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
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
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