1. Where now/where Next
We have explored the broad idea of doing historical deduction using present day DNA information: yours!
This is a huge, wondrous idea! You = walking fossil book
Now we’ll look at another case: when did your genes ‘learn’ to drink milk as an adult?
Then we’ll examine how we go fishing in your genes for the information they hold

2. Does milk make you gassy? Why/why not?
What’s normal? Which the ‘discovery’?

3. I see dead people: DNA is historical record
You got your genes at the Parent’s store
So did they
ad infinitum
Conclude: Your genes are not your own. They are a copy of a copy of a copy of a copy…
They contain a record of who (what) you were
You are a walking, talking historical record of where your genes have been, how you evolved
HUGE IDEA!!!!

4. Key fact: primary source is MILK
What’s milk got?
‘milk sugar’ = lactose =
I will later present the argument that C-C and C-H bonds have stored potential energy
But it’s more subtle than negative charges, and it’s harder to release than freeing a bond!
Key fact: primary source is MILK

5. Imagine a normal mammal...
not some bizarro cross-species-suckling human!
...where do you get milk?
...when do you get milk?
when do you no longer get milk?
Is it pointful to build milk-processing bioMachines (enzymes) for entire lifespan?

6. *Wikipedia’s descriptor
You & lactose
For millennia it was a sensible infancy vs. adulthood thing
Ability to digest milk was wasteful in adults!!!
If you don’t digest it… gut bacteria in your colon will!
result: ‘copious amounts’* of gas (CO2, methane, H2)
I will now argue that humans evolved extended lactase expression several times
This will entail allowing researchers to stare at people’s genes
*Wikipedia’s descriptor

7. Summary: lactose in humans
Heard of ‘lactose intolerance’? It’s the old normal!
Alternative = ‘lactase persistence’: production of lactose-digesting enzymes into adulthood
Ancestral: only babies need digest lactose b/c primary source is mother’s milk
‘Derived’ state: hey! I think I’ll suck on a cow/camel (ewww!)
So: who’s the mutant?
IN the paper, LP and LNP

8. Whence milk-induced gases?
How did humans ‘discover’ (genetically) how to drink milk?
How many times was this discovery made?
Where? When?

9. Caveat! This is ongoing research
So we’re discussing preliminary results based on initial datasets
TERMS:
LNP non-Persistence (LNP, the ‘ancestral’ state)
Lactase Persistence (LP, the ‘derived’ state)

10. Whodunnit? Smoking guns
We work from the PREMISES that…
If two sequences are identical, they will produce identical phenotypes
So we look for differences in genes and seek to CORRELATE with differences in phenotypes
Shown: DNA sequences with points of variability marked

11. Smoking & non-smoking gunsLP
sequences
EVERYBODY that can digest lactose has the red-dot changes. Some have some of the blues
NOBODY who canNOT digest lactose has the red dots.
EVERYBODY has the leftmost dot--a sign of something ancestral in everyone
LNP
sequences
Which mutations could be causal: lead to LNP phenotype?

12. Whodunnit? Smoking guns
ONE
bubble on the cow graph
EVERYBODY that can digest lactose has the red-dot changes. Some have some of the blues
NOBODY who canNOT digest lactose has the red dots.
EVERYBODY has the leftmost dot--a sign of something ancestral in everyone

13. Contrasting hypotheses
Single origin
All lac-expressors will have same change(s)
Multiple origins
All lac-expressors will NOT have same change(s)
Cause of change will be common to all sequences
Occam:
Simplest = most likely
Two hypotheses: single vs. multiple origins
Background: ‘the change’ will be inherited by all; irrelevant changes come & go

14. (chimpanzee sequence)
Camel or cow milk? Yes.
Circle: large group of people
Human
Green: digesters
Key idea: each string of letters represents a nucleotide change and the position at which it occurs. Those in RED are suspected of being CAUSAL of LP (lactase persistence = ongoing ability to digest milk)
Red: causes LP
Blue: nte, position
(chimpanzee sequence)

15. Sins of Omission
The female slave with whom Thomas Jefferson progenitored the descendants was Sally Hemings
Similar evidence has discovered the identity of the slain Russian Romanovs, established the innocence of death row inmates, convicted criminals

16. Their words
“This result would justify the hypothesis that the European T13910 and East African G13907 LP alleles might have arisen because of a common domestication event of the cattle whereas the C3712- G13915 allele in Arabia most likely arose due to the separate domestication event of camels.”

17. Think first
Why do I have more scars, more liver spots, more gray hairs than you?
Why does my 2004 Ford Focus have more dents, scratches, dings, missing pieces than one of my Lab Instructor’s new go-mobiles?

