Homology Activity

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OVERVIEW:

Lab #1:

• Part I: Exploring biological homology as it applies to morphology, DNA and proteins. Assignment –Activity 1-7 will be due at the end of the lab. The definition worksheet is due at the beginning of the next lab in your assignment blog and will count as a pre-lab.

 

 

Lab #2:

• Part II: Conducting a phylogenetic analysis on Vertebrates or Hominids or Hominoids using Mesquite software. Assignment – generate character matrixes and phylogenetic trees for presentations.

 

Lab #3:

• Part III: PowerPoint presentations on phylogenetic analyses. Due the week of Feb. 9th . This assignment will be completed by groups outside of scheduled lab time and presented during the scheduled lab section.

 

• Part IV: Final assignment - Review of student presentations. Due as a pre-lab in your  “Assignment Blog” – by the next lab meeting, week of Feb. 16th.

 

PART I: Homology –Molecules to Bones

 

The goal of Part I of this lab is to learn the important concept of biological homology.  In a series of 7 activities you will explore different aspects of this and related concepts.  By the end of this part of the lab you will turn in the completed questions to the activities. In addition, as your instructor lectures and you proceed through the activities, begin to fill in the definitions worksheet.

 

Figure 1: Homologous bones of the forelimb of 4 vertebrate organisms.  Can you guess the organisms to which they belong?
ACTIVITY #1: Limb Homology

 

1. Name the bone or bones labeled 153 in the specimen behind the display case.

 

 

 

1. On the organisms figured below, circle the bone or bones that you think are homologous to the bone labeled 153. How did you determine what bones were homologous?

 

 

 

 

 

 

1. From the evidence supplied at this activity and around the room at other activities, what bone or group of bones of the forelimb show the most variation?  Why do you think they are so variable?

 

 

 

 

 

 

 

 

1. What is the function of specimen A?  Why?

 

ACTIVITY #2: Vertebrate Skeletons

 

1. Which two specimens (A-Opossum, B-Mudpuppy, C-Bird, D-Monkey, E-Dog, F-Human, or G-Cat – in blue tape) look the most similar to you? Do you think that makes them the most closely related compared to all the other organisms? 

 

 

 

 

 

 

 

1. What evidence did you consider to make your determination?  You may answer generally, but give at least one specific line of evidence.

 

 

 

 

 

 

 

 

 

 

 

 

 

1. On the frog skeleton, the bone labeled #1 is homologous to which bone(s) labeled X, Y, Z, or none in the bird? …to which bone(s) labeled X, Y, Z or none in the human?

 

ACTIVITY #3: Ungulate Limbs

 

1. The bone labeled #1 on Specimen C is homologous to which labeled bone on (Write down the appropriate Letter):

 

1.  
   1. the cow leg  =

 

1.  
   1. the horse leg  =

 

1.  
   1. the human skeleton  =

 

 

1. Find the “heel bone” on the human and then the homologous bone on the horse.  Using the “heel bone” as a landmark to guide you, list the phalanx bones that are completely lost in the horse (specimen A). Use the Roman numeral system seen in Figure 3.2.

 

 

 

1. In specimen A, which bones are not lost, but are extremely reduced?

 

 

 

1. In specimen A, which bone has become enlarged and prominent?

 

 

 

1. Out of the four organisms (represented by the three leg specimens and human skeleton), which are the most closely related?  Draw a phylogenetic tree representing your proposed relationships. (Hint: Start by writing down the taxa. Connect the two most closely related).

 

                                                         

     Recent

 

 

 

            Time

 

 

 

 

    Ancient

           

                                                      Dissimilarity

 

ACTIVITY #4: Hominoids and out-group.

