David Gilichinsky
Duane Froese
Eske Willerslev
Sarah Stewart Johnson
Maria T. Zuber
Martin B. Hebsgaard
& Rasmus Nielsen
Kasper Munch
M. Thomas P. Gilbert
Regin Rønn
Tina Brand
Torben R. Christensen
& Mikhail Mastepanov
Michael Bunce
What got me interested?
image credit: http://ssed.gsfc.nasa.gov/tharsis/ngs.html
What got me interested?
image credit: http://ssed.gsfc.nasa.gov/tharsis/ngs.html
What may have gotten (two of) the authors interested?
• Has extracted and analyzed ancient DNA from:
• Siberian permafrost cores (chloroplast (300-400 ka) & vertebrate mtDNA
Eske Willerslev
(20-30 ka) (1.5-2 Ma, no amplification))1
• Siberian mammoth bone (melanocortin type 1 receptor gene(~43 ka))2
• Silty base of Greelander ice core (chloroplasts & invertebrate mtDNA
(~450-800 ka [wide range]))3
• Explored the discrepancy between theoretical calculations for DNA survival, and
published data showing the persistence of bacterial DNA.4
• Research projects broadly focus on:
• “modeling global processes on early Mars”5
• “detection of biosignatures in ancient environments” 5
Sarah Stewart Johnson
1) Willerslev et al. 2003. Science. 300: 791
2) Römpler et al. 2006. Science. 313: 62
3) Willerslev et al. 2007. Science. 317: 111
4) Willerslev et al. 2004. Curr. Biol. 14: R9
5) http://www.mit.edu/~ssj/bio.html
What may have gotten (two of) the authors interested?
• Has extracted and analyzed ancient DNA from:
• Siberian permafrost cores (chloroplast (300-400 ka) & vertebrate mtDNA
Eske Willerslev
(20-30 ka) (1.5-2 Ma, no amplification))1
• Siberian mammoth bone (melanocortin type 1 receptor gene(~43 ka))2
• Silty base of Greelander ice core (chloroplast & invertebrate mtDNA
(~450-800 ka [uncertain]))3
• Explored the discrepancy between theoretical calculations for DNA survival, and
studies showing the persistence of bacterial DNA.4
• Research projects broadly focus on:
• “modeling global processes on early Mars”5
• “detection of biosignatures in ancient environments” 5
Sarah Stewart Johnson
1) Willerslev et al. 2003. Science. 300: 791
2) Römpler et al. 2006. Science. 313: 62
3) Willerslev et al. 2007. Science. 317: 111
4) Willerslev et al. 2004. Curr. Biol. 14: R9
5) http://www.mit.edu/~ssj/bio.html
What may have gotten (two of) the authors interested?
• Has extracted and analyzed ancient DNA from:
• Siberian permafrost cores (chloroplast (300-400 ka) & vertebrate mtDNA
Eske Willerslev
(20-30 ka) (1.5-2 Ma, no amplification))1
mtDNA
present
in manytype
copies/nucleated
• •Siberian
mammoth
bone (melanocortin
1 receptor gene(~43 ka))2
• cell
Silty base of Greelander ice core (chloroplast & invertebrate mtDNA
(~450-800 ka [uncertain]))3
• high
copy number
facilitates
retrieval
• Explored
the discrepancy
between theoretical
calculations
for DNA survival, and
studies showing the persistence of bacterial DNA.4
•
• maternal mode of inheritance is ideal
for studying
ancestor/descendent
Research
projects broadly
focus on:
• relationships
“modeling global processes on early Mars”5
• “detection of biosignatures in ancient environments” 5
Pääbo et al. 1989. J. Biol. Chem. 9709
Sarah Stewart Johnson
1) Willerslev et al. 2003. Science. 300: 791
2) Römpler et al. 2006. Science. 313: 62
3) Willerslev et al. 2007. Science. 317: 111
4) Willerslev et al. 2004. Curr. Biol. 14: R9
5) http://www.mit.edu/~ssj/bio.html
4 Key Issues
1.
in vitro based predictions
4 Key Issues
1.
in vitro based predictions
2.
bacterial DNA and culturable
cells from million year old
specimens
4 Key Issues
1.
in vitro based predictions
2.
bacterial DNA and culturable
cells from million year old
specimens
3.
contamination
4 Key Issues
1.
in vitro based predictions
2.
bacterial DNA and culturable
cells from million year old
specimens
3.
