Mouse Molecular Genetics

MOLECULAR GENETICS OF COLOR MUTATIONS IN ROCK POCKET MICE INTRODUCTION THE ROCK POCKET MOUSE The rock pocket mouse, Chaetodipus intermedius, is a small, nocturnal animal found in the deserts of the southwestern United States. Because most rock pocket mice have a sandy, light-colored coat, they are able to blend in with the light color of the desert rocks and sand that they live on. But populations of primarily dark-colored rock pocket mice have been found living in areas where the ground is covered in a dark rock called basalt, which was caused by geologic lava flows thousands of years ago. Scientists have collected data from a population of primarily dark-colored mice living in an area of basalt in Arizona’s Pinacate lava flow, as well as from a nearby light-colored population. Researchers analyzed the data from these two populations to search for the genetic mutation responsible for the dark coat color. Through their analyses, they discovered a mutation in the Mc1r gene that is involved in coat-color determination. THE MC1R GENE The coat color of rock pocket mice is primarily determined by two pigments: eumelanin, which is dark colored, and pheomelanin, which is light colored. The synthesis of these pigments is controlled by the products of several genes, including the Mc1r gene. This gene encodes a protein called melanocortin 1 receptor (MC1R). This receptor is found embedded in the membrane of melanocytes, which are cells specialized for pigment production. The melanocytes of wild-type (nonmutant) mice produce more pheomelanin than eumelanin. The result is a sandy-colored mouse. The mutated version of the Mc1r gene, however, triggers melanocytes to increase the production of eumelanin, resulting in the dark coat-color phenotype. GENE MUTATION A gene mutation is any change in the DNA sequence of a gene. Gene mutations can change the structure of the resulting protein. A change in protein structure can change, negate, or have no effect on function. There are several types of mutations, and several results that mutations can have on the amino acid sequences of proteins. Types of Mutations x Substitution mutation: The replacement of one nucleotide of DNA for another. Mutations that affect a single nucleotide are called point mutations. x Insertion mutation: The addition of one or more nucleotides to the DNA gene sequence. The insertion of nucleotides can result in frameshift mutations. x Deletion mutation: The loss of one or more nucleotides from the DNA gene sequence. The deletion of nucleotides can result in frameshift mutations. Potential Results a Gene Mutation Has on a Protein x Silent mutation: This mutation does not cause a change in the amino acid sequence of the protein; therefore, there is no change in the resulting protein. x Missense mutation: This mutation causes an amino acid in the sequence to be changed to another amino acid. This type of mutation causes a change in the primary structure of the protein (the linear sequence of amino acids), which typically results in a change in the three-dimensional conformation of the protein. x Nonsense mutation: This mutation causes the protein to be truncated (cut short) due to the incorporation of a “stop” signal into the DNA sequence. This results in translation being stopped before the amino acid sequence of the protein is completed. MATERIALS genetic code chart (see page 7 of this handout or a biology textbook) blue, red, and green colored pencils PROCEDURE 1. Watch the short film The Making of the Fittest: Natural Selection and Adaptation. 2. Using the DNA nucleotide sequence in the gene tables (page 3), determine the complementary messenger RNA (mRNA) sequence for the portion of the Mc1r gene provided. (Note: You are only transcribing a small portion of the DNA sequence for this protein. The actual gene contains 951 base pairs.) The numbers above some of the columns indicate amino acid positions in the protein sequence. Also indicated is whether the sequence codes for an extracellular, intracellular, or transmembrane part of the protein. 3. Using the mRNA sequence determined in Step 2, determine the resulting amino acid sequence of the MC1R protein. (Note: This is only a portion of the 317 amino acids in the entire protein. The numbers above some of the columns in the tables indicate amino acid positions in the protein sequence.) You may use the genetic code chart provided in your textbook or the one on page 7 of this handout. 4. There are five mutations in the dark-color Mc1r mutant gene. Compare the DNA sequence of the wild-type Mc1r gene with the DNA sequence of the mutant Mc1r gene. Indicate the locations of the five mutations by circling the five single DNA nucleotides that are mutated in the mutant Mc1r gene table. 5. From the introduction, determine whether each of these mutations is a silent, missense, or nonsense mutation. a. Using the mutant Mc1r gene data, shade in the columns (including DNA, mRNA, and amino acid) in the mutant table that contain a silent mutation. Use a blue colored pencil to do this. b. Likewise, use a red colored pencil to shade in the columns that contain a missense mutation. c. Shade any columns that contain nonsense mutations by using a green colored pencil. 