Download YEAST GENETICS AND MOLECULAR BIOLOGY

© Stefan Hohmann 2000-2004
YEAST GENETICS AND MOLECULAR
BIOLOGY
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The yeast Saccharomyces cerevisiae: habitate and use
Other yeasts
Yeast is a eukaryote: the yeast cell
Yeast has a sexual cycle and an exciting sex life
Yeast genetics: basics
Yeast genetics: crossing yeast strains
Yeast genetics: making mutants
Cloning yeast genes: vectors
Cloning yeast genes by complementation
Deleting genes in yeast
Smart gene deletions and transposon mutagenesis
Getting further: more genes/proteins
Model systems studied in yeast
Yeast biotechnology
Yeast information resources WWW
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© Stefan Hohmann 2000-2004
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There is unfortunately no real text book on yeast genetics and molecular biology
Genetic Techniques for Biological Research by Corinne Michels gives a brief
overview on yeast genetics and summarises genetic approaches
Yeast Gene Analysis by Brown and Tuite is a book about methods
There are excellent resources on the WWW and many individual group pages with
interesting information and even movies! Check out the course link page
For instance, there is kind of a text book on the Internet:
http://www.phys.ksu.edu/gene/chapters.html
This site: http://genome-www.stanford.edu/Saccharomyces/VL-yeast.html links to
various types of basic information on yeast genetics
This site links to more than 700 hundred yeast labs all over the world
http://genome-www.stanford.edu/Saccharomyces/yeastlabs.html
The Stanford Saccharomyces Genome database under http://genomewww.stanford.edu/Saccharomyces has information on all yeast genes including
links and information to other yeast genome projects and global analysis projects
The yeast Saccharomyces cerevisiae:
habitate and use
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Yeast lives on fruits, flowers and other sugar containing substrates
Yeast copes with a wide range of environmental conditions:
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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 osmoti 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
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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
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Saccharomyces cerevisiae is the yeast used in baking
because it produces carbon dioxide from sugar very rapidly
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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
© Stefan Hohmann 2000-2004
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Saccharomyces cerevisiae is used for drug screening and functional analysis
because it is a eukaryote but can be handled as easily as bacteria
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Saccharomyces cerevisiae is the most important eukaryotic cellular model system
because it can be studied by powerful genetics and molecular and cellular biology; many important features of
the eukaryotic cell have first been discovered in yeast
Hence S. cerevisiae is used in research that aims to find out features and mechanisms of the function of the
living cell AND in to improve existing or to generate new biotechnological processes
Other important yeasts
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© Stefan Hohmann 2000-2004
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Schizosaccharomyces pombe, the fission yeast; important model organisms in molecular and
cellular biology; used for certain fermentations
Kluyveromyces lactis, the milk yeast; model organism
some biotech importance due to lactose fermentation
Candida albicans, not a good model since it lacks a sexual cycle;
but studied intensively because it is human pathogen
Saccharomyces carlsbergensis and Saccharomyces bayanus are species closely related to S.
cerevisiae; brewing and wine making
Pichia stipidis, Hansenula polymorpha, Yarrovia lipolytica have smaller importance for genetic
studies (specilaised features such as peroxisome biogenesis are studied), protein production
hosts
Filamentous fungi, a large group of genetic model organisms in genera like Cryptococcus,
Aspergillus, Neurospora...., biotechnological importance, includes human pathogens. Also S.
cerevisiae can grow in a filamentous form.
Saccharomyces cerevisiae is a eukaryote
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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 dividions
Cell has a eukaryotic structure with different
organelles:
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© Stefan Hohmann 2000-2004
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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 4-7mm large
The ”eyes” at the bottom are bud scars
© Stefan Hohmann 2000-2004
Life cycle of yeasts
Budding Yeast
Fission Yeast
Yeast has a sex life!
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© Stefan Hohmann 2000-2004
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Yeast cells can proliferate both as haploids (1n,
one copy of each chromosome) and as diploids
(2n, two copies of each chromosome); 2n cells
are 1.2-fold bigger
Haploid cells have one of two mating types:
a or alpha (a)
Two haploid cells can mate to form a zygote;
since yeast cannot move, cells must grow
towards each other (shmoos)
The diploid zygote starts dividing from the
junction
Under nitrogen starvation diploid cells undergo
meiosis and sporulation to form an ascus with
four haploid spores
Thus, although yeast is unicellular, we can
distinguish different cell types with different
genetic programmes:
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Haploid MATa versus MATalpha
Haploid versus Diploid (MATa/alpha)
Spores
Mothers and daughters
Yeast sex!
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© Stefan Hohmann 2000-2004
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Central to sexual communication is the pheromone
response signal transduction pathway
This pathway is a complex system that controls the
response of yeast cells to a- or alpha-factor
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 alpha-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
(animal and plant)
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
Yeast sex!
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© Stefan Hohmann 2000-2004
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Cought in the act: cell attachment, cell fusion and
nuclear fusion in an electron micrograph
Haploid cells produce mating peptide pheromones,
i.e. a-factor and alpha-factor, to which the mating
partner responds to prepare for mating
This means that yeast cells of different sex can be
distinguished genetically, i.e. by expression of
different sets of genes
Hence, haploid-specific genes are those that
encode proteins involved in the response to
pheromone as well as the RME1 gene encoding the
repressor of meiosis
A-specific genes are those needed for a-factor
production and the gene for the alpha-factor
receptor
Alpha-specific genes are those needed to produce
alpha-factor and the gene for the a-factor receptor
Genetic determination of yeast cell type
© Stefan Hohmann 2000-2004
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The mating type is determined by the allele of the mating type locus MAT on chromosome III
The mating type locus encodes regulatory proteins, i.e. transcription factors
The MATa locus encodes the a1 transcriptional activator (a2 has no known function)
The MATalpha locus encodes the alpha1 activator and the alpha2 repressor
The mating type locus functions as a master regulator locus: it controls expression of many
genes
Gene expression that determines the
mating type
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© Stefan Hohmann 2000-2004
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In alpha cells the alpha1 activator stimulates
alpha-specific genes and the alpha2 repressor
represses a-specific genes
In a cells alpha-specific genes are not activated
and a-specific genes are not repressed (they
use a different transcriptional activitor to
become expressed)
In diploid cells the a1/alpha2 heteromeric
repressor represses expression of alpha1 and
hence alpha-specific genes are not activated.
A-specific genes and haploid-specific genes
are repressed too.
One such haploid-specific gene is RME,
encoding the repressor of meiosis. Although it
is not expressed in diploids the meiosis and
sporulation programme will only start once
nutrients become limiting
Taken together, cell type is determined with
very few primary transcription factors that act
individually or in combination.
This is a fundamental principle and is
conserved in multicellular organisms for the
determination of different cell types: homeotic
genes (in fact, a1 is a homeobox factor)
Haploids and dipoids in nature and laboratory
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© Stefan Hohmann 2000-2004
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In nature, yeast cells always grow as diploids, probably because this increases their chance to
survive mutation of an essential gene (because there is another copy)
Under nitrogen starvation, diploid cells sporulate and then 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 can find a mating partner to form a diploid again? 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 prefered form, why then sporulate and have
mating types?
There are probably several reasons: (1) Spores are hardy and survive very 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, i.e. better than
the previous one (4) Sometimes cells may find a mating partner from a different tetrad 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
So how does this mysterious switch of sex work?
Haploids can switch mating type!
