Molecular Phylogeny

Wellcome trust: http://www.wellcometreeoflife.org/video/

http://www.youtube.com/watch?v=lI4d8KlfMvY&feature=channel_page

Phylogenetic Tree

Hypothetical graphic representation of the evolutionary relationships between organisms being studied

Only as good as the data chosen and the analysis performed

Species A

Species B

Species C

Species D

Species E

1

2

3

4

Common

ancestor

Phylogenetics in human health and medicine

Identifying emerging diseases:

West Nile virus

common house mosquito

Captive and wild birds in the NY

area carried virus.

tiger mosquito

Discovering snakebite antivenins (antivenoms)

•! Developing antivenins is especially important in Australia, which has more poisonous snakes than any other continent

•! Unfortunately little is known about the venoms of most snakes

•! Recently phylogenetic studies are helping identify closely related species of venomous snakes

Phylogenetics in human health and medicine:

Using phylogenetic analysis to protect

ecosystems from invasive species

•!What appeared to be an exotic alga along the California coast was examined by scientists.

•!Phylogenetic analysis of DNA sequences revealed the alga was Coulerpa taxifolia, one of two the most invasive species worldwide which is extremely toxic to herbiferous fish.

•!Early identification, eradication programs and continuous monitoring has reversed its spread

Coulerpa taxifolia Why study tree of life?: http://www.youtube.com/watch?v=ooLr8d_pDBc&NR=1

How are Phylogenetic Trees Generated?

Systematists use:

•!Morphological data: fossils records and bones and teeth

•!Molecular data: protein and DNA sequences

All available information is used to infer evolutionary relationships

Cytochrome C Oxidase

•!Determine how closely related species are based on evolutionary history

•!Do this by comparing a protein found in oxygen-utilizing organisms:

cytochrome c oxidase - subunit III or COX-III

Inner mitochondrial membrane

Cytochrome C Oxidase

•!COX-III is a protein

•!like all proteins, it is composed of building blocks - amino acids - linked together like beads on string.

•!The order or sequence of amino acids in protein dictates its structure and function like the colors and/or shapes of beads of a necklace will change its appearance

Gly Thr

Gly Glu

Ser Lys

Cys

Pro

Leu Met

Val

Lys

Val

Leu Asp Ala Val Arg Gly Ser

Pro

Ala

Ile

Asn Val

Ala

Val

His Val

Phe

Arg

Cytochrome C Oxidase

•!Was presumably present in first oxygen-utilizing ancestral organisms

•!Present today in modern descendants of this original ancestor

•!There have been mutations in gene for COX-III protein throughout evolutionary history

•!Compare the amino acid sequences for COX-III proteins: modern species that are most closely related will have most similar amino acid sequences

How are proteins made?

•! proteins - products of

genes

•! Information encoded in a

gene is first transcribed

into messenger RNA

•! Then the RNA message is

translated into a protein

How do proteins change

through evolutionary history?

•! Mutations in DNA may alter

proteins, the products of gene

expression

•! Resulting proteins may function

better or worse than the original

protein, or may be non-functional

•! Mutations accumulate over time

•! To determine how closely related two species are, compare either

the gene sequences or the

amino acid sequences for a

particular protein

mutation!

altered protein

Molecular Phylogeny Lab Exercise

Use bioinformatics tool called Geneious to compare evolutionary relationships between organisms

1.! Examine amino acid sequence in human COX-III

2.! Compare Norway rat sequence to human and determine how similar they are

3.! Compare human and rat sequences with those of other animals and plants, and construct phylogenetic tree

4.! Compare several ungulate (hoofed mammals) sequences and construct phylogenetic tree of ungulates

Before coming to lab today, you should

have followed the pre-lab directions.

On RPI-LMS in the folder, Intro to Lab:

•! Download the version of Geneious you need: www.geneious.com

•! Also download from RPI-LMS, the Geneious folder BIOL-1010 Fall 2010 to your desktop.

•! Open the application, Geneious & click on Local in the Sources menu (left side of screen).

•! Then, select Import and From Multiple Files. Select BIOL-1010 Fall 2010. You will be using the documents from these folders during the first lab.

Geneious Desktop

You might see

a help panel here

Create a folder and chose a Human COX-III Sequence

1. In the Service Panel, highlight Local Folder.

2. From File in the Menu Bar, select New Folder. Label it COX-III for cytochrome c oxidase subunit III.

3. Open the folder BIOL-1010 Fall

2010 and the COX-III Human &

Rat folder. Choose one of the documents for Homo sapiens and rename it by clicking on the alphanumeric accession number under Name and changing it.

4. Click on this document, and drag and drop it into the COX-III folder you created.

Examining Human COX-III Sequence

Document Viewer Panel

Compare COX-III Sequences from human and Norway rat

•! Select a COX-III document for rat

•! Rename the document as you did previously

•! Click, drag, drop it into your folder

•! Open COX-III folder and highlight both sequences by holding down shift key

•! Click on Alignment button on Tool bar to do a sequence alignment

Human and Rat

Examine identity bar

Count white blocks, locations of mismatches

Calculating % Match

Determine the % match between the human and rat

COX-III using the following formula:

% Match (% Identity) =

(total number AA – mismatches) x 100%

total number of AA

Compare your result with statistics; pairwise %

similarity should be the same

Determine % Identity for:

•!human - chimpanzee

•!human - fruit fly

How to go about comparing sequences from several organisms.

•! Collect sequences from one additional animal and two plants

•!Do sequence alignment for all species

•! Then construct a phylogenetic tree

Constructing a Phylogenetic Tree

•! Highlight the sequences for all of the organisms you are comparing by holding down the shift key while you click the first and last sequences. All should be highlighted.

•! Note: Do not highlight the protein alignment for human-rat comparison

•! Click the Alignment button on the tool bar. A new window will open. Click OK.

•! Highlight the Alignment entry in your folder; then, look in the Document View panel

•! All of the sequences you selected will appear there

•! Are the sequences all the same length? Are there regions with many mismatches?

•! Click on Tree in the Tool bar. A new window will open

•! Do a simple Neighbor-Joining (NJ) tree without Consensus by leaving the Resample Tree box tree unchecked

•! Click OK; your tree will appear in the bottom panel

Constructing a Phylogenetic Tree

Ungulate Tree

•! Use same steps to create a tree of ungulates

•! Collect sequences for cow, goat, deer, horse, pig, hippopotamus, and sheep

•! Also bowfin and Minke whales

•! Choose one animal to represent the outgroup

•! Next week you will use some of the other approaches used to determined evolutionary relationships in ungulates (hoofed mammals)

Tree of Ungulates

http://www.wellcometreeoflife.org/interactive/

For next week:

•! Logon to RPI-LMS. Go to: Lab Content,

1. Evolution, Wet Lab: Ungulates.

Click on Pre-Lab. Read the Pre-Lab

Directions and complete them.

•! There will be a quiz on Ungulate Terminology and Bones of Human

Foot at the beginning of lab.

Pre-Lab - Evolutionary Relationships

•! Write out definitions for ungulate terms – pre-lab assignment

•! Learn bones of human foot

•! How Bambi evolved into Moby Dick •! Describes Indohyus, ancient deer-like land mammal

•! Missing link in whales’ evolutionary history •! http://www.guardian.co.uk/science/2007/dec/19/whale.deer

•! http://blogs.guardian.co.uk/science/2007/12/are_whales_descended_from_deer.html

•! Establishes evolutionary relationship between cetaceans

(whales) and ungulates