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 Common Ancestor Years x10 6 ~800 ~350 ~80 Human Mouse Fly Worm Yeast Mam malian Model - transgenic m ice - disease phenotype Multicellular Biology - genetic analysis - organismal biology Unicellular Biology - Gene function classical genetics recombinant genetics ~30,000 genes ~15,000 genes ~6,000 genes QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. 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 Affymetrix whole genome yeast array 2 kb Gene 1 25mers • • • • Gene 2 25mers 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). Lisa Wodicka, Dave Lockhart, Affyme Mapping complex traits • • • • Construct a SNP genetic map Perform cross Analyze rare segregants Identify regions inherited solely from one parent YJM789 Laboratory strain YJM789 parent • Isolated from the lung of an AIDS patient. • Able to grow at 42 °C, form pseudohyphae and undergo colonymorphology switching. • Hypersensitive to cycloheximide. • Polymorphic – one difference every 150 bases relative to sequenced strain Allelic variation between two strains can be detected using arrays. Laboratory strain (non-pathogenic) 2 kb Gene 1 YJM789 (virulent wild isolate) Polymorphisms * 25mers * 2 kb Gene 1 * * 25mers Mismatch control probe (position 13 of 25) Yeast Array missing signals = markers Since probe locations are known, a genetic map can be constructed: interesting loci (virulence) can be mapped and positionally cloned for study. 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. Verification of markers by tetrad analysis Expect 90 cross-overs per genome. Expect clear recombination breakpoints Expect most markers to segregate 2:2. Markers segregate as expected 96 crossovers (90 expected). 96% of markers segregate 2:2. Clear breakpoints observed. Segregation of markers in one tetrad (one chromosome) Haploid 1 Haploid Diploid 16 ... 1 1 16 ... 1 16 16 ... Laboratory strain (S96) genotype: MATa, lys5, LYS2, ho, CYH Wild Isolate (YJM789) genotype MAT LYS5, ho::hisG, cyh 16 ... spore 1 ... spore 2 ... spore 3 (mat lys2, LYS5, ho, cyh) ... spore 4 Inheritance of markers in 10 lys2 segregants 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. 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. Mapping Complex Traits: Feasibility Summary • Identified 3808 genetic markers. • Demonstrated that traits can be mapped using these markers. • Next step: Map virulence loci. 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. 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 Construction of yeast deletion strains yeast sequence KanR Amplify selectable marker gene using primers with yeast gene homology at 5’ ends plasmid Deletion Cassette Chromosomal Gene Replace yeast gene by homologous recombination 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 Serial analysis of deletion strains Apply Selection 1 2 3 6,000 Identify deletion strains with growth defects Molecular tags as strain-identifiers Unique 20-mers Good hybridization properties Similar melting temperatures More than 5 base differences between each 1.1 x 10 12 possible 20mers 12,000 best Can be introduced during strain construction Two different tags (UPTAG and DOWNTAG) per strain Shoemaker et al., 1996. Nature Genetics, 14:450-456 Detecting molecular tags in yeast pools T G A KA R N T G A T G A T G A T G A T G A 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 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 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 Strains that are heterozygous for drug target are haploinsufficient in the presence of drug: 1. a) 0µg/ml tunicamycin b) 0.5µg/ml tunicamycinc) 2µg/ml tunicamycin 6 5 4 3 2 1 0 6 5 4 3 2 1 0 6 5 600 O. 4 3 D. 2 1 0 0 2 4 6 8 10 12 time (hrs) alg7/ALG7 ALG7/ALG7 0 2 4 6 8 10 12 time (hrs) 0 2 4 6 8 10 12 time (hrs) Giaever et al., 1999. Nature Genetics, 21:278-283 Tunicamycin sensitivity Analysis of pools of heterozygous (and homozygous) strains reveals primary and secondary drug targets G. Giaever, unpublished results Examples- global screens Synthetic lethals Synthetic dosage lethals Heterozygous diploids Haploinsufficiency modifiers Increased drug sensitivity- (target ID) Direct phenotype screening Method for genomic synthetic lethal (SL) screen MAT a deletion set YF mutation, plasmid,reporter,…… each deletion strain in quadruplicate Final double mutant selection no growth potential SL interaction Tong et al., 2001 Science,Vol. 294, 2364-2368--- (Boone Lab) Qui ckTi me™ and a TIFF ( Uncompressed) decompressor are needed to see this pictur e. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see t his picture.