 

 

This ACTIVITY has 7-8 skulls; 1.) human, 2.) gorilla, 3.) chimpanzee, 4.) baboon, 5.) gibbon, 6.)orangutan 7.) lemur and 8.) dog, which represents a distantly related out-group. The following questions will help “determine character polarity” for Foramen magnum (FM) location.  That is to say, by using a distantly related species (canine in this case) a researcher can make an educated guess as to which character states are ancestral (older) and which are derived (more recent).  Derived traits are used to help determine a new branch on a phylogenetic tree.  Ancestral traits are traits shared by all taxa or once shared.  Derived traits are new and can represent a new species diverging away from a common ancestor.  The character in this case was Foramen magnum location and the character states were medial vs. distal. 

 

 

1.      Carefully pick up skulls and locate the Foramen magnum (FM) on all specimens.  It is the empty, round hole (or filled in with black casting) on the underside of the skull.  What is the function of the FM?

 

 

 

 

 

2.      If the dog (labeled E) is the most distantly related taxon among all the specimens, what do you think is the more ancestral character state, a FM located more medially towards the center of gravity or a FM located more distally further away from the center of gravity?

 

 

 

3.      Considering that bipedalism is related to the location of the FM, do you think bipedalism is an ancestral or derived trait?

 

 

 

 

 

4.      List two other characters useful in analyzing skulls and identify the ancestral state for each?

 

 

 

 

 

ACTIVITY # 5:  Hominids – Bi-pedal, non-ape lineage including Homo sapiens

COMPLETE ACTIVITY #4 BEFORE ATTEMPTING ACTIVITY #5

 

1. Draw a phylogenetic tree of the specimens based solely on the character “size of skull”.  Use the capital letters as taxa names.

 

Small                                                                                       Large

 

 

 

      Recent

 

 

 

            Time

 

 

 

     Ancient

Dissimilarity

 

 

1. Do you think overall size is a useful character?  Why or why not?

 

 

 

 

1. Draw a final tree based on your gut instincts (Gestalt) as to the real relationship between the taxa represented by these 7 specimens. (For some ideas, see chapter 6, pp -205-230 in Photographic Atlas for Physical Anthropology, Whitehead et al.).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1. What character(s) most influenced your tree topology?

 

ACTIVITY # 6: Determining DNA homology begins with Aligning DNA

COMPLETE BEFORE ACTIVITY #7 AT ANY LAPTOP COMPUTER

 

The NCBI  (National Center for Biotechnology Information) has been charged with creating automated systems for storing and analyzing knowledge about molecular biology, biochemistry, and genetics.  If you are interested in the emerging field of Bioinformatics (genomics and proteomics) it all starts with learning to use the resources and tools at the NCBI website.

 

At this activity, you will use information and software tools from NCBI to ask some basic questions regarding DNA molecules.  Specifically, how is homology determined between different DNA molecules.  The DNA sequence #1 (below) was used in a search engine and software tool called Blastn.  Blastn is a search engine and software tool that analyzes the DNA sequence of interest and compares it to all other DNA sequences in the NCBI database. Blastn runs a pair wise comparison between each base in the DNA sequence and retrieves all other sequences that share a certain percent similarity.  Aligning and displaying the DNA molecules side by side helps researchers find similarities and differences.

 

Nucleotide sequence 1:  gggtgaacag ccgcacggga gtaggtacgc acctgacctc gctggcactg ccgggcaagg cagagggtgt ggcgtcgctc accagccagt gcagctacag cagcaccatc gtccatgtgg gagacaagaa gccgcagccg gagttagaga tggtggaaga     Comparison between 2 DNA sequences that have been aligned by Blastn at NCBI website.  

GGGTGAACAGCCGCACGGGAGTAGGTACGCACCTGACCTCGCTGGCACTGCCGGGCAAGG  Seq.#1

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

GGGTGAACAGCCGCACGGGAGTAGGTACGCACCTGACCTCGCTGGCACTGCCGGGCAAGG  Seq.#2

 

CAGAGGGTGTGGCGTCGCTCACCAGCCAGTGCAGCTACAGCAGCACCATCGTCCATGTGG  Seq.#1

|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

CAGAGAGTGTGGCGTCGCTCACCAGCCAGTGCAGCTACAGCAGCACCATCGTCCATGTGG  Seq.#2

 

GAGACAAGAAGCCGCAGCCGGAGTTAGAGATGGTGGAAGATGCTGCGAGTGGGCCAGAAT  Seq.#1

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

GAGACAAGAAGCCGCAGCCGGAGTTAGAGATGGTGGAAGATGCTGCGAGTGGGCCAGAAT  Seq.#2

 

 

Blastn will also align and show differences (Ignore the gray highlights for now).

