yeast genetics

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 tS© 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 tS© 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 Saccharomyces cerevisiae (carlsbergensis) is the beer yeast Saccharomyces cerevisiae is the yeast used in baking because it produces carbon dioxide from sugar very rapidly besides other yeasts; reason is the enormous fermentation capacity, low pH and high ethanol tolerance because it ferments sugar to alcohol even in the presence of oxygen, lager yeast ferments at 8°C Saccharomyces cerevisiae is used to produce commercially important proteins Saccharomyces cerevisiae is used for drug screening and functional analysis because it is a eukaryote but can be handled as easily as bacteria because it can be genetically engineered, it is regarded as safe and fermentation technology is highly advanced Saccharomyces cerevisiae is the most important eukaryotic cellular model system because it can be studied by powerful genetics 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 tS© 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.  tS© 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 4-7µ m large The ”eyes” at the bottom are bud scars tS© . cerevisiae divides by budding (hence: budding yeast) while Schizosaccharomyces pombe divides by fission (hence: fission yeast) Budding results in two cells of unequal size. yeast cells age and mothers die after about 30-40 dividions Cell has a eukaryotic structure with different organelles:         Cell wall consisting of glucans. a mother (old cell) and a daughter (new cell) Yeast life is not indefinite.Saccharomyces cerevisiae is a eukaryote       Belongs to fungi. ascomycetes Unicellular organism with ability to produce pseudohyphae S. Life cycle of yeasts Budding Yeast tS© Fission Yeast . two copies of each chromosome). one copy of each chromosome) and as diploids (2n. although yeast is unicellular.2-fold bigger Haploid cells have one of two mating types: a or alpha (α ) Two haploid cells can mate to form a zygote.Yeast has a sex life!       Yeast cells can proliferate both as haploids (1n. 2n cells are 1. since yeast cannot move. we can distinguish different cell types with different genetic programmes:     Haploid MATa versus MATalpha Haploid versus Diploid (MATa/alpha) Spores Mothers and daughters tS© . 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. Yeast sex!         tS© 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!       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 tS© 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 tS© 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)     tS© 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? . with possibly advantageous allele combination In order to do yeast genetics and to grow haploid cells in the laboratory. i. which may turn out to be advantageous. 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.Haploids and dipoids in nature and laboratory          tS© In nature. 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. diploid cells sporulate and then haploid spores germinate. probably because this increases their chance to survive mutation of an essential gene (because there is another copy) Under nitrogen starvation. 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. better than the previous one (4) Sometimes cells may find a mating partner from a different tetrad and form a new clone. yeast cells always grow as diploids.e. 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. 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   tS© .Haploids can switch mating type!     Mating type switch is due to two silent mating type loci on the same chromosome. 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. Yeast genetics: the genetic material     The S. 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. cerevisiae nuclear genome has 16 chromosomes In addition. there is a mitochondrial genome and a plasmid.500 kb and was completely sequenced as early 1996 (first complete genome sequence of a eukaryote) tS© . Yeast genetics: the genetic material         The yeast genome is predicted to contain about 6. 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.800bp (MUC1/FLO11) tS© .200 genes. very few have more than one. which originates from an ancient genome duplication This means that there are many genes for which closely related homologue exist. still ongoing There is substantial ”gene redundancy”. however.000bp The largest known regulatory sequences are spread over about 2. which often are differentially regulated The most extreme example are sugar transporter genes. mapping of introns is not complete The intergenic space between genes is only between 200 and 1. there are more than twenty Roughly 1/3 of the genes has been characterised by genetic analysis. annotation is. 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 tS© .Yeast genome analysis         A joint goal of the yeast research community: determination of the function of each and every gene For this. .Yeast genetics: nomenclature        Yeast genes have names consisting of three letters and up to three numbers: GPD1. where      Y stands for ”yeast” The second letter represents the chromosome (D=IV.. cdc28 Mutant alleles are designated with a dash and a number: tps1-1.. cdc28-2 If the mutation has been constructed. rho1-23. MATa. is written with a capital letter at the beginning and not in italics..e.e.. MATα tS© . RHO1. i.... a protein. by gene deletion. 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. Rho1p..) 