MARIA'S TRAVELS (Waterman & Stanley. 1998.)

Synopsis: An epidemic caused by a fungus has begun in the U.S with potentially devasting effects on the corn crop. Derrick Hernandez and Maria Santini are graduate students studying the epidemic and crop improvement. An important resource in their studies is wild corn, teosinte, which may be resistant to the fungus. Maria, a geneticist, and Derrick, an ecologist, are headed south for their first field season.

PART 1

While enroute to Durango from Mexico City, Maria was delayed several days by storms. Worse, she contracted diarrhea, which she began treating right away. After a day's rest in Durango, she made the steep and difficult hike to the research site where she soon collapsed. Her symptoms were abdominal and leg cramping, disorientation, and rapid shallow breathing. Alerted by radio, Dr. Frederico Stegnaro in Durango advised giving her small amounts of saline during the night. The next day she made her way down the mountain on a pack mule and with Derrick's help (he was feeling poorly himself) to see the doctor.

At the clinic, Dr. Stegnaro took Maria's blood samples and did a variety of procedures to test out several possibilities. Thin and thick blood slides showed no evidence of malaria,but her blood chemistry was more revealing. Her hematocrit was abnormally high, blood pH lower than normal and K+ very low. He also noted what might be one or two unusual red blood cells. He started rehydration therapy and sent her by ambulance jeep to Mazatlan Hospital.

PART 2

Dr. Luna said, "I wanted to talk with you about some of the findings. The doctor in Durango reported one or two odd erythrocytes. I did not find these. But I did do a special electrophoresis test on a sample of your blood. Senora Santini, are you aware that you have sickle cell trait?"

Maria, stunned, said, "What? What? I have sickle cell disease?"

"No, Senora Santini, you have sickle cell trait, not the disease. You will live normally with no restrictions."

"How could I have this? I'm Italian!"

"Most people think that only individuals with African ancestry can have sickle cell," the doctor replied, " but that is not true."

Maria turned to Marcus and quietly asked "Marcus, doesn't your cousin Leland have sickle cell?"

 

NEW PART 3.

Maria, interested in determining the molecular basis for her condition, decides to examine a list of Hb Beta-chain gene sequences that she has retrieved from GENBANK. During your preliminary research, you have isolated the hemoglobin beta sub-unit from Maria's red blood cells. With this molecular data you can use the appropriate software tools to help Maria understand her illness.

Getting

Started

Students begin by using the group problem board.

 

Group Problem Board

What do we know?	What do we need to know?	How will we find out?

Discussion by the groups on the problem board.

Read handout below.

Computer Lab: Homology Searching with Hemoglobin Proteins

Background Information

The transport of oxygen to the cells of the body depends upon the ability of hemoglobin, a complex protein found in red blood cells, to bind oxygen molecules that enter from the lungs. The protein itself consists of four major subunits, two alpha subunits and two beta subunits. Each subunit binds a heme group consisting of an iron atom that is the principal binding region for oxygen molecules entering red blood cells, and is also responsible for giving red blood cells their characteristic color. The oxygen molecules remain tightly bound to the hemoglobin protein until the red blood cell is transported to peripheral tissues where the lower oxygen tension causes its dissociation from the protein.

The formation of the hemoglobin protein is based upon the instructions from various genes found in the nucleus of red blood cells. Thus, inheritance of a correct hemoglobin protein depends upon proper sets of nucleotide sequences, which in turn code for specific amino acids, which further interact to produce the fully functional three-dimensional protein.

Various changes in these nucleotide sequences, commonly known as mutations, result in different proteins being produced that may result in a defect in the ability to transport oxygen. One of the most deleterious consequences of these mutations is a condition called sickle cell anemia (Hbs). In this case, a single nucleotide base substitution results in an improper amino acid being placed in the beta chain subunit that subsequently changes the critical shape of the protein, making it much more difficult to transport oxygen. Not only does a person with this condition have difficulties respiring, but also leads to a red blood cell that is distorted in shape (i.e. "sickle shape") that can lead to a group of harmful symptoms that can ultimately lead to death. The prevalence of this condition in the United States is less than 1%, whereas in Africa the prevalence can reach as high as 20%. Cross-culturally, the condition seems to arise more frequently in black populations in comparison to Caucasians.