contamination
4.
authenticity
in vitro based predictions & supporting data
Post-mortem DNA decay
• DNA normally degraded by endogenous nucleases1
• Dessication, low temperature or high salt can inactivate nucleases1
• leads to slower, but persistant, DNA decay through oxidation
and hydrolysis1
1) Hofreiter et al. 2001. Nat. Rev. Genetics. 2: 353
in vitro based predictions & supporting data
Post-mortem DNA decay
G
C
T
A
Lindahl, T. 1993. Nature. 362: 709
in vitro based predictions & supporting data
Post-mortem DNA decay
rate constant for depurination of DNA: 4 x 10-9 sec -1 (70° pH: 7.4)1
2
1) Lindahl and Nyberg. 1972. Biochemistry. 11: 3610
2) Pääbo and Wilson. 1991. Curr. Biol. 1:45
in vitro based predictions & supporting data
Post-mortem DNA decay
• DNA crosslinking also contributes to DNA decay
Pääbo. 1989. PNAS. 86:1939
in vitro based predictions & supporting data
Post-mortem DNA decay
• DNA crosslinking also contributes to DNA decay
• avg. time until dsDNA chain breaks at an apurinic site:190 h (37° pH: 7.4)1
• Taking above lesion frequency, and calculating rate constant, determine:
• max. survival time of amplifiable 120-bp fragments of bacterial 16S
rDNA is ~ 400 ka.2
• Also, assaying samples from different ages shows direct relationship
between DNA damage and sample age. 2
1) Lindahl and Andersson. 1972. Biochemistry. 11: 3618
2) Hansen et al. 2006. Genetics. 173: 1175
in vitro based predictions & supporting data
Post-mortem DNA decay
Summary & General Conclusions
• Short (less than 500 bp) pieces of DNA persist longer
Range of 4 years (A) to 13,000 years (C)
Pääbo. 1989. PNAS. 86:1939
in vitro based predictions & supporting data
Post-mortem DNA decay
Summary & General Conclusions
• Short (less than 500 bp) pieces of DNA persist longer
• DNA decay rates are slowed ~threefold with every 10°C drop in temp.1
1) Lindahl, T. 1993. Nature. 362: 709
in vitro based predictions & supporting data
Post-mortem DNA decay
Summary & General Conclusions
• Short (less than 500 bp) pieces of DNA persist longer
• DNA decay rates are slowed ~threefold with every 10° drop in temp.1
• The oldest authenticated DNA found is possibly ~ 800 ka
Eske Willerslev
origin of
samples
oldest authentic
DNA
largest amplicon
samples yielding
no ampilcons
Siberian
permafrost 2
300 - 400 ka
280 bp
1.5 - 2 Ma
Siberian
mammoth bone 3
43 ka
138 bp
n/a
Silty base of
Greelander ice 4
450 - 800 ka
120 bp
n/a
1) Lindahl, T. 1993. Nature. 362: 709
2) Willerslev et al. 2003. Science. 300: 791
3) Römpler et al. 2006. Science. 313: 62
4) Willerslev et al. 2007. Science. 317: 111
in vitro based predictions & supporting data
Post-mortem DNA decay
Summary & General Conclusions
• Short (less than 500 bp) pieces of DNA persist longer (show gel)
• DNA decay rates are slowed ~threefold with every 10° drop in temp.1
• The oldest authenticated DNA found is possibly ~ 800 ka
Eske Willerslev
origin of
samples
oldest authentic
DNA
largest amplicon
samples yielding
no ampilcons
Siberian
permafrost 2
300 - 400 ka
280 bp
1.5 - 2 Ma
Siberian
mammoth bone 3
43 ka
138 bp
n/a
Silty base of
Greelander ice 4
450 - 800 ka
120 bp
n/a
permafrost 5
400-600 ka
4 kb
740 ka- 1 Ma
1) Lindahl, T. 1993. Nature. 362: 709
2) Willerslev et al. 2003. Science. 300: 791
3) Römpler et al. 2006. Science. 313: 62
4) Willerslev et al. 2007. Science. 317: 111
5) Stewart Johnson et al. 2007. PNAS. 104:14401
in vitro based predictions & supporting data
Post-mortem DNA decay
Summary & General Conclusions
• mention:
origin of
samples
frozen antarctic
penguin bone
age of bone
amplicon size
how many bones
yielded DNA
523 yr
1600 bp
n/a
< 2000 yr
663 - 1042 bp
35%
> 2000 yr
390 bp
45%
Total number of bones yielding sequences: 96
image credit: http://wwwstaff.murdoch.edu.au/~mbunce/
Lambert et al. 2002. Science. 295: 2270
4 Key Issues
1.
in vitro based predictions
2.
bacterial DNA and culturable
cells from million year old
specimens
Bacterial cells or DNA from million year old specimens
origin of sample
source of
inoculum
result
age
bees preserved in
amber1
bee tissue
culture Bacillus sp.