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Mating type switch is due to two silent mating type loci on the same chromosome, which
become activated when translocated to the MAT locus
The mechanisms of silencing these two copies of the MAT locus has been studied in detail and
has conserved features to higher cells: heterochromatin formation
The translocation is a gene conversion initiated by the HO nuclease, that cuts like a restriction
enzyme within the active mating type locus in the chromosome
Laboratory yeast strains lack the HO nuclease and hence have stable haploid phases
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© Stefan Hohmann 2000-2004
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Interestingly, only mother
cells can switch
This ensures that after
cell devision two cells of
opposite mating type are
formed
This feature is due to
unequal inheritance of a
regulatory proteins
Also this is a strategy
that is conserved an
found in differentiation of
cell types in multicellular
organisms
Yeast genetics: the genetic material
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© Stefan Hohmann 2000-2004
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The S. cerevisiae nuclear genome has 16 chromosomes
In addition, there is a mitochondrial genome and a plasmid,
the 2micron 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)
Yeast genetics: the genetic material
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© Stefan Hohmann 2000-2004
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The yeast genome is predicted to contain about 6,200 genes,
annotation is, however, still ongoing
There is substantial ”gene redundancy”, which originates from an
ancient genome duplication
This means that there are many genes for which closely related
homologue exist, which often are differentially regulated
The most extreme example are sugar transporter genes; there are
more than twenty
Roughly 1/3 of the genes has been characterised by genetic
analysis, 1/3 shows homology hinting at their biochemical function
and 1/3 is not homologous to other genes or only to other
uncharacterised genes
Only a small percentage of yeast genes has introns, very few have
more than one; mapping of introns is not complete
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)
Yeast genome analysis
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© Stefan Hohmann 2000-2004
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A joint goal of the yeast research community: determination of the
function of each and every gene
For this, there are several large projects and numerous approaches
Micro array analysis: simultaneous determination of the expression
of all genes
Micro array analysis to determine the binding sites in the genome
for all transcription factors
Yeast deletion analysis: a complete set of more than 6,000 deletion
mutants is available for research
Various approaches to analyse the properties of these mutants
All yeast genes have been tagged to green fluorescent protein
(GFP) to allow protein detection and microscopic localisation
Different global protein interaction projects are ongoing
Yeast genetics: nomenclature
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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: tps1D::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
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© Stefan Hohmann 2000-2004
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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
Yeast genetics: markers and strains
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© Stefan Hohmann 2000-2004
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Genetic markers are used to follow chromosomes in genetic crosses, to
select diploids in genetic crosses, to select transformants in
transformation with plasmids or integration of genes into the genome
Commonly genetic markers cause auxotrophies: HIS3, URA3, TRP1,
LEU2, LYS2, ADE2
The ade2 mutation has a specific useful feature: cells turn red
The first markers in yeast genetics were fermentation markers, i.e. genes
that confer the ability to catabolise certain substrates: SUC, MAL, GAL
SUC genes (SUC1-7) encode invertase (periplasmic enzyme) and can be
located on different chromosomes in different yeast strains (telomere
location)
MAL loci (MAL1-6) encode each three genes: maltase, maltose
transporter and a transcriptional activator; also telomer location
GAL genes encode the enzymes needed to take up galactose and convert
it to glucose-6-phosphate
Like in E. coli also certain antibiotic resistance markers can be used in
transformation: kanamycin resistance, kanR
There are many yeast strains in use in the laboratories:
W303-1A, S288C, S1278b, SK1, BY4741....
Their specific properties can be quite different and are different to wild or
industrial strains
The full genotype of our favourite strain W303-1A reads like this:
MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal0
Yeast genetics: crossing strains
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© Stefan Hohmann 2000-2004
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Yeast genetics is based on the possibility to cross two haploid strains with different mutations
and of opposite mating type to a diploid strain
The diploid can then be investigated, for instance if one wants to find out if the two haploid
strains had mutations in the same or different genes
The diploid can be sporulated to form tetrads, tetrads can be dissected using a micromanipulator
and spores form individual colonies, and hence can be investigated
In the past, such genetic crosses were done a lot in order to map genes on chromosomes: the
frequency with which two mutations recombined (i.e. resulted in spores carrying both mutations
or spores without any of the two mutations) is a measure for the genetic distance
The last genetic map (before the genome was sequenced) encompassed more than 1,000 genes
and turned out to be very accurate (also thanks to the enormous capacity of yeast for genetic
recombination)
Today genetic crosses are used to generate yeast strains with new combination of mutations, for
instance double, triple....mutations – for this it is useful to know some principles of genetic
crosses and gene segregation
And even today with the genome fully sequenced we often perform genetic screens for new
mutations, for instance to find genes/proteins that function in the same pathway/molecular
system than an already known gene/protein – then genetic analysis of the mutants one obtained
is the first and essential step in characterisation
Yeast genetics: crossing strains
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© Stefan Hohmann 2000-2004
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In order to cross two strains they are mixed on agar
plates and allowed to mate, e.g.
MATa leu2 URA3 x MATalpha LEU2 ura3
Diploid cells will be heterozygous for both
complementing markers and can be selected on
medium lacking both leucine and uracil
Diploids will be grown and plated on sporulation
medium, where asci/tetrads form within some days
Sporulation occurs under nitrogen starvation, such as
on potassium acetate KAc medium
The ascus wall is digested with a specific enzyme mix
(e.g. from snail stomac) and spores are separated with
a micromanipulator on agar plates
Spores will germinate and each spore gives rise to a
colony, which can be studied individually
This means that the properties of the meiotic progeny
can be studied directly, because in yeast the individual
organism is the single cell: a unique advantage of
yeast, which has made yeast (and some other fungi)
highly useful in genetics
The trained geneticist often can see already from the
pattern of growth of the spore colonies how two
mutations separated, for instance if a double mutant
forms smaller colonies than either single mutants
Otherwise, the spore colonies are replicated to
different media in order to characterise the properties
of the spores and to follow the genetic markers
Yeast genetics: crossing strains
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© Stefan Hohmann 2000-2004
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The mating type of the spores is determined by replicated the spores on a lawn of tester strains with complementing
markers, allowed to form diploids and then replicated on medium selective for diploids: only those will grow that had
a different mating type then the tester strain
The records of a genetic cross in a lab book will look like below for a cross between two strains that are sensitive to
NaCl
Comparing markers pairwise one can see particular patterns where for instance all four spores are different or two
spores have the same marker combination – how is this interpreted ?
Tetrad
Spore
MAT
leu
ura
his
SUC
NaCl
1
A
a
+
+
-
-
-
1
B
alpha
+
-
+
-
-
1
C
a
-
-
-
+
-
1
D
alpha
-
+
+
+
+
2
A
a
-
-
-
-
-
2
B
a
+
+
+
+
+
2
C
alpha
+
-
+
-
-
2
D
alpha
-
+
-
+
-
Yeast genetics: meiosis
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© Stefan Hohmann 2000-2004
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We need to recapitulate first what happens
during meiosis: yeast tetrad analysis is
nothing else then just watching directly the
outcome of meiosis
The diploid is 2n and hence has two
chromosomes
DNA is replicated resulting in two
chromosomes with two identical chromatids
each
The chromosomes align and can undergo
recombination
The then first meiotic division will separate
the chromosomes from each each
The second meiotic division will separate the
chromatids, ie. each spore represents
essentially one chromatid
Yeast genetics: the outcome of a cross
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Let us now imagine that LEU2 and URA3 are close together on the same chromosome
LEU2
ura3
LEU2 ura3
LEU2
ura3
LEU2 ura3
leu2
URA3
leu2 URA3
leu2 URA3
leu2
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© Stefan Hohmann 2000-2004
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URA3
In the likely case that no cross-over occurs between the two markers all haploid spores will just
look like the parental haploid strains
There are only two different types of spores, i.e. (leu-plus ura-minus) and (leu-minus ura-plus)
spores
Hence such a tetrad is called a parental ditype PD
Yeast genetics: cross over
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Let us now imagine that LEU2 and URA3 are close together on the same chromosome and a
cross over occurs between them
LEU2
ura3
LEU2 ura3
LEU2
ura3
leu2
URA3
LEU2 URA3
leu2 ura3
leu2 URA3
leu2
© Stefan Hohmann 2000-2004
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URA3
In this case we will get spores that look like the parental haploids but also spores that have new
combinations of the two markers
There are four different types of spores
Hence such a tetrad is called a tetratype T
Yeast genetics: double cross over
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Let us now imagine that LEU2 and URA3 are close together on the same chromosome and two
cross over occur between them such that four DNA strands are involved
LEU2
ura3
LEU2 URA3
LEU2
ura3
leu2
URA3
LEU2 URA3
leu2 ura3
leu2 ura3
© Stefan Hohmann 2000-2004
leu2
URA3
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In this case we will get only spores that look different from the parental haploids
There are two different types of spores
Hence such a tetrad is called non parental ditype NPD
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Since with close linkage it is most likely that no cross over occurs and least likely that two
cross over occur the proportion of tetrads would be PD > T > NPD and the relative numbers can
be used to map genetic distances. For mapping one investigated hundreds of tetrads from the
same cross. This has been done extensively in the past and the last genetic map from 1995
comprised about 1,000 locations
To generate new combination of mutations (such as leu2 ura3) one will have to dissect the more
tetrads the closer the two genes are, and this can be estimated based on the physical distance
(in kb), which relates well to the genetic distance (in cM, centi Morgan). For two close genes
(1cM, i.e. 1% recombinant spores) one would have to dissect at least 25 tetrads, statistically
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Crossing with markers on different
chromosomes
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Let us now imagine that LEU2 and URA3 are on different chromosomes
LEU2
© Stefan Hohmann 2000-2004
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LEU2 ura3
LEU2 URA3
LEU2
ura3
LEU2 ura3
LEU2 URA3
leu2
URA3
leu2 URA3
leu2 ura3
leu2 URA3
leu2 ura3
leu2
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ura3
URA3
Different chromosomes assort randomly in the first meiotic division
For this reason two types of tetrads become equally frequent, the parental and the non-parental
ditype, PD and NPD
Hence, linked and unlinked genes can easily be distinguished in tetrad analysis because with
unlinked genes PD = NPD while with linked genes PD>>NPD.
