Assignments for majors
These assignments will enable you to study the effects of mutations in the globin gene for 17 patients and learn how these mutations affect the health of each patient. For your ease in completing each assignment, the background text relevant to the experiment that you will perform is in red, instructions for each assignment are indicated by plain text, and questions or activities that you will be asked to provide answers for are indicated by bold text.
The following assignment is designed to help you become familiar with the operation of HemoglobinLab by studying sickle-cell disease.
Getting to Know HemoglobinLab: Sickle-Cell Disease
Chills, fever, headache, and vomiting are but a few symptoms of the disease called malaria. Malaria is caused by a protozoan, Plasmodium vivax, that lives in tropical countries and reproduces in a mosquito called the Anopheles mosquito. Plasmodium is transmitted to humans when an infected mosquito bites a human and sporozoites, an infectious stage of Plasmodium, enter the human bloodstream and travel to the liver. Sporozoites reproduce in liver cells and release progeny called merozoites into the bloodstream which infect red blood cells, reproduce, and rupture these cells to infect more red blood cells. Individuals who are heterozygous for sickle-cell disease have a higher resistance to malaria than wild type individuals. This resistance occurs because the fragile structure of sickled red blood cells interrupts the life cycle of Plasmodium.
1. Select the Blood Samples view on the input screen for HemoglobinLab. Scroll down the Select Case list and choose patient Miriam Dembele. Read Miriam's case history. Note that her case history is consistent with increased resistance to malaria. Compare Miriam's blood sample with the healthy control sample. Are there any obvious differences? Select the Microscope view and make note of any obvious differences in red blood cell structure. Do any of the red blood cells show phenotypic characteristics of sickle-cell disease? If so, approximately what percentage of her cells show these characteristics?
2. Select the Gel Electrophoresis view to examine the electrophoretic migration pattern for the beta globin subunits of Miriam's hemoglobin as compared with a control sample from a healthy patient. Is the migration pattern of Miriam's hemoglobin indicative of a mutation in one of her globin genes? Is Miriam homozygous or heterozygous for this mutation? Explain your answer.
3. Select the Peptide Sequence view. Click the Find Difference button to identify the amino acid change in Miriam's hemoglobin compared with the normal control hemoglobin. Differences in the amino acid sequence of Miriam's hemoglobin protein compared with the normal protein will align at the far left of the screen. Which amino acid has been substituted for in Miriam's gene? Note the position of this amino acid change. This will be important for identifying the position of the nucleotide change in the globin gene.
4. Select the Edit DNA Sequence view. Miriam's globin gene sequence appears, compared with the normal, wild-type globin gene sequence. First, you will need to locate the DNA sequence with the triplet ATG that indicates the position of the start codon that would appear on globin mRNA produced by transcription of this gene. You can do this either by scanning the gene by clicking on the double arrows or (more easily) by typing ATG in the Search window and hitting the return key. This will take you to the ATG with the A in nucleotide 87 outlined with a red box. Click on the Bracket Codons button to outline codons beginning at the ATG. Use the single arrow to advance to codon 6 (nucleotides 105 - 107).
Click on nucleotide 106--it should now be outlined with a red--and change the A to a T. Click the Translate button to see a comparison of your custom-mutated protein to Miriam's protein sequence and the normal protein.
Is this mutation consistent with what you know about the most common mutation that causes sickle-cell disease?
Refer to a codon chart by clicking on the Genetic Code button at the top of the HemoglobinLab homepage and identify the normal codon and the mutated codon that you changed to simulate the amino acid change in Miriam's hemoglobin.
Gel Electrophoresis of Hemoglobin
The initial diagnosis of a mutation in a hemoglobin gene often involves the interpretation of a patient's clinical symptoms, patient histories, and the results of biochemical tests such as gel electrophoresis and DNA sequencing. The following assignments are designed to illustrate the importance of gel electrophoresis as a technique that can be used to study protein structure.
1. A number of mutations in the hemoglobin protein result in a mutant protein that demonstrates a faster electrophoretic migration pattern on a gel than the normal protein. Pretend you are a biochemist who is interested in identifying hemoglobin mutations of this kind. You have available to you 17 patients who have donated blood samples to your lab, and your laboratory technician has run gels on the hemoglobin samples from these patients. It is now your job to interpret these gels to identify which patients may contain the mutant forms of hemoglobin that you are interested in learning more about.
Select the Gel Electrophoresis view, and examine the electrophoresis pattern for the hemoglobin molecules from each of the patients by clicking on each patient's name.
Which one of the patients has hemoglobin molecules that show a faster electrophoretic migration pattern than the control molecules?
Select the Peptide Sequence view and click the Find Difference button to identify the altered amino acid sequence for this patient. What is the mutation that appears? Is this mutation at the N-terminus or C-terminus of the globin polypeptide?
Provide a possible explanation for why this change in amino acid sequence would cause the mutant protein to show a faster electrophoretic migration pattern than the normal protein.
Read this patient's case history. This patient's mother has symptoms indicative of a hemoglobin mutation, but the patient's father appears normal.
Is the gel electrophoresis pattern that you observed for this patient's hemoglobin consistent with his or her family history? Explain your answer.
After you meet with this patient and discuss your interest in his or her blood, the patient tells you that his or her mother was born and raised in Hiroshima, Japan.
