Are All Fish Related? A look at biochemical evidence for evolution by iyunp418

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									 • Analyze protein profiles
   from fresh muscle tissue
   by…
 • Using polyacrylamide
   electrophoresis to
   separate proteins by size
 • Examine phylogenetic
   relationships based on
   muscle protein profiles

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             Traditional Systematics and
                      Taxonomy

      • Traditional classification based upon
        traits, especially morphological
             characters.




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             Biochemical Similarities
• Traits are discrete characteristics. In biology we are
  concerned with “heritable traits.”
• Proteins are generally responsible for traits, most
   often as enzymes affecting biochemical pathways or
   as structural or dynamic elements of the cytoskeleton.

• DNA codes for proteins that confer traits


     DNA        RNA          Protein Trait
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• Experiment: Compare protein profiles various
  fresh muscle tissue.
• Procedure:
    –   Extract proteins from tissue
    –   Denature proteins
    –   Separate proteins by size using polyacrylamide gel
        electrophoresis
    –   Stain to visualize protein bands
    –   Analyze and interpret gels
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             Protein size comparison

       • Break protein complexes into individual
         proteins
       • Denature proteins using detergent and
         heat
       • Separate proteins based on size


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              Why use polyacrylamide
             gels to separate proteins?
• Acrylamide gel        tight matrix
• Ideal for protein separation
• Smaller pore size than agarose
• Proteins much smaller than DNA
      – average amino acid = 110 Da
      – average base pair = 649 Da


      – 1 kilobase of DNA = 650 kDa
      – 1 kilobase of DNA encodes 333 amino acids = 36 kDa
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                   Protein size
       • Size measured in kilodaltons (kDa)
       • Dalton = mass of hydrogen atom
               = 1.66 x 10 -24 gram
       • Average amino acid = 110 daltons



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        Muscle contains proteins of
                many sizes
  Protein                    kDa               Function
  titin                      3000              center myosin in sarcomere
  dystrophin                 400               anchoring to plasma membrane
  filamin                    270               cross-link filaments into gel
  myosin heavy chain         210               slide filaments
  spectrin                   265               attach filaments to plasma membrane
  nebulin                    107               regulate actin assembly
  a-actinin                  100               bundle filaments
  gelosin                    90                fragment filaments
  fimbrin                    68                bundle filaments
  actin                      42                form filaments
  tropomyosin                35                strengthen filaments
  myosin light chain         27                slide filaments
  troponin (T, I, C)         30, 19, 17       mediate regulation of contraction
  thymosin                   5                sequester actin monomers
  These are generalized molecular masses and may vary in different species….


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                       Actin and Myosin
• Actin:
             •   5% of total protein
             •   20% of vertebrate muscle mass
             •   375 amino acids = 42 kDa
             •   Forms filaments


• Myosin:
             • Tetramer of two heavy subunits
               (220 kDa)
               and two light subunits (20 kDa)
             • Breaks down ATP for muscle
                 contraction
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         What’s in the sample buffer?
  • Tris buffer to provide appropriate pH
  • SDS (sodium dodecyl sulfate) detergent to
      dissolve proteins and give them a negative
      charge
  • Glycerol to make samples sink into wells
  • Bromphenol Blue dye to visualize
      samples
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             Why heat the samples?
• Heating the samples
  denatures protein            s-s
                                     SDS, heat
  complexes, allowing
                                     proteins
  the separation of                  with SDS
  individual proteins by
                           -
  size

                           +
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       How does an SDS-PAGE gel
                work?
• Negatively charged proteins
 move to positive electrode
                                 s-s

• Smaller proteins move faster         SDS, ß-Me,
                                       heat
                                        proteins with
                                        SDS

• Proteins separate by size
                          -


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                          +                      14
             Gel Analysis
                   15% SDS-PAGE
                   • Lane 1: Marker
                   • Lane 2: Shark
                   • Lane 3: Salmon
                   • Lane 4: Rainbow trout
                   • Lane 5: Cod
                   • Lane 6: Sturgeon
                   • Lane 8: Croaker
                   • Lane 9: Weakfish
                   • Lane 10: Actin/myosin

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         Molecular Weight Analysis
             kDa   mm
             203     8.5
             135    12.0         250
              86    18.5         200
                                 150




                           kDa
              41   28.0
                                 100

              33   34.0           50
                                   0
                                       0   20       40    60
              19   41.5
                                           mm from well

              8    44.5
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                           Phylogenetic Tree
                                                                                                    TUNA
                             CARP                      SALMON                           SNAPPER     MACKEREL
                             MINNOW                    TROUT                            PERCH
                                       CATFISH                                COD       WALLEYE
                                                                 SMELT        HAKE
                       ANCHOVIES                                                        BASS
                                                                              POLLOCK                   FLOUNDER
                       HERRINGS                        PIKE
                                                                                                        SOLE
                       SARDINES                                                                         HALIBUT



                        STURGEON
                                                                                             GAR

                                  SHARK

              Agnatha         Chondrichthyes                  Ostheichthyes       Amphibia   Reptilia    Aves   Mammalia


             OYSTER
             CLAM
             MUSSEL
             SCALLOP       CRAB
             OCTOPUS       LOBSTER
             SQUID         SHRIMP

             Mollusk      Arthropod       Echinoderm      Chordate




                   Protostome                     Deuterostome




                        Metazoa

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