1. Announcements -First midterm exam will be in this room on Friday (4-25) from 10:30AM-12:20PM -Exam will cover material presented in lecture and quiz section through the end of last week, however… -this weeks material will reinforce some of the previous concepts you have learned -this weeks material WILL be covered on the next midterm exam

2. Leo Pallanck’s office hours: Friday afternoons by appointment (pallanck@u.washington.edu)

3. Today Inheritance Mutant analysis Genomics what can we learn by studying whole genomes? how are biological processes studied by analyzing mutants? Throughout the quarter... how and why are model organisms used in genetics? how does that information apply to humans? What this course is about how are unique physical traits determined by genes? how are traits transmitted to progeny? how is genetic information read within cells? From Lecture 1 More to come!

4. Mutant analysis (AKA Genetic Analysis) The use of mutants to understand how a biological process normally works* Very powerful - can be used to study metabolic pathways, animal development, neurobiology, cell division, etc. A simple analogy… *See the Salvation of Doug article at the following site: http://bio.research.ucsc.edu/people/sullivan/savedoug.html

5. Analysis of pizza synthesis Analagous to genes

6. Analysis of pizza synthesis No red sauce?! Analagous to a mutation The mutant phenotype Analagous to genes

7. What is a model organism? A species that one can experiment with to ask a biological question Why bother with model organisms? -All organisms are related at the molecular level -Not always possible to do experiments on the organism you want -If the basic biology is similar, it may make sense to study a simple organism rather than a complex one Mutant analysis involves model organisms

8. Which of these cameras do you think would be easier to understand? Box camera IMAX 3-D camera

9. Features of a good model organism Short generation time Small, easy to maintain Telomeres 96 million telomeres per cell! Large numbers of progeny Well-studied life cycle, biology Appropriate for the question at hand Mendel used a model organism—the garden pea -relatively short generation time—one per year -lots of progeny per cross -self-pollination and out-crossing possible -true-breeding varieties readily available from local merchant

10. Some commonly used model organisms -Bacteria — Escherichia coli -Budding yeast — Saccharomyces cerevisiae -Fruit fly — Drosophila melanogaster -Nematode — Caenorhabditis elegans -Mouse — Mus musculus

11. Quiz Section this week: Complementation analysis of yeast mutants An introduction to yeast...

12. Mutagenesis is easier in single-cell organisms with haploid lifestyles Budding yeast—a single-celled fungus that divides by budding Yeast cells can exist as haploids… Haploid life cycle: Yeast as a model “genetic” organism mitosis cytokinesis The haploid life cycle (1n) mutation

13. mating diploid zygote  haploid a haploid 1n The haploid life cycle (1n) The life cycle of “budding” yeast Yeast cells can also exist as diploids…

14. meiosis A tetrad with 4 haploid spores (“gametes”) Mendelian segregation occurs here 2  cells 2 a cells 1n The life cycle of “budding” yeast (cont) a/  diploid life cycle (2n)

15. Wild-type yeast can survive on ammonia, a few vitamins, a few mineral salts, some trace elements and sugar… They synthesize everything else they need, including adenine What genes does yeast need to synthesize adenine? Case study: analyzing the adenine biosynthetic pathway by generating and studying “ade” mutants (Why might we care about adenine?) Conducting a mutant analysis with yeast

16. -adenine plate “complete” plate sterile piece of velvet Adenine- requiring colonies (ade mutants) m2 m1 m3 “Replica-plating” plate cells Treat wt haploid cells with a mutagen: Identifying yeast mutants that require adenine

17. m1wild-type “complete” plate -adenine plate replica-plate using velvet That is, are they LOF mutations? Why do we care? What do you conclude? Genotypes: ADEade diploids ADE is dominant over ade “  ” mating type “a” mating type Are the adenine-requiring mutants recessive?

18. What would you predict if… only one enzyme is needed for synthesis of adenine? many enzymes are needed for synthesis of adenine? How to find out how many different genes we have mutated? Are m1 and m2 alleles of the same gene? Do complementation test to ask: are the mutations alleles of the same gene or of different genes? All mutants would be alleles of the same gene. Different genes might be mutant. Are all of the mutations in one gene?