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”. rho1. Recessive mutant genes are written with small letters in italics: tps1. i. indicate the strand or direction of the ORF  Some genes do not follow this nomenclature: you heard already about: HO. this is indicated and the genetic marker used for deletion too: tps1∆ ::HIS3 The gene product.Usually they are meaningful (or meaningless) abbreviations Wild type genes are written with capital letters in italics: TPS1. M=XIII.. often a ”p” is added at the end: Tps1p. HSP12. PDC6. CDC28. YML016W. ADE2 The ade2 mutation has a specific useful feature: cells turn red The first markers in yeast genetics were fermentation markers. LEU2. SK1. MAL. 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. kanR There are many yeast strains in use in the laboratories: W303-1A. genes that confer the ability to catabolise certain substrates: SUC. URA3. 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. to select transformants in transformation with plasmids or integration of genes into the genome Commonly genetic markers cause auxotrophies: HIS3.. S288C. 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 tS© .Yeast genetics: markers and strains            Genetic markers are used to follow chromosomes in genetic crosses. BY4741..e. Σ 1278b. coli also certain antibiotic resistance markers can be used in transformation: kanamycin resistance. to select diploids in genetic crosses. LYS2. i.. TRP1. 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... and hence can be investigated In the past. tetrads can be dissected using a micromanipulator and spores form individual colonies. 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. triple. such genetic crosses were done a lot in order to map genes on chromosomes: the frequency with which two mutations recombined (i. 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. 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    tS© .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.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 double.e.. g. which can be studied individually This means that the properties of the meiotic progeny can be studied directly. from snail stomac) and spores are separated with a micromanipulator on agar plates Spores will germinate and each spore gives rise to a colony. the spore colonies are replicated to different media in order to characterise the properties of the spores and to follow the genetic markers   tS© . such as on potassium acetate KAc medium The ascus wall is digested with a specific enzyme mix (e. for instance if a double mutant forms smaller colonies than either single mutants Otherwise. e. 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.Yeast genetics: crossing strains        In order to cross two strains they are mixed on agar plates and allowed to mate. 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. because in yeast the individual organism is the single cell: a unique advantage of yeast. where asci/tetrads form within some days Sporulation occurs under nitrogen starvation.g. 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 1 1 1 1 Spore A B C D MAT a alpha a alpha leu + + - ura + + his + + SUC + + NaCl + 2 2 2 tS© A B C D a a alpha alpha + + - + + + + - + + + - 2 . ie. each spore represents essentially one chromatid tS© .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. e. i. (leu-plus ura-minus) and (leu-minus ura-plus) spores Hence such a tetrad is called a parental ditype PD tS© .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 leu2 ura3 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. 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 leu2 ura3 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 tS© . 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.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 leu2 ura3 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. which relates well to the genetic distance (in cM. For mapping one investigated hundreds of tetrads from the same cross. and this can be estimated based on the physical distance (in kb). 1% recombinant spores) one would have to dissect at least 25 tetrads. i. centi Morgan). For two close genes (1cM.e. This has been done extensively in the past and the last genetic map from 1995 comprised about 1.  tS© . tS© .Crossing with markers on different chromosomes  Let us now imagine that LEU2 and URA3 are on different chromosomes LEU2 ura3 LEU2 ura3 LEU2 leu2 ura3 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. linked and unlinked genes can easily be distinguished in tetrad analysis because with unlinked genes PD = NPD while with linked genes PD>>NPD. PD and NPD Hence. in order to obain the new combination of genes (leu2 ura3) one only needs to dissect one tetrad. statistically . 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.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 leu2 ura3 URA3 LEU2 URA3 leu2 ura3 leu2 URA3 leu2 URA3       tS© 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. i.e. the properties of the mutant) can tell a lot about the function of a gene. 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). 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.e. 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 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  Induced versus spontaneous mutations   tS© .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. the intellectual challenge is to design conditions and /or strains such that the mutant grows. 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. 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 + 0. but the wild type does not A smart screening system allows one to go for spontaneous mutations.Yeast genetics: finding mutants  Screening versus selection     When screening for mutants one tests clone by clone to find interesting mutants For that.4M NaCl  To develop a new selection system is the art of genetic analysis      When selecting for mutants one has established a condition under which the mutant phenotype confers a growth advantage In other words.4M NaCl tS© . 