Crossing with markers on different
chromosomes
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Let us now imagine that LEU2 and URA3 are on different chromosomes and a crossing over
occurs between a centromere and a marker
LEU2
ura3
LEU2 ura3
LEU2
ura3
LEU2 URA3
leu2
URA3
leu2 ura3
leu2 URA3
leu2
© Stefan Hohmann 2000-2004
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URA3
Now the different alleles of URA3 will only be separated in the second meiotic division
The result is a tetratype tetrad T
The above situation means also that if markers are distant from the centromere many Ts will
occur while if both markers are close to the centromere few Ts will occur.
What is the outcome of double cross-overs with four or with three strands?
Due to the possibility of double cross-overs the proportion between different tetrad types for
unlinked genes that are not centromere-linked becomes 1:1:4 for PD:NPD:T
This also means that one out of four spores will be recombinant, i.e. in order to obain the new
combination of genes (leu2 ura3) one only needs to dissect one tetrad, statistically
Yeast genetics: making mutants
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Mutations that enhance or abolish the function of a certain
protein are extremely useful to study cellular systems
The phenotype of mutations (i.e. the properties of the
mutant) can tell a lot about the function of a gene, protein or
pathway
This approach is valid even with the genome sequenced and
even with the complete deletion set available: point
mutations can have different properties than deletion
mutants
Random versus targetted mutations
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© Stefan Hohmann 2000-2004
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In random mutagenesis one tries to link genes to a certain
function/role; this identifies new genes or new functions to
known genes
Hence in random mutagenesis usually the entire genome is
targetted
Random mutagenesis is also possible for a specific protein
(whose genes is then mutated in vitro); in this case one wishes
to identify functional domains
In targetted mutagenesis one knocks out or alters a specific
gene by a combination of in vitro and in vivo manipulation
Induced versus spontaneous mutations
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Mutations can be induced by treating cells with a mutagen; this
can of course give multiple hits per cell
Spontaneous mutations ”just occur” at a low frequency and it is
likely that there is only one hit per cell
Yeast genetics: finding mutants
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Screening versus selection
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To develop a new selection system is the art of genetic
analysis
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© Stefan Hohmann 2000-2004
When screening for mutants one tests clone by clone to find
interesting mutants
For that, one usually plates many cells and tries to find mutants
because they are unable to grow on a certain medium after
replica-plating or because they develop a colour
For screening, mutations are usually induced to increase their
frequency
Still: screening requires hundreds of perti dishes and commonly
more than 10,000 clones to be scored
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When selecting for mutants one has established a condition
under which the mutant phenotype confers a growth advantage
In other words, the intellectual challenge is to design conditions
and /or strains such that the mutant grows, but the wild type
does not
A smart screening system allows one to go for spontaneous
mutations, because up to 108 cells can easily be spread on one
plate
Selection systems are often based on resistance to inhibitors
We try to train our students to watch out for any such
opportunity to find conditions that allow to select for new
mutants with interesting properties to advance the understanding
of the system under study
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
Yeast genetics: characterising mutants
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© Stefan Hohmann 2000-2004
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Once mutants have been identified they need to be characterised and the genes
affected have to be identified; this requires the following steps
A detailed phenotypic analysis, i.e. testing also for other phenotypes than the one
used in screening/selection
Establishing if a mutant is dominant or recessive
Placing the mutants into complementation groups. Usually one complemetation
group is equivalent to one gene
Cloning the gene by complementation.
Dominant and recessive mutations
Recessive: wild type phenotype
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© Stefan Hohmann 2000-2004
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The dominant or recessive character is
revealed by crossing the mutant with the
wild type to form a diploid cell
Such diploids are heterozygous, because
one chromosome carries the wild type
allele and the other one the mutant allele
of the gene affected
A mutation is dominant when the mutant
phenotype is expressed in a heterozygous
diploid cell. The diploid has the same
phenotype as the haploid mutant
A mutation is recessive when the wild type
phenotype is expressed in a heterozygous
diploid cell. The diploid has the same
phenotype as the wild type
MUT1
MUT1
mut1
mut1
Dominant: mutant phenotype
Dominant and recessive mutations

A dominant character can have a number of important
reasons, which may reveal properties of the gene
product’s function:
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© Stefan Hohmann 2000-2004
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The mutations leads to a gain of function, e.g. a regulatory
protein functions even without its normal stimulus
The gene product functions as a homo-oligomere and the
non-functional monomere causes the entire complex to
become non-functional
The gene dosis of one wild type allele is insufficient to
confer the wild type phenotype, i.e. there is simply not
enough functional gene product (this is rare)
The recessive character of a mutation is usually due to
loss of function of the gene product
This means that recessive mutations are far more
common, because it is simpler to destroy a function
than to generate one
Further genetic analysis of the mutant depends on the
dominant/recessive character, that is one reason why
this step is taken first
In addition, it is useful to do a tetrad analysis of the
diploid in order to test that the mutant phenotype is
caused by a single mutation, i.e. that the phenotype
segregates 2:2 in at least ten tetrads studied; this is
important when mutations have been induced by
mutagenesis
Recessive: wild type phenotype
MUT1
MUT1
mut1
mut1
Dominant: mutant phenotype
Complementation groups
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© Stefan Hohmann 2000-2004
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After selection or screening for mutants with a certain
phenotype and after determination of the dominant/recessive
character of the underlying mutation one would like to know
if all mutants isolated are affected in the same or in different
genes
For recessive mutations, this is done by a complementation
analysis
This requires that mutants with different mating types are
available for generation of diploids (this can be achieved by
making the mutants already in two strains with opposite
mating type and complementing markers)
These mutants are then allowed to form diploids in all
possible combination; for instance if one has 12 mutants with
mating type a and 9 with mating type alpha 9x12=108 crosses
are possible
If two haploid mutants have recessive mutations in one and
the same gene the resulting diploid should have the mutant
phenotype too
If two haploids have recessive mutations in two different
genes (confering the same phenotype) then the diploid
should have wild type phenotype, i.e. the mutations
complement each other
Hence, mut1 and mut2 represent two different
complementation groups representing most likely different
genes
No functional gene product
of MUT1
mut1
mut1
mut1
mut1
mut2
mut2
MUT2
MUT1
MUT1
mut1
mut1
MUT2
Functional gene products
of MUT1 and MUT2
Intragenic complementation
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© Stefan Hohmann 2000-2004
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Intragenic complementation is rare, but is does occur
Two mutant alleles, like mut1-1 and mut1-2, cause a
clear mutant phenotype in haploid cells and are
recessive
The heterozygous mut1-1/mut1-2 however shows a
(partial) wild type phenotype
The explanation is that the two mutated protein
products Mut1-1p and Mut1-2p can form a heteromere
that at least has partial function
This has been demonstrated extensively with certain
metabolic enzymes (ILV1, encoding a feedback
regulated enzyme in amino acid biosynthesis)
The occurence of intragenic complementation means
that the gene product must be an oligomere
The ”opposite”, non-allelic non-complementation, can
of course also occur: two recessive mutations in two
different genes fail to complement. This occurs
sometimes when the gene products are involved in
the same process or complex and the two functional
alleles are just not enough to confer full functionality
mut1-1
No functional gene product
of MUT1
mut1-1
mut1-2
mut1-2
But a heteromere consisting of
Mut1-1p and Mut1-2p can be functional
Cloning in yeast
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The era of yeast molecular genetics started as early as 1978, when
S. cerevisiae was first transformed successfully with foreign DNA
There are numerous transformation protocols but all are at least
three orders of magnitude less efficient as transformation in E. coli
Yeast can maintain replicating plasmids but the copy number is
P(BLA)
much smaller than in E. coli, usually between one and 50 per cell Apa LI (2367)
Yeast can maintain more than one type of plasmid at the same
time. This can complicate gene cloning from a library. It can also
be very useful to transform yeast with two different plasmids
AP
simultaneously, for instance for a method called plasmid shuffling
Cloning and plasmid preparation from yeast is very ineffective
Therefore, cloning in yeast uses E. coli as a plasmid production
system:
Apa LI (178)
ALPHA
Hin d III (400)
Pst I (416)
Bam H I (430)
Ava I (435)
Xma I (435)
r
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© Stefan Hohmann 2000-2004
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Plasmids are constructed in vitro
Plasmids are transformed into E. coli and the constructions are
confirmed, just in the same way as when working with bacteria
Plasmids are produced in bacteria....
