YEAST GENETICS AND MOLECULAR

BIOLOGY

 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
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 Yeast information resources WWW

 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://genome-www.stanford.edu/Saccharomyces has information on all yeast genes
including links and information to other yeast genome projects and global analysis
projects
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 The yeast Saccharomyces cerevisiae:
habitate and use

 Yeast lives on fruits, flowers and other sugar containing substrates

 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 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
 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 used for drug screening and functional analysis
because it is a eukaryote but can be handled as easily as bacteria
 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
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 Other important yeasts

 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.
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 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 dividions
 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 A yeast cells is about 4-7µ m large
 Mitochondria for respiration The ”eyes” at the bottom are bud scars
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 Life cycle of yeasts

Budding Yeast Fission Yeast

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 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); 2n cells are
1.2-fold bigger
 Haploid cells have one of two mating types:
a or alpha (α )
 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:
 Haploid MATa versus MATalpha
 Haploid versus Diploid (MATa/alpha)
 Spores
 Mothers and daughters
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 Yeast sex!

 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
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 Yeast sex!

 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
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 Genetic determination of yeast cell type

 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
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 Gene expression that determines the
mating type

 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)
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 Haploids and dipoids in nature and laboratory

 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?
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 Haploids can switch mating type!

 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

 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
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 Yeast genetics: the genetic material

 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)
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 Yeast genetics: the genetic material

 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)
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 Yeast genome analysis

 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
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 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: tps1∆ ::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, MATα
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 Yeast genetics: markers and strains

 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, Σ 1278b, 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
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 Yeast genetics: crossing strains

 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
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 Yeast genetics: crossing strains

 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
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 Yeast genetics: crossing strains

 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 - + - + -
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 Yeast genetics: meiosis

 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
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 Yeast genetics: the outcome of a cross

 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 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
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 Yeast genetics: cross over

 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 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
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 Yeast genetics: double cross over

 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
leu2 URA3

 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

 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,
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 Crossing with markers on different
chromosomes

 Let us now imagine that LEU2 and URA3 are on different chromosomes

LEU2 ura3
LEU2 ura3 LEU2 URA3
LEU2 ura3 LEU2 ura3 LEU2 URA3

leu2 URA3 leu2 URA3 leu2 ura3

leu2 URA3 leu2 ura3
leu2 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.
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 Crossing with markers on different
chromosomes

 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 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
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 Yeast genetics: making mutants

 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
 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
 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
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 Yeast genetics: finding mutants

 Screening versus selection

 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 YPD YPD + 0.4M NaCl
because they are unable to grow on a certain medium after
Wild type
replica-plating or because they develop a colour
 For screening, mutations are usually induced to increase their hog1∆
frequency
sko1∆
 Still: screening requires hundreds of perti dishes and commonly
more than 10,000 clones to be scored aca1∆ aca2∆
 To develop a new selection system is the art of genetic hog1∆ sko1∆
analysis hog1∆ aca1∆
 When selecting for mutants one has established a condition aca2∆
under which the mutant phenotype confers a growth advantage hog1∆ sko1∆
 In other words, the intellectual challenge is to design conditions aca1∆ aca2∆
and /or strains such that the mutant grows, but the wild type YPD YPD + 0.4M NaCl
does not
 A smart screening system allows one to go for spontaneous Wild type
mutations, because up to 108 cells can easily be spread on one aca2∆
plate
 Selection systems are often based on resistance to inhibitors hog1∆
 We try to train our students to watch out for any such hog1∆ aca2∆
opportunity to find conditions that allow to select for new
mutants with interesting properties to advance the
understanding of the system under study
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 Yeast genetics: characterising mutants

 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.
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 Dominant and recessive mutations

Recessive: wild type phenotype

 The dominant or recessive character is
revealed by crossing the mutant with the
wild type to form a diploid cell
MUT1
 Such diploids are heterozygous, because
one chromosome carries the wild type allele
and the other one the mutant allele of the MUT1
gene affected
mut1
 A mutation is dominant when the mutant
phenotype is expressed in a heterozygous
diploid cell. The diploid has the same mut1
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 Dominant: mutant phenotype
phenotype as the wild type
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 Dominant and recessive mutations

 A dominant character can have a number of important

reasons, which may reveal properties of the gene
product’s function: Recessive: wild type phenotype
 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 MUT1
 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) MUT1
 The recessive character of a mutation is usually due to
mut1
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 mut1
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
Dominant: mutant phenotype
 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
tS©
 Complementation groups