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 YPD Wild type hog1∆ sko1∆ aca1∆ aca2∆ hog1∆ sko1∆ hog1∆ aca1∆ aca2∆ hog1∆ sko1∆ aca1∆ aca2∆ YPD Wild type aca2∆ hog1∆ hog1∆ aca2∆ YPD + 0. Usually one complemetation group is equivalent to one gene Cloning the gene by complementation. tS© . this requires the following steps A detailed phenotypic analysis. 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.e. i.Yeast genetics: characterising mutants      Once mutants have been identified they need to be characterised and the genes affected have to be identified. 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 wild type MUT1 MUT1 mut1 mut1 Dominant: mutant phenotype tS© . 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.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 Such diploids are heterozygous. e. a regulatory protein functions even without its normal stimulus The gene product functions as a homo-oligomere and the nonfunctional 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.g. that is one reason why this step is taken first In addition. there is simply not enough functional gene product (this is rare) MUT1 MUT1 mut1     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.e. which may reveal properties of the gene product’s function:   Recessive: wild type phenotype  The mutations leads to a gain of function. because it is simpler to destroy a function than to generate one Further genetic analysis of the mutant depends on the dominant/recessive character. 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.Dominant and recessive mutations  A dominant character can have a number of important reasons. that the phenotype segregates 2:2 in at least ten tetrads studied. this is important when mutations have been induced by mutagenesis mut1 Dominant: mutant phenotype tS© . i. the mutations complement each other Hence. 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.Complementation groups No functional gene product of MUT1        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. i. mut1 and mut2 represent two different complementation groups representing most likely different genes mut1 mut1 mut1 mut1 mut2 mut2 MUT2 MUT1 MUT2 MUT1 mut1 Functional gene products of MUT1 and MUT2 mut1 tS© .e. 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. 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. like mut1-1 and mut1-2. 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 tS© .Intragenic complementation        Intragenic complementation is rare. 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. but is does occur Two mutant alleles. coli. coli as a plasmid production system: r A p a L I (1 7 8 ) A LPH A H in d III (4 0 0 ) P st I (4 1 6 ) B a m H I (4 3 0 ) A v a I (4 3 5 )   pU C 18 2686 bp X m a I (4 3 5 ) S m a I (4 3 7 ) E co R I (4 5 1 ) P (L A C ) O R I     Plasmids are constructed in vitro Plasmids are transformed into E. coli Yeast can maintain replicating plasmids but the copy number is P (B LA ) much smaller than in E.. It can also be very useful to transform yeast with two different plasmids AP simultaneously.Cloning in yeast     The era of yeast molecular genetics started as early as 1978. for instance for a method called plasmid shuffling Cloning and plasmid preparation from yeast is very ineffective Therefore. . cloning in yeast uses E.... just in the same way as when working with bacteria Plasmids are produced in bacteria. yeast has a very efficient and reliable system for homologous recombination.. which can be used for cloning tS© . coli and the constructions are confirmed. usually between one and 50 per cell A p a L I ( 2 3 6 7 ) Yeast can maintain more than one type of plasmid at the same time.and then transformed into yeast A p a L I (1 1 2 1 )   Hence we work with so-called yeast-E. when S. coli shuttle vectors On the other hand. This can complicate gene cloning from a library. 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.. pUC19.Yeast-E. HIS3. they are propagated only through integration into the genome  Amp-resistance Pst I (4795) Tet-resistance YIp5 Apa LI (3971) PMB1 Nco I (1867) Apa LI (3473) Xma I (2541) Sma I (2543)   5541bp Pst I (1644) URA3 Ava I (2541) YIp5: pBR322 plus the URA3 gene tS© . coli vector such as pBR322. pBLUESCRIPT  of a yeast selection marker such as URA3. coli shuttle vectors EcoR I (2) Cla I (28) Apa LI (5217) Hind III (33) BamH I (379)  Integrative plasmids (YIp) consist of the backbone of a E. TRP1. LEU2 but are lacking any replication origin for yeast Hence. 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 X tS© genome ura3 genome URA3 ura3 .Integration of plasmids into the yeast genome      Integration occurs by homologous recombination. they are propagated relatively Ava I (4835) stably at high copy number. coli vector PMB1 such as pBR322. pUC19. LEU2 and have the replication origin of the yeast 2micron plasmid Hence. coli shuttle vectors Apa LI (7445) Amp-resistance Pst I (7023) EcoR I (2) Hind III (106) 2micron ORI Ava I (1391) Apa LI (6199) 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. TRP1. YEp24 pBLUESCRIPT 7769bp Apa LI (5701)  of a yeast selection marker such URA3. 