....and then transformed into yeast
Hence we work with so-called yeast-E. coli shuttle vectors
On the other hand, yeast has a very efficient and reliable system
for homologous recombination, which can be used for cloning
pUC18
Sma I (437)
2686 bp
Eco R I (451)
P(LAC)
ORI
Apa LI (1121)
Yeast-E. coli shuttle vectors
EcoR I (2)
Cla I (28)
Apa LI (5217)
 Integrative plasmids (YIp)
consist
Hind III (33)
Amp-resistance
BamH I (379)
Pst I (4795)
of the backbone of a E. coli vector such
as pBR322, pUC19, pBLUESCRIPT
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of a yeast selection marker such as
URA3, HIS3, TRP1, LEU2
but are lacking any replication origin for
yeast
Hence, they are propagated only through
integration into the genome
Tet-resistance
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YIp5
Apa LI (3971)
5541bp
Pst I (1644)
PMB1
Nco I (1867)
URA3
© Stefan Hohmann 2000-2004
Apa LI (3473)
Ava I (2541)
YIp5: pBR322 plus the URA3 gene
Xma I (2541)
Sma I (2543)
Integration of plasmids into the yeast
genome
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Integration occurs by homologous recombination, this means that a plasmid like YIp5 will
integrate into the URA3 locus
Integration results in the duplication of the target sequence
The duplicated DNA flanks the vector
If there is more than one yeast gene on the plasmid, integration can be targetted by
linearisation within one of the sequences: cut DNA is highly recombinogenic
Integrated plasmids are stably propagated but occasional pop-out by recombination between
the duplicated sequences
plasmid
© Stefan Hohmann 2000-2004
URA3
X
genome
ura3
X
genome
ura3
URA3
Yeast-E. coli shuttle vectors
Apa LI (7445)
Amp-resistance
EcoR I (2)
Hind III (106)
Pst I (7023)
 Replicative episomal
plasmids (YEp) consist
2micron ORI
Ava I (1391)
Apa LI (6199)
of the backbone of a E. coli vector
PMB1
such as pBR322, pUC19,
YEp24
pBLUESCRIPT
7769bp
Apa LI (5701)
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of a yeast selection marker such
URA3, HIS3, TRP1, LEU2
and have the replication origin of the yeast
2micron plasmid
Hence, they are propagated relatively
Ava I (4835)
stably at high copy number, typically 20-50
per cell
Their copy number can be pushed to 200
Tet-resistance
per cell by using as marker a partially
defective LEU2 gene
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© Stefan Hohmann 2000-2004
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Pst I (2001)
EcoR I (2242)
Cla I (2268)
Hind III (2273)
Pst I (2482)
Nco I (2705)
URA3
Xma I (3379)
Ava I (3379)
Sma I (3381)
Hind III (3439)
YEp24: pBR322 plus the URA3 gene, plus 2micron origin
BamH I (3785)
Yeast-E. coli shuttle vectors
EcoR I (2)
Apa LI (7626)
Cla I (28)
Hind III (33)
Amp-resistance
 Replicative centromeric
plasmids (YCp) consist
Pst I (7204)
BamH I (379)
Tet-resistance
of the backbone of a E. coli vector
Apa LI (6380)
such as pBR322, pUC19,
pBLUESCRIPT
PMB1
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of a yeast selection marker such
Apa LI (5882)
URA3, HIS3, TRP1, LEU2
and have a chromosomal replication origin
for yeast, ARS (for autonomously
Apa LI (5457)
replicating sequence)
Pst I (5451)
have the centromere CEN of a yeast
chromosome
ARS1
Hence, they are propagated stably at low
copy number, typically one per cell
POLY
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© Stefan Hohmann 2000-2004
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Pst I (1644)
YCp50
7950bp
Nco I (1867)
URA3
XmaI (2541)
Ava I (2541)
Sma I (2543)
POLY
Ava I (4703)
CEN4
YCp50: pBR322 plus the URA3 gene, plus CEN4, plus ARS1
Yeast-E. coli shuttle vectors
 Plasmid series
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are based on an E. coli cloning vector such as
pUC19 or pBLUESCRIPT
have one out of three or four different yeast
markers
come as YIp, YCp and
YEp for convenience
Apa LI (178)
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YIps are used for integration only
YCps are used for low copy
expression
YEps are used for overexpression
Apa LI (178)
ARSH4
CEN6
2 MICRON
Hin d III (809)
Apa LI (4134)
HIS3
Ava I (4680)
Hin d III (808)
HIS3
Hin d III (996)
Hin d III (995)
Pst I (1188)
F1 ORI
pRS423
APr
pRS313
5797 bp
Pst I (1187)
4967 bp
LACZ'
F1 ORI
T7 P
Apa LI (4137)
LACZ'
Ava I (2092)
T7 P
Cla I (2108)
Bam H I (2110)
APr
Hin d III (2113)
Xma I (2116)
© Stefan Hohmann 2000-2004
Eco R I (2125)
MCS
Pst I (2135)
PMB1
Xma I (2137)
Ava I (2137)
Sma I (2139)
Apa LI (2888)
Sma I (2118)
PMB1
MCS
Pst I (2126)
Eco R I (2128)
Hin d III (2140)
Bam H I (2143)
Cla I (2147)
T3 P
T3 P
P(LAC)
Apa LI (2891)
Ava I (2116)
Ava I (2161)
Cloning by complementation
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© Stefan Hohmann 2000-2004
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Frequently when one has isolated a number of mutants and classified them into
complementation groups the nature of the gene is not known (and this is still often the case
even though the genome sequence is known!)
To identify the gene it is cloned from a gene library by complementation of the mutation
A gene library is a large population of plasmids containing different fragments of genomic yeast
DNA, cumulatively representing the entire yeast genome
Such libraries are constructed by digesting the entire yeast DNA partially with a nuclease such
as Sau3A (cutting site GATC), which cuts frequently; this strategy generates many overlapping
fragments and it ensures that all genes are functionally represented; Sau3A fragments can be
cloned into BamHI (GGATCC) cut plasmids; all available yeast libraries are done that way
If the fragments cloned are 5-9kb on average, 2,000 plasmids represent the genome once and
10,000 plasmids give a more than 90% probability that all genes are functionally represented
The library is transformed into the yeast mutant of interest
Transformants are screened or selected for restoration of the wild type phenotype
Plasmids are prepared from positive clones, transformed into E. coli and further analysed;
some sequence information reveals the identity of the clone
Retransformation into the yeast mutant verifies that the plasmid contains a truly
complementing gene; this is necessary because yeast cells can take up more than one kind of
plasmid
Cloning by complementation
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© Stefan Hohmann 2000-2004
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Cloning by complementation sounds like a straightforward approach but there are quite a few
caveats to it
First of all, it can only be done with recessive mutants
For cloning of genes with dominant mutants, a gene library has to be prepared from each
mutant and transformed into the wild type strain; transformants showing the mutant phenotype
are then screened or selected
In addition, complementation of a mutation does not mean that the cloned gene is indeed the
one that is defective in the mutant – it could be a multi-copy suppressor
This can even happen with centromeric vectors, because selective pressure can drive up the
copy number of even these plasmids
A multi-copy suppressor is a gene that overcomes the primary defect in the mutant when
expressed at high levels; this is a common phenomenon
It is in fact so common that it is a useful approach to clone new genes starting from a certain
mutant – we return to that
To demonstrate that the cloned gene is the one that is mutated in the mutant, a deletion mutant
has to be constructed by homologous recombination using the cloned gene as template
If the original and the deletion mutant have the same phenotype, this is good evidence that the
two genes are the same
Final proof is obtained by crossing the two mutants; if the diploid has the mutant phenotype too
and all spores isolated form the diploid as well, this is proof that the two genes are the same
Deletion of genes by homologous recombination is one of the most powerful techniques in
yeast and one of the reasons why yeast is so popular; it works so well that systematic deletion
of all 6,200 genes has been done and we have this collection in the lab
Cloning by complementation
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© Stefan Hohmann 2000-2004
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If the original and the deletion mutant
have the same phenotype, this is good
evidence that the two genes are the
same
Final proof is obtained by crossing the
two mutants; if the diploid has the
mutant phenotype too (i.e. there is no
complementation between the original
and the deletion mutant) then one can be
very sure that the cloned gene is the one
orginally mutated.