19. m1m2 “complete” diploids -adenine replica-plate “  ” mating type “a” mating type Do m1 and m2 complement, or fail to complement? Are m1 and m2 alleles of the same gene, or alleles of different genes? 4 4 Performing a complementation test

20. What do you conclude from the pair-wise crosses shown below? m1m1 m2m2 m3m3 m4m4 m5m5 m6m6 m7m7 m1m1 m2m2 m3m3 m4m4 m5m5 m6m6 m7m7 o o o o o o o x o ++++ o Complementation tests with ade mutants m1, m5, m7 are mutations in one gene Conclusion? o = no growth on -ade + = growth on -ade

21. m1m1 m2m2 m3m3 m4m4 m5m5 m6m6 m7m7 m1m1 m2m2 m3m3 m4m4 m5m5 m6m6 m7m7 x ++++ o + o +++ ++++ +++ + + m1, m5, m7 are mutations in one gene Conclusion? m2, m4 are in one gene Four complementation groups o o o o o o o oo What do you conclude from the pair-wise crosses shown below? o = no growth on -ade + = growth on -ade m3 m6 Usually means four genes Complementation tests with ade mutants

22. Yeast cells can normally grow on a sugar called galactose as the sole carbon source. Seven mutant “  ” haploid yeast strains have been isolated that are unable to grow on galactose (“gal”) plates. Six of these mutant strains were each cross-stamped on a gal plate with a wild type “a” strain. The resulting pattern of growth on the gal plates is depicted below (shading = growth). In all plates, the wild type strain is in the horizontal streak. On the leftmost plate, mark the location of the a/  diploid with a circle. What is the mode of inheritance of mutant phenotype in mutants 1-6? How can you tell? Diploids grow on gal plate… so, wild type is dominant Practice Question

23. Each of the seven “  ” mutant strains was cross-stamped on gal plates against “a” versions of the seven mutants. The results are depicted below: Looking just at mutants 1–6 for now… group these six mutants by complementation group. m1, m2, m5 m3, m6 m4 Practice question (continued)

24. Now consider mutant 7. What is surprising about the result in the complementation table? Mutant 7 was cross-stamped on gal plate with wild type as you saw with the other six mutants earlier: What do you conclude about the mode of inheritance of mutant 7? How does that help you explain the complementation test result for mutant 7? What can you conclude about how many genes are represented in this collection of seven mutants? Fails to complement any of the others… how could it be an allele of 3 different genes? Complementation test fails with a dominant mutation… heterozygote will always show the mutant phenotype At least 3 genes (can’t tell about m7) Practice question (continued)

25. Complementation is relevant to humans Family AFamily B = deaf Within each family, does deafness look like it’s dominant or recessive? Assign genotypes (A, B, etc.) to the deaf individuals in these pedigrees. recessive aaBBAAbb AaBb

26. Complementation is relevant to humans Niemann Pick Type C disease (NPC): a recessive human neurodegenerative disease resulting in premature death Cellular cholesterol accumulation accompanies the disease (can be detected using a chemical called ‘filipin’ which fluoresces upon binding cholesterol) normalNPCNPC1:NPC2NPC1:NPC3 Do NPC1 & NPC2 complement?Do NPC1 & NPC3 complement?

27. Practice Question Hearing mice… independently assorting genes A and B, both needed for hearing: AaBb x WITHOUT drawing a Punnett square, predict the progeny phenotypes and proportions with respect to hearing ability. a, b: complete LOF, recessive

28. N R CH N HC C H2NH2N N R CH N C C H2NH2N - OOC N R CH N C C H2NH2N C HC HNHN O CH 2 COO - C O N HC HN NH CH N C C using mutants to order the steps in a pathway Adenine (AIR) (CAIR) (SAICAR)... For example, this molecule accumulates in an ade13 mutant. ADE13 encodes the enzyme that carries out the next step...

29. The Yeast Adenine Biosynthetic Pathway A B C DEFG H I J K Y

30. A second phenotype of some ade mutants… plate cells Mutagenize: -adenine plate Replica-plate complete plate Some of the adenine- requiring mutants are red!

31. Are the red ade mutations recessive? m8wild-type complete plate -adenine plate replica-plate using velvet Genotypes: ADEade White color is dominant Ability to make adenine is dominant

32. How can one LOF mutation generate two very different phenotypes? some intermediate “X” adenine ADE1 ade1 LOF mutation X red pigment UNK1* *not a real gene name! This gene has not yet been identified X Hypothesis: Two phenotypes/one LOF mutation another intermediate “Y” adenine Y

33. Suppose we isolate LOTS of independent red mutants: Are all red mutants defective in the SAME GENE? How to tell?

34. m9m10 mutations fail to complement = same gene mutations complement = different genes Must modify the hypothesis. All pairwise combinations reveal two complementation groups. diploids m9m11 white diploids Complementation tests of red mutants

35. X ADE2 ade2 X Y ADE1 red pigment “UNK1” Modified hypothesis for red phenotype adenine X Y But how do mutations in ADE1 result in a build-up of X?