No functional gene product

 After selection or screening for mutants with a certain
phenotype and after determination of the dominant/recessive of MUT1
mut1
character of the underlying mutation one would like to know
if all mutants isolated are affected in the same or in different
genes mut1
 For recessive mutations, this is done by a complementation
analysis mut1
 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 mut1
mating type and complementing markers)
 These mutants are then allowed to form diploids in all
mut2
possible combination; for instance if one has 12 mutants with
mating type a and 9 with mating type alpha 9x12=108 crosses
are possible
mut2
 If two haploid mutants have recessive mutations in one and MUT2
the same gene the resulting diploid should have the mutant
phenotype too
 If two haploids have recessive mutations in two different MUT2
genes (confering the same phenotype) then the diploid MUT1
should have wild type phenotype, i.e. the mutations
complement each other
 Hence, mut1 and mut2 represent two different MUT1 Functional gene products
complementation groups representing most likely different mut1 of MUT1 and MUT2
genes

mut1
tS©
 Intragenic complementation

 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 No functional gene product
recessive of MUT1
mut1-1
 The heterozygous mut1-1/mut1-2 however shows a
(partial) wild type phenotype
 The explanation is that the two mutated protein mut1-1
products Mut1-1p and Mut1-2p can form a heteromere
that at least has partial function mut1-2
 This has been demonstrated extensively with certain
metabolic enzymes (ILV1, encoding a feedback
regulated enzyme in amino acid biosynthesis) mut1-2

 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 But a heteromere consisting of
different genes fail to complement. This occurs Mut1-1p and Mut1-2p can be functional
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
tS©
 Cloning in yeast

 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
A p a L I (1 7 8 )
 Yeast can maintain replicating plasmids but the copy number is P (B LA ) A LPH A
much smaller than in E. coli, usually between one and 50 per cell A p a L I ( 2 3 6 7 ) H in d III (4 0 0 )

 Yeast can maintain more than one type of plasmid at the same P st I (4 1 6 )
B a m H I (4 3 0 )
time. This can complicate gene cloning from a library. It can also
A v a I (4 3 5 )
be very useful to transform yeast with two different plasmids AP r X m a I (4 3 5 )
simultaneously, for instance for a method called plasmid shuffling pU C 18
S m a I (4 3 7 )
2686 bp
 Cloning and plasmid preparation from yeast is very ineffective E co R I (4 5 1 )
P (L A C )
 Therefore, cloning in yeast uses E. coli as a plasmid production O R I
system:
 Plasmids are constructed in vitro
A p a L I (1 1 2 1 )
 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
tS©
 Yeast-E. coli shuttle vectors

EcoR I (2)
Cla I (28)
Apa LI (5217) Hind III (33)

 Integrative plasmids (YIp) Amp-resistance BamH I (379)

Pst I (4795)
consist Tet-resistance
 of the backbone of a E. coli vector such
as pBR322, pUC19, pBLUESCRIPT
 of a yeast selection marker such as
YIp5
URA3, HIS3, TRP1, LEU2
 but are lacking any replication origin for Apa LI (3971)
5541bp
yeast
Pst I (1644)
 Hence, they are propagated only through
PMB1
integration into the genome
Nco I (1867)
Apa LI (3473) URA3
Ava I (2541)
Xma I (2541)
YIp5: pBR322 plus the URA3 gene
Sma I (2543)
tS©
 Integration of plasmids into the yeast
genome

 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

URA3

X genome
ura3
X genome
ura3 URA3
tS©
 Yeast-E. coli shuttle vectors

Apa LI (7445) EcoR I (2)

Amp-resistance Hind III (106)
Pst I (7023)
2micron ORI
 Replicative episomal Ava I (1391)
plasmids (YEp) consist Apa LI (6199)
 of the backbone of a E. coli vector PMB1
such as pBR322, pUC19, YEp24 Pst I (2001)
pBLUESCRIPT
Apa LI (5701)
7769bp EcoR I (2242)
 of a yeast selection marker such
URA3, HIS3, TRP1, LEU2 Cla I (2268)
 and have the replication origin of the yeast Hind III (2273)
2micron plasmid
Pst I (2482)
 Hence, they are propagated relatively
stably at high copy number, typically 20-50 Ava I (4835) Nco I (2705)
per cell URA3
 Their copy number can be pushed to 200 Xma I (3379)
per cell by using as marker a partially Tet-resistance
defective LEU2 gene Ava I (3379)
Sma I (3381)
Hind III (3439)
YEp24: pBR322 plus the URA3 gene, plus 2micron origin BamH I (3785)
tS©
 Yeast-E. coli shuttle vectors

