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.
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.
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
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)
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