Stefan Hohmann 2000 2004 YEAST GENETICS AND MOLECULAR

© Stefan Hohmann 2000 -2004 YEAST GENETICS AND MOLECULAR BIOLOGY w w w w The yeast Saccharomyces cerevisiae: habitate and use Other yeasts Yeast is a eukaryote: the yeast cell Yeast has a sexual cycle and an exciting sex life Yeast genetics: basics Yeast genetics: crossing yeast strains Yeast genetics: making mutants Cloning yeast genes: vectors Cloning yeast genes by complementation Deleting genes in yeast Smart gene deletions and transposon mutagenesis Getting further: more genes/proteins Model systems studied in yeast Yeast biotechnology

Yeast information resources WWW w w w w © Stefan Hohmann 2000 -2004 w There is unfortunately no real text book on yeast genetics and molecular biology Genetic Techniques for Biological Research by Corinne Michels gives a brief overview on yeast genetics and summarises genetic approaches Yeast Gene Analysis by Brown and Tuite is a book about methods There are excellent resources on the WWW and many individual group pages with interesting information and even movies! Check out the course link page For instance, there is kind of a text book on the Internet: http: //www. phys. ksu. edu/gene/chapters. html This site: http: //genome-www. stanford. edu/Saccharomyces/VL-yeast. html links to various types of basic information on yeast genetics This site links to more than 700 hundred yeast labs all over the world http: //genome-www. stanford. edu/Saccharomyces/yeastlabs. html The Stanford Saccharomyces Genome database under http: //genomewww. stanford. edu/Saccharomyces has information on all yeast genes including links and information to other yeast genome projects and global analysis projects

The yeast Saccharomyces cerevisiae: habitate and use w w Yeast lives on fruits, flowers and other sugar containing substrates Yeast copes with a wide range of environmental conditions: n n n © Stefan Hohmann 2000 -2004 n Temperatures from freezing to about 55°C are tolerated Yeasts proliferate from 12°C to 40°C Growth is possible from p. H 2. 8 -8. 0 Almost complete drying is tolerated (dry yeast) Yeast can still grow and ferment at sugar concentrations of 3 M (high osmoti pressure) Yeast can tolerate up to 20% alcohol w Saccharomyces cerevisiae is the main organism in wine production w Saccharomyces cerevisiae (carlsbergensis) is the beer yeast w Saccharomyces cerevisiae is the yeast used in baking w Saccharomyces cerevisiae is used to produce commercially important proteins w Saccharomyces cerevisiae is used for drug screening and functional analysis w Saccharomyces cerevisiae is the most important eukaryotic cellular model system besides other yeasts; reason is the enormous fermentation capacity, low p. H and high ethanol tolerance because it ferments sugar to alcohol even in the presence of oxygen, lager yeast ferments at 8°C because it produces carbon dioxide from sugar very rapidly because it can be genetically engineered, it is regarded as safe and fermentation technology is highly advanced because it is a eukaryote but can be handled as easily as bacteria because it can be studied by powerful genetics and molecular and cellular biology; many important features of the eukaryotic cell have first been discovered in yeast Hence S. cerevisiae is used in research that aims to find out features and mechanisms of the function of the living cell AND in to improve existing or to generate new biotechnological processes

Other important yeasts w w © Stefan Hohmann 2000 -2004 w w Schizosaccharomyces pombe, the fission yeast; important model organisms in molecular and cellular biology; used for certain fermentations Kluyveromyces lactis, the milk yeast; model organism some biotech importance due to lactose fermentation Candida albicans, not a good model since it lacks a sexual cycle; but studied intensively because it is human pathogen Saccharomyces carlsbergensis and Saccharomyces bayanus are species closely related to S. cerevisiae; brewing and wine making Pichia stipidis, Hansenula polymorpha, Yarrovia lipolytica have smaller importance for genetic studies (specilaised features such as peroxisome biogenesis are studied), protein production hosts Filamentous fungi, a large group of genetic model organisms in genera like Cryptococcus, Aspergillus, Neurospora. . , biotechnological importance, includes human pathogens. Also S. cerevisiae can grow in a filamentous form.

Saccharomyces cerevisiae is a eukaryote w w w 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: n n © Stefan Hohmann 2000 -2004 n n n Cell wall consisting of glucans, mannans and proteins Periplasmic space with hydrolytic enzymes Plasma membrane consisting of a phospholipid bilayer and many different proteins Nucleus with nucleolus Vacuole as storage and hydrolytic organelle Secretory pathway with endoplasmic reticulum, Golgi apparatus and secretory vesicles Peroxisomes for oxidative degradation Mitochondria for respiration A yeast cells is about 4 -7 mm large The ”eyes” at the bottom are bud scars

© Stefan Hohmann 2000 -2004 Life cycle of yeasts Budding Yeast Fission Yeast

Yeast has a sex life! w w w © Stefan Hohmann 2000 -2004 w Yeast cells can proliferate both as haploids (1 n, one copy of each chromosome) and as diploids (2 n, two copies of each chromosome); 2 n cells are 1. 2 -fold bigger Haploid cells have one of two mating types: a or alpha (a) Two haploid cells can mate to form a zygote; since yeast cannot move, cells must grow towards each other (shmoos) The diploid zygote starts dividing from the junction Under nitrogen starvation diploid cells undergo meiosis and sporulation to form an ascus with four haploid spores Thus, although yeast is unicellular, we can distinguish different cell types with different genetic programmes: n n Haploid MATa versus MATalpha Haploid versus Diploid (MATa/alpha) Spores Mothers and daughters

Yeast sex! w w w © Stefan Hohmann 2000 -2004 w w w 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 G 1 phase of the cell cycle to fuse to a diploid cell) The pathway controls expression of genes important for mating

Yeast sex! w w © Stefan Hohmann 2000 -2004 w w 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 RME 1 gene encoding the repressor of meiosis A-specific genes are those needed for a-factor production and the gene for the alpha-factor receptor Alpha-specific genes are those needed to produce alpha-factor and the gene for the a-factor receptor

Genetic determination of yeast cell type © Stefan Hohmann 2000 -2004 w w w 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 a 1 transcriptional activator (a 2 has no known function) The MATalpha locus encodes the alpha 1 activator and the alpha 2 repressor The mating type locus functions as a master regulator locus: it controls expression of many genes

Gene expression that determines the mating type w w © Stefan Hohmann 2000 -2004 w w In alpha cells the alpha 1 activator stimulates alpha-specific genes and the alpha 2 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 a 1/alpha 2 heteromeric repressor represses expression of alpha 1 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, a 1 is a homeobox factor)

