylogenetic analysis usesequations Analyze mtDNA from distantly related individuals Primates, back to chimp and human CA Figure: Hasegawa et al. (1993) 19
- 20. Equations as estimates Use of equations for rate Transition rate: 𝑣 𝑆 = 2 𝜋 𝑇 𝜋 𝐶 + 𝜋 𝐴 𝜋 𝐺 𝛼 Transversion rate: 𝑣 𝑉 = 2 𝜋 𝑇 + 𝜋 𝐶 𝜋 𝐴 + 𝜋 𝐺 𝛽 Substitution rate: 𝑣 𝑎𝑣𝑒 = 𝑓 𝑣 𝑆 + 𝑣 𝑉 20
- 21. More time meansmore uncertainty Denominator more uncertain Phylogenetic analysis counts all substitutions since last CA Reversions will cause undercount in mutations Need methods of calibration 21
- 22. Nodes vs. tips 22 Figure:Rieux et al. (2014)
- 23. Calibration affects rates 23 Figure:Rieux et al. (2014)
- 24. Noncoding region ishigher than coding region Pedigree rate is higher by order of magnitude Rates are in substitutions per site per million years Method Noncoding Region Coding Region Pedigree (99.5% CI) 0.475 (0.265-0.785)a 0.15 (0.02-0.49)a Phylogenetic (±1 Std Error) 0.033 (0.027-0.039)b 0.0170 (--)c 24 Pedigree rates from Howell et al. (2003) Phylogenetic noncoding from Hasegawa et al. (1993) Phylogenetic coding from Ingman et al. (2000)
- 25. Pedigree is higherthan phylogenetic Method Weighted rate Pedigree 0.17 Phylogenetic, Tip 0.021 Phylogenetic, Node 0.018 25 Pedigree rate from Howell et al. (2003) Phylogenetic, tip-calibrated rate from Rieux et al. (2014) Phylogenetic, node-calibrated rate from Hasegawa et al. (1993) & Ingman et al. (2000) *Rates are in substitutions per site per million years
- 26. Context is everything(Pääbo, 1996) Phylogenetic rate: Common ancestor is >100,000 years ago Pedigree rate: Common ancestor in <10,000 years ago 26
- 27. IV. Improvement on Current Findings I.MITOCHONDRIA AND DNA II. MTDNA ANATOMY AND REPLICATION III. METHODS OF FINDING SUBSTITUTION RATE IV. IMPROVEMENT ON CURRENT FINDINGS V. USING THE RATE 27
- 28. Bringing the ratestogether 𝑅𝑎𝑡𝑒 𝑐𝑜𝑑𝑖𝑛𝑔 = 0.5204𝑒−2.042𝑡 + 0.0144 28 Figure: Ho et al. (2005)
- 29. Bringing the ratestogether 𝑅𝑎𝑡𝑒 𝑛𝑜𝑛𝑐𝑜𝑑𝑖𝑛𝑔 = 0.4535𝑒−6.408𝑡 + 0.0148 29 Figure: Ho et al. (2005)
- 30. A better outgroupis in the nucleus MtDNA integrated into nucleus 540 bp segment Identical in all tested genomes (Zischler et al., 1995) 30 Figure: Zischler et al. (1995)
- 31. V. Using theRate I. MITOCHONDRIA AND DNA II. MTDNA ANATOMY AND REPLICATION III. METHODS OF FINDING SUBSTITUTION RATE IV. IMPROVEMENT ON CURRENT FINDINGS V. USING THE RATE 31
- 32. Use in forensics Forensic applications focus on HV1 and HV2 Romanov identification (Butler and Levin, 1998) Tsarina, her daughters, Prince Philip were exact matches One mismatch for Tsar Nicholas II and relatives “Anastasia” did not match 32
- 33. Dating divergence Estimatingcommon ancestor of Neanderthals and Humans 33 Figure: Ho et al. (2005)
- 34. Unrelated to “Great”Grandma How far back in time do we need to go to be “unrelated” to our ancestors? 1.1% (12 bp) difference in unrelated control sequences (Piercy et al., 1993) Roughly 1000 generations before we are unrelated to our ancestors 34
- 35. Knowing this, welook deeper Substitution rate is effectively variable Temporally and spacially Allows a second look at archeological dates Could help us understand relationships better Methods used in mtDNA could be extended 35
- 36. Acknowledgements Thank you! Friends Family Chris Cole and rest of biology faculty Everyone else here 36
- 37.
Editor's Notes
- #2 Have you ever wondered how related you are to your ancestors?
- #5 Plant mtDNA is very different, as in much larger, but not necessarily more genes. Possibly baggage from evolution Plants have more than 150kb length Cats are smaller, like humans around 16kb Inherited by mainly seed parent, except in conifers (maybe all gymnosperms) Mitochondria are present in cytoplasm of ovum, not in sperm head
- #6 Each number is a sequence polymorphism at that site that is unique to each sample beneath it.
- #7 Green fibers are mitochondria Blue is actin filaments Orange is nucleus
- #9 Cytochrome c oxidase: 3 subunits from mitochondria, 11 from nucleus NADH dehydrogenase: 7 subunits from mitochondria, 37 from nucleus ATP Synthase: 2 subunits from mitochondria, others from nucleus All involved in electron transport chain
- #10 Origin of replication of Heavy strand Does not contain genes Two main area of hypervariation, mutate faster than rest of control region
- #11 Heavy strand is first replicated (high in purines, A and G) There is some lag until light strand is synthesized Displacement-loop structure forms D-loop is whole control region without promoters (HSP and LSP)
- #12 Transition: purine (A ↔ G) or pyrimidine (C ↔ T) Transversion: purine to pyrimidine or vice versa Transition Transition, Transversion Both Transversion Transition
- #13 Sequence differences are between two sequences of comparison (for example, chimps and humans)
- #15 93 bp segment that is statistically selected for (using Tajima’s D-value) Occurs when D-loop is single strand during replication lag
- #16 Fractions were normalized to fraction 3 Black bars: cytochrome-c oxidase activity - Gilford Response spectrophotometer White bars: homologous DNA recombination – mitochondrial DNA recombined with plasmids Homologous recombination between maternal mito molecules is invisible
- #17 Red is maternal sequence, blue is paternal sequence
- #18 Five generations, four females and current one LHON affects sensation at peripheral nerves, not mitochondrial disease, but using pedigree as if it was Circles are female, squares are male—filled shapes visibly suffer form LHON Numbers bolded, italicized had sequences analyzed Focus on 9 and 31
- #19 Due to brevity of pedigrees (five generations is quite a long pedigree)
- #20 Numerator is more certain, as there is more time for mutations to occur
- #21 where 𝜋 𝑋 is the frequency of nucleotide 𝑋, and 𝛼 and 𝛽 are parameters that determine transition rate and transversion rate, respectively. Denominator is less certain
- #22 Denominator often in millions of years, with significance to maybe .5 million years
- #23 Used BEAST, Bayesian Evolutionary Analysis Sampling Trees
- #24 Hasegawa et al. calibrated with external node of last common mtDNA ancestor
- #25 Noncoding is higher than coding
- #27 Extrapolation
- #31 MtDNA mutates so quickly phylogenetic information can be wiped out by so many substitutions Mutates very slowly since unchanged
- #33 Prince Philip’s great-grandmother was Tsarina’s sister Is the mismatch of Anastasia outside the realm of possibility, knowing mitochondria change through generations?
- #34 Using transitionary rate
- #35 22,000 years