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 BamH I (3785) . plus 2micron origin tS©  Replicative episomal plasmids (YEp) consist     of the backbone of a E.Yeast-E. HIS3. coli shuttle vectors EcoR I (2) Apa LI (7626) Amp-resistance Cla I (28) Hind III (33) BamH I (379) Tet-resistance  Replicative centromeric plasmids (YCp) consist  Pst I (7204)    of the backbone of a E. HIS3.Yeast-E. typically one per cell POLY Pst I (1644) Nco I (1867) URA3 YCp50 7950bp Xma I (2541) Ava I (2541) Sma I (2543) POLY CEN4 Ava I (4703) YCp50: pBR322 plus the URA3 gene. TRP1. pUC19. they are propagated stably at low copy number. coli vector Apa LI (6380) such as pBR322. ARS (for autonomously Apa LI (5457) replicating sequence) Pst I (5451) have the centromere CEN of a yeast chromosome ARS1 Hence. plus CEN4. LEU2 and have a chromosomal replication origin for yeast. plus ARS1 tS© . pBLUESCRIPT PMB1  of a yeast selection marker such Apa LI (5882) URA3. Yeast-E. YCp andI ( YEp for convenience Apa L 178) C EN 6    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 2 M IC R O N H in d III (8 0 9 ) H IS 3 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 A va I (4 6 8 0 ) H in d III (9 9 6 ) P st I (1 1 8 8 ) pRS423 5797 bp A p a L I (4 1 3 7 ) F1 O R I LA C Z' T7 P A v a I (2 0 9 2 ) C la I (2 1 0 8 ) pR S313 4967 bp P st I (1 1 8 7 ) 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 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 8 8 ) PM B1 S m a I (2 1 1 8 ) 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 ) tS© A p a L I (2 8 9 1 ) . coli cloning vector such as pUC19 or pBLUESCRIPT have one out of three or four different yeast markers come as YIp. coli shuttle vectors  Plasmid series    are based on an E. which cuts frequently. 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. 2. Sau3A fragments can be cloned into BamHI (GGATCC) cut plasmids. 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). this strategy generates many overlapping fragments and it ensures that all genes are functionally represented. all available yeast libraries are done that way If the fragments cloned are 5-9kb on average. transformed into E.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.000 plasmids represent the genome once and 10.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. this is necessary because yeast cells can take up more than one kind of plasmid tS© . it can only be done with recessive mutants For cloning of genes with dominant mutants. 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.Cloning by complementation            Cloning by complementation sounds like a straightforward approach but there are quite a few caveats to it First of all. if the diploid has the mutant phenotype too and all spores isolated form the diploid as well. a gene library has to be prepared from each mutant and transformed into the wild type strain. 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. this is good evidence that the two genes are the same Final proof is obtained by crossing the two mutants. 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. 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. transformants showing the mutant phenotype are then screened or selected In addition.200 genes has been done and we have this collection in the lab tS© . it works so well that systematic deletion of all 6. e. 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. if the diploid has the mutant phenotype too (i. it works so well that systematic deletion of all 6. 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.200 genes has been done and we have this collection in the lab mut1∆ mut1∆ mut1 mut1 mut2 mut2 MUT2 MUT1 MUT2 MUT1 mut1∆ Functional gene products of MUT1 and MUT2 mut1∆ tS© .Cloning by complementation No functional gene product of MUT1     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. ORF replaced by marker URA3 X in vivo URA3 tS© X Recombination in yeast Your favourite gene deleted from the genome .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. Doing the same in plants or mammalian cells takes years. 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. often a whole PhD thesis YFG1 Your favourite gene on a plasmid in vitro URA3 Your favourite gene on a plasmid. this requires the primers to be designed accordingly (see below) It can also be done with long PCR primers. the entire plasmid is amplified by PCR with the exception of the ORF.e. i. 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. as little as 30bp can be enough to mediate recombination. 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 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© .Deleting a yeast gene  There are a number of different ways to generate the piece of DNA for yeast transformation. 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. in which only the marker is amplified and recombination is mediated by the primer sequences. GFP or an immuno-tag for protein detection lacZ URA3 YFG1 Diploid cell tS© . with lacZ. 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. if the casette is inserted in frame to the end of the ORF it will generate a fusion protein. a gene can be tagged.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. For instance. GFP or an immuno-tag for protein detection and purification For instance. with lacZ. if the cassette is inserted in frame to the end of the ORF it will generate a fusion protein.Smart gene deletion   In a similar way. there are now sets of strains available in which each yeast has been tagged with GFP or TAP-tag YFG1 GFP URA3 tS© . a gene can be tagged. For instance. i.e.e. recombination just leaves behind a single loxP site   tS© . 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. recombination between the two loxP cassettes is stimulated by the Cre-recombinase (transformed on a separate plasmid). to pop-out the integrated DNA again An example for this are the loxP-kanR-loxP cassettes. 