Haploids and dipoids in nature and laboratory w w w © Stefan Hohmann 2000 -2004 w w w In nature, yeast cells always grow as diploids, probably because this increases their chance to survive mutation of an essential gene (because there is another copy) Under nitrogen starvation, diploid cells sporulate and then haploid spores germinate, provided that they have received functional copies of all essential genes This often means that only a single spore (if any) of a tetrad survives How to make sure that this single spore can find a mating partner to form a diploid again? The answer is mating type switch! After the first division the mother cell switches mating type and mates with its daughter to form a diploid, which then of course is homozygous for all genes and starts a new clone of cells If mating type can be switched and diploid is the prefered form, why then sporulate and have mating types? There are probably several reasons: (1) Spores are hardy and survive very harsh conditions (2) Sporulation is a way to ”clean” the genome from accumulated mutations (3) Meiosis is a way to generate new combinations of alleles, which may turn out to be advantageous, i. e. better than the previous one (4) Sometimes cells may find a mating partner from a different tetrad and form a new clone, with possibly advantageous allele combination In order to do yeast genetics and to grow haploid cells in the laboratory, mating type switch must be prevented: all laboratory strains are HO mutants and can not switch So how does this mysterious switch of sex work?

Haploids can switch mating type! w w 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 w w © Stefan Hohmann 2000 -2004 w w Interestingly, only mother cells can switch This ensures that after cell devision two cells of opposite mating type are formed This feature is due to unequal inheritance of a regulatory proteins Also this is a strategy that is conserved an found in differentiation of cell types in multicellular organisms

Yeast genetics: the genetic material w w w © Stefan Hohmann 2000 -2004 w The S. cerevisiae nuclear genome has 16 chromosomes In addition, there is a mitochondrial genome and a plasmid, the 2 micron circle The yeast chromosomes contain centromeres and telomeres, which are simpler than those of higher eukaryotes The haploid yeast genome consists of about 12, 500 kb and was completely sequenced as early 1996 (first complete genome sequence of a eukaryote)

Yeast genetics: the genetic material w w w © Stefan Hohmann 2000 -2004 w w 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, 000 bp The largest known regulatory sequences are spread over about 2, 800 bp (MUC 1/FLO 11)

Yeast genome analysis w w w w © Stefan Hohmann 2000 -2004 w A joint goal of the yeast research community: determination of the function of each and every gene For this, there are several large projects and numerous approaches Micro array analysis: simultaneous determination of the expression of all genes Micro array analysis to determine the binding sites in the genome for all transcription factors Yeast deletion analysis: a complete set of more than 6, 000 deletion mutants is available for research Various approaches to analyse the properties of these mutants All yeast genes have been tagged to green fluorescent protein (GFP) to allow protein detection and microscopic localisation Different global protein interaction projects are ongoing

Yeast genetics: nomenclature w w w w Yeast genes have names consisting of three letters and up to three numbers: GPD 1, HSP 12, PDC 6. . . Usually they are meaningful (or meaningless) abbreviations Wild type genes are written with capital letters in italics: TPS 1, RHO 1, CDC 28. . . Recessive mutant genes are written with small letters in italics: tps 1, rho 1, cdc 28 Mutant alleles are designated with a dash and a number: tps 1 -1, rho 1 -23, cdc 28 -2 If the mutation has been constructed, i. e. by gene deletion, this is indicated and the genetic marker used for deletion too: tps 1 D: : HIS 3 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: Tps 1 p, Rho 1 p, Cdc 28 p 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: YDR 518 C, YML 016 W. . . , where n n n © Stefan Hohmann 2000 -2004 n n w 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

Yeast genetics: markers and strains w w w w © Stefan Hohmann 2000 -2004 w w w 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: HIS 3, URA 3, TRP 1, LEU 2, LYS 2, ADE 2 The ade 2 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 (SUC 1 -7) encode invertase (periplasmic enzyme) and can be located on different chromosomes in different yeast strains (telomere location) MAL loci (MAL 1 -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, kan. R There are many yeast strains in use in the laboratories: W 303 -1 A, S 288 C, S 1278 b, SK 1, BY 4741. . Their specific properties can be quite different and are different to wild or industrial strains The full genotype of our favourite strain W 303 -1 A reads like this: MATa leu 2 -3/112 ura 3 -1 trp 1 -1 his 3 -11/15 ade 2 -1 can 1 -100 GAL SUC 2 mal 0

Yeast genetics: crossing strains w w w © Stefan Hohmann 2000 -2004 w w Yeast genetics is based on the possibility to cross two haploid strains with different mutations and of opposite mating type to a diploid strain The diploid can then be investigated, for instance if one wants to find out if the two haploid strains had mutations in the same or different genes The diploid can be sporulated to form tetrads, tetrads can be dissected using a micromanipulator and spores form individual colonies, and hence can be investigated In the past, such genetic crosses were done a lot in order to map genes on chromosomes: the frequency with which two mutations recombined (i. e. resulted in spores carrying both mutations or spores without any of the two mutations) is a measure for the genetic distance The last genetic map (before the genome was sequenced) encompassed more than 1, 000 genes and turned out to be very accurate (also thanks to the enormous capacity of yeast for genetic recombination) Today genetic crosses are used to generate yeast strains with new combination of mutations, for instance double, triple. . mutations – for this it is useful to know some principles of genetic crosses and gene segregation And even today with the genome fully sequenced we often perform genetic screens for new mutations, for instance to find genes/proteins that function in the same pathway/molecular system than an already known gene/protein – then genetic analysis of the mutants one obtained is the first and essential step in characterisation

Yeast genetics: crossing strains w w w © Stefan Hohmann 2000 -2004 w w w In order to cross two strains they are mixed on agar plates and allowed to mate, e. g. MATa leu 2 URA 3 x MATalpha LEU 2 ura 3 Diploid cells will be heterozygous for both complementing markers and can be selected on medium lacking both leucine and uracil Diploids will be grown and plated on sporulation medium, where asci/tetrads form within some days Sporulation occurs under nitrogen starvation, such as on potassium acetate KAc medium The ascus wall is digested with a specific enzyme mix (e. g. from snail stomac) and spores are separated with a micromanipulator on agar plates Spores will germinate and each spore gives rise to a colony, which can be studied individually This means that the properties of the meiotic progeny can be studied directly, because in yeast the individual organism is the single cell: a unique advantage of yeast, which has made yeast (and some other fungi) highly useful in genetics The trained geneticist often can see already from the pattern of growth of the spore colonies how two mutations separated, for instance if a double mutant forms smaller colonies than either single mutants Otherwise, the spore colonies are replicated to different media in order to characterise the properties of the spores and to follow the genetic markers

Yeast genetics: crossing strains w w © Stefan Hohmann 2000 -2004 w 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 Na. Cl 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 Na. Cl 1 A a + + - - - 1 B alpha + - - 1 C a - - - + - 1 D alpha - + + 2 A a - - - 2 B a + + + 2 C alpha + - - 2 D alpha - + -

