Using ADE Mutants to Study Respiration and Fermentation in Yeast Extensions to Basic Lab

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1 Using ADE Mutants to Study Respiration and Fermentation in Yeast Extensions to Basic Lab Introduction: David Form, Nashoba Regional High School And Jessica Forton, Melrose High School Bioinformatics Component We have experimentally determined the energy-yielding and color phenotypes of two adenine-requiring mutants in the presence of glucose, glycerol and excess adenine. We will now use bioinformatics to compare the two mutations and their position in the biochemical chain of reactions that produces adenine in yeast. Specifically, we will compare each of the genes and their enzyme products according to: 1. n which of the 16 yeast chromosomes each gene is located. 2. The name and function of each enzyme. 3. The size of each enzyme 4. The 3-D structure of each enzyme. Each student will be asked to research one of the genetic mutants associated with the ade biochemical pathway. Student groups will be formed so that you can compile the information in order to determine the position of each enzyme mutant in the biochemical pathway that leads to the production of adenine. Procedure: 1. Go to the Saccharomyces Genome yeastgenome.org. 2. Under Sequence, go Gene/Sequence Resources. 3. Under 1. Enter a Name enter Ade* and click on Submit Form. 4. You will see a list of each gene in the Ade pathway. 5. Select the gene in which you are interested. 6. Under Biology/Literature, we will be exploring Locus info, Protein Info and 3-D structure. 7. Under Locus Info, you will be able to determine: i. The chromosome on which this gene is located. ii. iii. The name of the enzyme. The function of the enzyme. 8. Under Protein info, you will be able to find the size of the protein in amino acids and the molecular weight of the protein. 9. Under 3-D structure, you will find the PDB code for the first homolog (variant) of this protein. This code will allow you to view the 3-D structure of the enzyme, using jmol.

2 10. Use the information gathered from SGD to fill in the information for your assigned gene/enzyme on Tables 1 and When you are done, to to FirstGlance in 12. Insert the PDB code for your enzyme in the box. 13. A new window will open up with a 3-D model of your enzyme. 14. Click on spin and background to stop the model from spinning and to make the background black. 15. bserve your model in cartoon (ribbon) and contact (space-fill) views. Feel free to play around with the other views and features of this program! Data Table 1 Mutant Symbol Name of enzyme Enzyme Function Ade1 Ade2 Ade3 Ade4

3 Ade5/7 Ade6 Ade5/7 Ade8 Data Table 2 Mutant Symbol Chromosome # Protein length (# of a.a) M.W. of protein PDB id # Ade1 Ade2 Ade3 Ade4 Ade5/7 Ade6 Ade5/7 Ade8 Critical Thinking Questions

4 1. Summarize all of the ways in which you have demonstrated that the various enzymes of the adenine-producing pathway in yeast are separate, distinct proteins, with different structures, specificities and encoded by different genes. 2. Did you find evidence that a single enzyme can catalyze more than one reaction? How do you think that this is possible? 3. Mutations in the ade1 and ade2 genes result in yeast that develop a pink phenotype under certain conditions. What are these conditions? 4. Why do these (Ade1 and Ade2) mutations yield a pink phenotype, while mutations in the other ade genes will not yield a pink phenotype?

5 Ade1 Mutant Symbol Data Table 1 (Teacher s Version) Name of enzyme N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthetase Enzyme Function required for 'de novo' purine nucleotide biosynthesis; red pigment accumulates in mutant cells deprived of adenine Ade2 Phosphoribosylaminoimidazole carboxylase catalyzes a step in the 'de novo' purine nucleotide biosynthetic pathway; red pigment accumulates in mutant cells deprived of adenine Ade3 C1-tetrahydrofolate synthase involved in single carbon metabolism and required for biosynthesis of purines, thymidylate, methionine, and histidine; null mutation causes auxotrophy for adenine and histidine Ade4 Phosphoribosylpyrophosphate amidotransferase (PRPPAT; amidophosphoribosyltransferase), catalyzes first step of the 'de novo' purine nucleotide biosynthetic pathway Ade5,7 aminoimidazole ribotide synthetase and Bifunctional enzyme of the 'de

6 glycinamide ribotide synthetase novo' purine nucleotide biosynthetic pathway Ade6 Formylglycinamidine-ribonucleotide (FGAM)- synthetase catalyzes a step in the 'de novo' purine nucleotide biosynthetic pathway Ade5,7 aminoimidazole ribotide synthetase and glycinamide ribotide synthetase Bifunctional enzyme of the 'de novo' purine nucleotide biosynthetic pathway Ade8 Phosphoribosyl-glycinamide transformylase catalyzes a step in the 'de novo' purine nucleotide biosynthetic pathway Data Table 2 (Teacher s Version) Mutant Symbol Chromosome # Protein length M.W. of protein PDB id # (# of a.a) Ade ,603 2CNQ Ade2 XV ,339 3K5H Ade3 VII ,204 3PZX Ade4 XIII ,719 1ECF Ade5,7 VII ,067 2QK4 Ade6 VII 1, ,904 1T3T Ade5,7 VII ,067 2QK4

