Molecular Biology

A.Y. 2020/2021
Overall hours
Learning objectives
The aim of this course is to provide the students of a solid background in Molecular biology. Beside the description of basic processes such as DNA replication and repair, transcription, translation, a part of the course will be devoted to examples of regulation of these processes.
In addition, the students will acquaint with the principles of basic molecular biology techniques and basic knowledge on genome-wide approaches and post-genomic analyses that will be further developed in future courses to prepare for professional development in advanced biotechnology.
Expected learning outcomes
At the end of the course, the student will acquire the following skills:
- Understanding the complexity of the information flux from genes to proteins.
- Ability to comprehend and interpret data related to biomolecular processes investigated with large-scale approaches.
- Development of an aptitude to deal with the continuous advancements of molecular biology, a discipline in continuous expansion
- Ability to transfer the acquired molecular biology knowledge to related problems in the frame of the multiple biotechnology applications.
Course syllabus and organization

Single session

Lesson period
First semester
There are no changes to the program due to the emergency.
"Synchronous" lessons are by streaming through Teams platform.
In case of emergency, exam session are by streaming through Zoom.
Course syllabus
A brief history of molecular biology from the discovery of DNA to the frontiers of current biology and the birth of biotechnology.

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 σ 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.

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.

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.

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.

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.

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.

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
The aim of this course is to provide the students of a solid background in molecular biology of the gene. Beside the description of basic processes such as DNA replication and repair, transcription, translation, a part of the course will be devoted to examples of regulation of gene expression.
In addition, the students will acquaint with the principles of basic molecular biology techniques and basic knowledge on genome-wide approaches and post-genomic analyses that will be further developed in future courses to prepare for professional development in advanced biotechnology.
Teaching methods
Lesson with projections of slides, videos or connection to web sites of database with attempts to interact with the class and to stimulate questions and curiosity toward the dealt topics.
Teaching Resources
Amaldi-Benedetti-Pesole-Plevani, Biologia Molecolare-terza edizione CEA

The slides of the course and other information will be available on the Portal ARIEL of University of Milan -, at the site of the course of Biologia molecolare - Linea unica. Moreover, lessons will be recorded and available in Teams.
Assessment methods and Criteria
At the end of the course the student will register to one of the exam sessions. The exam is written and consists of a combination of quiz, problems and open questions. The final exam mark is the sum of the scores earned for each answer.
BIO/11 - MOLECULAR BIOLOGY - University credits: 9
Lessons: 72 hours
Professor: Pellicioli Achille
Educational website(s)
Wednesday 10.30-12.30 and Friday 10.30-11.30