Yeast genetics: meiosis w w © Stefan Hohmann 2000 -2004 w w 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 2 n 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 The second meiotic division will separate the chromatids, ie. each spore represents essentially one chromatid

Yeast genetics: the outcome of a cross w Let us now imagine that LEU 2 and URA 3 are close together on the same chromosome LEU 2 ura 3 leu 2 URA 3 leu 2 w © Stefan Hohmann 2000 -2004 w w URA 3 In the likely case that no cross-over occurs between the two markers all haploid spores will just look like the parental haploid strains There are only two different types of spores, i. e. (leu-plus ura-minus) and (leu-minus ura-plus) spores Hence such a tetrad is called a parental ditype PD

Yeast genetics: cross over w Let us now imagine that LEU 2 and URA 3 are close together on the same chromosome and a cross over occurs between them LEU 2 ura 3 leu 2 URA 3 LEU 2 URA 3 leu 2 ura 3 leu 2 URA 3 leu 2 © Stefan Hohmann 2000 -2004 w w w URA 3 In this case we will get spores that look like the parental haploids but also spores that have new combinations of the two markers There are four different types of spores Hence such a tetrad is called a tetratype T

Yeast genetics: double cross over w Let us now imagine that LEU 2 and URA 3 are close together on the same chromosome and two cross over occur between them such that four DNA strands are involved LEU 2 ura 3 LEU 2 URA 3 LEU 2 ura 3 leu 2 URA 3 LEU 2 URA 3 leu 2 ura 3 © Stefan Hohmann 2000 -2004 leu 2 URA 3 w w w 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 w 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 leu 2 ura 3) 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 c. M, centi Morgan). For two close genes (1 c. M, i. e. 1% recombinant spores) one would have to dissect at least 25 tetrads, w

Crossing with markers on different chromosomes w Let us now imagine that LEU 2 and URA 3 are on different chromosomes LEU 2 © Stefan Hohmann 2000 -2004 w LEU 2 ura 3 LEU 2 URA 3 leu 2 URA 3 leu 2 ura 3 leu 2 w w ura 3 URA 3 Different chromosomes assort randomly in the first meiotic division For this reason two types of tetrads become equally frequent, the parental and the non-parental ditype, PD and NPD Hence, linked and unlinked genes can easily be distinguished in tetrad analysis because with unlinked genes PD = NPD while with linked genes PD>>NPD.

Crossing with markers on different chromosomes w Let us now imagine that LEU 2 and URA 3 are on different chromosomes and a crossing over occurs between a centromere and a marker LEU 2 ura 3 LEU 2 URA 3 leu 2 ura 3 leu 2 URA 3 leu 2 © Stefan Hohmann 2000 -2004 w w w URA 3 Now the different alleles of URA 3 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 (leu 2 ura 3) one only needs to dissect one tetrad, statistically

Yeast genetics: making mutants w w 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 n n n © Stefan Hohmann 2000 -2004 n w 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 n n Mutations can be induced by treating cells with a mutagen; this can of course give multiple hits per cell Spontaneous mutations ”just occur” at a low frequency and it is likely that there is only one hit per cell

Yeast genetics: finding mutants w Screening versus selection n n w To develop a new selection system is the art of genetic analysis n n n © Stefan Hohmann 2000 -2004 When screening for mutants one tests clone by clone to find interesting mutants For that, one usually plates many cells and tries to find mutants because they are unable to grow on a certain medium after replica-plating or because they develop a colour For screening, mutations are usually induced to increase their frequency Still: screening requires hundreds of perti dishes and commonly more than 10, 000 clones to be scored n n When selecting for mutants one has established a condition under which the mutant phenotype confers a growth advantage In other words, the intellectual challenge is to design conditions and /or strains such that the mutant grows, but the wild type does not A smart screening system allows one to go for spontaneous mutations, because up to 108 cells can easily be spread on one plate Selection systems are often based on resistance to inhibitors We try to train our students to watch out for any such opportunity to find conditions that allow to select for new mutants with interesting properties to advance the understanding of the system under study YPD YPD + 0. 4 M Na. Cl Wild type hog 1 D sko 1 D aca 2 D hog 1 D sko 1 D hog 1 D aca 2 D hog 1 D sko 1 D aca 2 D Wild type aca 2 D hog 1 D aca 2 D

Yeast genetics: characterising mutants w w © Stefan Hohmann 2000 -2004 w Once mutants have been identified they need to be characterised and the genes affected have to be identified; this requires the following steps A detailed phenotypic analysis, i. e. testing also for other phenotypes than the one used in screening/selection Establishing if a mutant is dominant or recessive Placing the mutants into complementation groups. Usually one complemetation group is equivalent to one gene Cloning the gene by complementation.

Dominant and recessive mutations Recessive: wild type phenotype w w w © Stefan Hohmann 2000 -2004 w The dominant or recessive character is revealed by crossing the mutant with the wild type to form a diploid cell Such diploids are heterozygous, because one chromosome carries the wild type allele and the other one the mutant allele of the gene affected A mutation is dominant when the mutant phenotype is expressed in a heterozygous diploid cell. The diploid has the same phenotype as the haploid mutant A mutation is recessive when the wild type phenotype is expressed in a heterozygous diploid cell. The diploid has the same phenotype as the wild type MUT 1 mut 1 Dominant: mutant phenotype

Dominant and recessive mutations w A dominant character can have a number of important reasons, which may reveal properties of the gene product’s function: n n n w w © Stefan Hohmann 2000 -2004 w w The mutations leads to a gain of function, e. g. a regulatory protein functions even without its normal stimulus The gene product functions as a homo-oligomere and the non-functional monomere causes the entire complex to become non-functional The gene dosis of one wild type allele is insufficient to confer the wild type phenotype, i. e. there is simply not enough functional gene product (this is rare) The recessive character of a mutation is usually due to loss of function of the gene product This means that recessive mutations are far more common, because it is simpler to destroy a function than to generate one Further genetic analysis of the mutant depends on the dominant/recessive character, that is one reason why this step is taken first In addition, it is useful to do a tetrad analysis of the diploid in order to test that the mutant phenotype is caused by a single mutation, i. e. that the phenotype segregates 2: 2 in at least ten tetrads studied; this is important when mutations have been induced by mutagenesis Recessive: wild type phenotype MUT 1 mut 1 Dominant: mutant phenotype