EcoR I (2)
Apa LI (7626) Cla I (28)
Amp-resistance Hind III (33)
Pst I (7204) BamH I (379)
 Replicative centromeric
Tet-resistance
plasmids (YCp) consist POLY
 of the backbone of a E. coli vector
such as pBR322, pUC19, Apa LI (6380) Pst I (1644)
pBLUESCRIPT PMB1 Nco I (1867)
 of a yeast selection marker such
Apa LI (5882)
YCp50
URA3, HIS3, TRP1, LEU2 URA3

7950bp
and have a chromosomal replication origin
for yeast, ARS (for autonomously Apa LI (5457) Xma I (2541)
replicating sequence)
 have the centromere CEN of a yeast Pst I (5451) Ava I (2541)
chromosome ARS1 Sma I (2543)
 Hence, they are propagated stably at low POLY
copy number, typically one per cell
Ava I (4703)
CEN4

YCp50: pBR322 plus the URA3 gene, plus CEN4, plus ARS1
tS©
 Yeast-E. coli shuttle vectors
 Plasmid series
 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
A p a L I (1 7 8 )
2 M IC R O N H in d III (8 0 9 )
H IS 3
A va I (4 6 8 0 ) H in d III (9 9 6 )
P st I (1 1 8 8 )
F1 O R I
pRS423
LA C Z'
5797 bp
T7 P
A p a L I (4 1 3 7 ) A v a I (2 0 9 2 )
C la I (2 1 0 8 )
A Pr H in d III (2 1 1 3 )
E c o R I (2 1 2 5 )
M C S
P st I (2 1 3 5 )
P M B1 X m a I (2 1 3 7 )
A v a I (2 1 3 7 )
S m a I (2 1 3 9 )
B a m H I (2 1 4 3 )
T3 P
P (LA C )
A p a L I (2 8 9 1 )
tS©
 YIps are used for integration only
 YCps are used for low copy
expression
 YEps are used for overexpression
A p a L I (1 7 8 )
A R SH 4
C EN 6
A p a L I (4 1 3 4 ) H in d III (8 0 8 )
H IS 3
H in d I II ( 9 9 5 )
A Pr
pR S313
P st I (1 1 8 7 )
4967 bp
F1 O R I
LAC Z'
T7 P
B a m H I (2 1 1 0 )
X m a I (2 11 6 )
A va I (2 1 1 6 )
A p a L I (2 8 8 8 ) S m a I (2 1 1 8 )
PM B1 M C S
P st I (2 1 2 6 )
E c o R I (2 1 2 8 )
H in d I I I ( 2 1 4 0 )
C la I ( 2 1 4 7 )
T3 P
A v a I (2 1 6 1 )
 Cloning by complementation

 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
tS©
 Cloning by complementation

 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
tS©
 Cloning by complementation

No functional gene product

of MUT1
mut1∆
 If the original and the deletion mutant
have the same phenotype, this is good
evidence that the two genes are the mut1∆
same
 Final proof is obtained by crossing the mut1
two mutants; if the diploid has the
mutant phenotype too (i.e. there is no
complementation between the original mut1
and the deletion mutant) then one can
be very sure that the cloned gene is the
one orginally mutated. mut2
 To be 100% sure, one sporulates the
diploid and dissects some ten tetrads: mut2
all spores should have the mutant MUT2
phenotype
 Deletion of genes by homologous
recombination is one of the most MUT2
powerful techniques in yeast and one of MUT1
the reasons why yeast is so popular; it
works so well that systematic deletion
of all 6,200 genes has been done and we MUT1 Functional gene products
have this collection in the lab mut1∆ of MUT1 and MUT2

mut1∆
tS©
 Deleting a yeast gene

 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 µ g of DNA. Doing the same in plants
or mammalian cells takes years, often a whole PhD thesis
YFG1
Your favourite gene on a plasmid

in vitro Your favourite gene on a plasmid,

URA3 ORF replaced by marker

URA3

X
Recombination in yeast

X
in vivo Your favourite gene deleted
URA3 from the genome
tS©
 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
 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

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
tS©
 Smart gene deletion

 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 β -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

URA3

YFG1
Diploid cell
tS©
 Smart gene deletion

 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

URA3
tS©
 Smart gene deletion

 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
tS©
 Smart gene deletion

 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

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
tS©
 How to deal with essential genes

 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 (yfg1∆ 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 (yfg1∆ pURA3::YFG1 pLEU2::yfg1-1).
tS©
 From gene disruption to transposon
mutagenesis

