David Hess
Abstract:
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To introduce us to proteins, which truly make one organism different from another in terms of phenotype, our instructor challenged us to study the physical and proteomic traits of salmon, catfish, turbot, halibut, and yellow-fin tuna and estimate how each fish is related on the evolutionary tree. To do so, our lab group first accessed online research websites to compare the phenotypes of the different Ichthyoids. We specifically researched sizes, swim types, biological features, habitat preferences, and taxonomic names that derive from the evolutionary tree. After this, we then obtained samples of the muscle tissue in each fish, which were provided by the instructor, and then extracted the proteins from each sample. By treating the samples with sodium dodecyl sulfate and applying heat. We were able to denature the[D1] tertiary and quaternary structures of the proteins, which left the proteins long, stringy, and negatively charged. Next, we were able to separate the proteins by length a la gel electrophoresis, and compare the different proteins in the fish as we observed the different bars that appeared on the gel. After comparing both the physical traits and the proteins in each fish, we were to predict which species preceded the next according to evolution.
Purpose:
The purpose of this lab is to study the physical attributes and proteomics of different species of fish to determine the potential genealogical tree connecting these species
Instructor/Background:
Proteins often bind together, forming polypeptide chains. Some atoms on these chains are hydrophilic, while others are hydrophobic. This is due to the fact that the different r-groups (the only part of an amino acids that distinguishes it from another), may or may not form hydrogen bonds with the water molecules that they are summered in. When a hydrophobic group enters the body of water, the hydrogen bonds in the water break apart, yet cannot bind to the r group on the amino acids, so the water forms bonds with itself again around the r-group, thus pushing the r-group away due to the magnetic forces that push similarly charged atoms away from eachother. However, if a hydrophilic group is exposed to water, hydrogen bonds are formed with the r-group, pulling the r-group out of the remaining protein structure due to magnetic forces pulling the two bodies together as they are oppositely charged. These two interactions cause the protein to bundle up, making it hard to perform accurate gel electrophoresis on. It becomes especially difficult when these proteins bind together with disulfide bonds. Heat and sodium dodecyl sulfate break apart the disulfide and hydrogen bonds. This allows us to separate the proteins in electrophoresis, which can then be compared. [A]
Data/Organization [D2]of Records:
The following data results from reseach using the Fishbase website to compare phenotypes between the studied fish
Common Name:
Salmon
Scientific Fish:
Oncorhynchus Keta
Taxonomic Classification:
Family
Salmonidae (Salmonids)
Order
Salmoniforms (Salmons)
Class
Actinopterygii (Ray-Finned Fish)
Size:
Max Published Weight: 15.9kg
Environment:
Marine; Freshwater; Brackish; Benthopelagic; Anadromous
Depth Range: 0-250m
Biology:
Inhabits Ocean and Coastal streams. Adults cease eating in freshwater. Die After Spawning. Migrating fry forms schools in estuaries, remain close to shore for a few months, and disperse and enter into the sea. Epilegic.
Swim Type:
Anguilliform (Moves Body and Caudal Fin)
Additional Factors:
Definitions of Unfamiliar Terms:
Epilogic-Living in the upper zone of the ocean from just below the surface to about 100m in depth
Common Name:
Halibut
Scientific Fish:
Hippoglossus Hippoglossus
Taxonomic Classification:
Family
Pleuronectidae (Right-Eye Flounders)
Order
Pleuronectiformes (Flatfish)
Class
Actinopterygii (Ray-finned Fish)
Size:
Max Recorded Length: 470.0cm
Max Recorded Weight: 320.0kg
Environment:
Marine; Demersal
Depth: 50-2000m
Biology:
Adults are Benthic, but occasionally Pelagic. Feeds mainly one other fishes, but also eats cephalopods, large crustaceans, and other bottom-living animals. Seriously affected by overfishing
Swim Type:
Anguilliform: Body and Caudal Fin
Additional Factors:
Dorsal Spines
Definitions of Unfamiliar Terms:
Benthic: Lives one the bottom of a body of water
Pelagic: Lives far away from land
Common Name:
Catfish
Scientific Fish:
Neoprius Graeffei
Taxonomic Classification:
Family
Arildae (Sea Catfishes)
Order
Siluriformes (Catfish)
Class
Actinopterygii (Ray-Finned Fishes)
Size:
Max Length 60.0cm
Environment:
Marine; Freshwater; Brackish; Demersal
PH Range 7.5-8.2
Anadromous
Biology:
Inhibit freshwater rivers and lagoons, Brackish estuaries, coastal marine waters. Feeds on arthropods, insects, aquatic plants, mollusks, prawns, crayfish, fishes, and bottom detritus
Swim Type:
Anguilliform (moves body and caudal fin)
Additional Factors:
1 Dorsal spine, 7 dorsal soft rays, and 15-19 soft anal spines
Definitions of Unfamiliar Terms:
Anadromous: Migrates from freshwater to spawn in salt-water
Common Name:
Yellowfin Tuna
Scientific Fish:
Thunaus Albacarares
Taxonomic Classification:
Family
Scombridae (Mackerels, Tunas, Bonitos)
Order
Perciformes (Perch-Likes)
Class
Actinopterygii (Ray-Finned Fishes)
Size:
Max Weight: 200.0kg
Max Length: 230.0cm
Environment:
Marine; Brackish; Pelagic-Oceanic; Oceandromous
Depth Range: 1-250m
Biology:
Lives above and below thermoclines, Pelagic in open water, rarely seen around reefs, school by size, large fish school with porpoise, sensitive to low concentrations of oxygen, resides near ocean debris
Swim Type:
Anguilliform (Movements of body and/or Caudal fin)
Additional Factors:
11-14 Dorsal Rays, 12-16 Dorsal soft rays,11-16 Anal Soft Rays, 39 Vertebrae
Definitions of Unfamiliar Terms:
Common Name:
Turbot
Scientific Fish:
Scophthalmus Maximus
Taxonomic Classification:
Family
Actinopterygii (Ray-Finned Fish)
Order
Pleuronectiformes (Flatfish)
Class
Actinopterygii (Ray-Finned Fish)
Size:
Max Published Weight: 25.0kg
Environment:
Marine; Brackish; Demersal; Oceandromous; Temperate
Depth Range: 20-70m
Biology:
Live one sand, rock, or mixed bottom. Almost Circular Bottom. Eye side without scales, but instead bony tubercles. Feeds one bottom-living fishes (sand eels, gobies, etc.) and larger crustaceans and bivalves. Lives especially in Brackish Waters
Swim Type:
Anguilliform: Movements of body and/or caudal fin
Additional Factors:
Larvae are initially systematic, but after 40-50 days, the right eye moves to its left side.