 

GGGTGAACAGCCGCACGGGAGTAGGTACGCACCTGACCTCGCTGGCACTGCCGGGCAAGG  Seq.#1

............................................................  Seq.#2

 

CAGAGGGTGTGGCGTCGCTCACCAGCCAGTGCAGCTACAGCAGCACCATCGTCCATGTGG  Seq.#1

.....A......................................................  Seq.#2

 

GAGACAAGAAGCCGCAGCCGGAGTTAGAGATGGTGGAAGATGCTGCGAGTGGGCCAGAAT  Seq.#1

............................................................  Seq.#2

 

1.      Above are two different ways to view the same alignment of sequence #1 and sequence #2. Do you think sequence #1 and #2 are homologous molecules of DNA?  Why or why not?

 

 

The Blast search and alignment also came back with another sequence (seq.#3) aligned below. (Dots represent the same DNA base, while letters in bold are how sequence #3 differs from sequence #1).

 

CACCTGACCTCGCTGGCACTGCCGGGCAAGGCAGAGGGTGTGGCGTCGCTCACCAGCCAG  Seq.#1

.......G...C...A.G.....A........C...A......T...C............  Seq.#3

 

TGCAGCTACAGCAGCACCATCGTCCATGTGGGAGACAAGAAGCCGCAGCCGGAGTTAGAG  Seq.#1

.......................G........C.....A.....A.....C...C.....  Seq.#3

 

ATGGTGGAAGAT  Seq.#1

.C...A......  Seq.#3

 

 

2.      Do you think sequence #1 and #3 are homologous molecules of DNA?

 

 

 

Notice that the length of the alignment between sequence 1 & 3 is shorter than the alignment between sequence 1 & 2.  In fact only a faction of the original sequence (highlighted in gray) is aligned with this third sequence.  This begs the question, what happened to the rest of the molecule of sequence #1?  This new alignment with sequence #3 just chops off some of the sequence.  How does that effect DNA homology?  Are sequence #1 and #3 actually homologous if only parts of their molecules are similar?  How similar do they have to be in order to be considered homologous?

 

3.      Considering the new questions raised above, do you still think these sequences represent homologous stretches of DNA?  What would you like to know before you would consider two DNA sequences homologous? (Hint: Imagine molecules are just small morphological features.)

 

 

ACTIVITY # 7: Determining Protein homology – viewing conserved domains

COMPLETE  ACTIVITY #6 BEFORE ACTIVITY #7 AT ANY LAPTOP COMPUTER

 

The DNA sequence #1 (below) used in the activity #6 has the following Amino Acid (AA) translation. 

 

Nucleotide sequence 1:  gggtgaacag ccgcacggga gtaggtacgc acctgacctc gctggcactg ccgggcaagg cagagggtgt ggcgtcgctc accagccagt gcagctacag cagcaccatc gtccatgtgg gagacaagaa gccgcagccg gagttagaga tggtggaaga tgctgcgagt gggccagaat   AA translation 1:  RTGVGTHLTSLALPGKAEGVASLTSQCSYSSTIVHVGDKKP     Look over the protein sequences below that were generated from the Conserved Domain Database – a software tool from NCBI.  3PYP is the protein retrieved from the database that has the most similarity with the AA #1 translation from the DNA sequence #1. 1DRM A is the heme domain of a protein from the bacterium, Bradyrhizobium japonicum.  These two proteins have been aligned side by side to aid in a pair wise comparison.  The capital letters were determined by the computer software to be the most chemically similar amino acids and have putative structural homology. (See Figure 7.1 for amino acid abbreviations and codons).                 3PYP          1 ~~mehvafgsedientlakmddgqldglafGAIQLDGDGNILQYNAAEGd~~iTGRDPKQ 56 1DRM_A        1 ~~~~~~~~~~~~~~~mrethlrsilhtipdAMIVIDGHGIIQLFSTAAEr~~lFGWSELE 43   3PYP         57 VIGKNFfkDVAPCTDspeFYGKFKEGvas~~~~gnlNTMF~EYTFDYQ~MTPTKVKVHMK 110 1DRM_A       44 AIGQNVn~ILMPEPDrsrHDSYISRYrttsd~phiiGIGR~IVTGKRRdGTTFPMHLSIG 100   3PYP        111 Kals~~~~~~gdsYWVFVKRV~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 125 1DRM_A      101 Emqsg~~~~gepyFTGFVRDLtehqqtqarlqelq~~~~~~~~~~~~~~~~~~~~~~~~~ 131

