The yeast Saccharomycescerevisiae

A model organism in genetics, genomics and

systems biology

Statement

“….the reason that yeast could serve as a model for all eukaryotic biology

derives from the facility with which the relation between gene structure and

protein function can be established.” (Botstein and Fink, Science 1988).

”…yeast has graduated from a position as the premier model for eukaryotic

cell biology to become the pioneer organism … of the entirely new fields of

study called “functional genomics” and “systems biology.” These new fields

look beyond the functions of individual genes and proteins, focusing on how

these interact and work together to determine the properties of living cells

and organisms.” (Botstein and Fink, Genetics 2011).

Content

Yeast is a eukaryotic organism

The yeast genome, definitions and gene ontology

Foundations for model organism: tools for genetic tractability

Defining function: functional genomics and networks

The concepts of yeast strains and variability

SGD: The Saccharomyces Genome Database

The yeast Saccharomyces cerevisiae:habitate, importance and use Yeast lives primarily on fruits, flowers and other sugar containing substrates

Free-living organism: yeast copes with a wide range of environmental conditions:

Temperatures from freezing to about 55°C are tolerated

Yeasts proliferate from 12°C to 40°C

Growth is possible from pH 2.8-8.0

Almost complete drying is tolerated (dry yeast)

Yeast can still grow and ferment at sugar concentrations of 3M (high osmotic pressure)

Yeast can tolerate up to 20% alcohol

Saccharomyces cerevisiae is the main organism in wine production besides other yeasts;

reason is the enormous fermentation capacity, low pH and high ethanol tolerance.

Saccharomyces cerevisiae (carlsbergensis) is the beer yeast because it ferments sugar to

alcohol even in the presence of oxygen, lager yeast ferments at 8°C.

Saccharomyces cerevisiae is the yeast used in baking because it produces carbon dioxide

from sugar very rapidly.

Saccharomyces cerevisiae is used to produce commercially important proteins because it can be

genetically engineered, it is regarded as safe and fermentation technology is highly advanced.

Saccharomyces cerevisiae is the most important eukaryotic cellular model system because it can be studied by

powerful genetics.

Pioneers of genomics, functional genomics, systems and synthetic biology employ S. cerevisiae.

Saccharomyces cerevisiae is a eukaryote Belongs to fungi, ascomycetes

Unicellular organism with ability to produce pseudohyphae

S. cerevisiae divides by budding (hence: budding yeast) while

Schizosaccharomyces pombe divides by fission (hence: fission

yeast).

Budding results in two cells of unequal size, a mother (old cell)

and a daughter (new cell).

Yeast life is not indefinite; yeast cells age and mothers die

after about 30-40 divisions.

Cell has a eukaryotic structure with different organelles:

Cell wall consisting of glucans, mannans and proteins

Periplasmic space with hydrolytic enzymes

Plasma membrane consisting of a phospholipid

bilayer and many different proteins

Nucleus with nucleolus

Vacuole as storage and hydrolytic organelle

Secretory pathway with endoplasmic reticulum, Golgi

apparatus and secretory vesicles

Peroxisomes for oxidative degradation

Mitochondria for respiration

A yeast cells is about 4-7µm large

The ”eyes” at the bottom are bud scars

Life cycle of yeasts: yeast has a sex life

Yeast cells can proliferate both as haploids (1n, one copy of

each chromosome) and as diploids (2n, two copies of each

chromosome).

Haploid cells have one of two mating types: a or alpha (α).

Two haploid cells can mate to form a zygote from which a

diploid cell buds off.

Under nitrogen starvation diploid cells undergo meiosis and

sporulation to form an ascus with four haploid spores.

Those germinate to form haploid cells.

Hence, the properties of the meiotic products can be studied

directly.

Yeast: a unicellular organisms with different cell types!

Thus, although yeast is unicellular, we can distinguish different cell types with different genetic programmes:

Haploid MATa versus MATα (can respond to pheromone, can mate; cannot do meiosis)

Haploid versus Diploid (MATa/alpha) (cannot respond to pheromone or mate, can sporulate)

Spores (survival structures)

Mothers and daughters (age n+1, can switch mating type, ago 0)

Yeast sex!

Central to sexual communication is the pheromone response signal

transduction pathway.

All modules of that pathway consist of components conserved from

yeast to human.

The pathway consists of a specific pheromone receptor, that binds a- or

α-factor; it belongs to the class of seven transmembrane G-protein

coupled receptors, like many human hormone receptors.

Binding of pheromone stimulates reorientation of the cell towards the

source of the pheromone (the mating partners).

Binding of pheromone also stimulates a signalling cascade, a so-called

MAP (Mitogen Activated Protein) kinase pathway, similar to many

pathways in human (animals and plants).

This signalling pathway causes cell cycle arrest to prepare cells for

mating (cells must be synchronised in the G1 phase of the cell cycle to

fuse to a diploid cell).

The pathway controls expression of genes important for mating.

In nature, yeast cells always grow as diploids: increases their chance to survive mutation of an essential gene (because there is always a second gene copy).

However, from time to time deleterious mutations need to be ”cleaned out” and advantageous mutations should eventually be manifested.

Under nitrogen starvation, diploid cells sporulate; under favourable conditions haploid spores germinate, provided that they have received functional copies of all essential genes.

This often means that only a single spore (if any) of a tetrad survives.

How to make sure that this single spore finds a mating partner to form a diploid? The answer is mating type switch!

After the first division the mother cell switches mating type and mates with its daughter to form a diploid, which then of course is homozygous for all genes and starts a new clone of cells.

If mating type can be switched and diploid is the preferred form, why then sporulate and have mating types?

There are probably several reasons: (1) Spores are hardy and survive harsh conditions (2) Sporulation is a way to ”clean” the genome from accumulated mutations (3) Meiosis is a way to generate new combinations of alleles, which may turn out to be advantageous (4) Sometimes cells may find a mating partner from a different population and form a new clone, with possibly advantageous allele combination.

In order to do yeast genetics and to grow haploid cells in the laboratory, mating type switch must be prevented: all laboratory strains are HO mutants and can not switch.

Haploids and dipoids in nature and laboratory

Yeast genetics: the genetic material

The S. cerevisiae nuclear genome has 16 chromosomes.

In addition, there is a mitochondrial genome and a plasmid, the 2μ circle.

The yeast chromosomes contain centromeres and telomeres, which are simpler than those of

higher eukaryotes.

The haploid yeast genome consists of about 12,500 kb and was completely sequenced as

early 1996 (first complete genome sequence of a eukaryote).

Since then, the genomes of numerous other yeast species and many different yeast strains

where sequenced.

Yeast genetics: the genetic material

The yeast genome is predicted to contain about 6,600 protein coding genes. 5056 or 76% are verified, 764 or 12% are uncharacterised and 787 or 12% are “dubious”.http://www.yeastgenome.org/cache/genomeSnapshot.html

Initial definition: likely protein coding is an ORF of at least 100 codons. Of course there are proteins smaller than 100 codons.

There are another 1,400 genetic elements (chromosome structure, RNA genes, transposons etc).

There is substantial ”gene redundancy”, which originates from an ancient genome duplication and subsequent reshuffling.

About 100 million years ago, a tetraploid was formed from a diploid. Tetraploids are viable but highly unstable. Perhaps extra copies of glycolytic genes provided a selective advantage. Ca 15% duplication remains.

This means that there are many genes for which closely related homologues (paralogues) exist, which often are differentially regulated and whose products are adapted to specific conditions.

The most extreme example are sugar transporter genes; there are more than twenty.

Only a small percentage of yeast genes has introns, very few have more than one; mapping of introns is still incomplete.

The intergenic space between genes is only between 200 and 1,000bp.

The largest known regulatory sequences are spread over about 2,800bp (MUC1/FLO11).

This means that the yeast genome is highly compact, about 1 gene per 2kb.

Extreme metabolic adaptation

Preferred carbon sources are glucose, fructose and sucrose.

Glucose and fructose mediate a gene expression programme called glucose repression;

genes required for utlisation of different carbon sources.

At high concentrations, glucose and fructose are fermented to ethanol and carbon

dioxide irrespective of the presence of oxygen: Crabtree effect.

A fermenting yeast cell consists to ca 50% of proteins involved in just the fermentative

pathway: glycolysis.

Together with high ethanol tolerance the fermentative capacity may confer a selective

advantage in high sugar containing environments.

Gene ontology

Gene Ontology, or GO, is a major bioinformatics initiative emerged from yeast

functional genomics to unify the representation of gene and gene product

attributes across all species.

More specifically, the project aims to:

Maintain and develop a controlled vocabulary of gene and gene product

attributes.

Annotate genes and gene products, and assimilate and disseminate annotation

data.

Provide tools for easy access to all aspects of the data provided by the project.

Three main attributes: molecular function, biological process and cellular

component.

GO annotation: molecular function

GO annotation: biological process

GO annotation: cellular component

Yeast genetics: nomenclature

Yeast genes have names consisting of three letters and up to three numbers:GPD1, HSP12, PDC6...Usually they are meaningful (or meaningless) abbreviations

Wild type genes are written with capital letters in italics: TPS1, RHO1, CDC28...

Recessive mutant genes are written with small letters in italics: tps1, rho1, cdc28

Mutant alleles are designated with a dash and a number: tps1-1, rho1-23, cdc28-2

If the mutation has been constructed, i.e. by gene deletion, this is indicated and the genetic marker used for deletion

too: tps1Δ::HIS3

The gene product, a protein, is written with a capital letter at the beginning and not in italics; often a ”p” is added at the end: Tps1p, Rho1p, Cdc28p

Many genes have of course only be found by systematic sequencing and as long as their function is not determined they get a landmark name: YDR518C, YML016W..., where

Y stands for ”yeast”

The second letter represents the chromosome (D=IV, M=XIII....)

L or R stand for left or right chromosome arm

The three-digit number stands for the ORF counted from the centromere on that chromosome arm

C or W stand for ”Crick” or ”Watson”, i.e. indicate the strand or direction of the ORF

Some genes do not follow this nomenclature: you heard already about: HO, MATa, MATa

Tools that made yeast the prime model organism

S. cerevisiae has two vegetative stages, haploids and diploids.

This allows generating mutations/mutants in haploids and study the consequences of such

mutations directly.

Furthermore, mutations can be allocated to genes by complementation in diploids

heterozygous for a mutation.

Genetic relationship can be studied directly in the haploid progeny of meiosis: gene mapping

and functional relationship of different genes.

Those were main features making yeast a model in the pre-genomic era, i.e. can 1960-1990.

Tools that made yeast the prime model organism S. cerevisiae combines many advantages of bacterial with eukaryotic genetics.

Yeast can be “transformed” with replicating plasmids.

Transformation is efficient, although not as efficient as in E. coli.

This enables genetic studies (e.g. functional complementation of

mutations with yeast or heterologous genes).

Complementation is an outstanding tools for functional analysis.

Plasmids also enable system perturbation such

as overexpression.

Since yeast does not produce plasmid in high amounts shuttle

vectors are used for cloning and production in E. coli and analysis in yeast.

Typical plasmid copy numbers are between 1/cell (centromeric

plasmids) and 20-50/cell (episomal plasmids) and up to 200/cell.

Yeast can be transformed with more than one plasmid at a time.

This enables further system perturbation but also advanced approaches

such as two-hybrid and FRET analysis for protein interactions.

pRS423

5797 bp

HIS3

APr

LACZ'

MCS

P(LAC)

T7 P

T3 P

2 MICRON

F1 ORI

PMB1

BamH I (2143)

Cla I (2108)

Eco R I (2125)

Sma I (2139)

Xma I (2137)

Pst I (1188)

Pst I (2135)

Apa LI (178)

Apa LI (2891)

Apa LI (4137) Ava I (2092)

Ava I (2137)

Ava I (4680)

Hind III (809)

Hind III (996)

Hind III (2113)

Tools that made yeast the prime model organism

S. cerevisiae has an incredibly efficient systems for

homologous recombination.

Homologous recombination occurs between two pieces

of DNA with same sequence, commonly between two

homologous chromosomes in meiosis, but also with

transformed DNA.

This recombination systems enables genome

manipulation with highest efficiency and of extreme

precision.

A piece of DNS generated in vitro and transformed into

yeast may find its right place in the genome based on

only a few bases sequence identity and initiate the

predicted genetic change.

Wild type

hog1D

sko1D

aca1D aca2D

hog1D aca1D

aca2D

hog1D sko1D

hog1D sko1D

aca1D aca2D

YPD YPD + 0.4M NaCl

Wild type

aca2D

hog1D

hog1D aca2D

YPD YPD + 0.4M NaCl

Deleting a yeast gene

There are a number of different ways to generate a piece of DNA for yeast transformation, i.e. the marker flanked by fragments with DNA from YFG1. Today commonly a PCR approach is employed.

In two separate PCR reactions the flanking regions of YFG1 are amplified and used in a second round as primers to amplify the marker gene; this requires the primers to be designed accordingly.

It can also be done with long PCR primers, in which only the marker is amplified and recombination is mediated by the primer sequences; as little as 30bp can be enough to mediate recombination when a heterologous marker is used to target integration.

The approach has been used to generate genome-wide deletion mutant collections.

YFG1 First PCR to amplify the flanking

parts of your favourite gene

URA3 Final PCR product ready

for transformation

URA3

Second PCR to amplify the marker

Tagging and reporters

In a similar way, a gene can be tagged. For instance, if the cassette is inserted in frame to the end of the ORF it will generate a fusion protein, with lacZ, GFP or an immuno-tag for protein detection and purification.

There are now sets of strains available in which each (almost) yeast gene has been tagged with GFP or TAP-tag.