6. Answer the questions following the gene tables. GENE TABLES DNA mRNA Amino Acid 015 TTG AGG TGG GCG TGT CCG CAA GGA GTG 024 GAG TGT CCG CAA GGA GTG 024 GAG CAC TGA CAC CAT 114 GTC CAC TGA CAC CAT 114 GTC GAC GGG GCC CGA GCC 163 ACC GAC GGG CGA GCC 163 ACC MUTANT MC1R GENE (DARK-COLORED COAT PHENOTYPE) DNA mRNA Amino Acid 015 TTG AGG TGG Extracellular Domain I WILD-TYPE MC1R GENE (LIGHT-COLORED COAT PHENOTYPE) DNA 105 CGG GAC CGG TGG GCC mRNA Amino Acid MUTANT MC1R GENE (DARK-COLORED COAT PHENOTYPE) DNA mRNA Amino Acid 105 CGG GAC CGG TGG Extracellular Domain III WILD-TYPE MC1R GENE (LIGHT-COLORED COAT PHENOTYPE) DNA mRNA Amino Acid 154 TCA TAA CAC TGT MUTANT MC1R GENE (DARK-COLORED COAT PHENOTYPE) DNA mRNA Amino Acid 154 TCA TAA CAC TGT Intracellular Domain I WILD-TYPE MC1R GENE (LIGHT-COLORED COAT PHENOTYPE) WILD-TYPE MC1R GENE (LIGHT-COLORED COAT PHENOTYPE) 208 CAC GTG TAC GAA 212 CGT MUTANT MC1R GENE (DARK-COLORED COAT PHENOTYPE) DNA mRNA Amino Acid 208 CAC GTG 212 CGT TAC Transmembrane V DNA mRNA Amino Acid DNA mRNA Amino Acid 230 GAA CAG GTG GTT CCA AAG GCT GAG TTT 239 CCG CCA AAG GCT GAG TTT 239 CCG MUTANT MC1R GENE (DARK-COLORED COAT PHENOTYPE) DNA mRNA Amino Acid 230 GAA CAG GTG Intracellular Domain III WILD-TYPE MC1R GENE (LIGHT-COLORED COAT PHENOTYPE) QUESTIONS 1. Using the amino acid numbers provided above the first and last column of each table, list the locations of the five amino acids that contain a mutation. _______ _______ _______ _______ _______ 2. Of the five mutations you identified in the Mc1r gene, how many are the following: _____ substitutions _____ insertions _____ deletions (Enter a number on each line.) 3. Of the five mutations you identified in the Mc1r gene, how many are the following: _____ silent _____missense _____ nonsense (Enter a number on each line.) 4. a. Which four amino acid locations (see Question 1 above) contain the missense mutations? _______, _______, _______, _______ b. Explain the link between DNA sequence and protein structure and function. ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ 5. Using the information on mutations in the introduction and your knowledge of proteins, develop a hypothesis to explain how the changes in the MC1R protein’s amino acid sequence might affect its function. _______________________________________________________________________ ____________ ____________________________________________________________________________________ ____________________________________________________________________________________ 6. Many proteins, including MC1R, contain several structural domains that can fold and function independently from the rest. The domain names were provided for each portion of DNA sequence you translated earlier. Answer the following questions. a. Where is the MC1R protein found, and what is its function? Be specific. ____________________________________________________________________________________ ____________________________________________________________________________________ b. Which protein domains contain the four Mc1r missense mutations? (Refer to the gene tables you completed earlier.) ____________________________________________________________________________________ ____________________________________________________________________________________ c. Define “extracellular.” ____________________________________________________________________________________ d. Define “intracellular.” ____________________________________________________________________________________ e. Why is it significant that the four missense mutations are found in the extracellular and intracellular domains of the protein? Explain your answer. (Hint: Think about MC1R’s function.) ____________________________________________________________________________________ ____________________________________________________________________________________ 7. Using the information on the Mc1r gene in the introduction and your knowledge of proteins, develop a hypothesis to explain how the change in MC1R protein function might directly affect a rock pocket mouse’s coat color. Be specific and consider both the light-colored and dark-colored phenotypes. ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ 8. Explain why the mutation at amino acid location 211 is not as significant as the other four mutations. ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ 9. Mutations are a source of genetic variation. In the film, Dr. Sean Carroll says that mutations occur randomly. What does this mean? ____________________________________________________________________________________ ____________________________________________________________________________________ 10. It is a common misconception that “all mutations are bad.” Use the example of rock pocket mice to explain why this statement is not true. In your answer, explain how the dark coat-color mutation can be an advantage to some mice and a disadvantage to others. Mutations can result in new traits and the selective advantage provided by a trait depends on the environment. For example, on a light substrate, individuals with ____________________________________________________________________________________ dark-colored coats would be at a disadvantage because they would stand out more than individuals with light-colored coats, making them easier for predators to spot. However, in the dark lava flow habitat, those same dark-colored individuals would have a selective advantage because they would be better camouflaged than ____________________________________________________________________________________ light-colored individuals. So the statement that “all mutations are bad” is incorrect, because there are different selective pressures on the traits produced by mutations depending on the habitat. There are also silent mutations that do not change the resulting protein meaning that they are neutral, neither good nor bad. L ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ 11. Use your understanding of evolution and the information in the film to explain how the dark-colored mutation came to be so common in some populations of rock pocket mice. Be specific. ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ ____________________________________________________________________________________ GENETIC CODE CHART Below is the standard genetic code: mRNA Æ amino acid. The inner circle represents the first letter of the codon, and the remaining letters follow in the second and third rings. Find the corresponding amino acid in the outer circle. GENETIC CODE CHART Below is the standard genetic code: mRNA Æ amino acid. The inner circle represents the first letter of the codon, and the remaining letters follow in the second and third rings. Find the corresponding amino acid in the outer circle.