|Teoria||9||II semestre||Massimiliano Perduca|
|Laboratorio [1° turno]||3||II semestre||Barbara Molesini|
|Laboratorio [2° turno]||3||II semestre||Barbara Molesini|
|Laboratorio [3° turno]||3||II semestre||Barbara Molesini|
|Laboratorio [4° turno]||3||II semestre||Barbara Molesini|
The aim of this course is to give the students the basic knowledge of the molecular mechanisms concerning transmission, variation and expression of the genetic information.
-> Genetic information and informational molecules
General introduction and historical hints. The chemical structure of DNA and RNA. Three dimensional structure of DNA. Physico-chemical properties of DNA.
-> Molecular Biology techniques
Agarose gel electrophoresis. Nucleic acid hybridization. Polymerase chain reaction (PCR). Restriction endonucleases. Cloning and sub-cloning. gene expression systems.
-> DNA, RNA and gene structure
Definition of gene coding and regulatory regions. From genes to proteins; messenger RNA, transfer RNA and ribosomal RNA.
-> Genome organization and evolution
DNA content and number of genes. Mutations, DNA rearrangement and genome evolution. The organelle genomes. Interrupted genes; introns. cDNA. Gene families and duplication. DNA repeats.
-> Transposable elements
Transposition mechanisms and control. Retroviruses and retrotransposones. Transposons.
-> Chromatin and chromosomes
Nucleosomes, histones and their modifications. Higher organization levels of chromatin. Heterochromatin and euchromatin. Eukaryotic chromosomes, telomeres and centromeres.
-> DNA replication
DNA polymerases. Proofreading activity of DNA polymerases. Replication mechanism in bacteria and eukaryotic cells.
-> Introns and RNA splicing
Features of spliceosomal introns. Spliceosome and splicing mechanism. Alternative splicing and trans-splicing. Other kinds of introns: group I and group II introns and tRNA introns. The intron movement. RNA editing. Ribozymes and riboswitch.
-> DNA mutation and repair
Spontaneous mutations and mutations caused by physical and chemical mutagens. Pre- and post-replicative repair systems. Recombination in the immunity system cells. Approaches to homologous recombination.
-> Regulation of gene expression
Bacterial promoters. The operon. Activators, repressors and coactivators. Signal transductions and two component regulation systems. Eukaryotic promoters. Activators, repressors and coactivators. Gene expression and chromatin modifications. Epigenetic mechanisms.
-> RNAs and transcription
Different types of RNA: synthesis and maturation. Bacterial RNA polymerase. Sigma factors. Eukaryotic RNA polymerases. Eukaryotic mRNAs: capping, polyadenylation, cytoplasmic localization. The transcription process in bacteria and in eukaryotic cells.
Ribosomes. tRNA structure and function. Aminoacyl-tRNA synthesis. Initiation in bacteria and eukaryotic cells. Polypeptide chain synthesis and translation end. Regulation of translation.
-> Protein localization.
One credit of the course (corresponding to 8 hours) will be kept for the students to discuss an important topic chosen from the research literature in Molecular Biology.
Introduction to the Laboratory Course:
-> Nucleic acids isolation: basis, comparison of several extraction protocols, nucleic acids isolation troubleshooting.
-> Nucleic acids electrophoresis: agarose gels, polyacrylamide gels, denaturing and non-denaturing gels, Pulsed-field gel electrophoresis.
-> Spectrophotometric quantitation of isolated nucleic acids.
1.What is PCR?
2. Reagents: efficiency, specificity, fidelity
3. PCR cycle. Final number of copies of the target sequence
4.Amplifying the correct product: detection and analysis of PCR products, how to avoid contamination (uracil N-glycosylase, UV, enzymatic treatment), hot start, nested PCR
5. Techniques and applications: 5’RACE-PCR and 3’RACE-PCR, RT-PCR, PCR mutagenesis (deletion of sequences, base substitutions, insertion mutagenesis), modification of PCR products (introduction of restriction sites, adding promoters and ribosome-binding sites), joining overlapping PCR products, quantitative PCR
Total RNA extraction from different plant tissues using methods based on acid guanidinium thiocyanate-phenol-chloroform and adsorption to silica-gel membranes, DNase treatment, qualitative and quantitative evaluation of isolated total RNA samples employing electrophoresis on microfabricated-chips, first strand cDNA synthesis, semiquantitative Reverse transcription polymerase chain reaction (RT-PCR) and Quantitative Real-Time PCR utilizing SYBR Green chemistry.
Oral examination preceded by a propaedeutic written exam concerning the Laboratory Course.
|Teoria||Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick||Lewin's Genes X (Edizione 10)||Jones & Bartlett Publishers||2009||0763766321|
|Teoria||Harvey Lodish, Chris A. Kaiser, Anthony Bretscher, Angelika Amon, Arnold Berk, Monty Krieger, Hidde Ploegh and Matthew P. Scott||Molecular Cell Biology (Edizione 7)||Freeman||2012||1464102325|
|Teoria||Alberts et al.||The Cell (Edizione 5)||Garland Science||2007||978-0-8153-4105-5|
|Teoria||Geoffrey M. Cooper, Robert E. Hausman||The cell: a molecular approach (Edizione 6)||Sinauer Associates, Inc||2013||978-1-60535-155-1|
|Title||Format (Language, Size, Publication date)|
|10 CPEB2–eEF2 interaction impedes HIF-1α RNA translation||pdf (en, 2156 KB, 08/03/12)|
|11 Identification of a Minimal Region of the HIV-1 5-Leader Required for RNA Dimerization NC Binding and Packaging||pdf (en, 1109 KB, 08/03/12)|
|1 Structural characterization of full-length NSF and 20S particles||pdf (en, 3013 KB, 08/03/12)|
|2 Akt-dependent Skp2 mRNA translation is required for exiting contact inhibition oncogenesis and adipogenesis||pdf (en, 2034 KB, 08/03/12)|
|3 The Structural Basis of the Kinetic Mechanism of a Gap-Filling X-Family DNA Polymerase That Binds Mg2 dNTP Before Binding to DNA||pdf (en, 2949 KB, 08/03/12)|
|4 Structure and mechanism of the UvrA–UvrB DNA damage sensor||pdf (en, 1932 KB, 08/03/12)|
|5 Mechanism of RNA synthesis initiation by the vesicular stomatitis virus polymerase||pdf (en, 806 KB, 08/03/12)|
|6 Micrococcal Nuclease Does Not Substantially Bias Nucleosome Mapping||pdf (en, 1558 KB, 08/03/12)|
|7 DNAPKcs-dependent arrest of RNA polymerase II transcription in the presence of DNA breaks||pdf (en, 1276 KB, 08/03/12)|
|8 dNTP pools determine fork progression and origin usage under replication stress||pdf (en, 1912 KB, 08/03/12)|
|9 Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice||pdf (en, 3109 KB, 08/03/12)|
|Gruppi Esposizione Articolo Biotecnologie 2011-1202||pdf (it, 22 KB, 19/03/12)|