MOLECULAR BIOLOGY: LIFE SCIENCES AND BIOTECHNOLOGY
A brief history of molecular biology from the discovery of DNA to the frontiers of current biology and the birth of biotechnology.
STRUCTURE OF NUCLEIC ACIDS
Nitrogenous bases, nucleosides and nucleotides. The phosphodiester linkage. DNA double helix: B,A and Z DNA. How the major and minor grooves are formed in the B DNA. Physical and chemical properties of DNA. Denaturation, renaturation and hybridization. Forces that stabilize DNA. The importance of the weak bonds. DNA topology: supercoiling and parameters that describe it (Linking number, Twist, Writhe and or density of the suphelix . Negative supercoiling is the most common: why? DNA topoisomerases. Catalytic mechanism of type I and II topoisomerases.
RNA structure. Ribose chemistry and RNA stability. Alkaline hydrolysis. Secondary and tertiary structures of RNA. The various functions of RNA in the cell. Ribozymes.
Outlines of protein structure and sequence-specific interactions of protein with DNA. The Helix-Turn-helix (HTH), Zinc-finger, Leucine zipper, bHLH motifs in DNA binding proteins. The interaction between proteins and RNA.
INTERACTION BETWEEN MACROMOLECULES
Assembling DNA into chromosomes: chromatin structure. The nucleosome: composition and structure. Features of the histones: primary, secondary and tertiary structure. Organization of chromatin. The remodeling of the nucleosomes and the modifications of the histones: writers, erasers and readers. The histone code. Summary on the dynamic structure of the chromatin.
Basic techniques of Molecular biology - 1
The recombinant DNA techniques. Type II restriction enzymes: their nomenclature, classification, frequency and type of cut. How to monitor DNA fragmentation through conventional electrophoresis in agarose gel. Virtual restriction maps. Identification of specific DNA sequences by hybridization of denatured DNA fragments transferred from gel to filter (Southern blot/ hybridization).
Characteristics of plasmid cloning vectors. Cloning principles. Strategies to prevent the re-closure of a cloning vector. Clone to express a protein of interest (expression vector). Construction of genomic libraries and their use.
The polymerase chain reaction (PCR): principle of in vitro amplification of DNA. Temperature cycles. Thermostable DNA polymerases allowed automation.
Rapid DNA sequencing: the traditional enzymatic method of DNA sequencing (Sanger) and its automation. Brief note on the Next generation Sequencing (NGS) technologies.
The -omics era. Comparison by size, organization and gene density among the main sequenced genomes of prokaryotes and eukaryotes and relations with the biological complexity of organisms. Organization of the human genome: genes, intergenic regions, repeated tandem and interdisperse sequences. Satellite DNA, microsatellites, minisatellites and sequences derived from transposable elements, pseudogenes and hypotheses about their origin.
TRANSCRIPTION: MOLECULAR MECHANISMS
Overview of transcription. Universal properties of RNA polymerase. The transcription in prokaryotes. The bacterial RNA polymerase and the canonical bacterial promoter. The function of the sigma factor. Parameters describing the reaction forming the closed binary complex, the stage of isomerization, the beginning of transcription and evasion from the promoter. Factors that influence the strength of a promoter.
Transcription in eukaryotes: the RNA polymerases I, II and III and their functional specialization. Promoter structures: DNA elements and trans-acting factors: the gene promoter for the rRNA precursor. Promoters recognized by RNA pol III. The structure of the Core promotor: cis-elements and general transcription factors for RNA pol II (TFIID, A, B, F, E, H). TATA box-binding protein (TBP) and its DNA binding. The assembly of the basal transcriptional machinery. Phosphorylation of RNA pol II CTD. Proximal and distal elements. The mediator. Transcriptional activators and co-activators. Influence of chromatin structure in the transcriptional control. Model of transcription initiation of protein-encoding genes.
THE CELLULAR RNAs AND THE MATURATION OF THE TRANSCRIPTS
Co-transcriptional modifications of primary transcripts produced by RNA pol II (capping, splicing and polyadenylation) and role of CTD. Structure of the Cap and its formation at 5 'of the transcript. Polyadenylation: polyadenylation signal and molecular mechanism of poly (A) tail formation at 3 'of the transcript. Coupling of transcription termination with polyadenylation.