25 - 40 Ma
Siberian
permafrost core2
fractured
permafrost
bacteria were
cultured and
characterized
1.8 - 3 Ma
brine inclusion
of a salt crystal3
brine water
culture Bacillus sp.
250 Ma
ancient halite4
halite, brine
inclusion, both?
16S rDNA
65 - 425 Ma
1) Cano and Borucki. 1995. Science 268:1060
2) Shi et al. 1997. Microb. Ecol. 33: 169
3) Vreeland. 2000. Nature. 407: 897
4) Fish. 2002. Nature. 417: 432
4 Key Issues
1.
in vitro based predictions
2.
bacterial DNA and culturable
cells from million year old
specimens
3.
contamination
Contamination?
A close look at Cano and Borucki. 1995. Science 268: 1060
image credit: R.J. Cano; http://www.apsnet.org/education/feature/ancientdna/
Contamination?
A close look at Cano and Borucki. 1995. Science 268: 1060
Procedure to recover bee tissue
1.Decontaminated, laminar flow hood
2.Amber chemically surface sterilized and cracked, and tissue harvested
3.Inoculate trypticase soy broth
image credit: R.J. Cano; http://www.apsnet.org/education/feature/ancientdna/
Contamination?
A close look at Cano and Borucki. 1995. Science 268: 1060
Procedure to test for contamination
1.Inoculate TSB with:
a. solutions used for sterilzation
b.pieces from exterior or interior of the amber
2.Expose 3 TSA plates in the hood throughout tissue removal process
3.Soak (overnight) 11 pieces of amber (without inclusions) in a sporulated B.
subtilis culture.
a.surface sterilize the amber and transfer to TSB
4.Surface sterilize two pieces of amber (without inclusions) and simulate tissue
removal procedure on exterior and interior of amber for ~45 min
incubate all aerobically: 2 weeks at 35°C
image credit: R.J. Cano; http://www.apsnet.org/education/feature/ancientdna/
Contamination?
A close look at Cano and Borucki. 1995. Science 268: 1060
Procedure to test for contamination
1.Inoculate TSB with:
a. solutions used for sterilzation
b.pieces from exterior or interior of the amber
2.Expose 3 TSA plates in the hood throughout tissue removal process
3.Soak (overnight) 11 pieces of amber (without inclusions) in a sporulated B.
subtilis culture.
a.surface sterilize the amber and transfer to TSB
4.Surface sterilize two pieces of amber (without inclusions) and simulate tissue
removal procedure on exterior and interior of amber for ~45 min
incubate all aerobically: 2 weeks at 35°C
is this convincing?
image credit: R.J. Cano; http://www.apsnet.org/education/feature/ancientdna/
is this convincing?
•If so, how did the cell(s), let alone the DNA survive?
is this convincing?
•If so, how did the cell(s), let alone the DNA survive?
•they were spores...
•are spores protected from DNA damage?
is this convincing?
•If so, how did the cell(s), let alone the DNA survive?
•they were spores...
•are spores protected from DNA damage?
Protecting spore DNA
•low permeability to DNA-damaging agents
•Three other key reasons:
Protecting spore DNA
Repairing spore DNA
•Spores contain enzymes for DNA repair; however, their action
in a dormant spore is highly unlikely
•dormant spores have no detectable metabolism
•they lack ATP and nucleoside triphosphates
•low water content limits enzyme activity
•DNA damage generated during dormancy is repaired only when
the spore germinates.
•balance between accumulated damage and repair capacity
So, spore DNA is protected, but for how long?
•Using previously discussed depurination rate (not accounting
for special spore adaptations), we would expect more spores to
die than what is observed.