To be 100% sure, one sporulates the
diploid and dissects some ten tetrads:
all spores should have the mutant
phenotype
Deletion of genes by homologous
recombination is one of the most
powerful techniques in yeast and one of
the reasons why yeast is so popular; it
works so well that systematic deletion of
all 6,200 genes has been done and we
have this collection in the lab
mut1D
No functional gene product
of MUT1
mut1D
mut1
mut1
mut2
mut2
MUT2
MUT1
MUT1
mut1D
mut1D
MUT2
Functional gene products
of MUT1 and MUT2
Deleting a yeast gene
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Using the cloned gene the open reading frame is deleted in vitro and replaced by a marker gene
The result of this is basically the marker gene flanked by sequences originating from the gene that has to
be deleted
This piece of DNA is transformed into yeast, where it replaces the gene on the chromosome by
homologous recombination; the marker is used for selection of transformants
Subsequent Southern blot or PCR analysis and phenotypic analysis of the yeast strain confirm the
deletion
The approach works faithfully and yields several transformants per mg of DNA. Doing the same in plants
or mammalian cells takes years, often a whole PhD thesis
YFG1
Your favourite gene on a plasmid
Your favourite gene on a plasmid,
ORF replaced by marker
in vitro
URA3
© Stefan Hohmann 2000-2004
URA3
X
X
in vivo
URA3
Recombination in yeast
Your favourite gene deleted
from the genome
Deleting a yeast gene

There are a number of different ways to generate the piece of DNA for yeast transformation, i.e.
the marker flanked by fragments with DNA from YFG1
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It can be done using restriction enzymes and DNA ligation
It can be done by PCR/restriction/ligation; the entire plasmid is amplified by PCR with the exception of the
ORF; restriction sites in the PCR primers generate a site where the marker can be cloned in
It can be done by PCR without any cloning step; 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 (see below)
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; in such
cases the use of a heterologous marker is recommended to make integration in the right place more
reliable
The latter two approaches do not even require the gene to be cloned!! A gene deletion project hence may
take only a couple of days
© Stefan Hohmann 2000-2004
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
Smart gene deletion
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There are very smart ways to make most out of a gene deletion/disruption approach,
depending on the marker cassette used
For instance, if the marker cassette contains in addition the lacZ reporter gene a
precise fusion can be generated that places the lacZ gene under control of the yeast
promoter of YFG1
If such a construct is used for gene deletion in a diploid, it can be used to study the
expression of the gene by monitoring b-galactosidase activity in that diploid and after
sporulation of the diploid the mutant phenotype can be studied in the haploid progeny
In a similar way, a gene can be tagged. For instance, if the casette 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
lacZ
© Stefan Hohmann 2000-2004
URA3
YFG1
Diploid cell
Smart gene deletion
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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
For instance, there are now sets of strains available in which each yeast has
been tagged with GFP or TAP-tag
YFG1
GFP
© Stefan Hohmann 2000-2004
URA3
Smart gene deletion
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© Stefan Hohmann 2000-2004
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There are some ways to delete a yeast gene without leaving any trace behind, i.e. no marker
gene
This is very important if one wants to re-use the marker in order to make many deletions in one
and the same strain (there are strains with more than 20 deletions!)
It is also important for industrial yeast strains; when one wants to engineer those at the end no
foreign DNA should be left behind (but for hardliners on genetic engineering the intermediate
presence of foreign DNA ina yeast is already ”dangerous”)
All these methods use homologous recombination a second time, i.e. to pop-out the integrated
DNA again
An example for this are the loxP-kanR-loxP cassettes; recombination between the two loxP
cassettes is stimulated by the Cre-recombinase (transformed on a separate plasmid);
recombination just leaves behind a single loxP site
Smart gene deletion
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A very useful marker to work with is URA3 because one can select for and against its presence
Selection for URA3 is of course done on medium lacking uracil
Selection against URA3 uses the drug 5-flouro-orotic acid, which is toxic to URA3 cells
An example is shown below
URA3
plasmid
YFG1
genome
© Stefan Hohmann 2000-2004
YFG1
URA3
Integration of the plasmid, which only contains YFG1 flanking regions, creates a duplication; recombination between
the blue sequences leads to a pop-out of the entire plasmid plus the YFG1 coding region
How to deal with essential genes
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© Stefan Hohmann 2000-2004
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We have discussed now random chemical and targetted mutagenesis; an obvious question is:
how can we identify and work with mutations in genes whose products are essential for the cell
(and that is about 1/3)? A mutation that knocks out the function of that protein kills the cell and
it is difficult to work with dead cells....
For chemical mutagenesis the most common approach is to work with conditional mutations;
usually these are mutations where the gene product functions at a lower temperature, like 25°C,
but not at higher temperature, like 37°C; the mutant is temperature-sensitive; many essential
cellular functions have been identified through ts mutants
To determine in gene deletion experiments if a gene is essential, the deletion is done in a
diploid; if after sporulation only two spores survive and if all living spores do not have the
marker used for the deletion, the gene is regarded as essential
One can work with mutants in essential genes. Principally, the mutant is transformed with
plasmid that expresses the relevant gene conditionally.
For instance a plasmid contains the essential gene under the control of the promoter of the
GAL1 gene; this promoter is ”on” on galactose medium but ”off” on glucose medium; when
shifting cells to glucose one can study at least for some time the properties of the cells...and
watch them dying (yfg1D pGAL1-YFG1)
To analyse the function of in vitro generated point mutants, one can use plasmid shuffling. For
this, the mutant is first transformed with the wild type gene and then with a mutant gene. The
plasmid with the wild type gene carries URA3 as selectable marker, which can be forced to be
lost on medium with 5-FOA. If the mutant grows on 5-FOA medium, the mutant allele is
functional (yfg1D pURA3::YFG1 pLEU2::yfg1-1).
From gene disruption to transposon
mutagenesis
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© Stefan Hohmann 2000-2004
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The gene deletion/disruption technique has been
taken a step further to be used in random
mutagenesis
For this a gene library is first constructed as
discussed before such that the inserted yeast
DNA can be cut out with NotI, an enzyme that
only cuts a very few times in the yeast genome
Then this library is mutagenised with a
transposon in E. coli, where the Tn randomly
integrates into the yeast DNA
Subsequently, the entire mix of NotI fragments is
transformed into yeast where it is expected to
replace genes; with about 30,000 yeast clones a
more then 90% coverage of the genome is
achieved
The Tn used is a quite sophisticated example of
such a transposon, that can be partially cut out
again through the lox-sites. This creates a tag,
which allows immunolocalisation of the gene
product
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TR: Tn3 terminal inverted repeats
Xa: Factor Xa cleavage recognition site
loxR: lox site, target for Cre recombinase
lacZ: 5'-truncated lacZ gene encoding bgalactosidase
URA3 gene from S. cerevisiae
tet: tetracycline resistance gene
res: Tn3 site for resolution of transposition
intermediate
loxP: lox site, target for Cre recombinase
3xHA: Hemagglutinin (HA) triple epitope tag
From gene disruption to transposon
mutagenesis
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© Stefan Hohmann 2000-2004
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The reason why transposon mutagenesis is so powerful
lies in the fact that the gene affected by the insertion can
be determined very easily
For this, the entire genomic DNA of the mutant is isolated
and cut with an enzyme that does not cut within the
transposon
In this way of course many fragments are generated but
only one will contain the transposon plus some flanking
yeast DNA
Ligation generates a circular plasmid that can be
transformed into E. coli and further analysed
Sequencing using a primer binding to the transposon but
directing into the yeast DNA will reveal exactly where the
transposon was integrated when the sequence is compared
to that of the yeast genome
This method works so well that it has been used for a
comprehensive genome analysis
For instance, we have recently screened 25,000 Tn-mutants
for a number of properties and could allocate functions to a
number of uncharacterised genes with relevance to stress
tolerance
Derivative of the transposon with antibiotic markers are
very useful tools to mutagenise and study industrial strains
BamHI Primer
LacZ-D
Ampr
EcoRI
Ori
Yeast DNA
Cloning in yeast by gap repair
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The powerful yeast recombination system can be used in different ways to clone genes by
repair of gapped plasmids
Basis for this approach is that gapped, linear plasmids are not propagated by yeast cells unless
repaired to a circular plasmid
Repair can occur by recombination with a co-transformed piece of (partially) homologous DNA;
this can be used to generate mutations, e.g. by error-prone PCR. Note that in fact none of the
involved pieces of DNA needs to be from yeast itself!!