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. i.Smart gene deletion    There are some ways to delete a yeast gene without leaving any trace behind. which only contains YFG1 flanking regions. creates a duplication. which is toxic to URA3 cells An example is shown below URA3 plasmid YFG1 genome YFG1 URA3 Integration of the plasmid. recombination between the blue sequences leads to a pop-out of the entire plasmid plus the YFG1 coding region 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. .. The plasmid with the wild type gene carries URA3 as selectable marker. but not at higher temperature. this promoter is ”on” on galactose medium but ”off” on glucose medium.. which can be forced to be lost on medium with 5-FOA. the mutant is temperature-sensitive. usually these are mutations where the gene product functions at a lower temperature. when shifting cells to glucose one can study at least for some time the properties of the cells. if after sporulation only two spores survive and if all living spores do not have the marker used for the deletion. For instance a plasmid contains the essential gene under the control of the promoter of the GAL1 gene. Principally.. 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. If the mutant grows on 5-FOA medium. For this. like 37°C. the mutant is transformed with plasmid that expresses the relevant gene conditionally. one can use plasmid shuffling. For chemical mutagenesis the most common approach is to work with conditional mutations. the mutant is first transformed with the wild type gene and then with a mutant gene. 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. tS© .and watch them dying (yfg1∆ pGAL1-YFG1) To analyse the function of in vitro generated point mutants. like 25°C. the mutant allele is functional (yfg1∆ pURA3::YFG1 pLEU2::yfg1-1).How to deal with essential genes       We have discussed now random chemical and targetted mutagenesis.. the gene is regarded as essential One can work with mutants in essential genes. where the Tn randomly integrates into the yeast DNA Subsequently.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. with about 30. that can be partially cut out again through the lox-sites.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. cerevisiae tet: tetracycline resistance gene res: Tn3 site for resolution of transposition intermediate loxP: lox site. This creates a tag. which allows immunolocalisation of the gene product             tS© TR: Tn3 terminal inverted repeats Xa: Factor Xa cleavage recognition site loxR: lox site. an enzyme that only cuts a very few times in the yeast genome Then this library is mutagenised with a transposon in E. coli. target for Cre recombinase 3xHA: Hemagglutinin (HA) triple epitope tag . the entire mix of NotI fragments is transformed into yeast where it is expected to replace genes. target for Cre recombinase lacZ: 5'-truncated lacZ gene encoding β galactosidase URA3 gene from S. 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.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. we have recently screened 25. 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.000 Tnmutants 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 B a m H rI i m P e r E c o R I   L a c ∆ Z AY mr p O D r i N A e a s t  tS© . the template is duplicated . this can be used to generate mutations. 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 clone mutant alleles from the genome X YFG1 repair fragment gapped plasmid X gapped plasmid YFG1 X tS© X genomic copy is used to repair the gap. e. by error-prone PCR.g. 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.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. YFP.. BFP. This allows simultaneous observation of several proteins in the cell and even protein-protein interaction 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. RFP.. 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 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  and we have discussed some methods to study and engineer genes in yeast      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© . 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.e. obviously. wild-type situation is restored. 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. i. a suppressor either activates (or represses) the system affected by the primary mutation in another way or activates (or represses) an alternative. again. something one has to be aware of when working with such mutants tS© . a deletion mutant never can revert Definition: a suppressor is a gene or mutation that (partially) overcomes the effect caused by a given mutation.Getting further: suppressors        Definition: a reversion of a mutation means that the primary lesion is repaired and hence the orginal. hence a suppressor is a second-site genetic alteration that somehow restores (partially) the wild type situation Suppressors can be intragenic. a second mutation in the same gene/protein can restore (partial) functionality of the gene product. however. X common target tS© . Overexpressed Hog1p may confer sufficient activity to mediate the required function even in the absence of Hog1p. Overexpression and hence higher activity of the parallel pathway may be sufficient to activate the target.. Generally. X Two parallel pathways share one or several common targets.. 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. which.Getting further: multi-copy suppression       Multi-copy suppression is based on overexpression of a gene. when expressed at high levels.. usually on a multi-copy plasmid or via ectopic expression from a strong promoter A multi-copy (or gene dosage) suppressor is a gene.. 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. if one knows from other genetic experiments that two genes are functionally related. 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. the mutation is most likely recessive X X Sko1 tS© .g. A mutation that renders Hog1p active even without activation would suppress the pbs2 mutation and is probably dominant X The pathway ultimately inactivates a negative regulator. hence this is a method to identify interacting proteins Pbs2p and Hog1p are in the same pathway and Hog1p is activated by Pbs2p. 