Complementation groups w w w © Stefan Hohmann 2000 -2004 w w After selection or screening for mutants with a certain phenotype and after determination of the dominant/recessive character of the underlying mutation one would like to know if all mutants isolated are affected in the same or in different genes For recessive mutations, this is done by a complementation analysis This requires that mutants with different mating types are available for generation of diploids (this can be achieved by making the mutants already in two strains with opposite mating type and complementing markers) These mutants are then allowed to form diploids in all possible combination; for instance if one has 12 mutants with mating type a and 9 with mating type alpha 9 x 12=108 crosses are possible If two haploid mutants have recessive mutations in one and the same gene the resulting diploid should have the mutant phenotype too If two haploids have recessive mutations in two different genes (confering the same phenotype) then the diploid should have wild type phenotype, i. e. the mutations complement each other Hence, mut 1 and mut 2 represent two different complementation groups representing most likely different genes No functional gene product of MUT 1 mut 1 mut 2 MUT 1 mut 1 MUT 2 Functional gene products of MUT 1 and MUT 2

Intragenic complementation w w w © Stefan Hohmann 2000 -2004 w Intragenic complementation is rare, but is does occur Two mutant alleles, like mut 1 -1 and mut 1 -2, cause a clear mutant phenotype in haploid cells and are recessive The heterozygous mut 1 -1/mut 1 -2 however shows a (partial) wild type phenotype The explanation is that the two mutated protein products Mut 1 -1 p and Mut 1 -2 p can form a heteromere that at least has partial function This has been demonstrated extensively with certain metabolic enzymes (ILV 1, encoding a feedback regulated enzyme in amino acid biosynthesis) The occurence of intragenic complementation means that the gene product must be an oligomere The ”opposite”, non-allelic non-complementation, can of course also occur: two recessive mutations in two different genes fail to complement. This occurs sometimes when the gene products are involved in the same process or complex and the two functional alleles are just not enough to confer full functionality mut 1 -1 No functional gene product of MUT 1 mut 1 -2 But a heteromere consisting of Mut 1 -1 p and Mut 1 -2 p can be functional

Cloning in yeast w w w The era of yeast molecular genetics started as early as 1978, when S. cerevisiae was first transformed successfully with foreign DNA There are numerous transformation protocols but all are at least three orders of magnitude less efficient as transformation in E. coli Yeast can maintain replicating plasmids but the copy number is much smaller than in E. coli, usually between one and 50 per cell Yeast can maintain more than one type of plasmid at the same time. This can complicate gene cloning from a library. It can also be very useful to transform yeast with two different plasmids simultaneously, for instance for a method called plasmid shuffling Cloning and plasmid preparation from yeast is very ineffective Therefore, cloning in yeast uses E. coli as a plasmid production system: n © Stefan Hohmann 2000 -2004 n w w n n Plasmids are constructed in vitro Plasmids are transformed into E. coli and the constructions are confirmed, just in the same way as when working with bacteria Plasmids are produced in bacteria. . . . and then transformed into yeast Hence we work with so-called yeast-E. coli shuttle vectors On the other hand, yeast has a very efficient and reliable system for homologous recombination, which can be used for cloning

Yeast-E. coli shuttle vectors Eco. R I (2) Cla I (28) Apa LI (5217) w Integrative plasmids (YIp) consist Hind III (33) Amp-resistance Bam. H I (379) Pst I (4795) of the backbone of a E. coli vector such as p. BR 322, p. UC 19, p. BLUESCRIPT n of a yeast selection marker such as URA 3, HIS 3, TRP 1, LEU 2 but are lacking any replication origin for yeast Hence, they are propagated only through integration into the genome Tet-resistance n w w YIp 5 Apa LI (3971) 5541 bp Pst I (1644) PMB 1 Nco I (1867) URA 3 © Stefan Hohmann 2000 -2004 Apa LI (3473) Ava I (2541) YIp 5: p. BR 322 plus the URA 3 gene Xma I (2541) Sma I (2543)

Integration of plasmids into the yeast genome w w w Integration occurs by homologous recombination, this means that a plasmid like YIp 5 will integrate into the URA 3 locus Integration results in the duplication of the target sequence The duplicated DNA flanks the vector If there is more than one yeast gene on the plasmid, integration can be targetted by linearisation within one of the sequences: cut DNA is highly recombinogenic Integrated plasmids are stably propagated but occasional pop-out by recombination between the duplicated sequences plasmid © Stefan Hohmann 2000 -2004 URA 3 X X genome ura 3 URA 3

Yeast-E. coli shuttle vectors Apa LI (7445) Amp-resistance Eco. R I (2) Hind III (106) Pst I (7023) w Replicative episomal plasmids (YEp) consist 2 micron ORI Ava I (1391) Apa LI (6199) of the backbone of a E. coli vector PMB 1 such as p. BR 322, p. UC 19, YEp 24 p. BLUESCRIPT 7769 bp Apa LI (5701) n of a yeast selection marker such URA 3, HIS 3, TRP 1, LEU 2 and have the replication origin of the yeast 2 micron plasmid Hence, they are propagated relatively Ava I (4835) stably at high copy number, typically 20 -50 per cell Their copy number can be pushed to 200 Tet-resistance per cell by using as marker a partially defective LEU 2 gene n w © Stefan Hohmann 2000 -2004 w w Pst I (2001) Eco. R I (2242) Cla I (2268) Hind III (2273) Pst I (2482) Nco I (2705) URA 3 Xma I (3379) Ava I (3379) Sma I (3381) Hind III (3439) YEp 24: p. BR 322 plus the URA 3 gene, plus 2 micron origin Bam. H I (3785)

Yeast-E. coli shuttle vectors Eco. R I (2) Apa LI (7626) Cla I (28) Hind III (33) Amp-resistance w Replicative centromeric plasmids (YCp) consist Pst I (7204) Bam. H I (379) Tet-resistance of the backbone of a E. coli vector Apa LI (6380) such as p. BR 322, p. UC 19, p. BLUESCRIPT PMB 1 n of a yeast selection marker such Apa LI (5882) URA 3, HIS 3, TRP 1, LEU 2 and have a chromosomal replication origin for yeast, ARS (for autonomously Apa LI (5457) replicating sequence) Pst I (5451) have the centromere CEN of a yeast chromosome ARS 1 Hence, they are propagated stably at low copy number, typically one per cell POLY n w © Stefan Hohmann 2000 -2004 w w Pst I (1644) YCp 50 7950 bp Nco I (1867) URA 3 Xma I (2541) Ava I (2541) Sma I (2543) POLY Ava I (4703) CEN 4 YCp 50: p. BR 322 plus the URA 3 gene, plus CEN 4, plus ARS 1

Yeast-E. coli shuttle vectors w Plasmid series w w © Stefan Hohmann 2000 -2004 w are based on an E. coli cloning vector such as p. UC 19 or p. BLUESCRIPT have one out of three or four different yeast markers come as YIp, YCp and YEp for convenience w w w YIps are used for integration only YCps are used for low copy expression YEps are used for overexpression

