Molecular biology (2016/2017)



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 sem. Massimiliano Perduca
laboratorio [1° turno] 3 II sem. Massimiliano Perduca
laboratorio [2° turno] 3 II sem. Massimiliano Perduca

Lesson timetable

II sem.
Activity Day Time Type Place Note
teoria Monday 10:30 AM - 12:30 PM lesson Lecture Hall F  
teoria Wednesday 9:30 AM - 11:30 AM lesson Lecture Hall D  
teoria Thursday 9:30 AM - 11:30 AM lesson Lecture Hall F  
laboratorio [1° turno] Tuesday 2:30 PM - 7:30 PM laboratorio Laboratory Laboratorio di Biotecnologie genetiche from Mar 21, 2017  to Jun 9, 2017
laboratorio [2° turno] Friday 8:30 AM - 1:30 PM laboratorio Laboratory Laboratorio di Biotecnologie genetiche from Mar 24, 2017  to Jun 9, 2017

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.
At the end of the course the students will be able to recognize the major molecular mechanisms of life in prokaryotic and eukaryotic cells.

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

Practical:
Subcloning and protein expression:
PCR
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.

Assessment methods and criteria

Oral examination.
The final exam will be on three questions concerning any of the topics treated during the course and it is passed if both answers are positive.

Reference books
Activity Author Title Publisher Year ISBN Note
teoria Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick Lewin's Genes XII (Edizione 12) Jones & Bartlett Pub 2017 1284104494
teoria Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff Molecular Biology of the Cell (Edizione 7) Garland Science 2017 0815344643
teoria Nancy Craig, Rachel Green, Carol Greider, Gisela Storz, Cynthia Wolberger, Orna Cohen-Fix Molecular Biology: Principles of Genome Function (Edizione 2) OUP Oxford 2014 0199658579
teoria Arnold Berk, Chris A. Kaiser, Harvey Lodish, Angelika Amon, Hidde Ploegh, Anthony Bretscher, Monty Krieger, Kelsey C. Martin Molecular Cell Biology (Edizione 8) WH Freeman 2016 1464187444
laboratorio Erik Pierre Molecular Cloning (Edizione 1) Ml Books International 2015 1632394685
laboratorio Michael R. Green e Joseph Sambrook Molecular Cloning: A Laboratory Manual, Fourth Edition (Edizione 4) CSHL Press 2012 978-1-936113-42-2
laboratorio Erik Pierre Molecular Cloning (Edizione 1) Ml Books International 2015 1632394685
laboratorio Michael R. Green e Joseph Sambrook Molecular Cloning: A Laboratory Manual, Fourth Edition (Edizione 4) CSHL Press 2012 978-1-936113-42-2
Teaching aids
Title Format (Language, Size, Publication date)
10-ProQ-dependent small RNA  pdfpdf (en, 4898 KB, 03/04/17)
11-RdRP-synthesized antisense ribosomal siRNAs  pdfpdf (en, 2245 KB, 03/04/17)
12-Genome maintenance in Saccharomyces cerevisiae  pdfpdf (en, 3166 KB, 03/04/17)
13-TLR4-induced NF-kB and MAPK  pdfpdf (en, 8660 KB, 03/04/17)
14-Folate deficiency facilitates recruitment of binding factors  pdfpdf (en, 8876 KB, 03/04/17)
15-SIRT7-dependent deacetylation of CDK9  pdfpdf (en, 2277 KB, 03/04/17)
16-Mcm2–7 replicative helicase loading  pdfpdf (en, 4137 KB, 03/04/17)
1-STAT2  pdfpdf (en, 2591 KB, 03/04/17)
2-The evolutionary capacitor HSP90  pdfpdf (en, 1559 KB, 03/04/17)
3-Cdt1–Mcm2–7 complex  pdfpdf (en, 3655 KB, 03/04/17)
4-Mcm2–7 ring closure  pdfpdf (en, 1098 KB, 03/04/17)
5-Novel players in X inactivation  pdfpdf (en, 5939 KB, 03/04/17)
6-Signal Recognition Particle Targeting Complex  pdfpdf (en, 3525 KB, 03/04/17)
7-Sub1 contacts the RNA polymerase II  pdfpdf (en, 1861 KB, 03/04/17)
8-Cullin E3 Ligase Activity  pdfpdf (en, 3229 KB, 03/04/17)
9-G-quadruplex structures within the 3 UTR of LINE-1  pdfpdf (en, 777 KB, 03/04/17)
Gruppi Esposizione Articolo Biotecnologie 2016-17  pdfpdf (it, 11 KB, 09/03/17)
Laboratorio Didattico Biologia Molecolare I Turno 16-17  pdfpdf (it, 8 KB, 14/03/17)
Laboratorio Didattico Biologia Molecolare II Turno 16-17  pdfpdf (it, 8 KB, 14/03/17)

STUDENT MODULE EVALUATION - 2016/2017