Part 1: Understanding Lactose Intolerance What is it?

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1 Part 1: Understanding Lactose Intolerance What is it? A defining characteristic of mammals is that mothers produce milk for their infants through a process called lactation. Mother s milk is packed with the proteins, fats, and carbohydrates that support the baby s growth and development. The main carbohydrate in milk is the sugar lactose, which is a disaccharide. Infant mammals produce the enzyme lactase in their small intestines. Lactase breaks down lactose into glucose and galactose. These two simpler sugars, or monosaccharides, are easily absorbed through the small intestine and into the bloodstream. The blood delivers the sugars throughout the body to provide cells with a source of energy. At around the time that children stop drinking their mother s milk, most of them also stop producing lactase. If lactase is not produced and a person drinks milk, undigested lactose travels from the small intestine to the large intestine, where it is digested by bacteria, which produce lactic acid, carbon dioxide, hydrogen, and methane gas in the process. When this happens, a person can have abdominal pain, bloating, flatulence, and diarrhea. Individuals with these symptoms are lactose intolerant. Only a minority of human adults 35% of the global human population continues to produce lactase into adulthood and can drink milk without any problems. These individuals are lactose tolerant (lactase persistent). Genetic studies suggest that lactose tolerance arose among human populations in the last 7,000 to 9,000 years. This is also when humans began domesticating animals like cows, goats, and camels and started drinking their milk.

2 Part 2: Testing for the Lactase Enzyme Benedict s solution is used to test for simple sugars, or monosaccharaides. When Benedict s is added to a solution and heated (to increase the rate of the reaction), the solution will turn yellow/orange in the presence of a simple sugar. 1) Predict the color (orange/not) if you add Benedict s to the following solutions, and explain your reasoning for each why do you think that? a. Milk (contains the enzyme lactose): b. Lactose- free milk (it has the enzyme lactase added to it): c. Sucrose solution (sucrose is a disaccharide): 2) Knowing that the active ingredient in Lactaid is the lactase enzyme, predict the color (orange/not) if you add Lactaid and Benedict s to the following solutions. Again, explain your reasoning. a. Milk + Lactaid: b. Lactose- free milk + Lactaid: c. Sucrose solution + Lactaid (sucrose is a disaccharide): Follow the instructions to complete the testing: Materials: (per group) 6 test tubes Graduated cylinder 250 ml beaker and large beaker (water bath) Hot plate Lactaid tablet Benedict s solution and dropper Milk Lactose- free milk Sucrose solution Stirring rod Labels

3 Procedure: 1. Create enzyme solution by pouring 200mL of water into a clean beaker. Add one Lactaid tablet to the water and stir until the tablet is dissolved. Write the name of this solution on the side of the beaker. 2. Label your test tubes (A, B, C, etc) and add the following. Gently swirl contents to stir. A. 2 ml Milk and 1 ml water B. 2 ml Milk and 1 ml enzyme solution C. 2 ml Lactose- free milk and 1 ml water D. 2 ml Lactose- free milk and 1 ml enzyme solution E. 2 ml Sucrose solution and 1 ml water F. 2 ml Sucrose solution and 1 ml enzyme solution 3. Let each test tube sit for 5 minutes. While they are sitting, start your water bath. Fill a beaker ½ full of water, and warm it on a hot plate. You will be placing all your test tubes in this beaker. 4. Add 10 drops of Benedict s solution to each test tube. *Be sure not to get Benedict s solution on you* Heat the test tubes in a hot water bath for about five minutes. 5. For each test tube, look for a color change you may need to gently swirl contents. A color change from blue, through murky green to yellow/orange to red indicates the presence of a simple sugar (glucose, fructose, galactose).the more reddish the precipitate, the more glucose is present. Fill out the data table with your results: Milk + Water Milk + Enzyme Lactose- free Milk + Water Lactose- free milk + enzyme Sucrose Solution + water Sucrose Solution + enzyme Color with Benedict s Glucose Present? (Y/N) indicates break down of complex sugar (lactose or sucrose) Clean up and continue on to Part 3 to look at how lactose intolerance is inherited.

4 Part 3: Determine the Pattern of Inheritance of Lactose Intolerance Researchers examined nine extended Finnish families for five generations. Below is one pedigree, exactly how it appeared in the original paper. 1) Based on the pedigree above, which of the following best describe the inheritance of the lactose- intolerance (filled- in symbols) trait? Recessive Dominant Inherited Sex- Linked 2) Use the data in the pedigree and terms listed above to make a claim about how lactose intolerance is inherited. Provide at least two pieces of evidence to support your claim. (Hint: examine 5+6 in generation II, and their offspring) 3) What additional information might increase your confidence in making this claim?

5 Now examine three additional pedigrees from the Finnish study, all as they appeared in the original publication. 4) After examining the three additional pedigrees from the Finnish study, does this data support your initial claim of how lactose intolerance is inherited? 5) Based on your claim, write the genotype of someone who is lactose tolerant and someone who is lactose intolerant.

6 Part 4: Finding the Responsible Mutation: 1) Study the two tables below. Using the pedigrees from Part3, fill in the phenotype (e.g. lactose tolerant or lactose intolerant) of each individual in the second column of the table. 2) Identify and circle all the nucleotides that differ between at least two sequences in each table.

7 3) If you were the researcher who discovered the above variations, a. How would you describe the variation(s) you found in Sequence 1? b. How would you describe the variation(s) you found in Sequence 2? 4) Based on the sequence data, which variation is associated with lactose intolerance? Is this variation found on one chromosome or both chromosomes in individuals with the trait? Explain your answer. 5) Based on the pedigrees and DNA sequencing, how is the variation associated with lactose intolerance is inherited? Part 5: Analysis and Conclusions Analysis: 1) What type of sugar is lactose? 2) What reaction is catalyzed by the enzyme lactase, and what is the effect of adding lactase enzyme to milk? 3) When the body does not secrete lactase enzyme in the intestine, the lactose sugar is not digested. Bacteria that are a normal part of the colon use the lactose for food and produce gas. How does this contribute to the symptoms of lactose intolerance?

8 4) During the past 10,000 years agriculture has been important to human populations. In some isolated areas crops did not perform as well or the climate did not permit relying on them year round. In these places animals and their milk were the main food supply. Use your knowledge of evolution and natural selection to explain how some populations may have become lactose tolerant. 5) Looking at the above map, distribution of lactose intolerance is not consistent throughout the world. Propose a feasible hypothesis to explain these geographical differences, including the fact that approximately 30% of all Americans are lactose intolerant compared to other parts of the world where that number is more than 80%?