Because you are a well-rounded person with a strong knowledge of world history, what might you consider to be the cause of this patient's hemoglobin mutation?
2. Certain mutations in the beta globin gene result in altered amino acid sequences in the hemoglobin molecule that produce a protein with an increased affinity for binding to oxygen. One example of such a mutation produces a molecule called hemoglobin Yakima. Yakima involves an amino acid substitution mutation at position 99, where aspartic acid is replaced by histidine. Individuals with these mutant forms of hemoglobin often show redder-than-average complexions. Select the Blood Samples view and scroll through the patient case histories searching for the patient whose complexion matches this description.
Once you think you have found this patient, select the Microscope view and evaluate the patient's red blood cells for any obvious defects. Select the Gel Electrophoresis view.
Does the migration pattern of this patient's hemoglobin indicate a mutation in the protein? If your answer is yes, does this patient appear to be homozygous or heterozygous for this mutation?
Select the Peptide Sequence view and click the Find Difference button to identify the altered amino acid sequence for this patient.
What is the mutation that appears? Is this mutation indicative of hemoglobin Yakima? Provide reasons why you think this mutation may increase the affinity of oxygen for binding to hemoglobin Yakima.
Peptide Sequence Analysis of Hemoglobin
As powerful as gel electrophoresis is as a technique, changes in the amino acid sequence of a protein can be definitively determined only by analyzing DNA and peptide sequences. The following assignments will help you understand how these techniques can be applied to study protein structure.
1. Select the Blood Samples view and click on patient Rhonda Emolina. Compare the color of Rhonda's blood with that of the healthy control blood. Is the color of Rhonda's blood consistent with the conditions described in her patient history?
To determine the cause of Rhonda's anemia (abnormally low number of red blood cells), select the Gel Electrophoresis view.
Does Rhonda's hemoglobin migrate differently than the healthy control sample?
Because the results of this gel electrophoresis experiment are inconclusive in determining whether the cause of her anemia is due to a hemoglobin mutation or another problem such as an iron deficiency, more information about the sequence of Rhonda's hemoglobin protein needs to be considered.
Select the Peptide Sequence view and click on the Find Difference button to determine whether Rhonda may contain a mutated version of hemoglobin. Is there a mutation? If so at which position and what amino acid is changed?
2. Many invariant amino acid positions around the heme group have been identified in vertebrate and invertebrate hemoglobins. Changes in these invariant amino acids, many of which form the hydrophobic pocket around the heme group, result in a number of very serious blood diseases including a wide variety of anemias. Anemia is a condition that involves an abnormally low oxygen-carrying capability of the blood. Anemias can occur if the number of red blood cells in an individual is low. Anemias are also caused by the production of abnormal hemoglobin molecules (such as sickle-cell anemia, and thalassemia) and inadequate hemoglobin content in red blood cells. Common symptoms of anemias are decreased blood oxygen content, fatigue, shortness of breath, pale skin, and cool body temperature.
One of the invariant amino acids in beta globin is mutated in the form of hemoglobin called hemoglobin Hammersmith. This invariant amino acid is located at position 42 in the beta globin polypeptide. This mutation results in an unstable hemoglobin that cannot hold and position the heme group in the proper orientation for oxygen binding.
Select the Peptide Sequence view and compare the hemoglobin sequence for each female patient with the control sequence by clicking on the Find Difference button until you identify the female patient with hemoglobin Hammersmith. (Be sure to begin your search by starting at the first amino acid in the protein).
Which amino acid is substituted for in this patient?
Use the Edit DNA Sequence view to identify the codon for this amino acid. Alter this codon by changing positions in the codon until you have recreated the Hammersmith mutation. Refer to the codon chart in HemoglobinLab to confirm the mutation that you created.
In the previous exercises we have considered alterations in hemoglobin structure created by base-pair substitution mutations in the globin gene. Working together in a group of four or five students complete the following assignment which considers other types of mutations.
1. Select patient Juan Rodriquez. Click on the Blood Samples view and read his history. Note any differences in the color of Juan's blood compared with the control cells. Select the Microscope view and note the appearance of Juan's red blood cells compared with the control cells. Select the Gel Electrophoresis view and describe the electrophoretic pattern of Juan's hemoglobin.
Based on this electrophoretic pattern, develop at least two hypotheses that could explain this observation.
2. To determine if any of your hypotheses are correct, select the Peptide Sequence view. Click on the Find Differences button.
Compare the amino acid sequence of Juan's hemoglobin with the control sequence.
a. Once you have determined the first amino acid difference, refer to the codon chart in HemoglobinLab and identify the codon for the amino acid on the normal protein sequence. Select the Edit DNA Sequence view to modify this codon and recreate this mutation.
b. Return to the Peptide Sequence view. Use the double arrows to examine the rest of Juan's hemoglobin. Record all differences in amino acid sequence that you observe.
Do the results of this examination confirm or refute your hypothesis? If necessary, formulate a new hypothesis to account for this observation.
c. Return to the Edit DNA Sequence view. Based on your hypothesis, alter the codon sequence of Juan's globin gene until you have identified the nucleotide change(s) that have occurred in Juan's globin gene.
d. What is an aplastic crisis? What are common causes of aplastic anemia? Consider Juan's patient history. How might his aplastic crisis have resulted in the hemoglobin mutation that he has? If necessary, refer to an anatomy and physiology textbook in your library to answer this question.