Sickle Cell Anemia

1. 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

2. 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 it’s review for you, use you intellect to hear it in a new way. • Write down your answers or consolidate to print.

3. Central Dogma DNA RNA Protein

5. 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?

6. Symptoms of Sickle Cell Anemia • kidney damage and loss of body water in urine • painful erections in men (priapism) • blood blockage in the spleen or liver (sequestration) • eye damage • low red blood cell counts (anemia) • delayed growth • pain episodes • strokes • increased infections • leg ulcers • bone damage • yellow eyes or jaundice • early gallstones • lung blockage

8. Proteins synthesized from amino acids

9. Circle; Triangle; Square; Bond; Amino terminal; Carboxy terminal

10. * 4 classes of structure.

11. Website for Amino acid interactive Workshop • Amino acids – everyone open to this page • http://www.biomed.curtin.edu.au/biochem/tutorials/AAs/AA.html

12. Power of the R Groups • Note the one letter and 3 letter abbreviations for your 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?

14. Amino acid characteristics

15. Beta sheet Branched R groups Val, Thr, Ile Helix disrupters with close H bond participants: Ser, Asp, Asn Alpha helix is the “default” Ala, Glu, Leu Turns (not shown): Gly, Asp, Pro

17. The Nucleic Acids: DNA and RNA DNA synthesized from deoxynucleotide triphosphates (dNTPs) RNA synthesized from nucleotide triphosphates (NTPs)

19. OH OH dNTP NTP OH 5’ and 3’

21. 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

22. DNA RNA  PROTEINReplication

23. Central Dogma DNA Transcription RNA Translation Protein

24. DNA  RNA PROTEINTranscription

25. RNA, but NOT mRNA RNA, but NOT mRNA Mature mRNA DNA  RNA PROTEINRNA processing

26. Central Dogma DNA Transcription RNA Translation Protein

27. Translation

28. DNA RNA  PROTEINTranslation 5’ UTR Etc.

29. DNA RNA (with ribosomes)

30. 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?

32. *

33. 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.

34. Summary DNA (mutated = changed) RNA (mutated) Protein (possibly mutated) Remember: Mutation is not always “bad”! For example: Mutation → Evolution → An additional normal genome

35. 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

36. FASTA format for a protein sequence in single letter code Hemoglobin HBB1 >gi|4504349|ref|NP_000509.1| beta globin [Homo sapiens] MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVAN ALAHKYH

37. 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

38. Workshop due as email to me by 9AM Wednesday, 6/24. • Give the answers to questions/challenges from slides within today’s PowerPoint. • Print out your ClustalW results and add 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. • What is the chromosomal location of the gene that causes sickle cell anemia? • What is 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 can’t?