Cloning by complementation w w w © Stefan Hohmann 2000 -2004 w w 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 Sau 3 A (cutting site GATC), which cuts frequently; this strategy generates many overlapping fragments and it ensures that all genes are functionally represented; Sau 3 A fragments can be cloned into Bam. HI (GGATCC) cut plasmids; all available yeast libraries are done that way If the fragments cloned are 5 -9 kb on average, 2, 000 plasmids represent the genome once and 10, 000 plasmids give a more than 90% probability that all genes are functionally represented The library is transformed into the yeast mutant of interest Transformants are screened or selected for restoration of the wild type phenotype Plasmids are prepared from positive clones, transformed into E. coli and further analysed; some sequence information reveals the identity of the clone Retransformation into the yeast mutant verifies that the plasmid contains a truly complementing gene; this is necessary because yeast cells can take up more than one kind of plasmid

Cloning by complementation w w w w © Stefan Hohmann 2000 -2004 w w w Cloning by complementation sounds like a straightforward approach but there are quite a few caveats to it First of all, it can only be done with recessive mutants For cloning of genes with dominant mutants, a gene library has to be prepared from each mutant and transformed into the wild type strain; transformants showing the mutant phenotype are then screened or selected In addition, complementation of a mutation does not mean that the cloned gene is indeed the one that is defective in the mutant – it could be a multi-copy suppressor This can even happen with centromeric vectors, because selective pressure can drive up the copy number of even these plasmids A multi-copy suppressor is a gene that overcomes the primary defect in the mutant when expressed at high levels; this is a common phenomenon It is in fact so common that it is a useful approach to clone new genes starting from a certain mutant – we return to that To demonstrate that the cloned gene is the one that is mutated in the mutant, a deletion mutant has to be constructed by homologous recombination using the cloned gene as template If the original and the deletion mutant have the same phenotype, this is good evidence that the two genes are the same Final proof is obtained by crossing the two mutants; if the diploid has the mutant phenotype too and all spores isolated form the diploid as well, this is proof that the two genes are the same Deletion of genes by homologous recombination is one of the most powerful techniques in yeast and one of the reasons why yeast is so popular; it works so well that systematic deletion of all 6, 200 genes has been done and we have this collection in the lab

Cloning by complementation w w w © Stefan Hohmann 2000 -2004 w If the original and the deletion mutant have the same phenotype, this is good evidence that the two genes are the same Final proof is obtained by crossing the two mutants; if the diploid has the mutant phenotype too (i. e. there is no complementation between the original and the deletion mutant) then one can be very sure that the cloned gene is the one orginally mutated. To be 100% sure, one sporulates the diploid and dissects some ten tetrads: all spores should have the mutant phenotype Deletion of genes by homologous recombination is one of the most powerful techniques in yeast and one of the reasons why yeast is so popular; it works so well that systematic deletion of all 6, 200 genes has been done and we have this collection in the lab mut 1 D No functional gene product of MUT 1 mut 1 D mut 1 mut 2 MUT 1 mut 1 D MUT 2 Functional gene products of MUT 1 and MUT 2

Deleting a yeast gene w w w Using the cloned gene the open reading frame is deleted in vitro and replaced by a marker gene The result of this is basically the marker gene flanked by sequences originating from the gene that has to be deleted This piece of DNA is transformed into yeast, where it replaces the gene on the chromosome by homologous recombination; the marker is used for selection of transformants Subsequent Southern blot or PCR analysis and phenotypic analysis of the yeast strain confirm the deletion The approach works faithfully and yields several transformants per mg of DNA. Doing the same in plants or mammalian cells takes years, often a whole Ph. D thesis YFG 1 Your favourite gene on a plasmid, ORF replaced by marker in vitro URA 3 © Stefan Hohmann 2000 -2004 URA 3 X X in vivo URA 3 Recombination in yeast Your favourite gene deleted from the genome

Deleting a yeast gene w There a number of different ways to generate the piece of DNA for yeast transformation, i. e. the marker flanked by fragments with DNA from YFG 1 n n n 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 YFG 1 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 30 bp can be enough to mediate recombination; in such cases the use of a heterologous marker is recommended to make integration in the right place more reliable The latter two approaches do not even require the gene to be cloned!! A gene deletion project hence may take only a couple of days © Stefan Hohmann 2000 -2004 YFG 1 First PCR to amplify the flanking parts of your favourite gene Second PCR to amplify the marker URA 3 Final PCR product ready for transformation

Smart gene deletion w w 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 lac. Z reporter gene a precise fusion can be generated that places the lac. Z gene under control of the yeast promoter of YFG 1 If such a construct is used for gene deletion in a diploid, it can be used to study the expression of the gene by monitoring b-galactosidase activity in that diploid and after sporulation of the diploid the mutant phenotype can be studied in the haploid progeny In a similar way, a gene can be tagged. For instance, if the casette is inserted in frame to the end of the ORF it will generate a fusion protein, with lac. Z, GFP or an immuno-tag for protein detection lac. Z © Stefan Hohmann 2000 -2004 URA 3 YFG 1 Diploid cell

Smart gene deletion w w 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 lac. Z, 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 YFG 1 GFP © Stefan Hohmann 2000 -2004 URA 3

Smart gene deletion w w © Stefan Hohmann 2000 -2004 w 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 lox. P-kan. R-lox. P cassettes; recombination between the two lox. P cassettes is stimulated by the Cre-recombinase (transformed on a separate plasmid); recombination just leaves behind a single lox. P site

Smart gene deletion w w A very useful marker to work with is URA 3 because one can select for and against its presence Selection for URA 3 is of course done on medium lacking uracil Selection against URA 3 uses the drug 5 -flouro-orotic acid, which is toxic to URA 3 cells An example is shown below URA 3 plasmid YFG 1 genome © Stefan Hohmann 2000 -2004 YFG 1 URA 3 Integration of the plasmid, which only contains YFG 1 flanking regions, creates a duplication; recombination between the blue sequences leads to a pop-out of the entire plasmid plus the YFG 1 coding region

How to deal with essential genes w w © Stefan Hohmann 2000 -2004 w w 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 GAL 1 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 (yfg 1 D p. GAL 1 -YFG 1) 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 URA 3 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 (yfg 1 D p. URA 3: : YFG 1 p. LEU 2: : yfg 1 -1).