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. knock out of the repressor could overcome inactivation of the pathway.Getting further: suppressor mutations       An extragenic suppressor mutation alters a different gene product such that the. or one of the. e. extragenic suppressor mutations might occur such that protein interactions are restored. the repressor Sko1p. 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. the gene is either expressed through the GAL1 promoter (i. 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. not on 5-FOA) or only when the gene is expressed (i.e.200 x 4.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.e. mutation of PBS2 alone causes only a moderate phenotype. The second mutation in the parallel pathway leads to lethality X X common target .200 crosses.e. 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. ”on” on galactose and ”off” on glucose) or is on a plasmid with URA3 as marker. the primary mutant is transformed with a plasmid that carries the corresponding gene. sporulations and tetrad analyses done by robotics X common target tS© The two pathways control some common targets.  Let us first assume mutation in all these four proteins cause similar phenotypes. though parallel pathways X X common target tS©   . we would conclude that Hog1p and Cba1p work in different. 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. 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. 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. 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.Getting further: epistasis I    The concepts of suppressor analysis and synthetic lethality are also the basis for a powerful tool of genetics. i. to generate a double mutant.e. i. 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 tS© .e.Getting further: epistasis II X X Sko1     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. 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. an activating mutation of HOG1 can suppress the salt sensitivity of a pbs2∆ mutant. but not vice versa. closer to the physiological effect . as indicated here for Hog1p.e.Getting further: epistasis III X     X tS© 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. i. overexpression of PBS2 would not suppress a hog1∆ mutation In a similar way. is cloned in fusion with a DNA binding domain. the target or prey. coli lexA protein The potential binding partner. 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 tS© . 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. is cloned in fusion to a transcriptional activation domain. the bait. such as that of the E. nowadays there are also other methods The method is so powerful since it is not restricted to yeast proteins. which may be a library. the interacting partners can origin from any organism.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. such as that from VP16. . 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.... this can be done by mutagenesis and the use of a counterselectable reporter.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........... tS© .. using laboratory robots 6000 bait strains are crossed to 6000 prey strains to study all possible protein intercations etc. which is not osmosensitive The link between Rck2p and Hog1p and between Hog1p and Hot1p was found in two-hybrid screens        tS© . on which we work. which has a similar lethal effect) Parts of the SHO1-branch were found as synthetic osmosensitive mutants in combination with an ssk2 ssk22 mutant. survival of a sln1 mutant (or commonly used an ssk2∆ N.Genetic analysis in action: the HOG pathway  The analysis of the osmosensing HOG pathway. 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. i. although S. that of a multicellular organism Note. that even yeast has different cell types that can be distinguished by expressing different sets of proteins. i. however. yeasts are not just simple human cells Another limitation is the fact that yeasts are unicellular and hence lack an important level of complexity. certain modules are however used in different context reflecting the evolution in specific environments Hence. a hallmark of cellular differentiation By the way.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.e. cerevisiae and S. pombe are both yeasts. they are as distinct from each other than each is from human S.e. across eukaryotes This is of course only true to a certain extent but many principal molecular mechanisms are indeed conserved. pombe Human tS© . cerevisiae S. in S.e. i.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. 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. where the completion of certain events is monitored before the next one is started The relative importance of these check points is species specific. G2 and M In addition. 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 tS© . G1. S. there are crucial check points. such as G-protein couples receptors. all eukaryotic cells have common classes of signalling proteins. these are modules of three protein kinases that typically control gene expression. the yeast pheromone receptors belong to this class A prototypical eukaryotic signalling system are MAP (mitogen activated protein) kinase cascades. animals and fungi use cAMP as a second messenger and it seems that cAMP mediates nutritional signals For instance. 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. cerevisiae is lacking receptor tyrosine kinases or nuclear receptors. cerevisiae has at least six such pathways. the module is used in many signalling pathways responsive to different stimuli and hence controlled by different sensing mechanisms S. a type of hormone receptors. important classes of mammalian hormone receptors tS© . for instance S.Model character: signal transduction        The principles of signal transduction are well conserved among eukaryotic cells For instance. Model character: signal transduction tS© . a morphological switch is associated with pathogenesis for instance of Candida albicans and hence much research is focussed on the basic mechanisms S.