Definitions of Unfamiliar Terms:
Oceandromous: migratory one salt-water
Upon the conclusion of our lab, we obtained a gel with protein bands that looked like this:
The following graph shows a standard curve based on the distance that the bars travelled and the weight of said bars:
The following table describes the distances various bands of proteins moved down their wells. We would use this information to calculate the weight of these bands by comparing them to our standard curve:[D3]
Fish Designation
Distance Travelled: Band 1
Distance Travelled: Band 2
Distance Travelled: Band 3
Distance Travelled: Band 4
Fish A
34mm
31mm
28mm
23mm
Fish B
32.5mm
28.5mm
27mm
24.5mm
Fish C
34mm
31.5mm
27mm
24.5mm
Fish D
34mm
31.5mm
26.5mm
23.5mm
Fish E
35mm
32.5mm
30mm
28mm
By using the band distances and the standard curves that we made, we were able to calculate the weight of these protein bands in Kilo Daltons:
Fish Designation
Protein Weight: Band 1
Protein Weight: Band 2
Protein Weight: Band 3
Protein Weight: Band 4
Fish A
12.5 kD
17 kD
23.5 kD
40 kD
Fish B
14.5 kD
20 kD
26 kD
34.5 kD
Fish C
12.5 kD
16.25 kD
26 kD
33.3 kD
Fish D
12.5 kD
26.25 kD
27.5 kD
37.5 kD
Fish E
10.6 kD
16.5 kD
19.3 kD
22.5 kD
By comparing the bands on the gel, our lab group made the following tables showing which fish had certain proteins in their muscles tissue.
A
B
C
D
E
3.5mm
X
3.4 mm
X
X
X
32.5 mm
X
X
31.5 mm
X
X
31 mm
X
30 mm
X
29.5 mm
X
28.5 mm
X
28 mm
X
27 mm
X
X
26.5 mm
X
24.5 mm
X
24 mm
X
23.5 mm
X
23 mm
X
*Each, “X” represents the presence of the mentioned protein on the left-hand side of the table in the fish
This table compares the proteins located in the chart above, and shows the similarities of proteins between the species.
A
B
C
D
E
A
4
0
1
1
0
B
0
4
1
0
1
C
1
1
4
2
0
D
1
0
2
4
0
E
0
1
0
0
4
Results:
Upon the completion of the analysis of our results, we obtained the following Celptogram[D4]. We knew that Species E only shared a common protein with species B, so it needed to be on one of the ends of the Cleptogram[D5]. We also noted that species C and D shared multiple common proteins in common, so they needed to be close together on the tree. During our analysis of the proteins, our teacher identified which letter represented each fish (it had remained a blind experiment up till this point) as the following:
Fish A-Salmon
Fish B-Yellow fin Tuna
Fish C-Halibut
Fish D-Turbot
Fish E-Catfish
With this extra information, we were able to analyze both our results and the evolutionary tree to create the cleptogram. For example, we noticed that species C and D both had a similarity with D, so we looked at the evolutionary tree to measure whether Tuna or Halibut were closer to Salmon evolutionarily to finish our prediction.
Discussion:
When reviewing the data once more, we noticed some discrepancies in our cladogram compared to the evolutionary tree. For example, our Yellow-fin Tuna found its way onto the beginning of the tree, when it should have landed near the end according to the evolution tree in our packet. Otherwise, we believe this experiment [D6]was a success, as we learned about how proteins can be used to supplement genetics and give us another tool in understanding our history. This could possibly be result of contamination in the fish muscle samples, due to touching the muscles with the same pair of gloves when transferring them into the tunes for protein extraction. If we were ever to do this experiment again, we would be sure to use tweezers of another similar tool to handle the muscles.
Work cited
“Hydrophobic_Interactions”http://chemwiki.ucdavis.edu/Physical_Chemistry/Physical_Properties_of_Matter/Atomic_and_Molecular_Properties/Intermolecular_Forces/Hydrophobic_interactions