From gene disruption to transposon mutagenesis w w © Stefan Hohmann 2000 -2004 w 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 Not. I, 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 Not. I fragments is transformed into yeast where it is expected to replace genes; with about 30, 000 yeast clones a more then 90% coverage of the genome is achieved The Tn used is a quite sophisticated example of such a transposon, that can be partially cut out again through the lox-sites. This creates a tag, which allows immunolocalisation of the gene product w w w w w TR: Tn 3 terminal inverted repeats Xa: Factor Xa cleavage recognition site lox. R: lox site, target for Cre recombinase lac. Z: 5'-truncated lac. Z gene encoding bgalactosidase URA 3 gene from S. cerevisiae tet: tetracycline resistance gene res: Tn 3 site for resolution of transposition intermediate lox. P: lox site, target for Cre recombinase 3 x. HA: Hemagglutinin (HA) triple epitope tag

From gene disruption to transposon mutagenesis w w w © Stefan Hohmann 2000 -2004 w w The reason why transposon mutagenesis is so powerful lies in the fact that the gene affected by the insertion can be determined very easily For this, the entire genomic DNA of the mutant is isolated and cut with an enzyme that does not cut within the transposon In this way of course many fragments are generated but only one will contain the transposon plus some flanking yeast DNA Ligation generates a circular plasmid that can be transformed into E. coli and further analysed Sequencing using a primer binding to the transposon but directing into the yeast DNA will reveal exactly where the transposon was integrated when the sequence is compared to that of the yeast genome This method works so well that it has been used for a comprehensive genome analysis For instance, we have recently screened 25, 000 Tn-mutants for a number of properties and could allocate functions to a number of uncharacterised genes with relevance to stress tolerance Derivative of the transposon with antibiotic markers are very useful tools to mutagenise and study industrial strains

Cloning in yeast by gap repair w w w 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 repair fragment gapped plasmid © Stefan Hohmann 2000 -2004 YFG 1 gapped plasmid YFG 1 genomic copy is used to repair the gap; the template is duplicated

Localising proteins with the cell: GFP w w w © Stefan Hohmann 2000 -2004 w w The green-fluorescent protein is used now systematically to localise proteins within the yeast cells A main advantage of the GFP technology is that it allows watching processes in the living cell ! Usually the coding sequence of GFP is fused to the end of the coding region of the gene of interest This can be done on a plasmid but also within the genome The resulting construct is tested for functionality by complementing the corresponding deletion mutant GFP shines green in the fluorescence microscope and the subcellular localisation can be deduced using control staining of different compartments There are now many different versions of GFP with different detection threshold and different emission colours: CFP, BFP, RFP, YFP. . . This allows simultaneous observation of several proteins in the cell and even protein-protein interaction

Getting further: isolating more genes w So far we have discussed different ways to generate mutations in yeast: n n n w and we have discussed some methods to study and engineer genes in yeast n n w w w n n © Stefan Hohmann 2000 -2004 by fusion with a reporter gene to monitor gene expression by fusion with an epitope or with GFP to study the protein level or protein localisation The power of genetic analysis lies in the possibility to use one gene/mutant to isolate further genes, which encode proteins involved in the same or in parallel or related cellular processes The same genetic approaches can be used to allocate different genes/proteins to the same (or to different) cellular functions and to sort them in an order, for instance within a signalling pathway Such approaches to get further include n n w chemical random mutagenesis random targetted mutagenesis with transposon-tagged DNA targetted deletion/disruption of yeast genes Multi-copy suppression Suppressor mutation Synthetic lethality The yeast two-hybrid system All these systems are used in multiple variations; the intellectual challenge is to find the conditions that allow the approach to be used

Getting further: suppressors w w © Stefan Hohmann 2000 -2004 w w w Definition: a reversion of a mutation means that the primary lesion is repaired and hence the orginal, wild-type situation is restored; obviously, a deletion mutant never can revert Definition: a suppressor is a gene or mutation that (partially) overcomes the effect caused by a given mutation; hence a suppressor is a second-site genetic alteration that somehow restores (partially) the wild type situation Suppressors can be intragenic, i. e. a second mutation in the same gene/protein can restore (partial) functionality of the gene product; again, this is only possible with point mutations and not with deletion mutants More common are extragenic suppressor and we will discuss multi-copy suppressors and suppressor mutations How a suppressor functions differs of course a lot from system to system but usually the analysis of the suppressor function provides a lot of important information Principally, a suppressor either activates (or represses) the system affected by the primary mutation in another way or activates (or represses) an alternative, partially redundant system Suppressors are useful as we discuss them here but at the same time can be annoying: yeast mutants that poorly grow can easily generate suppressors, something one has to be aware of when working with such mutants

Getting further: multi-copy suppression w w © Stefan Hohmann 2000 -2004 w w 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 Pbs 2 p and Hog 1 p are in the same pathway and Hog 1 p is activated by Pbs 2 p. Overexpressed Hog 1 p may confer sufficient activity to mediate the required function even in the absence of Hog 1 p. X Two parallel pathways share one or several common targets. Overexpression and hence higher activity of the parallel pathway may be sufficient to activate the target. X common target

Getting further: suppressor mutations w w w © Stefan Hohmann 2000 -2004 w 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 Pbs 2 p and Hog 1 p are in the same pathway and Hog 1 p is activated by Pbs 2 p. A mutation that renders Hog 1 p active even without activation would suppress the pbs 2 mutation and is probably dominant X The pathway ultimately inactivates a negative regulator, e. g. the repressor Sko 1 p; knock out of the repressor could overcome inactivation of the pathway; the mutation is most likely recessive X X Sko 1

Getting further: synthetic lethality w w 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 GAL 1 promoter (i. e. ”on” on galactose and ”off” on glucose) or is on a plasmid with URA 3 as marker, which can be counter selected with 5 -FOA Mutations are then screened that cause the yeast to grow only in the presence of the plasmid (i. e. not on 5 -FOA) or only when the gene is expressed (i. e. not on glucose) The principle approach is so powerful that synthetic lethality screens are now done at a genome wide scale using the yeast deletion mutant collection: this means 4, 200 x 4, 200 crosses, sporulations and tetrad analyses done by robotics © Stefan Hohmann 2000 -2004 X common target The two pathways control some common targets; mutation of PBS 2 alone causes only a moderate phenotype. The second mutation in the parallel pathway leads to lethality X X common target

Getting further: epistasis I w w w 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. w © Stefan Hohmann 2000 -2004 X w X common target w Let us first assume mutation in all these four proteins cause similar phenotypes, such as moderate sensitivity to salt When we combine the hog 1 and the pbs 2 in a hog 1 pbs 2 double mutant then we would expect that the double mutant has the same level of sensitivity as each single mutant; we would conclude that they function in the same pathway When we combine the pbs 2 and the cba 1 mutation in a pbs 2 cba 1 double mutant we would expect a strongly enhanced sensitivity of the double mutant as compared to the single mutants; we would conclude that Hog 1 p and Cba 1 p work in different, though parallel pathways