7 Ade8 IV ,540 3TQR

8 Using ADE Mutants to Study Respiration and Fermentation in Yeast David Form, Nashoba Regional High School And Jessica Forton, Melrose High School Extensions to Basic Lab Genetic Analysis of Ade1 and Ade2 Mutants: Demonstrating Genetic Dominance and Complementarity with Living Punnet Squares Introduction: The wild type, as well as the ade1 and ade2 mutant strains of yeast, are haploid. So, the wild type have one copy of the version of ade1 and ade2 that produce functional enzymes. The ade1 mutants have a gene that produces a functional copy of the ade 2 enzyme, but a mutant copy of the ade1 gene, which does not produce a functional enzyme. In contrast, the ade2 yeast contain a single copy of a normal ade1 gene and a mutant version of the ade2 gene. They produce a functional ade1 enzyme, but a non-functional ade2 enzyme. When we mate these yeast, they will produce diploid offspring that contain two copies of each ade gene, one from each parent. The dominant allele will determine the phenotype (white or red) of the diploid offspring. By mating individuals who have mutations on different genes that affect the same process, we will produce offspring with a normal, or wild-type phenotype. This is because the offspring have one copy of the normal allele for both genes. This is known as genetic complementation. In this experiment, we will cross the ade 1 and ade2 mutants with the wild type to determine if these mutations are dominant or recessive. We will cross the ade1 and ade2 mutants to determine if the ade1 and ade2 alleles are on separate genes. Procedure: 1. Prepare streak plates of the following yeast cultures: i. Ade1 alpha strain ii. iii. iv. Ade1 a strain Ade2 alpha strain Ade2 a strain v. Wild type alpha strain vi. Wild type a strain 2. Pour YED agar into sets of four Petri dishes. Pairs of students will work on ne of the four dishes: i. Draw a Punnett square on the bottom of each dish as follows:

9 1 WT alpha Ade1 alpha Ade1 a 2 WT alpha Ade2 alpha Ade2 a 3 WT alpha Ade1 alpha Ade2 a

10 4 WT alpha Ade2 alpha Ade1 a 3. For each plate: i. bserve and record the phenotypes (color) of each parental haploid yeast strain. ii. iii. iv. n the agar, over each circle, you will mix two strains of haploid yeast, represented by the closest box to the side and above the circle. For example, in plate 1 in the circle on the upper left, you will mix the strains WT alpha and. To mix the strains, use a sterile Q-tip, or inoculating loop, to pick a single colony of yeast and spread it evenly over the area of the circle. Use a second sterile Q-tip to pick a colony of the second yeast strain and spread it evenly over the area of the same circle. v. The yeast need to be well mixed, so that they can mate and form diploid offspring. It is also important to use a small amount of yeast, as you want to observe the phenotypes of the offspring, and not the parental strains. vi. Incubate the plates for 2-3 days and record the phenotypes of the offspring.

11 Data Use colored pencils to record the phenotypes (pink or white) of each cross in the four circle in each Punnett square. Write the genotypes of the diploid offspring above each circle. 1 WT alpha Ade1 alpha Ade1 a 2 WT alpha Ade2 alpha Ade2 a 3 WT alpha Ade1 alpha

12 Ade2 a 4 WT alpha Ade2 alpha Ade1 a Data Table Strain Ploidy Phenotype Genotype WT alpha haploid white WT Ade1 alpha haploid Ade1 a WT/WT diploid pink Ade1 WT/Ade1 WT/Ade2 Ade1/Ade1 Ade2/Ade2 Ade1/Ade2

13 Critical Thinking Exercises: 1. When combined with the wild type allele, is the Ade1 mutation dominant or recessive? How do you know? 2. When combined with the wild type allele, is the Ade2 mutation dominant or recessive? How do you know? 3. From the data discussed in q. 1 and 2, what color did you expect for the Ade1/Ade2 diploid hybrids? Explain your choice. 4. Do the Ade1/Ade1, Ade2/Ade2 and the Ade1/Ade2 diploid yeast have at least one good copy of the Ade1 and Ade2 enzymes? 5. Explain why the Ade1/Ade2 diploid yeast differed from the Ade1/Ade1 and Ade2/Ade2 diploid yeast in phenotype. 6. Why are the Ade1 and Ade2 mutations called complementary mutations? What does this have to do with the position of these enzymes in the biochemical pathway for the production of adenine?

14 TEACHER S VERSIN Data Use colored pencils to record the phenotypes (pink or white) of each cross in the four circle in each Punnett square. Write the genotypes of the diploid offspring above each circle. 1 WT alpha o white Ade1 alpha o red white Ade1 a red 2 WT alpha o white Ade2 alpha o o Ade2 a o 3 WT alpha o white Ade1 alpha o red

15 white Ade2 a red 4 white WT alpha o white white Ade2 alpha o red white Ade1 a red white white Data Table Strain Ploidy Phenotype Genotype WT alpha haploid white WT haploid white WT Ade1 alpha haploid pink Ade1 Ade1 a haploid pink Ade1 WT/WT diploid white WT/WT WT/Ade1 diploid white WT/Ade1 WT/Ade2 diploid white WT/Ade2 Ade1/Ade1 diploid pink Ade1/Ade1 Ade2/Ade2 diploid pink Ade2/Ade2

16 Ade1/Ade2 diploid white Ade1/Ade2

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