•>50% of spores survive in water for one year at 10°C
•There are no direct experimental measures for spore survival
over extended time periods, however:
•data indicates spores remain viable for decades
•possibly centuries or millenia
•Millions of years....”controversial”
Nicholson et al. 2000. MMBR. 64: 548
Setlow, P.. 1995. Annu. Rev. Microbiol. 49: 29
4 Key Issues
1.
in vitro based predictions
2.
bacterial DNA and culturable
cells from million year old
specimens
3.
contamination
4.
authenticity
Authenticity
• Contamination may confound results, so develop a
set of criteria such that results may be “authentic”.
Willerslev et al. 2004. Trends Ecol. Evol. 19: 141
Authenticity
• Contamination may confound results, so develop a
set of criteria such that results may be “authentic”.
Willerslev et al. 2004. Trends Ecol. Evol. 19: 141
• concerned with contamination
• previous studies showed no relationship between sample age and
microbial population
• however, different strains resist DNA degradation differently
• This study aimed to test DNA durability for diverse strains preserved
under “optimal frozen conditions”
A) DNA conc. (using Picogreen fluorescence
assay) as a function of permafrost age
• Results:
• Non-sporforming Actinobacteria (genus: Arthrobacter) are most durable
• more so than gram-positive spore-formers
• more so than gram-negatives, (mostly Proteobacteria)
A) DNA conc. (using Picogreen fluorescence
assay) as a function of permafrost age
• Results:
• Non-sporforming Actinobacteria (genus: Arthrobacter) are most durable
• more so than gram-positive spore-formers
• more so than gram-negatives, (mostly Proteobacteria)
• Surprising! Why is a non-sporulating organism most durable
• 3 suggestions: (i) DNA repair, (ii) a dormancy adaptation, (iii) structural
feature of the DNA.
David Gilichinsky
Duane Froese
Eske Willerslev
Sarah Stewart Johnson
Maria T. Zuber
Martin B. Hebsgaard
& Rasmus Nielsen
Kasper Munch
M. Thomas P. Gilbert
Regin Rønn
Tina Brand
Torben R. Christensen
& Mikhail Mastepanov
Michael Bunce
Can cold bacteria repair their DNA to remain
viable over geologic timescales?
David Gilichinsky
Duane Froese
1) Look at size of DNA fragments
rationale: long fragment = repair
Eske Willerslev
2) Look for signs of metabolic activity
rationale: repair = life
Sarah Stewart Johnson
Maria T. Zuber
Martin B. Hebsgaard
& Rasmus Nielsen
Kasper Munch
M. Thomas P. Gilbert
Regin Rønn
Tina Brand
Torben R. Christensen
& Mikhail Mastepanov
Michael Bunce
Permafrost Samples
Age range: modern to ≥ 1 Ma
Depth range: 0.5 - 41.6 m
DNA Fragment size
• Extract DNA from sediment
• Try to amplify 4-kb fragment of rDNA (“universal” primers)
• 4 x larger than largest fragment retrieved from ancient DNA
• 20 x larger than ancient DNA from plants and mammals
• Decide not to try culturing since:
• less than <1% of cells can be cultured
• low-temperature growth is a pain (contaminant prone)
• Result: 4-kb amplicons retrieved from 400-600 Ka samples!
Denise Aslett
Evidence for viability
DNA Repair
• Amplicon size (4 kb) from bacterial, but not plant DNA
• Decreasing sequence diversity with age (expected)
• no change in amount of preserved cellular structures
(except DNA) over time...suggests cells are not reproducing
Evidence for viability
DNA Repair
• Treatment of DNA with uracil-N-glycosylase (UNG) before PCR
• rationale: UNG breaks damaged DNA so only undamaged
(repaired) DNA will be amplified
deamination
cytosine
uracil
Evidence for viability
DNA Repair
• Treatment of DNA with uracil-N-glycosylase (UNG) before PCR
• rationale: UNG breaks damaged DNA so only undamaged
(repaired) DNA will be amplified
Evidence for viability
Method
CO2 production
• Undisturbed permafrost cores (25 ka, 500 ka & 740 ka)
• incubate (anaerobic), 9 months at -10°C & remove all CO2
• use stainless steal chamber and tubing
• controls: 2 cores without soil
• Entrapped CO2 discharged for 3 months, followed by 6 months of
constant CO2 production
~0.8
~0.14
Conclusion
DNA repair may be superior to dormancy for long
term survival. Surprised?
Future work may target
Extraterrestrial Life
image credit: http://ssed.gsfc.nasa.gov/tharsis/ngs.html
Cryopreservation?
Aging