Through the advances in biotechnology, the exact nucleotide sequence of the gene encoding the beta-globulin protein has become known. It is now possible, through the use of a large database of gene sequences to study the beta-globulin chain. In this manner, you will be able to trace the "flow of information" from this gene sequence through to a sequence of amino acids that comprise the final beta-globlin protein. In this way, you can use the appropriate software tools in order to help Maria understand her condition.

Biology Workbench Activities

The first activity using Biology Workbench allows groups of students to focus on different ways that molecular data can be used. Students will have to analyze the results they find and then report these findings in the form of a poster. During the poster session, students will try to make sense of other students focus area and then make sense of all the data as a whole. The important aspect in this activity is that the students will be self-directed in their learning and in addition to this, they will be teaching each other through group collaboration.

 

What ideas should come out of this activity?

Through this activity, students should come to the understanding that different variants of nucleotide sequence are present. This will allow them to see that some mutations in an organism's DNA do not end up affecting protein synthesis. This will allow the facilitator to bring up the idea that some DNA is not essential for the final product. Rather, only various regions (exons) are actually needed for when the cell goes to the protein synthesis stage. Upon comparison of the normal DNA with the sickle cell DNA, students will find out that the sequences look very much the same, in terms of their similarities and differences. However, they should be able to see that there is a substitution in the sickle DNA that is dramatically different than the normal DNAs. When considering the human and gorilla, students will also see that when we begin to compare differences between species, the number of differences becomes greater, thus bringing up the idea that the DNA has changed during the course of evolution to form the human. However, they will also notice that a lot of DNA is similar. Thus, they will see that some portions of DNA have been conserved through evolution, and these sequences are probably the most important in facilitating the protein's structure. This idea will be confirmed in the students minds when they go and analyze the amino acid sequence between the human and gorilla, which basically shows the same Hb protein aside from a single neutral mutation.
Students should discover that the sequences of mRNA (cDNA) are actually a lot smaller than the original DNA (gDNA). Hence, similar sequences from the gDNAs have been used to construct the final mRNA product. Upon analysis of the protein structure, they should see even more so, that the amino acid structure is basically the same. When comparing normal versus sickle protein structure they will see one amino acid change that sticks out. When they follow this back to the RNA, they will see that this nucleotide base substitution was crucial in determining the amino acid to be placed in the protein. Questions should also arise concerning the nature of the thalassemia patient's Hb. The effect of a deletion in the DNA causes a frameshift mutation that completely changes the translating reading frame, thus resulting in a truncated Hb polypeptide. Hence, students will come to the realization that certain single nucleotide changes can have a dramatic effect on the final product produced. Last, when comparing the mRNA and amino acid sequence from the human and gorilla, students will see that, despite the fact there are differences in their mRNAs, the same basic protein results. Thus, they will be able to see the redundacy in the genetic code. Stemming from this, the teacher may want to bring up the point that certain species tend to only use certain codons for a particular amino acid and then ask students why this may be.

Protein Explorer

The activity using Protein Explorer is a logical extension to see the relationship between molecular sequence and how this determines three-dimensional structure. There are questions in the activity package that allow students not only to see the differences between normal and sickle Hb, but also to discover some other general ideas about protein structure.

What ideas should come out of this activity?

In using protein explorer, students will see firsthand, the dramatic effects that a single nucleotide change (=amino acid change) can have on protein structure. This is illustrated perfectly with sickle Hb since the change in shape results in the aggregation of two Hb molecules. Students can now speculate as to what effects this dimer complex will have on oxygen transport. Through discussion, the teacher can bring out the idea of positive cooperativity and how this cannot happen if Hb is in the sickle form. After looking at the effects on oxygen transport, one can then ask the class to relate the Hb clumping to the effect it will have on cell shape and the symptomology that is shown in the disease. Students are bound to ask what would happen in Maria's case since she is only a heterozygote. Thus, a discussion can be led in this direction. This discussion can also be related to the higher prevalence in Africa and why this may be the case.

