|teoria||9||II semestre||Massimiliano Perduca|
|laboratorio [1° turno]||3||II semestre||Massimiliano Perduca|
|laboratorio [2° turno]||3||II semestre||Massimiliano Perduca|
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
Subcloning and protein expression:
Electrophoresis and DNA gel extraction
Plasmidic DNA purification
Digestion with restriction enzymes and ligation
Transformation and colony PCR
Heterologous expression of a recombinant protein in E. coli and analysis by SDS PAGE and Western Blot.
|teoria||Nancy L. Craig, Orna Cohen-Fix, Rachel Green, Carol W. Greider, Gisela Storz, Cynthia Wolberger||Biologia molecolare Principi di funzionamento del genoma (Edizione 1)||Pearson||2013||9788871928111|
|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-Codon Usage and 3′ UTR Length Determine Maternal mRNA Stability in Zebrafish||pdf (en, 2886 KB, 22/03/16)|
|11-piRNA adhesion traps mRNAs to the germ plasm||pdf (en, 7847 KB, 22/03/16)|
|12-Tissue-independent and tissue-specific patterns of DNA methylation alteration in cancer||pdf (en, 1672 KB, 22/03/16)|
|13-Crystal structure of a substrate-engaged SecY||pdf (en, 12586 KB, 22/03/16)|
|14-A novel cytoprotective function for the DNA repair protein Ku in regulating p53 mRNA translation and function||pdf (en, 1108 KB, 22/03/16)|
|1-Crystal structures of the M1 and M4 Muscarinic Acetylcholine Receptors||pdf (en, 12962 KB, 22/03/16)|
|2-Two-Stage Synapsis of DNA Ends during Non-homologous End Joining||pdf (en, 1950 KB, 22/03/16)|
|3-A new MCM modification cycle regulates DNA replication initiation||pdf (en, 1994 KB, 22/03/16)|
|4-The Nuclear Pore Complex as a Flexible and Dynamic Gate||pdf (en, 1930 KB, 22/03/16)|
|5-A U1 snRNP–specific assembly pathway reveals the SMN complex as a versatile hub for RNP exchange||pdf (en, 1152 KB, 22/03/16)|
|6-Hepatitis B virus X protein identifies the Smc5-6||pdf (en, 23996 KB, 22/03/16)|
|7-Chromatin accessibility a window into the genome||pdf (en, 958 KB, 22/03/16)|
|8-Distal Alternative Last Exons Localize mRNAs to Neural Projections||pdf (en, 3247 KB, 22/03/16)|
|9-Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation||pdf (en, 4323 KB, 22/03/16)|
|Gruppi Esposizione Articolo Biotecnologie 2015-16||pdf (it, 10 KB, 11/03/16)|
|Laboratorio Didattico Biologia Molecolare I Turno 15-16||pdf (it, 7 KB, 30/03/16)|
|Laboratorio Didattico Biologia Molecolare II Turno 15-16||pdf (it, 7 KB, 30/03/16)|