Molecular biology (2012/2013)



Course code
4S00800
Credits
12
Coordinator
Massimiliano Perduca
Academic sector
BIO/11 - MOLECULAR BIOLOGY
Language of instruction
Italian
Teaching is organised as follows:
Activity Credits Period Academic staff Timetable
Teoria 9 II semestre Massimiliano Perduca
Laboratorio [1° turno] 3 II semestre Barbara Molesini
Laboratorio [2° turno] 3 II semestre Barbara Molesini

Lesson timetable

II semestre
Activity Day Time Type Place Note
Teoria Monday 11:30 AM - 1:30 PM lesson Lecture Hall F  
Teoria Wednesday 10:30 AM - 12:30 PM lesson Lecture Hall F  
Teoria Thursday 9:30 AM - 11:30 AM lesson Lecture Hall F  
Laboratorio [1° turno] Tuesday 1:30 PM - 7:30 PM lesson Laboratory Laboratorio di Biotecnologie genetiche  
Laboratorio [2° turno] Friday 1:30 PM - 7:30 PM lesson Laboratory Laboratorio di Biotecnologie genetiche  

Learning outcomes

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.

Syllabus

Theory:
-> 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.
-> Translation
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.
-> PCR
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

Experiments:
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. Rapid Amplification of cDNA Ends: 5'RACE-PCR; 3'RACE-PCR.

Assessment methods and criteria

Oral examination preceded by a propaedeutic written exam concerning the Laboratory Course.

Reference books
Activity Author Title Publisher Year ISBN Note
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
Teaching aids
Title Format (Language, Size, Publication date)
1 Circular RNAs are a large class of animal RNAs with regulatory potency  pdfpdf (en, 1090 KB, 03/04/13)
2 End-joining inhibition at telomeres requires the translocase and polySUMO-dependent ubiquitin ligase Uls1  pdfpdf (en, 1480 KB, 03/04/13)
3 Allosteric Inhibition of a Zinc-Sensing Transcriptional Repressor Insights into the Arsenic Repressor (ArsR) Family  pdfpdf (en, 1695 KB, 03/04/13)
4 Signal recognition initiates reorganization of the presequence translocase during protein import  pdfpdf (en, 1106 KB, 03/04/13)
5 ATPase-dependent quality control of DNA replication origin licensing  pdfpdf (en, 648 KB, 03/04/13)
6 FOXA1 mediates p16INK4a activation during cellular senescence  pdfpdf (en, 1325 KB, 03/04/13)
7 Dispersed Sites of HIV Vif-Dependent Polyubiquitination in the DNA Deaminase APOBEC3F  pdfpdf (en, 993 KB, 03/04/13)
8 Structural basis for the nuclear export activity of Importin13  pdfpdf (en, 2216 KB, 03/04/13)
Gruppi Esposizione Articolo Biotecnologie 2012-1013  pdfpdf (it, 16 KB, 13/03/13)