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. cerevisiae may use the switch and co-expression of polysaccharide degrading enzymes to penetrate plant tissues tS© . 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. there are counterparts for most if not all subunits in yeast and human The mechanisms of transcriptional activation seem to be conserved.e. 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. i. 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     tS© . Model character: vesicular transport    Vesicular transport. transport to the vacuole and endocytosis are studied by genetic analysis combined with biochemistry and cell biology tS© . 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. i.e. 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 tS© . e. the genes are homologous and mutations causes premature ageing in human and yeast. i. WRN (Werner’s syndrom) in human and SGS1 in yeast. alleles of known genes. 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. respectively As discussed earlier.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. for instance the accumulation of rDNA circles There is also a ”common” gene. a regulator of nitrogen metabolism Is a process very much assocated with multicellular organisms Yeast cells have a pre-determined life span. yeast develops different cell types determined by different gene expression pattern  Ageing      Cell type determination  tS© . that behave as non-Mendelian genetic elements: PSI+ (Sup35p). a protein involved in translation termination and URE3 (Ure2p). 200 genes and an initial phenotypic characterisation. 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.200 yeast genes has now become reality allowing a comprehensive picture of transcriptional changes depending on conditions or in certain mutants tS© . the set is complete Functional information can also come through other approaches. which can resolve some 1. for instance.Functional genomics         The term functional genomics is not very well defined.000 different yeast proteins Analysis of the expression of all 6. 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. their goal is the construction of deletion strains for all 6. 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 tS© . 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.g. protein interaction data to connect proteins with each other Dynamic modelling and experimentation aims at decribing the overriding rules how e. metabolism and signalling dynamically operate We use such approaches to understand how signalling pathways operate      tS© . which is normally performed by lactic acid bacteria (faster and more reliable production). where the biology of yeast is the limiting factor. polyploid or even aneuploid. ability to degrade polysaccharides that disturb filtration.Yeast biotechnology: fermentation industry    The yeast fermentation industry. high osmotolerance (high-sugar doughs)  In the food industry attempt are done in parallel using classical genetics (where possible) and genetic engineering. hence there are many attempts to improve yeasts     Wine yeasts: ability to perform the malolactic fermentation. osmotic and alcohol tolerance. public perception has so far not allowed to use genetically engineered yeasts in the food industry tS© . better productivity and less byproducts during starvation Beer yeast: ability to degrade polysaccharides (better filtration and low calory beer). wine making and industrial alcohol production. ability to kill competing bacteria and yeasts (cleaner fermentation and wine taste). freeze-tolerance after fermentation initiation (frozen doughs). ability to hydrolyse saccharides. 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). reduced production of acetoin and butanediol (reduced maturation time). brewing. comprising baking. is still the biggest BioTech business world-wide Industrial yeast strains are usually difficult to work with because they are diploid. which contain flavour compounds in glycosidic bonds (improved flavour). many appear to be cross-species hybrids There are many possible improvements to the fermentation processes. 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. we try to market that strain through a start-up company tS© . 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. 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. such as bacteria and yeasts Yeast have the advantage that they may (or may not) perform the same or at least similar posttranslation modifications.Yeast biotechnology: heterologous expression  The production of proteins is of interest for several purposes:    For research. it catabolises methanol and the promoter for methanol oxidase is extremely strong and can be induced by methanol In S. 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. 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. 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. 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 tS© . expression of the cDNAs is driven by a strong yeast promoter. a cDNA library is typically cloned into a yeast expression vector. i.e. the reporter gene will be activated tS© . 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.Heterologous expression in yeast: onehybrid system     The yeast one-hybrid system is basically a half two-hybrid system To clone a transcription factor gene. for instance by applying transcriptional profiling. tS© .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.