This works extremely well and we have used it in the lab quite a lot
Repair can also occur by recombination and gene conversion with genomic DNA; this can be
used to clone mutant alleles from the genome
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repair fragment
gapped plasmid
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© Stefan Hohmann 2000-2004
YFG1
YFG1
gapped plasmid
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genomic copy is used to repair the gap; the template is duplicated
Localising proteins with the cell: GFP
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© Stefan Hohmann 2000-2004
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The green-fluorescent protein is used now
systematically to localise proteins within the yeast
cells
A main advantage of the GFP technology is that it
allows watching processes in the living cell !
Usually the coding sequence of GFP is fused to the
end of the coding region of the gene of interest
This can be done on a plasmid but also within the
genome
The resulting construct is tested for functionality by
complementing the corresponding deletion mutant
GFP shines green in the fluorescence microscope
and the subcellular localisation can be deduced
using control staining of different compartments
There are now many different versions of GFP with
different detection threshold and different emission
colours: CFP, BFP, RFP, YFP...
This allows simultaneous observation of several
proteins in the cell and even protein-protein
interaction
Getting further: isolating more genes
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So far we have discussed different ways to generate mutations in yeast:
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and we have discussed some methods to study and engineer genes in yeast
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© Stefan Hohmann 2000-2004
by fusion with a reporter gene to monitor gene expression
by fusion with an epitope or with GFP to study the protein level or protein localisation
The power of genetic analysis lies in the possibility to use one gene/mutant to isolate further
genes, which encode proteins involved in the same or in parallel or related cellular processes
The same genetic approaches can be used to allocate different genes/proteins to the same (or
to different) cellular functions and to sort them in an order, for instance within a signalling
pathway
Such approaches to get further include
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chemical random mutagenesis
random targetted mutagenesis with transposon-tagged DNA
targetted deletion/disruption of yeast genes
Multi-copy suppression
Suppressor mutation
Synthetic lethality
The yeast two-hybrid system
All these systems are used in multiple variations; the intellectual challenge is to find the
conditions that allow the approach to be used
Getting further: suppressors
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© Stefan Hohmann 2000-2004
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Definition: a reversion of a mutation means that the primary lesion is repaired and
hence the orginal, wild-type situation is restored; obviously, a deletion mutant
never can revert
Definition: a suppressor is a gene or mutation that (partially) overcomes the effect
caused by a given mutation; hence a suppressor is a second-site genetic alteration
that somehow restores (partially) the wild type situation
Suppressors can be intragenic, i.e. a second mutation in the same gene/protein can
restore (partial) functionality of the gene product; again, this is only possible with
point mutations and not with deletion mutants
More common are extragenic suppressor and we will discuss multi-copy
suppressors and suppressor mutations
How a suppressor functions differs of course a lot from system to system but
usually the analysis of the suppressor function provides a lot of important
information
Principally, a suppressor either activates (or represses) the system affected by the
primary mutation in another way or activates (or represses) an alternative, partially
redundant system
Suppressors are useful as we discuss them here but at the same time can be
annoying: yeast mutants that poorly grow can easily generate suppressors,
something one has to be aware of when working with such mutants
Getting further: multi-copy suppression
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© Stefan Hohmann 2000-2004
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Multi-copy suppression is based on overexpression of a gene, usually on a multi-copy plasmid or
via ectopic expression from a strong promoter
A multi-copy (or gene dosage) suppressor is a gene, which, when expressed at high levels,
overcomes (some of) the effects of a certain mutation
Multi-copy suppression as a tool in gene discovery is exciting in a way: you hardly ever know what
you will get.....
Generally, however, one expects genes whose products function downstream in the same pathway
or in a parallel pathway
A nice thing about multi copy suppression: you get to the gene right away!
Turning the argumentation around, if one knows from other genetic experiments that two genes are
functionally related, multi-copy suppression is a way to sort two proteins within a pathway within an
epsitasis analysis: only a gene whose product functions downstream of the mutation can suppress
in multi-copy
Pbs2p and Hog1p are in the
same pathway and Hog1p is
activated by Pbs2p.
Overexpressed Hog1p may
confer sufficient activity to
mediate the required function
even in the absence of Hog1p.
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Two parallel pathways share
one or several common targets.
Overexpression and hence
higher activity of the parallel
pathway may be sufficient to
activate the target.
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common target
Getting further: suppressor mutations
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© Stefan Hohmann 2000-2004
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An extragenic suppressor mutation alters a different gene product such that the, or one of the, effects
of a certain mutation are overcome
Like with multi-copy suppression there are many ways in which this can happen and the outcome of
such an approach is often quite surprising but very informative
Typical suppressor mutations are those that activate a gene product downstream of the primary lesion
in the same pathway; since such mutations cause a gain of function they are usually dominant
Other typical suppressor mutations knock out a repressor downstream in the same or in a parallel
pathway; since such mutations cause a loss of function they are recessive
A suppressor mutation may also activate or inactivate pathways/systems that affect in some way the
same physiological system than the primary lesion
If a given protein is part of a multimeric complex and the primary mutation is a point mutation,
extragenic suppressor mutations might occur such that protein interactions are restored; hence this is
a method to identify interacting proteins
Pbs2p and Hog1p are in the
same pathway and Hog1p is
activated by Pbs2p. A mutation
that renders Hog1p active even
without activation would
suppress the pbs2 mutation and
is probably dominant
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The pathway ultimately
inactivates a negative regulator,
e.g. the repressor Sko1p; knock
out of the repressor could
overcome inactivation of the
pathway; the mutation is most
likely recessive
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Sko1
Getting further: synthetic lethality
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Synthetic lethality is a powerful method to identify genes whose products operate (in a
pathway) parallel to the one that is affected by the primary mutation
Typically, the primary mutant is transformed with a plasmid that carries the corresponding
gene; the gene is either expressed through the GAL1 promoter (i.e. ”on” on galactose and ”off”
on glucose) or is on a plasmid with URA3 as marker, which can be counter selected with 5-FOA
Mutations are then screened that cause the yeast to grow only in the presence of the plasmid
(i.e. not on 5-FOA) or only when the gene is expressed (i.e. not on glucose)
The principle approach is so powerful that synthetic lethality screens are now done at a
genome wide scale using the yeast deletion mutant collection: this means 4,200 x 4,200
crosses, sporulations and tetrad analyses done by robotics
© Stefan Hohmann 2000-2004
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common target
The two pathways control some
common targets; mutation of
PBS2 alone causes only a
moderate phenotype. The
second mutation in the parallel
pathway leads to lethality
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common target
Getting further: epistasis I
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The concepts of suppressor analysis and synthetic lethality are also the basis for a powerful tool of
genetics, epistasis analysis
In a way it is similar to complementation analysis (How many different genes in the mutant collection
cause the same phenotype?) as epistasis analysis asks the question: how many genes/proteins are
involved in the same genetic system/pathway and in which order do they function?
The basic idea is to combine two mutations in the same cell, i.e. to generate a double mutant; the
phenotype of the double mutant may reveal if the two gene products work in the same or in parallel
pathways and they may reveal the order within a pathway.
