1. A - 1 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display PowerPoint to accompany Genetics: From Genes to Genomes Fourth Edition Hartwell ● Hood ● Goldberg ● Reynolds ● Silver Reference A Prepared by Malcolm Schug University of North Carolina Greensboro

2. A - 2 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Saccharomyces cerevisiae: Genetic Portrait of a Yeast

3. A - 3 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Yeast Cells Showing Small and Large Buds Characteristic of Different Phases of Mitotic Cell Cycle Fig. A. 1

4. A - 4 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display The Life Cycle of Yeast Fig. A. 2

5. A - 5 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Outline of Reference A Overview of yeast in laboratory Overview of yeast in laboratory Current knowledge of the yeast genome Current knowledge of the yeast genome Basic tools for studying yeast Basic tools for studying yeast Significant details of yeast life cycle Significant details of yeast life cycle Cell differentiation Cell differentiation Molecular mechanisms of determining cell type Molecular mechanisms of determining cell type Mating Mating How cell-cell communication through pheromones promotes conversion of haploid cells to diploid cells How cell-cell communication through pheromones promotes conversion of haploid cells to diploid cells

6. A - 6 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display The Nuclear Genome of Yeast 16 linear chromosomes 16 linear chromosomes Each chromosome contains Each chromosome contains One centromere One centromere Two termini with longer subtelomeric repeats followed by short telomere repeat at very ends Two termini with longer subtelomeric repeats followed by short telomere repeat at very ends Multiple origins of replication at 30-40 kb intervals Multiple origins of replication at 30-40 kb intervals Packed into nucleosomes with core histones H2A, H2B, H3, and H4 Packed into nucleosomes with core histones H2A, H2B, H3, and H4 Fig. A.3a

7. A - 7 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Comparison of Yeast and Human Genomes YeastHuman Genome size 12 Mb 3200 Mb Number of Chromosomes 16 22 pairs of autosomes 1 pair of sex chromosomes Number of genes 600020,000-30,000 Ploidy Haploid or Diploid Diploid

8. A - 8 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Physical Maps and Complete Sequence of Genome Karyotypes – pulse- field gel electrophoresis Karyotypes – pulse- field gel electrophoresis Fig. A.3 b

9. A - 9 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Genetic Maps > 1000 markers determined by tetrad analysis > 1000 markers determined by tetrad analysis 4400 cM – very long compared to other fungi 4400 cM – very long compared to other fungi 3 kb/ cM 3 kb/ cM Genetic and physical maps proportional Genetic and physical maps proportional No meiotic recombination in rDNA repeats and around centromere No meiotic recombination in rDNA repeats and around centromere Fig. A.4

10. A - 10 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Determining Number of Yeast Genes ~ 7505 ORFs estimated using arbitrary cutoff of 100 amino acids/protein ~ 7505 ORFs estimated using arbitrary cutoff of 100 amino acids/protein Some ORFs may be shorter, some longer Some ORFs may be shorter, some longer Likely 6000-6500 genes Likely 6000-6500 genes 2000 protein products have no function 2000 protein products have no function 20% of genes lethal when disrupted 20% of genes lethal when disrupted Introns in 4% - 5% of genes Introns in 4% - 5% of genes Also genes for rRNAs, 274 tRNA genes, 71 sncRNAs, RNAs of unknown function, 3 RNAs functional subunits of RNase P, endoribonuclease MRP, and telomerase Also genes for rRNAs, 274 tRNA genes, 71 sncRNAs, RNAs of unknown function, 3 RNAs functional subunits of RNase P, endoribonuclease MRP, and telomerase

11. A - 11 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Gene Arrangement, Genome Organization, and Genome Function 75% of genome transcribed into RNAs 75% of genome transcribed into RNAs One gene every 1.5 – 2.0 kb of DNA One gene every 1.5 – 2.0 kb of DNA Some coregulated but not clustered according to function like bacteria operons Some coregulated but not clustered according to function like bacteria operons Few reiterated sequences Few reiterated sequences 100-200 copies of rDNA 100-200 copies of rDNA 5-7 copies of tRNA genes 5-7 copies of tRNA genes 4-5 copies of subtelomeric repeats 4-5 copies of subtelomeric repeats 50 copies of retrotransposons called Ty elements 50 copies of retrotransposons called Ty elements LTRs at either end LTRs at either end 5.4-6.3 kb in length 5.4-6.3 kb in length Can pair and recombine even if on different chromosomes Can pair and recombine even if on different chromosomes Source of reciprocal translocations, inversions, and deletions Source of reciprocal translocations, inversions, and deletions

