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YEAST CELL STRUCTURE © Stefan Hohmann 2000-2004 Yeast taxonomy Yeast is a eukaryote: the yeast cell Yeast has a sexual cycle Yeast cell duplication: budding vs fission Yeast has a cell wall (functions) Yeast genetics © Stefan Hohmann 2000-2004 YEAST TAXONOMY YEAST IS A EUKARYOTE: THE CELL Golgi apparatus Simili alle cellule vegetali •Vacuolo •Parete cellulare ma.... •Non hanno cloroplasti! © Stefan Hohmann 2000-2004 peroxysome Mostrano alcune peculiarità •Permanenza della membrana nucleare durante la mitosi •Gemmazione YEAST IS A EUKARYOTE: THE CELL 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: © Stefan Hohmann 2000-2004 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 (2-50) mm long 1-10 mm large © Stefan Hohmann 2000-2004 Life cycle of yeasts Budding Yeast Fission Yeast Life cycle of yeasts © Stefan Hohmann 2000-2004 Molti lieviti hanno la capacità di replicarsi sia in forma aploide (n) che diploide (2n) Yeast has a sex life! © Stefan Hohmann 2000-2004 Yeast cells can proliferate both as haploids (1n, one copy of each chromosome) and as diploids (2n, two copies of each chromosome); 2n cells are 1.2-fold bigger Haploid cells have one of two mating types: a or alpha (a) Two haploid cells can mate to form a zygote; since yeast cannot move, cells must grow towards each other (shmoos) The diploid zygote starts dividing from the junction Under nitrogen starvation diploid cells undergo meiosis and sporulation to form an ascus with four haploid spores Thus, although yeast is unicellular, we can distinguish different cell types with different genetic programmes: Haploid MATa versus MATalpha Haploid versus Diploid (MATa/alpha) Spores Mothers and daughters Yeast sex! © Stefan Hohmann 2000-2004 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! © Stefan Hohmann 2000-2004 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 afactor receptor Genetic determination of yeast cell type © Stefan Hohmann 2000-2004 The mating type is determined by the allele of the mating type locus MAT on chromosome III The mating type locus encodes regulatory proteins, i.e. transcription factors The MATa locus encodes the a1 transcriptional activator (a2 has no known function) The MATalpha locus encodes the alpha1 activator and the alpha2 repressor The mating type locus functions as a master regulator locus: it controls expression of many genes Gene expression that determines the mating type © Stefan Hohmann 2000-2004 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 haploidspecific 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) © Stefan Hohmann 2000-2004 Yeast has a cell wall (structure) © Stefan Hohmann 2000-2004 Yeast has a cell wall (structure) © Stefan Hohmann 2000-2004 Yeast has a cell wall (structure) © Stefan Hohmann 2000-2004 Yeast has a cell wall (functions) Yeast has a cell wall (functions) Coinvolgimento della parete in processi industriali: la produzione della birra © Stefan Hohmann 2000-2004 La flocculazione (Bottom yeast) e la flottazione (top yeast) sono due fenomeni di aggregazione asessuale cellula cellula che generano ammassi di cellule le cui dimensioni globali determinano la localizzazione nel mosto di birra E’ un fenomeno aggregativo generalmente associato all’ingresso della coltura in fase stazionaria E’ un fenomeno Ca++ dipendente, legato alla comparsa sulla superfice della parete di Lectine di superfice. Può quindi essere controllato perché se Avviene troppo presto, la fermentazione si arresta precocemente e la birra sarà instabile microbiologicamente (nonché avrà un tasso alcolico troppo basso) perché ci sono zuccheri fermentabili residui ad alta concentrazione Avviene troppo tardi si avranno problemi di chiarificazione del prodotto Yeast genetics: the genetic material © Stefan Hohmann 2000-2004 The S. cerevisiae nuclear genome has 16 chromosomes In addition, there is a mitochondrial genome and a plasmid, the 2micron circle The yeast chromosomes contain centromeres and telomeres, which are simpler than those of higher eukaryotes The haploid yeast genome consists of about 12,500 kb and was completely sequenced as early 1996 (first complete genome sequence of a eukaryote) © Stefan Hohmann 2000-2004 Yeast genetics: the genetic material Yeast genetics: the genetic material © Stefan Hohmann 2000-2004 The yeast genome is predicted to contain about 6,200 genes, annotation is, however, still ongoing There is substantial ”gene redundancy”, which originates from an ancient genome duplication This means that there are many genes for which closely related homologue exist, which often are differentially regulated The most extreme example are sugar transporter genes; there are more than twenty Roughly 1/3 of the genes has been characterised by genetic analysis, 1/3 shows homology hinting at their biochemical function and 1/3 is not homologous to other genes or only to other uncharacterised genes Only a small percentage of yeast genes has introns, very few have more than one; mapping of introns is not complete The intergenic space between genes is only between 200 and 1,000bp The largest known regulatory sequences are spread over about 2,800bp (MUC1/FLO11)
