1. Saccharomyces cerevisiae a model system representing a simple eukaryote Hörður Guðmundsson og Guðrún P. Helgadóttir

2. Advantages of S. cerevisiae Nonpathogenic Rapid growth (generation time ca. 80 min) Dispersed cells Ease of replica plating and mutant isolation Can be grown on defined media giving the investigator complete control over environmental parameters Well-defined genetic system Highly versatile DNA transformation system

3. Therapeutic products from yeasts Prokaryotic products: Tetanus toxin fragment C; Streptokinase Surface antigens of viruses: Hepatitis B; HIV; Foot and mouse disease; Influenza; Polio; Polyoma; Epstein-Barr; Oncogenic retroviruses Malaria antigen Animal products: Hirudin; porcine interferon; interleukin; trypsin inhibitor Human hormones: Insulin; parathyroid hormone; growth hormone, chorionic gonadotropin Human growth factors: IGF1; NGF; EGF; tissue factor; CSF; GM- CSF; TNF Human blood proteins: Hemoglobin; factors VIII and XIII; alpha-1- antitrypsin; antithrombin III; serum albumin Various human enzymes; CFTR; estrogen receptor; INF-alpha; INF- beta1

4. General advantages of S. cerevisiae Strains have both a stable haploid and diploid state Viable with a large number of markers → recessive mutations are conveniently manifested in haploid strains and complementation tests can be carried out with diploid strains

5. General advantages of S. cerevisiae The ease of gene disruptions and single step gene replacements offers an outstanding advantage for experimentation Yeast genes can functionally be expressed when fused to the green fluorescent protein (GFP) thus allowing to localize gene products in the living cell by fluorescence microscopy The yeast system has also proven an invaluable tool to clone and to maintain large segments of foreign DNA in yeast artificial chromosomes (YACs) being extremely useful for other genome projects and to search for protein-protein interactions using the two- hybrid approach

6. General advantages of S. cerevisiae Transformation can be carried out directly with short single-stranded synthetic oligonucleotides, permitting the convenient productions of numerous altered forms of proteins Extensively exploited in the analysis of gene regulation, structure–function relationships of proteins, chromosome structure, and other general questions in cell biology

7. The yeast genome S. cerevisiae contains a haploid set of 16 well-characterized chromosomes, ranging in size from 200 to 2,200 kb Total sequence of chromosomal DNA is 12,8 Mb 6,183 ORFs over 100 amino acids long

8. Advantage of using S.cerevisiae in this study Parallell analysis of yeast strains with heterozygous deletions of drug target genes can be used to monitor compound activities in vivo The cellular targets of clinically proven small molecules are used to identify proteins that can be safely and effectively targeted in humans Many human disease-associated genes have highly conserved yeast counterparts and therefore S. cerevisiae has been proven to be a powerful tool for mechanistic studies of clinically relevant compounds In this study reported targets for many well-characterized compounds were correctly identified Many potentially novel drug targets were also identified Since this study was completed tagged heterozygous deletion strains have been made available for virtually every gene in the yeast genome

9. Greinin Discovering Modes of Action for Therapeutic Compounds Using a Genome-Wide Screen of Yeast Heterozygotes Lum et al., Cell, 116, pp. 121–137

10. Yeast Genome Deletion Project Generating a library of gene deletion strands Using a PCR-based gene deletion strategy Homologus recombination Four different collections generated: Haploids Mat a Haploids Mat  Homozygous diploids (for non-essential genes) Heterozygous diploids (used in this experiment) Reducing the number of copies of a gene results in sensitization to drugs targeting the protein product of that gene

11. PCR-based gene deletion (in YGDP)

12. Replacement DNA (in YGDP) Two specific 20 bp barcode sequenses for each insertion, you only need to sequence the barcode to know which strain you have Using primers in Kan R and common it is possible to PCR all the strands using the same primers (vital to this exp.) 5´ barcodeKan R 3´ barcodeCommon (18bp) (20 bp) (18 bp)

13. Competitive growth Reducing number of copies of a gene results in sensitization to drugs targeting the protein product of that gene Constructed a heterozygous deletion pool containing 3503 deletion strands (about ½ the yeast genes) Grown for 10 generations in normal medium then grown for 10 generations in medium with drug

14. Isolation of genomic DNA DNA isolated G-0 PCR with Cy3 markt primer G-20 PCR with Cy5 markt primer

15. Hybridization

16. Identification Tags printed in triplicate on the microarray Color intesity for G-0 and G-20 compared

17. Fitness profiles of 78 compounds Assess the cellular effects of 78 compounds Three groups of compounds: Group I: showed no drug- specific fitness changes (18) Group II: a small number of significant outliers (56) Group III: showed wide spread fitness changes (4)

18. Advantages of fitness assay for analyzing drug activities Requires no prior knowledge of compounds mode of action Allows truly novel drug activities to be uncovered in a systematic and unbiased fashion Biological processes that are affected by a given compound are identified in addition to the precise protein targets

19. Limitations Compound of interest must be able to affect the growth rate of the cell The activity level of the targeted protein must be influenced by the dosage level of the corresponding gene Compounds that exert their effects through direct interaction with non-protein elements in the cell do not appear suitable for this approach

20. Thank you