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© Stefan Hohmann 2000-2004
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common target
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Let us first assume mutation in all these four proteins cause similar
phenotypes, such as moderate sensitivity to salt
When we combine the hog1 and the pbs2 in a hog1 pbs2 double mutant
then we would expect that the double mutant has the same level of
sensitivity as each single mutant; we would conclude that they function in
the same pathway
When we combine the pbs2 and the cba1 mutation in a pbs2 cba1 double
mutant we would expect a strongly enhanced sensitivity of the double
mutant as compared to the single mutants; we would conclude that Hog1p
and Cba1p work in different, though parallel pathways
Getting further: epistasis II
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© Stefan Hohmann 2000-2004
Sko1
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Let us now assume that deletion of PBS2 (and of HOG1) causes sensitivity
to high salt concentrations while deletion of SKO1 causes higher tolerance
to salt in the medium
If those proteins act in the same pathway there are different possibilities
for the phenotype of the pbs2 sko1 or hog1 sko1 double mutant
If Sko1p were downstream of Pbs2p and Hog1p we would expect that the
double mutant is tolerant, i.e. has the same phenotype as the sko1 single
mutant: sko1 would be epistatic (”dominant over”) to pbs2 and hog1 (and
this is really the case)
If Sko1p were upstream of Hog1p and Pbs2p we would expect that the
double mutant pbs2 sko1 and hog1 sko1 is sensitive to salt
Getting further: epistasis III
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© Stefan Hohmann 2000-2004
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Also multi copy suppression or activating mutations are
useful tools in epistasis analysis
Suppression by overexpression can only work for a
gene/protein functioning downstream of the primary lesion,
as indicated here for Hog1p; overexpression of PBS2 would
not suppress a hog1D mutation
In a similar way, an activating mutation of HOG1 can
suppress the salt sensitivity of a pbs2D mutant, but not vice
versa, and this is indeed exactly how it works
The epistasis concept has been used in very many examples
to analyse the order of events in signalling pathways and
other cellular systems: if the phenotype of the double mutant
resembles that of one of the single mutants the latter gene
product functions further downstream in the system, i.e.
closer to the physiological effect
Getting further: two-hybrid system
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© Stefan Hohmann 2000-2004
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The yeast two hybrid system is a method to detect the interaction of two proteins in the yeast
cell and it can be used to select for an interacting partner of a known protein
The original version uses a transcriptional read-out to monitor interaction, nowadays there are
also other methods
The method is so powerful since it is not restricted to yeast proteins; the interacting partners
can origin from any organism; in fact some versions do not use any yeast sequences
Basis for the system is the modular nature of transcription activators that consist of
exchangeable DNA binding and transcriptional activation domains
The gene of interest, the bait, is cloned in fusion
with a DNA binding domain, such as that of the E.
coli lexA protein
The potential binding partner, the target or prey,
which may be a library, is cloned in fusion to a
transcriptional activation domain, such as that
from VP16, a viral protein
Only when bait and target interact, a reporter
gene whose only promoter is a lexA binding site
will be activated
reporter
lexA
site
Application of the yeast two-hybrid
system
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© Stefan Hohmann 2000-2004
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The possible applications to the two-hybrid system are absolutely tremendous
The system can be used to detect interaction between two proteins
The system can be used to characterise the domains and residues in the two proteins that
mediate interaction; this can be done by mutagenesis and the use of a counterselectable
reporter, such as URA3
The system can of course be used to find interaction partners
The system can be used to find proteins that regulate the interaction between two proteins
The system can be used to screen for drugs that inhibit the interaction between two proteins
The system is actually used to construct an genome-wide map of protein interactions in yeast;
using laboratory robots 6000 bait strains are crossed to 6000 prey strains to study all possible
protein intercations
etc................
Genetic analysis in action: the HOG
pathway
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© Stefan Hohmann 2000-2004
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The analysis of the osmosensing HOG pathway, on
which we work, is a good example how different
genetic tools work in action
PBS2 and HOG1 were first identified in a genetic
screen for salt sensitive mutants
Deletion of SLN1 is lethal because this sensorhistidine kinase is a negative regulator of the
pathway and overactivation is deleterious
Downstream kinases were identified as recessive
suppressor mutations
Protein phosphatases were found as multi-copy
suppressors
Targets are defined because their deletion allows, to
different extent, survival of a sln1 mutant (or
commonly used an ssk2DN, which has a similar
lethal effect)
Parts of the SHO1-branch were found as synthetic
osmosensitive mutants in combination with an ssk2
ssk22 mutant, which is not osmosensitive
The link between Rck2p and Hog1p and between
Hog1p and Hot1p was found in two-hybrid screens
The model organisms
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© Stefan Hohmann 2000-2004
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The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are regarded as
model organisms in molecular biology
This means that it is anticipated that certain – or perhaps most – principal cellular systems
function in a similar way in yeasts and human, i.e. across eukaryotes
This is of course only true to a certain extent but many principal molecular mechanisms are
indeed conserved; certain modules are however used in different context reflecting the
evolution in specific environments
Hence, yeasts are not just simple human cells
Another limitation is the fact that yeasts are unicellular and hence lack an important level of
complexity, i.e. that of a multicellular organism
Note, however, that even yeast has different cell types that can be distinguished by expressing
different sets of proteins, a hallmark of cellular differentiation
By the way, although S. cerevisiae and S. pombe are both yeasts, they are as distinct from each
other than each is from human
S. cerevisiae
S. pombe
Human
Model character: eukaryotic cell cycle
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Cell cycle control is a prime example where genetic analysis in yeasts has provided fundamental insight
The eukaryotic cell cycle is set up of four distinct phases, G1, S, G2 and M
In addition, there are crucial check points, where the completion of certain events is monitored before the next
one is started
The relative importance of these check points is species specific, in S. cerevisiae START is a crucial point
Nutrient starvation and pheromone cause cell cycle arrest at this point
A key feature of budding yeast is that the stage of the cell cycle can simply be deduced from the cell’s
morphology, i.e. bud size
This has been used to order a large number of cdc according to the stage of the cycle where they are affected: the
foundation of genetic analysis of cell cycle control
© Stefan Hohmann 2000-2004
The actin cytoskeleton
during the cell cycle
Model character: signal transduction
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© Stefan Hohmann 2000-2004
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The principles of signal transduction are well conserved among eukaryotic cells
For instance, animals and fungi use cAMP as a second messenger and it seems
that cAMP mediates nutritional signals
For instance, all eukaryotic cells have common classes of signalling proteins, such
as G-protein couples receptors, a type of hormone receptors; the yeast pheromone
receptors belong to this class
A prototypical eukaryotic signalling system are MAP (mitogen activated protein)
kinase cascades; these are modules of three protein kinases that typically control
gene expression; the module is used in many signalling pathways responsive to
different stimuli and hence controlled by different sensing mechanisms
S. cerevisiae has at least six such pathways, which together control cellular
morphology and responses to pheromone and environmental stress
Genetic analysis in yeast has and is contributing greatly to the understanding of
how these pathways function
There are of course also limitations to the model character; for instance S.
