Sickle Cell Anemia

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Sickle Cell Anemia

• An example of why
• a change in protein can lead to disease
• a change in DNA can lead to a change in protein

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Ground Rules for Class Discussions and Workshops

• Be on time.
• Speak so that everyone from front to back can
  hear you.
• Listen when others are speaking.
• If its review for you, use you intellect to hear
  it in a new way.
• Write down your answers or consolidate to print.

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Central Dogma
DNA
RNA
Protein
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What do you already know about hemoglobin?

• What is the function of hemoglobin?
• What class of biomolecules does hemoglobin belong
  to?
• What are the symptoms of sickle cell anemia?
• Is sickle cell anemia hereditary?
• What does that tell us?

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Proteins

• synthesized from amino acids

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Circle Triangle Square Bond Amino terminal
Carboxy terminal
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4 classes of structure.
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Website for Amino acid interactive Workshop

• Amino acids everyone open to this page
• http//iws.ohiolink.edu/chemistry/biochemistry/aat
  ut.html

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Power of the R Groups

• Note the one letter and 3 letter abbreviations
  for you amino acid(s).
• Identify the atoms in red, blue, white, gray, and
  other colors
• Find the carboxy group, amino group, beta carbon,
  R group
• Categorize the amino acids and be able to say
  why some fit in more than one category!
• Aromatic
• Aliphatic, unbranched
• Aliphatic, branched
• Polar
• Positively charged (basic)
• Negatively charged (acidic)
• Small
• Has a sulfur atom in the R group
• Hmm?
• Which are hydrophobic vs. hydrophilic?
• Which would attract each other if brought
  together?
• Which would repel?
• Which would likely fold to the interior in an
  aqueous environment?
• Which would likely fold to the exterior in a
  lipid environment?

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The Nucleic Acids DNA and RNA

• DNA
• synthesized from deoxynucleotide triphosphates
  (dNTPs)
• RNA
• synthesized from nucleotide triphosphates
• (NTPs)

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OH
5 and 3
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Website for interactive workshop for DNA analysis

• DNA sequence
• Write the primary sequence of the DNA displayed
  in 3B from the 5 to the 3 end of both strands

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Central Dogma
DNA
Transcription
RNA
Translation
Protein
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DNA
RNA (with ribosomes)
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Translation
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Translation exercise

• Translate the following sequence using the codon
  table
• ATG GTG CAC CTG ACT CCT GAG GAG AAG TCT GCC GTT
  ACT
• Perform same procedure on the sequence below
  using a software program
• ATG GTG CAC CTG ACT CCT GTG GAG AAG TCT GCC GTT
  ACT http//us.expasy.org/tools/dna.html
• How many nucleotides have changed in the codon in
  boldface?
• What is the amino acid difference in the two
  sequences?
• What is the quality of that difference with
  respect to R groups?

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The early evidence that sickle cell anemia is
caused by an amino acid change in hemoglobin.
Tryptic digest the protease trypsin cleaves C
terminal to lysine and arginine.
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Summary
DNA (mutated changed)
RNA (mutated)
Protein (mutated)
Remember Mutation is not always bad! For
example Mutation ? Evolution ? An
additional normal genome
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Multiple sequence alignment for cytochrome C
mutation and conservation

• Human protein accession number AAA35732 (see next
  slide)
• Dog protein accession number XP_532493
• Yeast protein number from structure database
• 1YCC
• CLUSTAL W PROGRAM

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FASTA format for a protein sequence in single
letter code
Hemoglobin HBB1 gtgi4504349refNP_000509.1
beta globin Homo sapiens MVHLTPEEKSAVTALWGKVNVDE
VGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFS
DGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEF
TPPVQAAYQKVVAGVAN ALAHKYH
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FASTA format for a protein sequence in single
letter code
gtgi1244762gbAAA98563.1 p53 tumor suppressor
homolog MSQGTSPNSQETFNLLWDSLEQVTANEYTQIHERGVGYEYHE
AEPDQTSLEISAYRIAQPDPYGRSESYD LLNPIINQIPAPMPIADTQNN
PLVNHCPYEDMPVSSTPYSPHDHVQSPQPSVPSNIKYPGEYVFEMSFAQ
PSKETKSTTWTYSEKLDKLYVRMATTCPVRFKTARPPPSGCQIRAMPIYM
KPEHVQEVVKRCPNHATAKE HNEKHPAPLHIVRCEHKLAKYHEDKYSGR
QSVLIPHEMPQAGSEWVVNLYQFMCLGSCVGGPNRRPIQLV FTLEKDNQ
VLGRRAVEVRICACPGRDRKADEKASLVSKPPSPKKNGFPQRSLVLTNDI
TKITPKKRKIDD ECFTLKVRGRENYEILCKLRDIMELAARIPEAERLLY
KQERQAPIGRLTSLPSSSSNGSQDGSRSSTAFS TSDSSQVNSSQNNTQM
VNGQVPHEEETPVTKCEPTENTIAQWLTKLGLQAYIDNFQQKGLHNMFQL
DEFT LEDLQSMRIGTGHRNKIWKSLLDYRRLLSSGTESQALQHAASNAS
TLSVGSQNSYCPGFYEVTRYTYKHT ISYL
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How to prepare the sequences for the MSA on
ClustalW

• For Human and Dog
• Go to NCBI
• Select to search the protein database from the
  dropdown menu
• Enter the Accession Number (previous slide) and
  GO
• Click on the link
• Change the display to a FASTA file
• Copy the FASTA output for both species into a
  single text file. Make sure the header is
  separate from the sequence.
• For Yeast
• Clink on the link, find the FASTA format and copy
  into the same file
• Copy or upload the file into ClustalW

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Workshop due as hardcopy when you arrive Thursday
AM.

• Include answers from within todays class. Email
  to me by 9 AM Wed.
• Print out your ClustalW results and attach a
  short paragraph discussing how Clustal W gives
  you a clue as to which part(s) of the Cytochrome
  C protein you would hypothesize are most
  important to its function (which is/are the same
  in all 3 organisms). Start your paragraph as a
  hypothesis as to which parts are most important,
  and write your discussion as a defense of your
  hypothesis.
• Find out the chromosomal location of the gene
  that causes sickle cell anemia.
• Give the name of the gene.
• State the nucleotide change and amino acid change
  that leads to sickle cell anemia (there may be
  more than one change that gives rise to the
  disease)
• If sickle cell anemia is so devastating, why has
  it lasted in the population for such a long time?
  Give a molecular, mechanistic, evolutionary
  explanation (you may have to do a little research
  to get this). What does the sickled molecule do
  that the normal molecule cant?