Molecular biology (2010/2011)

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 3 II semestre Barbara Molesini

Lesson timetable

II semestre
Activity Day Time Type Place Note
Teoria Monday 10:30 AM - 12:30 PM lesson Lecture Hall C  
Teoria Tuesday 10:30 AM - 12:30 PM lesson Lecture Hall C  
Teoria Thursday 9:30 AM - 11:30 AM lesson Lecture Hall C  
Laboratorio Tuesday 1:30 PM - 7:30 PM lesson Laboratory Laboratorio di Biotecnologie genetiche  
Laboratorio Wednesday 8:30 AM - 12:30 PM lesson Laboratory Laboratorio di Biotecnologie genetiche  
Laboratorio 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:
-> Genomic DNA extraction from different plant tissues. Amplification by PCR of selected genes and visualization of PCR products on agarose gels.
-> Total RNA extraction from prokaryotes (bacteria) and eukaryotes (plants), spectrophotometric quantitation, denaturing gels. Synthesis of cDNA and visualization of cDNA population on gel. 5’ RACE-PCR and 3’ RACE-PCR.

Assessment methods and criteria

Oral examination.
An individual final report, concerning the Laboratory Course, must be prepared and positively evaluated before taking the final oral examination.

Reference books
Activity Author Title Publisher Year ISBN Note
Teoria WATSON James D , BAKER Tania A , BELL Stephen P , GANN Alexander , LEVINE Michael , LOSICK Richard Biologia molecolare del gene (Edizione 7) Zanichelli 2015 978-88-08-36480-7
Teoria LEWIN Benjamin Il Gene VIII Zanichelli 2006 978-8808-17902-9
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