Getting further: epistasis II X w w w X © Stefan Hohmann 2000 -2004 Sko 1 w Let us now assume that deletion of PBS 2 (and of HOG 1) causes sensitivity to high salt concentrations while deletion of SKO 1 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 pbs 2 sko 1 or hog 1 sko 1 double mutant If Sko 1 p were downstream of Pbs 2 p and Hog 1 p we would expect that the double mutant is tolerant, i. e. has the same phenotype as the sko 1 single mutant: sko 1 would be epistatic (”dominant over”) to pbs 2 and hog 1 (and this is really the case) If Sko 1 p were upstream of Hog 1 p and Pbs 2 p we would expect that the double mutant pbs 2 sko 1 and hog 1 sko 1 is sensitive to salt

Getting further: epistasis III X w w w © Stefan Hohmann 2000 -2004 w 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 Hog 1 p; overexpression of PBS 2 would not suppress a hog 1 D mutation In a similar way, an activating mutation of HOG 1 can suppress the salt sensitivity of a pbs 2 D mutant, but not vice versa, and this is indeed exactly how it works The epistasis concept has been used in very many examples to analyse the order of events in signalling pathways and other cellular systems: if the phenotype of the double mutant resembles that of one of the single mutants the latter gene product functions further downstream in the system, i. e. closer to the physiological effect

Getting further: two-hybrid system w w w © Stefan Hohmann 2000 -2004 w w 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 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 lex. A protein The potential binding partner, the target or prey, which may be a library, is cloned in fusion to a transcriptional activation domain, such as that from VP 16, a viral protein Only when bait and target interact, a reporter gene whose only promoter is a lex. A binding site will be activated reporter lex. A site

Application of the yeast two-hybrid system w w w © Stefan Hohmann 2000 -2004 w w w 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 URA 3 The system can of course be used to find interaction partners The system can be used to find proteins that regulate the interaction between two proteins The system can be used to screen for drugs that inhibit the interaction between two proteins The system is actually used to construct an genome-wide map of protein interactions in yeast; using laboratory robots 6000 bait strains are crossed to 6000 prey strains to study all possible protein intercations etc. . . .

Genetic analysis in action: the HOG pathway w w w © Stefan Hohmann 2000 -2004 w w w The analysis of the osmosensing HOG pathway, on which we work, is a good example how different genetic tools work in action PBS 2 and HOG 1 were first identified in a genetic screen for salt sensitive mutants Deletion of SLN 1 is lethal because this sensorhistidine kinase is a negative regulator of the pathway and overactivation is deleterious Downstream kinases were identified as recessive suppressor mutations Protein phosphatases were found as multi-copy suppressors Targets are defined because their deletion allows, to different extent, survival of a sln 1 mutant (or commonly used an ssk 2 DN, which has a similar lethal effect) Parts of the SHO 1 -branch were found as synthetic osmosensitive mutants in combination with an ssk 22 mutant, which is not osmosensitive The link between Rck 2 p and Hog 1 p and between Hog 1 p and Hot 1 p was found in two-hybrid screens

The model organisms w w w © Stefan Hohmann 2000 -2004 w The yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe are regarded as model organisms in molecular biology This means that it is anticipated that certain – or perhaps most – principal cellular systems function in a similar way in yeasts and human, i. e. across eukaryotes This is of course only true to a certain extent but many principal molecular mechanisms are indeed conserved; certain modules are however used in different context reflecting the evolution in specific environments Hence, yeasts are not just simple human cells Another limitation is the fact that yeasts are unicellular and hence lack an important level of complexity, i. e. that of a multicellular organism Note, however, that even yeast has different cell types that can be distinguished by expressing different sets of proteins, a hallmark of cellular differentiation By the way, although S. cerevisiae and S. pombe are both yeasts, they are as distinct from each other than each is from human S. cerevisiae S. pombe Human

Model character: eukaryotic cell cycle w w w w 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, G 1, S, G 2 and M In addition, there are crucial check points, where the completion of certain events is monitored before the next one is started The relative importance of these check points is species specific, in S. cerevisiae START is a crucial point Nutrient starvation and pheromone cause cell cycle arrest at this point A key feature of budding yeast is that the stage of the cell cycle can simply be deduced from the cell’s morphology, i. e. bud size This has been used to order a large number of cdc according to the stage of the cycle where they are affected: the foundation of genetic analysis of cell cycle control © Stefan Hohmann 2000 -2004 The actin cytoskeleton during the cell cycle

Model character: signal transduction w w w © Stefan Hohmann 2000 -2004 w w The principles of signal transduction are well conserved among eukaryotic cells For instance, animals and fungi use c. AMP as a second messenger and it seems that c. AMP mediates nutritional signals For instance, all eukaryotic cells have common classes of signalling proteins, such as G-protein couples receptors, a type of hormone receptors; the yeast pheromone receptors belong to this class A prototypical eukaryotic signalling system are MAP (mitogen activated protein) kinase cascades; these are modules of three protein kinases that typically control gene expression; the module is used in many signalling pathways responsive to different stimuli and hence controlled by different sensing mechanisms S. cerevisiae has at least six such pathways, which together control cellular morphology and responses to pheromone and environmental stress Genetic analysis in yeast has and is contributing greatly to the understanding of how these pathways function There are of course also limitations to the model character; for instance S. cerevisiae is lacking receptor tyrosine kinases or nuclear receptors, important classes of mammalian hormone receptors

© Stefan Hohmann 2000 -2004 Model character: signal transduction

Model character: morphology switch w w © Stefan Hohmann 2000 -2004 w We have already pointed out that yeast cells can switch their morphology This switch requires a MAP kinase pathway and nutritional signals; also c. AMP plays a role The yeast pseudohyphal switch (or invasive growth in haploids) is a model system for morphogenesis Most importantly, a morphological switch is associated with pathogenesis for instance of Candida albicans and hence much research is focussed on the basic mechanisms S. cerevisiae may use the switch and co-expression of polysaccharide degrading enzymes to penetrate plant tissues

Model character: control of gene expression w w w © Stefan Hohmann 2000 -2004 w w The principles of the control of transcription are well conserved across eukaryotes and many proteins function across species borders as we have already noted for transcription factors The organisation of the transcription initiation machinery seems to be conserved, i. e. there are counterparts for most if not all subunits in yeast and human The mechanisms of transcriptional activation seem to be conserved, but certain classes of activators (prolineand glutamine-rich) do not seem to function in yeast Although chromatin organisation seems to be more simple in yeast, aspects of its involvement in the control of gene expression are similar Control of gene expression means that signals and molecules have to traverse the nuclear membrane and these mechanisms seem to be well conserved