18. Concept: mutation clock
Things fall apart
Some things are irrelevant
Irrelevant/non-functional things can be reasonably be presumed to fall apart at constant rate
Calibrate to reliable externals: fossil record, ancestries, migrations… and you get the mutation clock
‘Ticks’ of the clock are changes in DNA sequence
By counting the number of differences between genome X and genome Y, we infer amount of time they have been diverging (growing more different)

19. Two paths 1000 years ago 750 years ago 500 years ago 250 years ago *A*B *C *D *E *F *G *H *I *J *K *L *M *N
Jen
Tom
Ann
Sam
Justin Timberlake

20. Do wild animals resemble cats, dogs, cattle?
OK, maybe cats...
Where did these docile, man-serving little blessings come from?
The same place as corn, broccoli, tomatoes…
We made that

21. Domestication

22. References (not trivial!)
Blue eyes
“Blue eye cool in humans may be caused by a perfectly associated founder mutation in a regulatory element located within the HERC2 gene inhibiting OCA2 expression”
Human Genetics 123:
Milk
“Independent Introduction of Two Lactase-Persistence Alleles into Human Populations Reflects Different History of Adaptation to Milk Culture”
American Journal of Human Genetics 82:

23. Resources HHMI: lactase movie

24. Learning about genes sequences & expression

25. Attack of the wild -OH (3’)
RECALL: This is the mechanism of addition
3’ OH ‘liberates’ PPi
What would happen if there were no 3’OH?
Fig. 14.6

26. How do we learn gene sequences?
Figure 4-2
The shoe is off the other foot!! H
Remember how adding -CH3 does not change functionality of Ura?
We can add other stuff. Colored stuff
DIDEOXY SEQUENCING 3′ 5′
Normal
dNTP
(extends
DNA strand)
ddNTP
(terminates
synthesis)
ddGTP’s
Template DNA
No OH
Labeled primer
Non-template DNA
1. Incubate a large number of normal dNTP’s with a small number of ddNTP’s (in this case starting with ddGTP’s), template DNA, a primer for the target sequence, and DNA polymerase.
2. Collect DNA strands that are produced. Each strand will end with a ddGTP (corresponding to a C on the template strand).
Create nucleotides with modified riboses--no oxygen at <<3’>> position. So they cannot be ADDED TO. Once in place, growing DNA chain is ended
Figure 20.9

27. But Bruce… GpGpGpCpCpCpApApAp TpApTpApTpTpApGpGp
How can I ‘target’ one gene
in 3 x 109 nucleotides (1 meter of DNA)?
Why are the sequences depicted upside down & backwards?
GpGpGpCpCpCpApApAp
TpApTpApTpTpApGpGp

28. Where now/where Next
We established that a nucleotide can be rendered ‘un- extendable’ by converting 3’ -OH to -H
We considered that ‘stuff’ can be added to non- pairing positions, just as Thy has a -CH3 vs. Ura
We found we could ‘pinpoint’ any specific subsequence of DNA we desired
I asserted that DNA Polymerase needs a ‘running start’—a primer that it can add TO
FYI: textbook covers DNA sequencing pp

29. Terminology: ‘dideoxy’C OH p
Where do we stop? C OH p T OH p C OH p T OH p C OH p T H p C H p
Terminology: ‘dideoxy’
G G G p OH
primer C OH p T OH p C OH p C OH p T H p
This is analogous to the case of figuring out the genetic material. Two pieces
1) What is a useful ‘handle’
2) DETECTION: how do we find out what happened?
G G A G G A G C C C
template
Two cases:
1) get a purple ‘dideoxy’ T (no 3’OH) => STOP
2) get a normal uncolored T => continue

30. Where do we stop? C T C C C C T T G G G C T C C T COH p
Where do we stop? T OH p C OH p C OH p C OH p C H p T OH p T OH p
G G G p OH
primer C OH p T OH p C OH p C OH p T OH p C H p
This is analogous to the case of figuring out the genetic material. Two pieces
1) What is a useful ‘handle’
2) DETECTION: how do we find out what happened?
G G A G G A G C C C
template
So: molecules that stop at total size of 9 nucleotides will be colored…?

31. The property of bigness
You’re 6’ tall, weigh 165
I’m 6’ tall, tip the 300
Which of us makes it through the huge crowd to the open subway doors the fastest?
Central idea of gel electrophoresis: the bigger they are, the slower they navigate the maze

32. And it goes like this

33. Keys All molecules of length 9 will have a red dye molecule attached
This is b/c G in 9 chain either
continued, and didn’t get a color
terminated with a colored ‘di-deoxy’ nucleotide
All molecules of length 8 are purple