1. Lizard skin color
Instr, Prep, Pep
Like blue eyes, like human skin color, like ‘learning’ to drink milk: sometimes a different way of doing things is just a nucleotide change away

2. Goals for this sequence
Give you a crack at primary scientific literature
Let you feel your powers!
Summarize many aspects of the semester
Think about some cool stuff

3. What is evolution? Dark demon that will destroy your soul?
Explanation of how varied forms of life are derived from pre-existing forms?
Technically: change in allele frequency over time. Pieces:
Variation (mutation, alleles)
Reproduction
Success & failure

4. Skin color in lizards
Argument: several species of lizard adapting ‘discover’ the same target of mutational attack--a transmembrane receptor in the melanin signaling pathway
Each solution clearly different--different nucleotide changes; different effects on expression/function

5. Now you see them... Which is easiest to predate?
The authors make the argument that this could also be a first step in speciation (separation of the two groups into two non-mating populations) because some visual signals are important in mate selection
Which is easiest to predate?
The white dunes happened ‘recently’--6,000 years ago
How did new lizard color appear so quickly?

6. Where are you: alleles vs. environments
Some words: derived is the more EvoBio way of putting it; ‘mutant’ will do for us
‘ecotone’ = transitional area
*ecotone: transition region between 2 different environments
Implicit argument: Don’t be inappropriately pigmented.
Does the data support this?

7. Duplicate, diverge, discover
Phenotypes
&
mutations
MCR1R
Yes, this protein looks familiar. Opsin, MCR1 both ‘serpentine receptors’, G-protein signalers that cross membrane like snake!
Duplicate, diverge, discover

8. This figure assumes we’re leaving
What could go wrong?
This figure assumes we’re leaving
Note that if a protein were EMBEDDED in the membrane, it would end up in the outer the end
Fig. 7-24

9. I’ll bring the promoter
DNA
lizard coding sequence
lizard coding sequence
Lizard
Promoter
Sequences
Extensive studies have been done on human cells for obvious reasons--we’re interested in ourselves
Thus, tools exist for growing human cells, producing proteins in human cells, studying responses and activities in human cells
DNA
Human
Promoter
Sequences

10. Expressing in human cells
What was this whole ‘expression in COS-7 cells’ thing?
What does this test? What does it not test?
What inputs did they measure, what outputs, and what do these mean?
Does the regulatory region get tested in the heterologous* system?
*hetero- = different, i.e. a system made of mixed human and lizard parts in this case

11. On the evils of ‘heterologous’ systems
What isn’t being tested?
How long has it been since your cell-sorting apparati were a lizard?
heterologous--DIFFERENT
Like chromosomes--a homologous pair is the matching set of mom’s + the highly similar, corresponding one you got from Dad

12. measuring amounts of protein--using the immune system!
A time for techniques
measuring amounts of protein--using the immune system!

13. Business end of an antibody
Green = antibody (defense protein)
Orange = something it ‘recognizes’ (sticks to)
Just a brief visit to get the key concept of an antibody--your body (and those of many organisms) makes millions/billions of variants, each with its own shape & surface
Each kind thus ‘matches to’ a different potential shape your body can encounter--shapes found on viruses, foreign particles, bacteria...
Molecular Biology of the Cell Fig

14. ELISA: Detecting expression
Enzyme Linked Immunosorbent Assay
USING antibodies for our purposes (abusing mice en route)
Red: protein in question
Yellow: antibody raised in convenient source
Blue: antibody attached to an enzyme (*), raised in 1st antibody in 2nd source
Amount of enzyme action = amount of red protein initially present
Key idea: This is a method of detecting or quantitating the presence of anything to which you can raise an antibody

15. Things that could go wrong: getting there
This figure assumes we’re leaving
Note that if a protein were EMBEDDED in the membrane, it would end up in the outer the end
Fig. 7-24

16. Phenotype I: sorting/delivery
Lost in transit
Total: grind up cell; measure antibody attachment
Surface expression: keep cells intact; measure surface attachment of the antibody
What does the difference in these values measure?

17. Things that could go wrong: saying something*
External signal ‘tweaks’ membrane protein (think hemoglobin + oxygen, tubulin + GTP)
Altered shape acts different-- influences another protein
Second protein is released Paul Revere style to spread the message
Others hear, respond by initiating their own actions (in the current case, reshape ATP into cAMP)
I can’t get in!
alphaMSH
Ow! Someone is kicking me in my butt!
Feel my pain!!
Look what I can do!
cAMP
Fig. 8-14
*Terminolologists: “Signal transduction”

18. Phenotype II: Signaling!
Oho!
No signal!
One of these things is not like the other
H. maculata shows no effect--IN THIS ARTIFICIAL SYSTEM USING ONLY THE CODING SEQUENCE!!!
Not surprising; receptor levels decreased
First, we’ll deal with the two on the left, where we successfully detect a ‘failure to communicate*’
*cultural reference: “Cool Hand Luke”

19. Dominance: dancing with Mc1r
A. inornata: dom or rec?
Implication?
S. undulatus: dom or rec?
Meaning?
What does recessive phenotype mean about the mutant’s effect on the cell or the other allele?
S. undu: let’s combine 2 ideas
1) It is dominant
2) It only poorly makes it to the cell surface
So--what MIGHT we infer about what it does to WT alleles
Suppose I told you this protein functions as a dimer?
Indeed, multimeric proteins are common sources of dominant mutations
Experiment: shine light on lizard skin; assess how much bounces off
(which they report as ‘transmission’)

20. Finishing lizards S. undulatus mutant is dominant
mutant form doesn’t make it to the surface well
MC1R is a dimer!!!

21. The other one H. maculata amino acid change: val to ile
not the whole gene

22. Other possibilities We get complaints from the folks in your apartment
When we go to your address, we hear the noise, but it’s not coming from your apt.
What do we conclude?
The key idea is that the mutation studied in Holbrookia (which had no phenotype in the sorting assay, no phenotype in the signaling assay, and was one of the most subtle amino acid changes possible) may NOT have been the cause--but the cause may have been nearby. I hope to re-visit this topic on Wed.

23. Relevant segments of Ch. 19, “Analyzing and engineering genes”.
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Relevant segments of Ch. 19, “Analyzing and engineering genes”.
Only as a supplement; material NOT introduced in this lecture will NOT be tested
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