 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
 TR: Tn3 terminal inverted repeats
 The Tn used is a quite sophisticated example of  Xa: Factor Xa cleavage recognition site
such a transposon, that can be partially cut out  loxR: lox site, target for Cre recombinase
again through the lox-sites. This creates a tag,  lacZ: 5'-truncated lacZ gene encoding β -
which allows immunolocalisation of the gene galactosidase
product  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
tS©
 From gene disruption to transposon
mutagenesis

 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 B a m HP r I i m e r E c o R I
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- L a c ∆ Z A- mr p O r i
mutants for a number of properties and could allocate
functions to a number of uncharacterised genes with
relevance to stress tolerance Y e a s t D N A
 Derivative of the transposon with antibiotic markers are
very useful tools to mutagenise and study industrial
strains
tS©
 Cloning in yeast by gap repair

 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

X repair fragment

gapped plasmid
YFG1
X

YFG1
gapped plasmid

X X
genomic copy is used to repair the gap; the template is duplicated
tS©
 Localising proteins with the cell: GFP

 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
tS©
 Getting further: isolating more genes

 So far we have discussed different ways to generate mutations in yeast:

 chemical random mutagenesis
 random targetted mutagenesis with transposon-tagged DNA
 targetted deletion/disruption of yeast genes
 and we have discussed some methods to study and engineer genes in yeast
 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
 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
tS©
 Getting further: suppressors

 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
tS©
 Getting further: multi-copy suppression

 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.
X Two parallel pathways share
Overexpressed Hog1p may
confer sufficient activity to
mediate the required function
even in the absence of Hog1p.
one or several common targets.
Overexpression and hence
X
higher activity of the parallel
pathway may be sufficient to
activate the target.

common target
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 Getting further: suppressor mutations

 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 The pathway ultimately X

same pathway and Hog1p is
activated by Pbs2p. A mutation
that renders Hog1p active even
without activation would
X inactivates a negative
regulator, e.g. the repressor
Sko1p; knock out of the
repressor could overcome
suppress the pbs2 mutation and inactivation of the pathway; the
is probably dominant mutation is most likely

X
recessive
Sko1
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 Getting further: synthetic lethality

 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

X The two pathways control some

common targets; mutation of
PBS2 alone causes only a
X
moderate phenotype. The
second mutation in the parallel
pathway leads to lethality X
common target common target
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 Getting further: epistasis I

 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.

 Let us first assume mutation in all these four proteins cause similar
phenotypes, such as moderate sensitivity to salt

X  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

X
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
common target
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 Getting further: epistasis II

 Let us now assume that deletion of PBS2 (and of HOG1) causes sensitivity

X
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)

X
Sko1  If Sko1p were upstream of Hog1p and Pbs2p we would expect that the
double mutant pbs2 sko1 and hog1 sko1 is sensitive to salt
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 Getting further: epistasis III

X  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 hog1∆ mutation
 In a similar way, an activating mutation of HOG1 can
suppress the salt sensitivity of a pbs2∆ 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

X
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
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 Getting further: two-hybrid system

 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 reporter
transcriptional activation domain, such as that
from VP16, a viral protein
lexA
 Only when bait and target interact, a reporter site
gene whose only promoter is a lexA binding site
will be activated
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 Application of the yeast two-hybrid
system

 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................
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 Genetic analysis in action: the HOG
pathway

 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 sensor-
histidine 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 ssk2∆ N, 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
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 The model organisms

 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
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 Model character: eukaryotic cell cycle

 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

The actin cytoskeleton

during the cell cycle
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 Model character: signal transduction

 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
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 Model character: signal transduction
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 Model character: morphology switch

 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
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 Model character: control of gene
expression

 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 (proline-
and 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
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 Model character: vesicular transport

 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
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 Model character: proteasome

 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
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 Model character: the unexpected

 Prions
 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
 Ageing
 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
 As discussed earlier, yeast develops different cell types determined by different gene expression pattern
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 Functional genomics

 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 two-
hybrid 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
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 Functional genomics: transcriptional
profiling

 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
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 From functional genomics to systems
biology

 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
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 Yeast biotechnology: fermentation
industry

 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
 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
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 Yeast biotechnology: heterologous
expression

 The production of proteins is of interest for several purposes:

 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 post-
translation 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
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 Heterologous expression in yeast: gene
cloning and functional analysis

 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
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 Heterologous expression in yeast: one-
hybrid system

 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
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 Heterologous expression in yeast: drug
screening

 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.
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