1 Lizard skin color Like blue eyes like

1 Lizard skin color 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

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 2

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 3

Skin color in lizards 4 http: //www. nsf. gov/news_summ. jsp? cntn_id=116093

Now you see them. . . 5 Which is easiest to predate? The white dunes happened ‘recently’--6, 000 years ago How did new lizard color appear so quickly? http: //www. nsf. gov/news_summ. jsp? cntn_id=116093

Where are you: alleles vs. environments *ecotone: transition region between 2 different environments Implicit argument: Don’t be inappropriately pigmented. Does the data support this? 6

7 Phenotypes & mutations ❖ MCR 1 R Yes, this protein looks familiar. Opsin, MCR 1 both ‘serpentine receptors’, G-protein signalers that cross membrane like snake! Duplicate, diverge, discover

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

I’ll bring the promoter DNA Lizard Promoter Sequences DNA Human Promoter Sequences lizard coding sequence 9

Expressing in human cells 10 ❖ 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

On the evils of ‘heterologous’ systems ❖ What isn’t being tested? ❖ How long has it been since your cell-sorting apparati were a lizard? 11

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

Business end of an antibody 13 Green = antibody (defense protein) Orange = something it ‘recognizes’ (sticks to) Molecular Biology of the Cell Fig. 15 -14

ELISA: Detecting expression Enzyme Linked Immunosorbent Assay 14 ❖ 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 1 st antibody in 2 nd source ❖ Amount of enzyme action = amount of red protein initially present http: //www. al 3 malka. com/up/uploads/653 fb 003 fd. jpg

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

Phenotype I: sorting/delivery 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? 16

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 c. AMP) I can’t get in! 17 alpha. MSH Ow! Someone is kicking me in my butt! Feel my pain!! Look what I can do! c. AMP Fig. 8 -14 *Terminolologists: “Signal transduction”

Phenotype II: Signaling Oho! No signal! ! 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” 18

Dominance: dancing with Mc 1 r 19 Experiment: shine light on lizard skin; assess how much bounces off (which they report as ‘transmission’) A. inornata: dom or rec? Implication? S. undulatus: dom or rec? Meaning?

20 Finishing lizards ❖ S. undulatus mutant is dominant ❖ mutant form doesn’t make it to the surface well ❖ MC 1 R 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?

You might also enjoy. . . 23 Relevant segments of Ch. 19, “Analyzing and engineering genes”. Only as a supplement; material NOT introduced in this lecture will NOT be tested More on ELISA: http: //www. sumanasinc. com/webcontent/animations/content/ELISA. html