Learning outcomes of the course unit
KNOWLEDGE AND UNDERSTANDING
The main goal of the course is the understanding of the fundamental biochemical aspects of nucleic acids. The first part focusses on the DNA structural features that are the key aspects of stability, informational content and expression of the genetic material. The molecular mechanisms of DNA replication, DNA repair, transcription and translation are analysed in depth. A large part of the course is dedicated to the understanding of fundamental regulatory strategies operating in bacteria and bacteriophages and their possible implications for the control of gene expression in more complex organisms.
APPLYING KNOWLEDGE AND UNDERSTANDING
The educational objective of the course is to attain the skills necessary for a critical analysis of the molecular and biochemical mechanisms of life, as well as for the understanding of the basic elements of the main cellular processes.
The course is aimed at increasing the ability to critically analyze the molecular mechanisms of life.
The course includes significant activity of classroom discussion aimed at developing the ability of students to trasmit the acquired competence in support of their arguments.
The many advancements in scientific research, particularly in the field of molecular biology require a continuous updating of skills. For this reason, the course aims to provide the necessary tools to achieve a wider knowledge and to align skills to the advancement in biological research.
The student is asked to understand and learn the basics of that genetic information transmission. At the end of the course the student must acquire skills to describe, communicate the main techniques used for nucleic acid manipulation. The student will learn how to apply these techniques to answer a diagnostic question.
For a better understanding of the course it is essential for the student to know the basic principles of chemistry, biochemistry and genetics.
Course contents summary
STRUCTURE OF NUCLEIC ACIDS
GENE REGULATION IN PROKARYOTES
MOLECULAR BIOLOGY TECHNIQUES
The course provides basic knowledge on the molecular aspects of the transmission of genetic information. The course initially introduces the basics of the concept of inheritance, and then introduces nucleic acids as molecules able to ensure the transmission of the genetic information through DNA replication, RNA transcription and proteins translation. Finally the course will provide the basis for the knowledge of the main techniques used for DNA/RNA manipulation, and their application in various fields of research, diagnosis, forensic medicine and treatment of human diseases.
STRUCTURE OF NUCLEIC ACIDS
Properties of genetic material: The transforming principle, Avery's discovery, Chargaff rule; chemico-physical properties of nucleotides; chemical modifications, protonation and keto-enolic tautomerism of nitrogenous bases; the primary structure of DNA; the thermodynamics of the phosphodiester bond; weak bonds and strong bonds; the double helix and base pairing according to the Watson and Crick model; semi-conservative DNA replication; alternative secondary DNA structures (DNA A, DNA Z, DNA H); repeated direct, inverted and specular sequences; and intrinsic curvature of DNA; structural DNA recognition elements (read-out); specific DNA-protein interactions; stability of the double helix: fusion and renaturation of DNA; primary and secondary structure and distinguishing characteristics of RNA; alkaline hydrolysis of RNA and mechanism of action of RNase A; topology elements: supercoiling, bond number and conformational variations of DNA; topoisomerase I and II; condensation of nucleic acids: histones, nucleosome, fibres and higher-order chromatin structures.
General outline of replication: DNA thermodynamics and synthesis mechanism; structure of the active DNA polymerase site; processivity and sliding clamp; DNA polymerase proof-reading activities; semi-discontinuous DNA synthesis: leading strand, lagging strand, Okazaki fragments and removal of primers; mechanism of action of DNA ligase; origins of replication; DNA polymerase III, replisome structure and assembly; DNA primase, DNA helicase, DNA topoisomerase and other proteins involved in replication; bidirectional replication of the E. coli genome; replication of eukaryotic genomes; the problem of DNA ends.
Point mutations; hydrolytic damage and chemical modifications of nitrogenous bases; Ames test; mismatch repair; repair by photoreactivation; repair by excision of bases; repair by nucleotide excision; repair by homologous recombination; translesion DNA synthesis; induction of SOS response.
General structure of genes and prokaryotic operons; bacterial promotors: regions -10 and -35, UP elements, extended element -10; bacterial RNA polymerase structure; the sigma factor; general outline of the transcription process: initiation, elongation, termination. Transcription in eukaryotes: promotors, the pre-initiation complex, the mediator, RNA polymerase II; capping and polyadenylation of RNA; RNA polymerases I and III.
Introns, exons and post-transcriptional processing of the primary transcripts; splicing chemistry; the spliceosome; splicing of group I and II introns; alternative splicing; RNA editing; transport of mRNA out of the nucleus.
The genetic code; messenger RNA (mRNA) structure; transfer RNA (tRNA) structure; attachment of amino acids to tRNA; aminoacyl-tRNA synthetases; the ribosome; peptide bond formation; molecular mechanism and functional phases of translation: initiation, elongation, termination; translation fidelity and energetics; the problem of broken RNAs.
GENE REGULATION IN PROKARYOTES
General principles of transcriptional regulation; positive and negative regulation of transcription; remote action; lactose operon: LacI, Cap; alternative sigma factors; NtrC, MerR and AraC; tryptophan operon (TrpR) and attenuation; transcriptional regulation and the lysis-lysogeny decision of lambda phage; structure a function of cI and cro repressors; cooperativity in the repressor bond; positive and negative transcriptional control, antitermination, antisense regulation. Lambda integration and excision by site-specific recombination.
MOLECULAR BIOLOGY TECHNIQUES
Polymerase (PCR) chain reaction; DNA sequencing by the Sanger method.
Molecular basis of genetic information. Composition and structure of nucleic acids. Chromatine, chromosomes and genes. DNA replication, DNA recombination, DNA damage and mutation, DNA repair. Types of RNA involved in protein synthesis: structures and functions. RNA transcription: enzymatic machinery and processes. Post transcriptional maturation of the eukaryotic mRNA. Genetic code and its properties. Protein synthesis: enzymatic machinery and processes. Molecular biology techniques: DNA isolation, DNA gel electrophoresis, restriction enzymes and restriction patterns, Southern Blot. DNA sequencing, cloning, amplification. PCR reaction. Application of molecular biology techniques in clinical diagnosis and forensic medicine.
BIOLOGIA MOLECOLARE - Principi di funzionamento del genoma
Craig, Cohen-Fix, Green, Greider, Storz, Wolberger.
REGOLAZIONE GENICA - autore Mark Ptashne, casa ed. Zanichelli
Understanding DNA - autore Calladine, case ed. Academic Press Terza edizione
"Fondamenti di biologia molecolare"
di Lizabeth A. Allison
The course will be held with oral lectures and will make use of multimedia systems. During the lessons, students will have the opportunity to discuss the key aspects of the course. The teachers will be available throughout the duration of the course to answer questions and support students during their training with individual meetings by appointment.
Assessment methods and criteria
At the end of the course there is a written and oral examination. The written exam consists of ten questions: nine with an open answer and one with a closed answer, generally an exercise or a DNA sequence analysis. The written exam has to be completed within two hours. At each answer is assigned a score from 0 to 3 points. The sum of the scores of the ten answers represents the mark of the written exam. The oral exam consists of questions regarding unanswered parts of the written exam, plus two other theoretical questions to verify the complete preparation of the student. The oral exam can be sustained only if the mark obtained in the written exam is at least 15/30. The average of the marks obtained in the written and oral exams determines the final mark of the exam.
Written examination: multiple choice questions on the topics illustrated during the course.