Lab Activity Instructions and Worksheets

Using Biology Workbench

1. Open Netscape Navigator application.

2. Type the following URL into the Location box into the navigator window.

http://workbench.sdsc.edu

SETTING UP AN ACCOUNT
3. Click on the hotlink "Set up a free account," which is in blue and is underlined. This will bring you to a new screen.

4. Supply the information requested. Use your full name without spaces (for example: blairwinchproject) and for a password, use "biology."

5. Click on the button "REGISTER."

6. At the next screen, click on "Biology Workbench," which should be blue and underlined.

STARTING A NEW SESSION
7. Scroll down and click on the button called "Session Tools."

1. At the next screen, click on the title screen where it says "Click here to toggle between menus and buttons." This will make all the tools show up as buttons.

2. Click on the button labeled "NEW."

3. At the next screen, name the session " Maria's Travels" in the white box to the right of the words "Session Description."

4. Click on the button "Start New Session". If you have done this correctly, you will see the name of your session under the column starting with "Default Session".

UPLOADING PROTEIN SEQUENCES

5. Click on the button "Protein Tools"

6. Scroll down and click on the button "ADD"

7. Click on the "RUN" button.

8. Click "BROWSE" button.

9. Retrieve protein variants from the desktop in a file called "Protein Sequence Variants".

Protein Sequence Variants

Normal Hb beta chain protein

MVHLTPEEKSAVTALWGKVNVDEVGGEALGRL
LVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHG
KKVLGAFSDGLAHLDNLKGTFATLSELHCDKLH
VDPENFRLLGNVLVCVLAHHFG

 

Maria's Hb beta chain protein

MVHLTPVEKSAVTALWGKVNVDEVGGEALGRL
LVVYPWTQRFFESFGDLSTPDAVMGNPKVKAHG
KKVLGAFSDGLAHLDNLKGTFATLSELHCDKLH
VDPENFRLLGNVLVCVLAHHFG

 

Thalassemia beta Hb protein

MVHLTPEEKSALLPCGAR

 

Gorilla Hb beta chain protein

MVHLTPEEKSAVTALWGKVNVDEVGGEALGRL
LVVYPWTQRFFESFGDLSTPDAVMGNPKVKAH
GKKVLGAFSDGLAHLDNLKGTFATLSELHCDKL
HVDPENFKLLGNVLVCVLAHHFG

10. Click on the "Upload File" button.

11. At this point, you will be able to see the sequences on your screen.

12. IMPORTANT!!!! Click the "SAVE" button at the top or the bottom of the screen.

ALIGNING SELECTED SEQUENCES

13. Click on 4 hemoglobin beta chain protien sequences to activate them. Be sure that a small checkmark is in the box to the left of all the sequences that you will want to analyze.

14. Click on the "CLUSTALW" button.

15. Click the "RUN" button.

16. On the next screen, click the "SUBMIT" button.

17. On the next screen go to "Import Allignments" and then click on "CLUSTALW-Protein " box.

18. Scroll down and highlight "BOXSHADE" and press "RUN". On the next screen go to "Residue Numbering" and select ruler. Press "SUBMIT". This will allow you to see differences in sequence by the highlighting in colors.

19. Go to the "File" menu and print your group's sequence alignment. Write your names on the printout

20. Now scan the sequences that you have just printed.

Complete multiple comparisons of the amino acid sequences to answer some of the questions your group has posed:

Case:

Waterman, M.A. and E.D. Stanley. 1998. Investigative cases and case-based learning in biology. In Jungck, J.R. and V. Vaughan, eds. BioQUEST Library V, on CD-ROM, San Diego: Academic Press.

 

Data file:

Protein Sequence Variants

 

Internet :

Biology WorkBench - http://workbench.sdsc.edu

Protein Explorer

Poster Session