The course is aimed to give insight about regulatory mechanisms of gene expression in eukaryots and to provide information about the molecular biology techniques currently employed in diagnostic and research laboratories.
The course aims to equip the student with a basic knowledge of the structural and functional organization of eukaryotic genomes. The course covers the aspects related to the stability of the genomes, including the accuracy of the replication machinery, the mechanisms of DNA repair and disorders related to their defects. Particular emphasis is dedicated to regulation of gene expression both in prokaryotes and eukaryotes. Information about the most common molecular biology techniques are provided.
Lectures
The complexity of eukaryotic genomes: the multiple coding levels of genetic information. The composition of the human genome. The satellite DNA and its dynamic.
Gene expression control. Checkpoints in eukaryotes: a comparison with regulative schemes of prokaryotes.
Regulation of eukaryotic RNA-polymerases. Basal promoters for RNA-Pol-II. General and tissue-specific transcription factors. Enhancers and silencers. The eukaryotic promoter as computer. Covalent modification of histones and chromatin remodeling. Wide range control elements: LCR and insulators.
Regulation of splicing. Coupling of transcription and identification of splicing junctions. Alternative splicing as gene expression checkpoint. Consensus sequences of intron boundaries and splicing pattern.
Post-transcriptional controls and mRNA stability. microRNA as gene expression regulators.
Protein translation. Processes granting the translation accuracy: the role of EF1 and EF2. Mechanisms controlling the start of translation.
How to study genes and genomes. Techniques for gene isolation and cloning. The recombinant DNA. Plamids and restriction enzymes. Flow chart of a typical gene cloning. Genomic and cDNA libraries how to make and exploit them.
Nucleic acid hybridization: screening of DNA libraries. Southern and northern-blot. In situ hybridization and FISH. Microarrays to analyze gene expression profiles and their use in diagnostics. Classifiers and hierarchical clustering.
An overview on PCR and its use. A typical PCR cycle and relevant parameters to design a cycle. Primer design. Real time PCR to quantify the amount of specific nucleic acids. Calibration curves and how to select the "right" housekeeping genes. Digital PCR.
DNA sequencing: chemical and enzymatic methods. The automation of Sanger sequencing. New generation sequencing and differences between sequencing techniques. Emulsion PCR and Bridged PCR. IonTorrent, pyrosequencing and reversible terminator sequencing.
Manipulating gene expression. Transfecting eukaryotic cells. Fluorescent protein as reporter genes. Somatic gene transfer with retroviral and lentiviral vectors. How to silence a gene: artificially designed miRNAs. Gene KO with CRISPR-CAS. The Homologous recombination and site-specific recombination. Production of KO mouse lines.
L'essenziale di Biologia Molecolare della Cellula (Alberts et al.)
Ricevimento: Tuesday h. 12:00.
PAOLO MALATESTA
SARA TAVELLA (President and Coordinator of Integrated Course)