Model character: vesicular transport w w © Stefan Hohmann 2000 -2004 w Vesicular transport, i. e. the mechanisms that control the trafficking of proteins and membranes is another feature that is highly conserved across eukaryotes Temperature sensitive sec mutants have been sorted according to the stage where transport stops (using electron micoscopy) and this has been the foundation for genetic analysis In addition, transport to the vacuole and endocytosis are studied by genetic analysis combined with biochemistry and cell biology

Model character: proteasome w w © Stefan Hohmann 2000 -2004 w 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 26 S proteasome consist of a 20 S catalytic and a 19/22 S regulatory subunit The 20 S proteasome is composed of 14 different proteins and all genes are known in yeast The yeast 20 S complex has been purified and the X-ray structure has been determined

Model character: the unexpected w Prions n n w Ageing n n w 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 r. DNA circles There is also a ”common” gene, WRN (Werner’s syndrom) in human and SGS 1 in yeast; the genes are homologous and mutations causes premature ageing in human and yeast, respectively Cell type determination n © Stefan Hohmann 2000 -2004 Have of course been in the focus of interest through mad cow disease Yeast also has two systems that seem to have all features of prions! This means they are genetic elements, alleles of known genes, that behave as non-Mendelian genetic elements: PSI+ (Sup 35 p), a protein involved in translation termination and URE 3 (Ure 2 p), a regulator of nitrogen metabolism As discussed earlier, yeast develops different cell types determined by different gene expression pattern

Functional genomics w w w w © Stefan Hohmann 2000 -2004 w The term functional genomics is not very well defined; since it is a nice term to attract funding these days many people call functional genomics what they have done for ages Strictly, it should probably mean ”the determination of the function of previously uncharacterised genes identified by genome sequencing” This aspect is indeed addressed in a systematic way in yeast by at least two different projects; their goal is the construction of deletion strains for all 6, 200 genes and an initial phenotypic characterisation; the set is complete Functional information can also come through other approaches; for instance, the yeast twohybrid system is used to construct a complete protein interaction map Transposon mutagenesis is used to tag a large number of yeast proteins to determine their localisation Functional information also comes from expression analysis Expression of proteins is studied by 2 D gel electrophoresis, which can resolve some 1, 000 different yeast proteins Analysis of the expression of all 6, 200 yeast genes has now become reality allowing a comprehensive picture of transcriptional changes depending on conditions or in certain mutants

Functional genomics: transcriptional profiling w w w © Stefan Hohmann 2000 -2004 w Transcriptional profiling in yeast is reality now and a number of articles using the technology have appeared A large data collection is generated in Stanford covering a number of growth conditions Another large collection generated by Rick Young’s lab concerns effects of mutations in certain components of the transcription initiation machinery We have used transcriptional profiling to study signal transdution in stress responses

From functional genomics to systems biology w w w © Stefan Hohmann 2000 -2004 w w Systems biology goes a step further then functional analysis: the goal of systems biology is to describe the operation of the entire cell with all its proteins In a more narrow definition, systems biology combines mathematical and experimental approaches to achieve a better understanding of biological networks and systems Systems biology is a multidisciplinary approach involving biologists, engineers and mathematicians There are two principle goals within systems biology: (1) to describe the wiring network of all proteins in the cell and (2) to decsribe the dynamic operation in the cell Reconstruction of the wiring network uses all available data such as genetic, gene expression, protein interaction data to connect proteins with each other Dynamic modelling and experimentation aims at decribing the overriding rules how e. g. metabolism and signalling dynamically operate We use such approaches to understand how signalling pathways operate

Yeast biotechnology: fermentation industry w w w The yeast fermentation industry, comprising baking, brewing, wine making and industrial alcohol production, is still the biggest Bio. Tech 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 n n n © Stefan Hohmann 2000 -2004 n w Wine yeasts: ability to perform the malolactic fermentation, which is normally performed by lactic acid bacteria (faster and more reliable production); ability to degrade polysaccharides that disturb filtration; ability to hydrolyse saccharides, which contain flavour compounds in glycosidic bonds (improved flavour); ability to kill competing bacteria and yeasts (cleaner fermentation and wine taste); osmotic and alcohol tolerance; better productivity and less byproducts during starvation Beer yeast: ability to degrade polysaccharides (better filtration and low calory beer); reduced production of acetoin and butanediol (reduced maturation time); increased osmotolerance (high gravity brewing leading to less tank volume) Distiller’s yeast: increased alcohol yield (less glycerol) and tolerance Baker’s yeast: ability to degrade different sugars at once through diminished catabolite repression (better leavening); freeze-tolerance after fermentation initiation (frozen doughs); high osmotolerance (high-sugar doughs) In the food industry attempt are done in parallel using classical genetics (where possible) and genetic engineering; public perception has so far not allowed to use genetically engineered yeasts in the food industry

Yeast biotechnology: heterologous expression w The production of proteins is of interest for several purposes: n n n w w w © Stefan Hohmann 2000 -2004 w w 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 a number of different expression hosts, such as bacteria and yeasts Yeast have the advantage that they may (or may not) perform the same or at least similar posttranslation modifications, such as glycosylation Yeast usually reaches only a lower level of expression: up to more than 50% of the cellular protein have been obtained in E. coli systems but no more than 10 -20% even in the yery best yeast system The apparently most productive known yeast is the species Pichia pastoris; it catabolises methanol and the promoter for methanol oxidase is extremely strong and can be induced by methanol In S. cerevisiae one usually uses the promoters of genes encoding glycolytic enzymes such as PGK 1 and TPI 1 or a regulated promoter such as that of GAL 1 The advantage of S. cerevisiae is that so much is known about its molecular biology and one can device genetic screens to improve protein production and secretion Recently we have developed a yeast strain that does not make ethanol but rather more biomass; we try to market that strain through a start-up company

Heterologous expression in yeast: gene cloning and functional analysis w w w © Stefan Hohmann 2000 -2004 w 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 c. DNA library is typically cloned into a yeast expression vector, i. e. expression of the c. DNAs is driven by a strong yeast promoter, such as that from PGK 1 The library is then used to complement a yeast mutant This approach has been especially successful with plant c. DNA: a number of genes encoding transport proteins and metabolic enzymes have been cloned in this way Successfull functional expression in yeast opens the possibility to do a functional analysis using yeast genetics of proteins derived from other organisms

Heterologous expression in yeast: onehybrid system w w w © Stefan Hohmann 2000 -2004 w The yeast one-hybrid system is basically a half two-hybrid system To clone a transcription factor gene, a c. DNA library is constructed such that it is linked to a yeast transcriptional activation domain and expressed in yeast As a reporter system a hybrid gene is used that contains fragments from the mammalian or plant promoter of interest If the fusion protein contains a DNA binding domain that recognises that heterologous promoter fragment, the reporter gene will be activated

Heterologous expression in yeast: drug screening w w w © Stefan Hohmann 2000 -2004 w w 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.