12. A - 12 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Genome Evolution Evolved through duplications of large chromosome segments 53 clustered gene duplications among 16 chromosomes Fig. A.6

13. A - 13 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Tools of the Yeast Geneticist Mutant Isolation and Characterization Mutant Isolation and Characterization Transformation and integration of DNA are very efficient Transformation and integration of DNA are very efficient Gene replacement creates directed mutations Gene replacement creates directed mutations Genome-wide analysis Genome-wide analysis

14. A - 14 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Mutant Isolation in a Diploid Cell A mutation on one homolog produces 2+ and 2- spores if the mutation is: A mutation on one homolog produces 2+ and 2- spores if the mutation is: Recessive Recessive In a nonessential gene In a nonessential gene Fig. A.7a

15. A - 15 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Mutant Isolation in a Diploid Cell If mutation is in gene essential for growth: If mutation is in gene essential for growth: Two live spores are recovered after meiosis Two live spores are recovered after meiosis Fig. A.7b

16. A - 16 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Power of Yeast Genetics Isolate large numbers of mutants, even rare mutants Isolate large numbers of mutants, even rare mutants Easily characterize dominance and recessiveness Easily characterize dominance and recessiveness Order gene products in a pathway using epistasis testing Order gene products in a pathway using epistasis testing Identify interacting gene products via suppressor analysis Identify interacting gene products via suppressor analysis

17. A - 17 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Transformation and Integration of DNA are Very Efficient Transformation of linear or circular DNA from external sources Transformation of linear or circular DNA from external sources High level of homologous recombination in yeast facilitates integration of DNA into chromosome High level of homologous recombination in yeast facilitates integration of DNA into chromosome

18. A - 18 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Gene replacement makes it possible to alter genes without genetic crosses. Fig. A.8

19. A - 19 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Genome-wide Analysis Basic cell processes are conserved from yeast to humans Basic cell processes are conserved from yeast to humans Large numbers of cells can easily be grown and assayed Large numbers of cells can easily be grown and assayed Mutations can be isolated in diploids, then analyzed in haploids Mutations can be isolated in diploids, then analyzed in haploids Conditional mutants can be isolated Conditional mutants can be isolated Recombination frequencies are high, facilitating molecular manipulations Recombination frequencies are high, facilitating molecular manipulations Genome size is small and number of genes low, so analysis and manipulations of the whole genome are possible Genome size is small and number of genes low, so analysis and manipulations of the whole genome are possible

20. A - 20 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Cell Differentiation in Yeast: Mechanisms for Determining Cell Type A, a, and a/a cells of yeast are differentiated cells that express different sets of genes A, a, and a/a cells of yeast are differentiated cells that express different sets of genes Differ in how they respond to signals from the environment and developmental potential Differ in how they respond to signals from the environment and developmental potential Different genetic programs establish pathways leading to each cell type Different genetic programs establish pathways leading to each cell type

21. A - 21 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Sex is Determined by Mating Type Locus MAT on chromosome III MAT on chromosome III Mating types a and  Mating types a and  Segregates 2:2 in tetrads derived from MATa/MAT  heterozygous diploids Segregates 2:2 in tetrads derived from MATa/MAT  heterozygous diploids MATa/MAT  are sterile MATa/MAT  are sterile MATa/MATa and MAT  /MAT  cells will mate with cells of opposite type MATa/MATa and MAT  /MAT  cells will mate with cells of opposite type

22. A - 22 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display MAT Locus Controls Expression of Genes that Determine Cell Type MATa and MAT  share some DNA sequences and some are unique MATa and MAT  share some DNA sequences and some are unique Presence of Ya, Y , or Ya+Y  had different consequences on expression of a-specific,  - specific, haploid-specific and meiosis (RME) genes due to activator and repressor differences Presence of Ya, Y , or Ya+Y  had different consequences on expression of a-specific,  - specific, haploid-specific and meiosis (RME) genes due to activator and repressor differences Fig. A.9

23. A - 23 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Mem1 is Both a Positive and Negative Regulator In a  cell, Mcm1 acts together with  1 to activate transcription of  -specific genes but acts to repress a-specific genes Fig. A-10a

