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

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.

Central Dogma

DNA

RNA

Protein

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?

Symptoms of Sickle Cell Anemia

pain episodes strokes increased

infections leg ulcers bone damage yellow eyes or

jaundice early gallstones lung blockage

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

Proteins

synthesized from amino acids

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

*

4 classes of structure.

Website for Amino acid interactive Workshop

Amino acids – everyone open to this page

http://www.biomed.curtin.edu.au/biochem/tutorials/AAs/AA.html

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?

Amino acid characteristics

Alpha helix is the “default”Ala, Glu, Leu

Beta sheetBranched R groupsVal, Thr, IleHelix disrupters with close H bond participants:Ser, Asp, Asn

Turns (not shown): Gly, Asp, Pro

The Nucleic Acids: DNA and RNA

DNAsynthesized from deoxynucleotide triphosphates

(dNTPs)

RNAsynthesized from nucleotide triphosphates

(NTPs)

OHOH OH

dNTP NTP

5’ and 3’

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

Website for interactive workshop for DNA analysis

DNA RNA PROTEIN

Replication

Central Dogma

DNA

RNA

Protein

Transcription

Translation

DNA RNA PROTEIN

Transcription

Mature mRNA

RNA, but NOT mRNA

RNA, but NOT mRNA

DNA RNA PROTEIN RNA processing

Central Dogma

DNA

RNA

Protein

Transcription

Translation

Translation

DNA RNA PROTEIN Translation

Etc.5’ UTR

DNA

RNA (with ribosomes)

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?

*

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.

Summary

DNA (mutated = changed)

RNA (mutated)

Protein (possibly mutated)

Remember: Mutation is not always “bad”! For example: Mutation → Evolution → An additional normal genome

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

Hemoglobin HBB1

>gi|4504349|ref|NP_000509.1| beta globin [Homo sapiens] MVHLTPEEKSAVTALWGKVNVDEVGGEALGRLLVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHGKKVLGAFSDGLAHLDNLKGTFATLSELHCDKLHVDPENFRLLGNVLVCVLAHHFGKEFTPPVQAAYQKVVAGVAN ALAHKYH

FASTA format for a protein sequence in single letter code

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

Workshop due as email to me by 9AM Wednesday, 6/24.

1. Give the answers to questions/challenges from slides within today’s PowerPoint.

2. 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.

3. What is the chromosomal location of the gene that causes sickle cell anemia?

4. What is the name of the gene? 5. 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)6. 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?