36. Y X ADE2ade1 YX Mutations in either ADE1 or ADE2 lead to a defect in adenine biosynthesis and lead to the build-up of intermediates in the pathway. Excess “X” is converted to a red pigment. red pigment “UNK1” adenine Modified hypothesis for red phenotype

37. Y X ade2 ade1 YX red pigment Same as ade2 single mutation! Red and adenine-requiring. “UNK1” 1. Phenotype of ade1 ade2 double mutation? Test your understanding adenine ADE7 ADE13 2. Phenotype of ade2 ade7 double mutation? 3. Phenotype of ade2 ade13 double mutation? Same as ade7 single mutation! White and adenine-requiring. Same as ade2 single mutation! Red and adenine-requiring. 4. Phenotype of unk1? White and able to grow on -ade plates.

38. Practice Questions Y X ADE2 ADE1 YX red pigment “UNK1” adenine ADE3 1A. A MATa ade2 ADE3 mutant was mated to a MAT  ADE2 ade3 mutant to create a diploid. What are the phenotypes of the three strains? Assume all other genes are wild type.

39. red no white no white yes Practice Questions Y X ADE2 ADE1 YX red pigment “UNK1” adenine ADE3 1A. A MATa ade2 ADE3 mutant was mated to a MAT  ADE2 ade3 mutant to create a diploid. What are the phenotypes of the three strains? Assume all other genes are wild type.

40. 1B. ADE2 and ADE3 assort independently. Draw the chromosomes at metaphase of meiosis I such that the two WILD TYPE alleles face the same pole. Place a crossover on the other chromosome arm relative to the ADE2 and ADE3 genes. A C D B Tetrad on complete plates 1C. Recall that each tetrad contains the products of a single meiosis. Predict the genotypes and growth properties of each spore resulting from this meiosis. 1D. Analysis of many tetrads demonstrates that three types are found, depending on the behavior of the chromosomes in meiosis. Which tetrad best fits the meiosis you just drew? Letter the spores below to match the genotypes in your table. #1#2#3 red Spore grow without adenine? complete genotype? A B C D no ade2 ade3 yes ADE2 ADE3 yes ADE2 ADE3 no ade2 ade3 ADE2 ade2 ADE3 ade3 A B C D

41. 1E. Now draw the chromosomes at metaphase of meiosis I such that one wild type and one mutant allele face each pole. Place a crossover on the other chromosome arm relative to the Adenine genes. Spore grow without adenine?complete genotype? A B C D Tetrad on complete plates 1F. Predict the genotypes and growth properties of each spore resulting from this meiosis. 1G. Which tetrad best fits the meiosis you just drew? Letter the spores below to match the genotypes in your table. #1#2#3 red ADE2 ade3 A B

42. 1E. Now draw the chromosomes at metaphase of meiosis I such that one wild type and one mutant allele face each pole. Place a crossover on the other chromosome arm relative to the Adenine genes. Spore grow without adenine?complete genotype? A B C D Tetrad on complete plates 1F. Predict the genotypes and growth properties of each spore resulting from this meiosis. 1G. Which tetrad best fits the meiosis you just drew? Letter the spores below to match the genotypes in your table. #1#2#3 red ADE2 ade2 ade3 ADE3 no ade2 ADE3 ADE2 ade3 ade2 ADE3 ADE2 ade3 A C B D A C D B

43. 1H. Now draw the chromosomes at metaphase of meiosis I such that one wild type and one mutant allele face each pole. On one chromosome, place a crossover on the other chromosome arm relative to the Adenine gene. On the other chromosome, place a crossover BETWEEN the centromere and the Adenine gene. Spore grow without adenine? complete genotype? A B C D Tetrad on complete plates 1I. Predict the genotypes and growth properties of each spore resulting from this meiosis. #1#2#3 red 1J. Which tetrad best fits the meiosis you just drew? Letter the spores below to match the genotypes in your table.

44. 1H. Draw the chromosomes at metaphase of meiosis I in either independently assorting orientation. On the first chromosome, place a crossover on the opposite chromosome arm relative to the Adenine gene. On the second chromosome, place a crossover BETWEEN the centromere and the Adenine gene. Spore grow without adenine?complete genotype? A B C D Tetrad on complete plates 1I. Predict the genotypes and growth properties of each spore resulting from this meiosis. #1#2#3 A C D B red 1J. Which tetrad best fits the meiosis you just drew? Letter the spores below to match the genotypes in your table. ADE2 ade2 ADE3 ade3 no ade2 ade3 no ade2 ADE3 no ADE2 ade3 yes ADE2 ADE3 A B C D