 

Question:

1.      From the pair wise comparison of AA’s in both sequences do you think these are homologous proteins? Why or why not?

 

 

 

 

 

 

 

Procedure for using Visualizing Protein Structure:

1.  
   • Go to Entrez Protein - it may be already bookmarked in your web browser - (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein) to search their protein database for the period 2 protein that is most similar to the AA translation #1 and use their molecular modeling software to visualize the three dimensional structure of proteins.

 

1. Entrez Protein - retrieve the protein sequence record for the PER2 gene with a search such as: “per2 NP_073728”. This search will bring up the file that has a Link (in the right column) to "Domains" or “Conserved Domain”. Clicking the Domains link shows that Per2 has a conserved PAS domain. Click on the PAS hyperlink (red box) to get to a full description of the PAS conserved domain.

 

1. At the Conserved Domain Database summary page of the PAS domain - Click "View 3D Structure" with Cn3D chosen from the pull down menu (default setting).

 

The program Cn3D will show you two windows.  One showing the 3D structure of two aligned proteins and another just below it that show the amino acid sequences of several proteins: 3PYP, 1DRM A are the ones in the 3D viewer.

 

 

1. To remove one protein to observe the structure of only one at a time go to Show/Hide à Pick Structures and unhighlight the 3PYP text. Click Done. Please return to the default before you move on to complete this activity.

 

1. 3D view (PAS - Cn3D) - To see how viewing options are chosen, go to Style -> Coloring Shortcuts -> Sequence Conservation -> Weighted variety option. ALSO go to Style -> Rendering Shortcuts -> Tubes.  These are the default settings, but you may play with other options.  Please return to the default to finish remaining steps.

 

 

1. Sequence Viewer (PAS - Sequence/Alignment Viewer) - PAS domain in caps and colored to correspond to 3D view. Highlight several of the colored letters to see them highlighted in the 3D viewer. Or click

 

 

1. 3D view - Double click on ligand (six-sided ring with Fe atom) in center of 3D structure.

 

 

1. 3D view - Show/Hide -> Select by Distance -> Residues Only -> Distance: 5, click OK. Highlighting residues within 5 Angstroms of the ligand allows users to help identify likely binding sites and/or residues involved in the protein's active site.

 

Question:

2.      As seen from the above exercise, very different looking amino acid sequences can have very similar 3D structure.  Do you think 3PYP and 1DRM A are homologous proteins?  Do you think they have a similar function? 

 

 

 

 

 

 

 

 

3.      What evidence would lead you to believe two amino acid sequences are homologous?

 

 

 

 

 

 

 

 

 

 

4.      Now consider the proteins are coded by DNA sequences.  What would convince you that two DNA sequences are homologous?

Definitions Worksheet:

 

1. Phylogeny:
2. Phylogenetic tree:
3. Cladogram:
4. Clade:
5. Taxa/Taxon:
6. Topology:
7. Character:
8. Character state:
9. Derived character:
10. Ancestral character:
11. Homologous character:
12. Analogous character:
13. Convergence:
14. Monophyletic taxon:
15. Polyphyletic taxon:
16. Outgroup:
17. Parsimony: