Download The yeast Saccharomyces cerevisiae A model organism in genetics

The yeast Saccharomyces
cerevisiae
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.
Haploids and dipoids in nature and
laboratory
 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.
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.
Apa LI (178)
 This enables genetic studies (e.g. functional complementation 2of
MICRON
mutations with yeast or heterologous genes).
Hin d III (809)
HIS3
Ava I (4680)
Hin d III (996)
 Complementation is an outstanding tools for functional analysis.
 Plasmids also enable system perturbation such
as overexpression.
Pst I (1188)
F1 ORI
pRS423
LACZ'
5797 bp
T7 P
Apa LI (4137)
Ava I (2092)
Cla I (2108)
 Since yeast does not produce plasmid in high amounts shuttle
vectors are used for cloning and production in E. coli and analysis in yeast.
APr
Hin d III (2113)
Eco R I (2125)
MCS
Pst I (2135)
 Typical plasmid copy numbers are between 1/cell (centromeric
plasmids) and 20-50/cell (episomal plasmids) and up to 200/cell.
PMB1
Xma I (2137)
Ava I (2137)
Sma I (2139)
BamH I (2143)
 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.
T3 P
P(LAC)
Apa LI (2891)
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.
YPD
YPD + 0.4M NaCl
YPD
YPD + 0.4M NaCl
Wild type
hog1D
sko1D
aca1D aca2D
hog1D sko1D
hog1D aca1D
aca2D
hog1D sko1D
aca1D aca2D
Wild type
aca2D
hog1D
hog1D aca2D
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
Second PCR to amplify the marker
URA3
URA3
Final PCR product ready
for transformation
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
GFP
URA3
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 twohybrid analysis: the global protein network
 Different types of proteomics approaches employing mass spectrometry approaches:
phosphoproteome; protein levels.
Genetic interaction network
5.4 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. cerevisiae
contributes 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.