cerevisiae is lacking receptor tyrosine kinases or nuclear receptors, important
classes of mammalian hormone receptors
© Stefan Hohmann 2000-2004
Model character: signal transduction
Model character: morphology switch
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© Stefan Hohmann 2000-2004
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We have already pointed out that yeast cells can switch their morphology
This switch requires a MAP kinase pathway and nutritional signals; also cAMP plays a role
The yeast pseudohyphal switch (or invasive growth in haploids) is a model system for
morphogenesis
Most importantly, a morphological switch is associated with pathogenesis for instance of
Candida albicans and hence much research is focussed on the basic mechanisms
S. cerevisiae may use the switch and co-expression of polysaccharide degrading enzymes to
penetrate plant tissues
Model character: control of gene
expression
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© Stefan Hohmann 2000-2004
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The principles of the control of
transcription are well conserved
across eukaryotes and many
proteins function across species
borders as we have already noted for
transcription factors
The organisation of the transcription
initiation machinery seems to be
conserved, i.e. there are counterparts
for most if not all subunits in yeast
and human
The mechanisms of transcriptional
activation seem to be conserved, but
certain classes of activators (prolineand glutamine-rich) do not seem to
function in yeast
Although chromatin organisation
seems to be more simple in yeast,
aspects of its involvement in the
control of gene expression are
similar
Control of gene expression means
that signals and molecules have to
traverse the nuclear membrane and
these mechanisms seem to be well
conserved
Model character: vesicular transport
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© Stefan Hohmann 2000-2004
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Vesicular transport, i.e. the mechanisms
that control the trafficking of proteins
and membranes is another feature that is
highly conserved across eukaryotes
Temperature sensitive sec mutants have
been sorted according to the stage
where transport stops (using electron
micoscopy) and this has been the
foundation for genetic analysis
In addition, transport to the vacuole and
endocytosis are studied by genetic
analysis combined with biochemistry
and cell biology
Model character: proteasome
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© Stefan Hohmann 2000-2004
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The proteasome is a multi protein
complex conserved in eukaryotes
It is located in the cytoplasm and
the nucleus and controls
degradtion of proteins that have
been ubiquitinated
The 26S proteasome consist of a
20S catalytic and a 19/22S
regulatory subunit
The 20S proteasome is composed
of 14 different proteins and all
genes are known in yeast
The yeast 20S complex has been
purified and the X-ray structure
has been determined
Model character: the unexpected
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Prions
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Ageing
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Is a process very much assocated with multicellular organisms
Yeast cells have a pre-determined life span, i.e. mother cells die after a certain number of divisions
The ageing process in yeast seems to have some features in common with that of human, for instance the
accumulation of rDNA circles
There is also a ”common” gene, WRN (Werner’s syndrom) in human and SGS1 in yeast; the genes are
homologous and mutations causes premature ageing in human and yeast, respectively
Cell type determination
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© Stefan Hohmann 2000-2004
Have of course been in the focus of interest through mad cow disease
Yeast also has two systems that seem to have all features of prions! This means they are genetic
elements, alleles of known genes, that behave as non-Mendelian genetic elements: PSI+ (Sup35p), a
protein involved in translation termination and URE3 (Ure2p), a regulator of nitrogen metabolism
As discussed earlier, yeast develops different cell types determined by different gene expression pattern
Functional genomics
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The term functional genomics is not very well defined; since it is a nice term to attract funding
these days many people call functional genomics what they have done for ages
Strictly, it should probably mean ”the determination of the function of previously
uncharacterised genes identified by genome sequencing”
This aspect is indeed addressed in a systematic way in yeast by at least two different projects;
their goal is the construction of deletion strains for all 6,200 genes and an initial phenotypic
characterisation; the set is complete
Functional information can also come through other approaches; for instance, the yeast twohybrid system is used to construct a complete protein interaction map
Transposon mutagenesis is used to tag a large number of yeast proteins to determine their
localisation
Functional information also comes from expression analysis
Expression of proteins is studied by 2D gel electrophoresis, which can resolve some 1,000
different yeast proteins
Analysis of the expression of all 6,200 yeast genes has now become reality allowing a
comprehensive picture of transcriptional changes depending on conditions or in certain
mutants
Functional genomics: transcriptional
profiling
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Transcriptional profiling in yeast is
reality now and a number of articles
using the technology have appeared
A large data collection is generated in
Stanford covering a number of growth
conditions
Another large collection generated by
Rick Young’s lab concerns effects of
mutations in certain components of the
transcription initiation machinery
We have used transcriptional profiling to
study signal transdution in stress
responses
From functional genomics to systems
biology
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© Stefan Hohmann 2000-2004
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Systems biology goes a step further then functional analysis: the goal of systems
biology is to describe the operation of the entire cell with all its proteins
In a more narrow definition, systems biology combines mathematical and
experimental approaches to achieve a better understanding of biological networks
and systems
Systems biology is a multidisciplinary approach involving biologists, engineers
and mathematicians
There are two principle goals within systems biology: (1) to describe the wiring
network of all proteins in the cell and (2) to decsribe the dynamic operation in the
cell
Reconstruction of the wiring network uses all available data such as genetic, gene
expression, protein interaction data to connect proteins with each other
Dynamic modelling and experimentation aims at decribing the overriding rules how
e.g. metabolism and signalling dynamically operate
We use such approaches to understand how signalling pathways operate
Yeast biotechnology: fermentation
industry
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The yeast fermentation industry, comprising baking, brewing, wine making and industrial
alcohol production, is still the biggest BioTech business world-wide
Industrial yeast strains are usually difficult to work with because they are diploid, polyploid or
even aneuploid; many appear to be cross-species hybrids
There are many possible improvements to the fermentation processes, where the biology of
yeast is the limiting factor; hence there are many attempts to improve yeasts
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Wine yeasts: ability to perform the malolactic fermentation, which is normally performed by lactic acid bacteria
(faster and more reliable production); ability to degrade polysaccharides that disturb filtration; ability to hydrolyse
saccharides, which contain flavour compounds in glycosidic bonds (improved flavour); ability to kill competing
bacteria and yeasts (cleaner fermentation and wine taste); osmotic and alcohol tolerance; better productivity and
less byproducts during starvation
Beer yeast: ability to degrade polysaccharides (better filtration and low calory beer); reduced production of acetoin
and butanediol (reduced maturation time); increased osmotolerance (high gravity brewing leading to less tank
volume)
Distiller’s yeast: increased alcohol yield (less glycerol) and tolerance
Baker’s yeast: ability to degrade different sugars at once through diminished catabolite repression (better
leavening); freeze-tolerance after fermentation initiation (frozen doughs); high osmotolerance (high-sugar doughs)
In the food industry attempt are done in parallel using classical genetics (where possible) and
genetic engineering; public perception has so far not allowed to use genetically engineered
yeasts in the food industry
Yeast biotechnology: heterologous
expression
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The production of proteins is of interest for several purposes:
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For research, such as for purification and structural analysis
For industry, such as for the production of enzymes for the food and paper industry or for research and diagnostics
For the pharmaceutical industry for the production of vaccines
There are a number of different expression hosts, such as bacteria and yeasts
Yeast have the advantage that they may (or may not) perform the same or at least similar posttranslation modifications, such as glycosylation
Yeast usually reaches only a lower level of expression: up to more than 50% of the cellular
protein have been obtained in E. coli systems but no more than 10-20% even in the yery best
yeast system
The apparently most productive known yeast is the species Pichia pastoris; it catabolises
methanol and the promoter for methanol oxidase is extremely strong and can be induced by
methanol
In S. cerevisiae one usually uses the promoters of genes encoding glycolytic enzymes such as
PGK1 and TPI1 or a regulated promoter such as that of GAL1
The advantage of S. cerevisiae is that so much is known about its molecular biology and one
can device genetic screens to improve protein production and secretion
Recently we have developed a yeast strain that does not make ethanol but rather more
biomass; we try to market that strain through a start-up company
Heterologous expression in yeast: gene
cloning and functional analysis
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Heterologous expression in yeast can be used to functionally
clone genes form other organisms
Quite a large number of genes from mammals and from plants
have been cloned by complementation of yeast mutants
For this, a cDNA library is typically cloned into a yeast
expression vector, i.e. expression of the cDNAs is driven by a
strong yeast promoter, such as that from PGK1
The library is then used to complement a yeast mutant
This approach has been especially successful with plant cDNA:
a number of genes encoding transport proteins and metabolic
enzymes have been cloned in this way
Successfull functional expression in yeast opens the
possibility to do a functional analysis using yeast genetics of
proteins derived from other organisms
Heterologous expression in yeast: onehybrid system
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© Stefan Hohmann 2000-2004
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The yeast one-hybrid system is basically a
half two-hybrid system
To clone a transcription factor gene, a
cDNA library is constructed such that it is
linked to a yeast transcriptional activation
domain and expressed in yeast
As a reporter system a hybrid gene is used
that contains fragments from the
mammalian or plant promoter of interest
If the fusion protein contains a DNA
binding domain that recognises that
heterologous promoter fragment, the
reporter gene will be activated
Heterologous expression in yeast: drug
screening
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Yeast can be grown easily and reproducibly even in microtitre
plates
Together with the possibility of genetic engineering and
heterologous expression this makes yeast a useful tool for high
throughput drug screening
An example of a very important class of human drug targets are the
G-protein coupled receptors
The yeast mating pheromone response is also controlled by such a
receptor, the pheromone receptors are GPCRs
The pathway has been engineered such that human GPCR control
the pathway and that the pathway controls the expression of
reporter genes
This has and is being used to screen for compounds that work as
agonists or antagonists to human hormones and hence are lead
compounds in drug design
Yeast can even be used for a preliminary assessment of seconday
effects confered by the compounds, for instance by applying
transcriptional profiling.