Splicing of pre-mRNAs: characteristics of the GU-AG class of introns of pre-mRNAs. Splicing reactions from the chemical point of view. Splicing catalyzed by the spliceosome (snRNPs and proteins). The introns of group I, II and III. Self-splicing. Splicing defects can lead to diseases. Alternative splicing of pre-mRNAs. Splicing regulators: SR and hnRNP proteins. The hypotheses on the origin of the introns. Relationship between the number of introns per gene and the biological complexity. Summary on eukaryotic gene and mRNA structures: the example of the beta-globin encoding gene and the corresponding mRNA. The 5'-UTR and 3'-UTR of eukaryotic mRNAs.
Nucleus-cytoplasm transport of mRNA.
The maturation of other transcripts. Notes on the maturation of rRNA and tRNA precursors. The RNA interference phenomenon. Maturation of miRNAs. primary pre-RNA, pre-miRNA and miRNA and the RISC complex formation. Functions of miRNAs in the cell. Small interference RNA (siRNA) and hints to their applications in gene expression shutdown.
Basic techniques of Molecular biology - 2
Isolation of (polyA) + RNA. Synthesis of cDNA. The cDNA libraries.
Techniques for the evaluation of the expression of single genes. Northern blot, RT-PCR, overview of qReal time-RT PCR. Large-scale techniques to study the transcriptomes: microarray and RNA seq.
TRANSLATION: MOLECULAR MECHANISMS
The machinery of translation. Ribosomes. The tRNAs: splicing of the tRNA precursors. Structure of the tRNA. The aminoacyl-tRNA synthetases. The mechanism of translation into prokaryotes as a paradigm. Translation initiation into eukaryotes. G protein and their role in the various phases of translation. Energy cost of protein synthesis.
Example of a negative feed-back translation control in prokaryotes: ribosomal proteins. General translational control in eukaryotes: the eIF4BP and eIF2 proteins and their regulation by phosphorylation. Adjustment of the translation of single mRNAs: the case of uORF in yeast GCN4 mRNA, control of ferritin mRNA translation: role of 5'-UTR and 3'-UTR on the translatability and stability of an mRNA. Modulation by miRNAs.
POST-TRANSLATIONAL MODIFICATIONS OF PROTEINS
The repertoire of post-translational modifications increases the diversity of the proteome. Sorting and localization of proteins. Nuclear-cytoplasm transport: the Ran protein. The co-translational translocation at the ER level. The secretory pathway. Post-translational modifications: N-glycosylation, phosphorylation, acetylation. The protein kinases and phosphatases. The importance of protein glycosylation in the production of glycoproteins of therapeutic interest.
DNA REPLICATION: MOLECULAR MECHANISMS
Universal properties of DNA polymerases. Structure, domains and catalytic mechanism. The thermodynamics of the polymerization reaction. How selectivity is achieved: the kinetic control. Proof-reading activity. Replicative and specialized DNA polymerases. The enzymology of DNA replication in prokaryotes and eukaryotes: the replisome. DNA replication initiation. The replicon-initiator model. Isolation of replicators by genetic approaches and their features. The initiation of DNA replication in bacteria and yeast. Replication termination. Telomeres. Telomerases and their function.
The various types of DNA damage. Main mechanisms of DNA repair: direct repair, mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER). The mechanism of MMR and NER in E. coli. Mechanism of DNA damage tolerance. The translesion DNA synthesis (TLS) carried out by specialized polymerases. Features of the TLS DNA polymerases. Repair of the double strand break (DSB) by non-homologous end joining (NHEJ) or homologous recombination.
Prerequisites for admission
Recommended Prerequisites: General and Cell Biology, Genetics and Biochemistry
-Watson, Baker, Bell, Gann, Levine and Losick "Molecular biology of the gene" 7th edition Cold Spring Harbor Laboratory Press.
-J. Dale, M. Von Schantz e N. Plant «From genes to genomes» 3rd edition
- B. Alberts, A. Johnson et al. "Molecular biology of the cell" Garland Science, 6th edition (2014).
- Biochemistry textbook adopted in the course of Line 1.
The slides of the course and other information are available on the Portal ARIEL of University of Milan - https://ariel.unimi.it/
, at the site of the course of Biologia molecolare - Linea 1
Assessment methods and Criteria
The general exam consists of a combination of quiz, problems and open questions. It can also be divided in a first written exam that is held approximately at the end of November and has the same structure of the general exam and a second written exam or total exam in January or at any dates of exam sessions.