42894 - Yeast Molecular Biology

Learning outcomes

The course objective is to focus on a number of crucial aspects of the molecular biology of the eucaryotic cell, in particular on cell cycle mechanisms and secretion. When possible the analysis will be extended to specific topics regarding the yeast Saccharomyces cerevisiae.

Course contents

The cell cycle. General features. Citologic aspects. Chechkpoints and restriction points. Cyclins, kinases, Cks proteins, ubiquitin-related enzymes. Cyclin/kinase complexes: structures and modulation by phosphorylation.
DNA damage checkpoint. Repair systems pathways. DNA repair foci. Sensors, adapters, effectors. Pathways in yeast and mammals. The spindle checkpoint. Main pathway. Interactions of Mad2 and Cdc20.
Restriction point in mammals and Ras. Cell cycle in mammals. Involved cyclins and kinases. Cell cycle activation and rest exit. pRB, cyclin D and cyclin E roles in G1-S transition. CIP and INK4.
Controlled proteolysis and cell cycle. G1 proteolysis, Sic1p (p27). M proteolysis, APC and regulation thereof. Pds1, Cdc20. Pre-RC complexes and DNA replication control. ORC cycle. Mitosis exit network and FEAR.
Molecular dynamics of mitosis. SMC complexes, Cohesin, condensin. Separase and securin and their role in sister-chromatid segregation. SMC comlpexes regulation in itosis and meiosis.
p53: overall structure, negative feedback with Mdm2, role in G1 and M.
Nuclear membrane traffiking. Nuclear localization sequences. Action cycles of importins and exportins. Structural main features. Ran and RanGTP gradient across the membrane.
Killer phenotype in S.cerevisiae. Genomes and structural features of of RNA viruses. Gag and Pol synthesis, frameshifting. Toxins production, secretiona and action. K28 toxin: secretion, endocitosis, immunity mechanisms.
Prion are abnormal genetic elements. Phenotypes and mechanisms of yeast prions PSI and URE3. Genetic criteria for prion definition and identification. Prion propagation and its molecular bases. Prion domains. Aggragtes formation in vivo and aggregation kinetics in vitro. Amyloid fibers. Structure hypotesis. Chaperones action in prion propagation. Prion strains, species barriers. Hypotesis and experimental evidence of species barrier crossing.
Main secretory pathway in the eucaryotic cell. Translocation of proteins into the rough endoplasmic reticulum. Co- and post-translational translocation. Role, structure and and action cycle of the Signal Recognition Particle. Structural and functional features of the interaction between SRP, SR and ribosome. Luminal chaperones and post translational translocation. Structure and composition of the "translocon". Interaction between soluble and membrane proteins.
Glicosylations. N-glycosylation as protein maturation marker. Synthesis of the oligosaccharidic chain main donor. Lectin cycle into the ER: calnexin and calreticulin. Erp57. Glicosyltransferase as a folding sensor. Misfolded proteins elimination: ERAD. Secreted protein "Quality control" concept.
Protein trafficking into the cell. Lysosomes targeting. Golgi processing of K28 toxin. Transport vesicles. Clathrin and others components of TGN vesicles. CopII Vesicles. CopI vesicles. Signals and cofactors in vesicle assembly. Targeting and fusion of vesicles: SNARES and cytoskeleton.
Mitochondrial heritage. Structure and features of the mitochondrial genome.
Specific seminar: meiosis in S.cerevisiae. S.cerevisiae life cycle, mating type. Diploid state preference. HO, RE and Ash1. Sporulation and meiosis in S.cerevisiae: experimental conditions. Nuclear and cytoplasmic events during spore formation. Overview on FRAP and FRET techniques.
Case study: complementation studies in S.cerevisiae. Overview on rRNA maturation. Roles of H/ACA snoRNPs and Naf1p. Study of a human protein in yeast. Tetrads analysis. Complementation study in a conditional mutant strain. Complementation analysis at the molecular level.

Readings/Bibliography

The reccomended bibliography will be available in digital format on the "biotecnologie" website for free download (http://www.biotecnologie.unibo.it/). The lessons presentations will be the main guide for bibliography exploration.

Teaching methods

Lessons. Seminars on specific topics.

Assessment methods

Written exam made of tests and free answer questions.

Teaching tools

Digital powerpoint presentations.

Links to further information

http://www.biotecnologie.unibo.it/

Office hours

See the website of Mattia Bonoli