24. A - 24 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Mem1 is Both a Positive and Negative Regulator In an a cell, Mcm1 alone is the transcriptional activator of  -specific genes. Fig. A-10b

25. A - 25 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Mating Type Switch Yeast strains differ according to stability of mating type Yeast strains differ according to stability of mating type Heterothallic strains Heterothallic strains stable haploid mating types stable haploid mating types Homothallic strains (HO) Homothallic strains (HO) switch mating type switch mating type Fig. A. 11 a

26. A - 26 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Cassette Model of Mating Type Switch HML, MAT, and HMR HML, MAT, and HMR Mating information on chromosome III Mating information on chromosome III MAT – information expressed MAT – information expressed HML, HMR – silent information HML, HMR – silent information Can be copied to MAT Can be copied to MAT Fig. A.11 b

27. A - 27 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Roles of HML, HMR, and HO in Mating Type Switch Wild-type strains Wild-type strains HML contains  information HML contains  information HMR contains a information HMR contains a information a/  do not switch even in HO strains a/  do not switch even in HO strains a1 and  2 regulators repress transcription of HO gene a1 and  2 regulators repress transcription of HO gene Cell can receive a cassette of mating type information located in HML or HMR and insert it into the MAT playback locus Cell can receive a cassette of mating type information located in HML or HMR and insert it into the MAT playback locus

28. A - 28 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Steps for Retrieving Information from Storage Locus Product of HO gene initiates process Product of HO gene initiates process HO-encoded enzyme makes double- stranded cut in chromosome III at a specific 18 bp sequence to right of Y segment in MAT HO-encoded enzyme makes double- stranded cut in chromosome III at a specific 18 bp sequence to right of Y segment in MAT Double-stranded break is repaired Double-stranded break is repaired Results in replacement of MAT DNA Results in replacement of MAT DNA

29. A - 29 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Gene Products that Function to Keep Storage Loci Silent Loss-of-function mutation in four SIR genes cancel repression and activate the  1,  2, or a1 genes located at HML or HMR Loss-of-function mutation in four SIR genes cancel repression and activate the  1,  2, or a1 genes located at HML or HMR Haploid cells Haploid cells Phenotype resembles that of a/  diploid because MATa and MAT  alleles are expressed Phenotype resembles that of a/  diploid because MATa and MAT  alleles are expressed sir - lines – HO-mediated transposition can occur from MAT to storage loci sir - lines – HO-mediated transposition can occur from MAT to storage loci Wild-type cells – mating type information flows only from storage loci to MAT Wild-type cells – mating type information flows only from storage loci to MAT

30. A - 30 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Role of HO in Mating Type Determination and Stability Serves as Paradigm for Development Pedigrees of cells undergoing HO- mating-type switch reveal rules governing mitotic cell lineages during development Pedigrees of cells undergoing HO- mating-type switch reveal rules governing mitotic cell lineages during development Rule 1 - cells always switch in pairs Rule 1 - cells always switch in pairs Fig. A.12 a,b

31. A - 31 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Rule 2 - Cells must gain competence to switch Rule 2 - Cells must gain competence to switch Do so through experience of switching at least once Do so through experience of switching at least once Fig. A.12c Role of HO in Mating Type Determination and Stability Serves as Paradigm for Development (cont.)

32. A - 32 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Cell-to-cell Communication Through Pheromones Triggers Conversion of Haploid a and  cells to a/  Diploids Pheromones bind to receptors and activate a signal transduction pathway Fig. A.13

33. A - 33 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Sterile mutants that produce no zone of growth inhibition either fail to secrete mating pheromone, or secrete mating pheromone but are unresponsive to pheromone from opposite cell type. Fig. A.14

34. A - 34 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Signal transduction cascade in mating response Signal transduction cascade in mating response Binding of pheromone to receptor causes conversion of GDP to GTP Binding of pheromone to receptor causes conversion of GDP to GTP Activation of MAP kinase cascade Activation of MAP kinase cascade Activation of STE12 transcriptional activator Activation of STE12 transcriptional activator STE12 turns on genes involved in G1 arrest of cells, cell fusion, and nuclear fusion STE12 turns on genes involved in G1 arrest of cells, cell fusion, and nuclear fusion Fig. A.15