1. Yeast as a model organism Model eukaryote –Experimental genetics –Gene function – Orthologs, family members –Pathway function - “Biological synteny” Testbed for genomic technologies –Genome sequenced (4/96) relatively less complex –Ability to assess biological relevance of the data

4. Genomics technology development Yeast as a testbed Gene expression patterns –DNA microarrays, SAGE Genomic DNA scans –Mapping complex traits (SNPs) Phenotype screening –Genome-wide knockouts Genetic interaction networks –Synthetic lethals Protein interaction networks –Two-hybrid, mass spectrometry

5. Affymetrix whole genome yeast array Each gene is probed by multiple oligonucleotide probes (>19). A control probe is synthesized adjacent to each actual probe ~120,000 different oligonucleotide sequences for the entire genome. Entire yeast genome is on 5 arrays (~ 65,000 25 mers on each). 2 kb Gene 1 Gene 2 25mers Lisa Wodicka, Dave Lockhart, Affymetrix

6. Mapping complex traits Construct a SNP genetic map Perform cross Analyze rare segregants Identify regions inherited solely from one parent

7. YJM789 Isolated from the lung of an AIDS patient. Able to grow at 42 °C, form pseudohyphae and undergo colony- morphology switching. Hypersensitive to cycloheximide. Polymorphic –one difference every 150 bases relative to sequenced strain Laboratory strain YJM789 parent

8. Allelic variation between two strains can be detected using arrays. Laboratory strain (non-pathogenic) YJM789 (virulent wild isolate) 2 kb Gene 1 25mers Mismatch control probe (position 13 of 25) 2 kb Gene 1 25mers * * ** missing signals = markers Polymorphisms Yeast Array Since probe locations are known, a genetic map can be constructed: interesting loci (virulence) can be mapped and positionally cloned for study.

9. Allelic variation in YJM789 3808 markers detected by automated analysis of scanned images. –Largest gap = 56 kb –Average frequency = 3000 bases (1.0 cM) More markers identified in one hybridization than in the past 40 years of yeast genetics.

10. Verification of markers by tetrad analysis Expect 90 cross-overs per genome. Expect 90 cross-overs per genome. Expect clear recombination breakpoints Expect clear recombination breakpoints Expect most markers to segregate 2:2. Expect most markers to segregate 2:2.

11. Segregation of markers in one tetrad (one chromosome) 96 crossovers (90 expected). 96% of markers segregate 2:2. Clear breakpoints observed. Markers segregate as expected

12. spore 1 spore 2 spore 4 spore 3 Laboratory strain (S96) genotype: MATa, lys5, LYS2, ho, CYH Wild Isolate (YJM789) genotype MAT  LYS5, lys2, ho::hisG, cyh Diploid Haploid 1 16 1 1 1... (mat  lys2, LYS5, ho, cyh)

13. Inheritance of markers in 10 lys2 segregants

14. Results of mapping five phenotypic loci in 10 segregants. Five regions identified that were inherited solely from one parent. Four encompassed known locations of MAT, LYS5, LYS2, and HO. Minimum intervals ranged from 12 to 90 kb.

15. Cycloheximide sensitivity = pdr5 Cycloheximide sensitivity maps to remaining 56 kb interval on Chromosome XV adjacent to pdr5. PDR5 is deleted in YJM789. Wildtype strain, deleted for pdr5 is unable to complement YJM789.

16. Mapping Complex Traits: Feasibility Summary Identified 3808 genetic markers. Demonstrated that traits can be mapped using these markers. Next step: Map virulence loci.

17. Virulence in YJM789 Virulence is a multigenic trait with 5 loci contributing. –Only 5 of 200 segregants from crosses between YJM789 and laboratory strain are virulent. Genes cannot be cloned by complementation. Hybridization with arrays is an appropriate way to map all contributing loci simultaneously.

18. Assigning Function through Mutational Analysis Inactivate gene product (delete gene). Grow mutant strain under different selective or stress conditions. Identify mutants with growth defects. Function of gene product may be revealed. –UV sensitivity = DNA repair protein –Adenine auxotrophy = Adenine biosynthesis

19. Construction of yeast deletion strains KanR plasmid Deletion Cassette Chromosomal Gene Amplify selectable marker gene using primers with yeast gene homology at 5’ ends Replace yeast gene by homologous recom- bination yeast sequence

20. International Deletion Consortium Members Mike Snyder, Jasper Rine, Mark Johnston, Jef Boeke, Howard Bussey, Rosetta, Acacia, Peter Philippsen, Hans Hegemann, Francoise Foury, Guido Volckaert, Bruno Andre, Giogio Valle, Jose Revuelta, Steve Kelly, Bart Scherens 24,000 strains in 3 years

22. Serial analysis of deletion strains Apply Selection Identify deletion strains with growth defects 1 2 3 6,000

23. Molecular tags as strain-identifiers Unique 20-mers Unique 20-mers Good hybridization properties Good hybridization properties Similar melting temperatures Similar melting temperatures More than 5 base differences between each More than 5 base differences between each 1.1 x 10 12 possible 20mers12,000 best Shoemaker et al., 1996. Nature Genetics, 14: 450 -456 Can be introduced during strain construction Can be introduced during strain construction Two different tags (UPTAG and DOWNTAG) per strain Two different tags (UPTAG and DOWNTAG) per strain

24. Detecting molecular tags in yeast pools PCR-amplify tags from pooled genomic DNA using fluorescently-labeled primers Hybridize labeled tags to oligonucleotide array containing tag complements Each tag has unique location

25. Tags can be used to perform negative selections on pools Growth in minimal media identifies all known auxotrophic strains Winzeler et., 1999 Science 285:901-906

26. Genomic profiling of drug sensitivities via “induced haploinsufficiency” Decreased gene dosage from two copies to one copy in heterozygous strains results in increased sensitivity, or drug- induced haploinsufficiency

29. Strains that are heterozygous for drug target are haploinsufficient in the presence of drug: Giaever et al., 1999. Nature Genetics, 21: 278-283

30. Tunicamycin sensitivity Analysis of pools of heterozygous (and homozygous) strains reveals primary and secondary drug targets G. Giaever, unpublished results

32. Examples- global screens Synthetic lethals Synthetic dosage lethals Heterozygous diploids Haploinsufficiency modifiers Increased drug sensitivity- (target ID) Direct phenotype screening

33. Method for genomic synthetic lethal (SL) screen Tong et al., 2001 Science,Vol. 294, 2364-2368--- (Boone Lab) YF mutation, plasmid,reporter,…… each deletion strain in quadruplicate Final double mutant selection MAT a deletion set no growth potential SL interaction