MOLECULAR BIOLOGY
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- Versione italiana
- Academic year
- 2018/2019
- Teacher
- MIRKO PINOTTI
- Credits
- 9
- Didactic period
- Primo Semestre
- SSD
- BIO/11
Training objectives
- Molecular Biology is a branch of life science which deals with living organisms at the level of molecular mechanisms governing their physiological processes, with a focus on interactions among macromolecules and particularly between proteins and nucleic acids (DNA/RNA). The mechanisms finely modulating the genetic information flow are certainly one the main topics.
Students will be guided through the course to understand the mechanisms underlying the control of gene expression at the different levels, from transcription to translation and beyond. Particular attention is paid to the logical connection among pathways.
The experimental activities are aimed at understanding the rationale behind the basic molecular biology protocols developed to investigate the nucleic acids and their features, and how DNA can be manipulated for biotechnological purposes.
KNOWLEDGE AND UNDERSTANDING
The student:
- knows the proper molecular biology terminology;
- knows the molecular mechanisms governing the biological processes;
- knows the different molecular mechanisms regulating gene expression, and their integration;
- knows the main experimental approaches to study nucleic acids.
ABILITY TO APPLY KNOWLEDGE AND UNDERSTANDING
The student is able to:
- use the proper molecular biology terminology;
- to understand and evaluate the impact of gene mutations/variations on the different processing that regulate gene expression
- to understand and design approaches to modulate gene expression for biotechnological or therapeutic purposes;
- to exploit the knowledge of techniques to investigate nucleic acids to plan a basic experimental research. Prerequisites
- No formal propedeuticity is required. However, basic knowledge of physics, mathematics, general and organic chemistry as well as of cellular biology and biochemistry, with particular focus on animal biology.
Course programme
- IN THE FIRST PART (10 hours) the student will be guided through the structure of nucleic acids and chromatin, and of its modifications. In detail:
DNA structure: physical and chemical properties, B, A and Z forms. Topology of DNA and Topoisomerases.
RNA structure: structure, roles and conformation. Non-conventional base pairing.
Structure of the genome: Nucleosomes and chromatin. Chromatin remodelling complexes. Histones chemical modifications and functional relevance.
IN THE SECOND PART (20 hours) the student will be guided on mechanisms of DNA replication, repair and recombination. In detail:
DNA replication: DNA polymerase: processivity, fidelity and mechanism. Component of the replicative fork. DNA polymerases in prokaryotes and eukaryotes. DNA pol III holoenzyme and replisome.
Initiation and regulation of replication in prokaryotes and eukaryotes.
Translation termination. Telomerase and telomeric proteins.
Polymerase chain reaction (PCR) and exploitation in applied molecular biology. Recombinant DNA, plasmids and cloning.
DNA repair: mutation types, mismatch repair system. DNA damage and repair; NER, BER.
Double strand break, non-homologous end-joining, homologous recombination.
Homologous recombination: Hollyday structure and resolution. Molecular machines involved and mechanisms.
Site-specific recombination: structure and mechanism of recombinases (Cre loxP system).
Transposons: classes of transposons and mechanisms.
IN THE THIRD PART (20 hours) the student will be guided through the mechanisms underlying transcription and its regulation, with some examples of approaches to modulate them for therapeutic purposes. In details:
Transcription in prokaryotes: RNA pol., mechanism and role of Sigma 70. Consensus sequences in the promoter: recognition and denaturation. Elongation and editing. Termination; Rho-dependent and Rho-independent mechanisms.
Transcription in eukaryotes: RNA pol. I, II and III, and features of promoters.
Transcription by pol II, CTD and its modifications. General Transcription factors, mediator Complex. Capping and poly-adenylation, PAP.
Splicing: reaction, donor and acceptor splice sites and branch site. The spliceosome and its mechanism. Role of ESE, ESS, ISS, ISE. Alternative splicing and trans-splicing. Self-splicing type I and II. mRNA editing. Nuclear export.
RT-PCR technique.
Transcriptional regulation in prokaryotes: activators, repressors and operons. Initiation regulation: Lac and Trp operons.
Transcriptional regulation in eukaryotes. Transcription factors, DNA binding domains and motifs, activation domain. Two hybrid system. Transcription factors and chromatin modifications. Loop, LCR and insulators. DNA methylations and imprinting.
Reporter genes and study of promoters.
Regulatory small RNAs.
IN THE FORTH PART (14 hours) the student will be guided through protein translation and its regulation as well as elements on post-translational modifications and protein trafficking. In detail:
Translation: recognition of the initiation codon in prokaryotes and eukaryotes. tRNA structure and function. Ammynoacyl-tRNA sinthetase, activity and specificity determinants.
Ribosomes and their cycle.
Initiation in prokaryotes: IF1, IF2 e IF3 and assembly of the 70S complex. Initiation in prokaryotes and role of the poli-A. Elongation and role of EF-Tu, EF-Ts and EF-G.
Termination, releasing and recycling factors.
Regulation of translation: an example from Ferritin. Nonsense and nonstop decay. Ribosome readthrough and amynoglycosides.
Overview of mechanisms governing protein trafficking and post-translational modifications.
Experimental activities: PCR, electrophoresis, basic cloning procedures. The students will be guided in the design of basic experiments and in the interpretation of experimental data. Didactic methods
- The course is structured in frontal lectures and guided activities in the laboratory of biochemistry. More specifically, the course comprises 76 hours divided in 64 hours of frontal lectures and 12 hours of experimental activity. Lesson will be provided on a weekly basis by taking advantage of power-point slides and, particularly when teaching enzyme reactions and modulation, of the classical backboard. Computational molecular modelling and videos is also exploited to explain the structure of macromolecules and their interactions, respectively. The last 4 hours of the course are devoted to summarize the main contents of the program. To conduct the experimental courses, students are divided in groups.
Learning assessment procedures
- The aim of the exam is to test the level of knowledge and deepening of the topics of the course program.
The assessment is expressed in thirtieths (minimum grade 18).
With the exception of students manifesting problems, the exam is written, and consists of 31 multiple-choice questions. To pass the exam (score 18/30) the student has to correctly respond to at least 18 questions. With 31 positive responses the score is 30/30 with honours.
The students have 45 minutes for the exam. Reference texts
- Scheme and Figures provided.
Free choice of one among these two books:
"Molecular Biology of the gene" Watson et al, Zanichelli
"Molecular Biology" Cox, Doudna and O’Donnell, Zanichelli
