Saccharomyces cerevisiae

a model system representing a simple

eukaryote

Hörður Guðmundsson og Guðrún P. Helgadóttir

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

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

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

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

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

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

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

Greinin

Discovering Modes of Action for Therapeutic Compounds Using a Genome-Wide Screen of Yeast Heterozygotes

Lum et al., Cell, 116, pp. 121–137

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

PCR-based gene deletion (in YGDP)

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 KanR and common it is possible to PCR all the strands using the same primers (vital to this exp.)

5´ barcode KanR 3´ barcode CommonCommon

(18bp) (20 bp) (20 bp) (18 bp)

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

Isolation of genomic DNA

DNA isolated G-0 PCR with Cy3 markt primer G-20 PCR with Cy5 markt primer

Hybridization

Identification

Tags printed in triplicate on the microarray

Color intesity for G-0 and G-20 compared

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)

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

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

Thank you