YFG1

URA3

GFP

Functional analysis tools

Microarray analysis: simultaneous determination of the expression of all genes.

Tiling arrays covering the entire genome to map transcribed regions. Being replaced by deep sequencing approaches.

Microarray analysis combined with chromatin immunoprecipitation (CHIP-ChIP or CHIP-seq) to determine the binding sites for all transcription factors.

Yeast deletion analysis: a complete set of more than 6,000 deletion mutants is available for research (haploid a and alpha, diploid homo- and heterozygous).

Various approaches to phenotypically analyse the properties of these mutants in high throughput.

All genes available in overexpression plasmids for scoring the effects of gene overexpression; genetic networks.

Functional analysis tools

All yeast genes have been tagged to green fluorescent protein (GFP) to allow protein detection and microscopic localisation.

All proteins have been tagged for quantification and purification or individual proteins or protein complexes.

All proteins have been tagged for different types of protein interaction studies, such a two-hybrid analysis: the global protein network

Different types of proteomics approaches employing mass spectrometry approaches: phosphoproteome; protein levels.

Genetic interaction network5.4 million gene pairs for synthetic genetic interactions

Genetic interaction network

The principle of synthetic lethality

or synthetic enhancement.

Two gene products affect the

same function but act in a parallel

pathway.

Mutation of each one of those

has no or only a minor effect on

that function.

Mutation of both genes in the

same cell, however, strongly

affects or abolishes that function.

Synthetic enhancement by two

mutations is a rather simple case.

Many diseases are result of

interaction of more than one

genes….

….and in many cases of

quantitative interactions rather

then gene deletion.

Genetic interaction network

Automatic crossing of yeast deletion mutants to generate

double mutants.

A genome-scale genetic interaction map was constructed

by examining 5.4 million gene-gene pairs for synthetic

genetic interactions, generating quantitative genetic

interaction profiles for ~75% of all genes in the budding

yeast.

A network based on genetic interaction profiles reveals a

functional map of the cell in which genes of similar

biological processes cluster together in coherent subsets,

and highly correlated profiles delineate specific pathways

to define gene function.

The global network identifies functional cross-connections

between all bioprocesses, mapping a cellular wiring

diagram of pleiotropy.

The genetic interaction degree correlated with a number

of different gene attributes, which may be informative

about genetic network hubs in other organisms.

Mapping of the genetic landscape provides a key for

interpretation of chemical-genetic interactions and drug

target identification.

The concept of yeast strains

A strain can be compared to ”race”: same species and can produce

viable offspring, but clearly distinct properties.

Natural yeast strain variation: S. cerevisiae can be found all over the

globe but there are clearly distinct local populations/strains with

different properties and genetic differences.

Yeast evolution has also experienced a strong influence of human

association and domestication.

Yeast strains for different purposes with different properties: baking,

brewing and wine making.

Today hundreds of strains in wine making and brewing. Rather few

strains for baking.

The concept of yeast strains Laboratory yeast strains derive from rather few natural yeast

strains and have been exposed to selective pressure and

genetic manipulation since the 50ies and 60ies.

Comparing to an average behaviour reveals that the most

common reference strain S288C is most atypical for the

species……

Different strains used for different purposes and all labs have

their favourite strains.

Reference strains used for large-scale collections.

Standardisation. What if all researchers used the same strain?

Probably, some important biology would never have been

discovered but datasets for systems biology would be more

comparable.

Comparing the global deletion mutant set in two widely used

yeast strains, BY4741 and Σ1278b, revealed that most

essential genes were essential in both strains, but some were

unique to each strain.

The reason commonly were more than one modifying mutation.

Examples were S. cerevisiaecontributes or contributed

Genetic analysis of the cell cycle. Made use of the fact that the cell cycle stage

can be revealed by presence and size of the bud.

Genetic analysis of protein trafficking.

Genetic analysis of mitochondrial biogenesis.

Understanding basic principles of eukaryotic gene expression and its regulation.

Understanding the principles of signal transduction through conserved pathways

like MAPK, TOR, AMPK, PKA and more.

Development of new technology for functional genomics and systems biology.

Study of human disease genes: very many such genes have functional

homologues in yeast: functional studies.

The Saccharomyces Genome Database SGD

A yeast community effort.

A unique resource on all yeast genes and proteins and access to numerous

datasets and curated literature.

A model for other organism-specific databases.

The Saccharomyces Genome Database (SGD) provides comprehensive

integrated biological information for the budding yeast Saccharomyces cerevisiae

along with search and analysis tools to explore these data, enabling the discovery

of functional relationships between sequence and gene products in fungi and

higher organisms.