12946 - Molecular Biology

Learning outcomes

After completing this course, the student has adequate knowledge of the fundamental principles and basic mechanisms of molecular biology, and of the fundamental nuclear mechanisms of gene expression in the eukaryotic and prokaryotic cells. In particular, the student is able to understand and discuss issues of gene expression, replication and DNA repair, and the structure of the gene at the molecular level. Moreover, the student knows basic mechanisms operating in the eukaryotic cell, including transcription and its regulation, transcript maturation and its regulation, the structure and regulation of chromatin, gene organization and general principles of genome organization. At the end of the laboratory the student knows how to perform basic molecular biology techniques, such as PCR, DNA restriction and ligation, construction of recombinant plasmids, Escherichia coli transformation and heterologous protein expression.

Course contents

Module 1 (5 cfu, 44 hr)

Structure, components and properties of nucleic acids. Gene structures. Central dogma. Genetic code. tRNA structure and synthetases. Ribosome. Protein synthesis: stages of translation initiation factors in prokaryotes and eukaryotes, elongation,translocation, termination. Codon-anticodon interactions. Transcription in prokaryotes: The RNA polymerase and transcription. Structure and function of sigma factors. Structure of the promoter. Termination. Antitermination. Bacterial operons andcoordinated regulation: lac, trp. Structure and function of the Lacrepressor. Operators. Catabolite repression. Antisense RNA. Phage strategies. Phage lambda. Structure of the CI repressor and lysogeny. Comparison of lytic and lysogenic cycles. Transcription in eukaryotes: RNA polymerase, transcription factors and regulatory elements. Replication. DNA polymerase I and III of E. coli. Other DNA polymerases. Repair systems. Recombination. Rec and Ruv systems. DNA topology.

Module 2 (20 hr)

Gene organization. Introns and exons, pseudogenes, gene clusters (globin genes and ribosomal genes). Structure and function of repeted DNA sequences. Transcription. Classes of RNA polymerases. Structure of RNA polymerase II. Promoter organization of class I, II and III. General transcription factors and their funztion. TBP structure in the DNA complex. Transcription regulation. Inducile transcription factors. Enhancers. Protein intaraction domains with DNA: structure and function.

Module 3 (20 hr)

Chromatin. Chromatin structure. Nucleosome: X-ray structure and molecular components. Post-translational modification of histones. Translational and rotational nucleosomal positioning. Chromatin role in transcription regulation. Transcript maturation. Intron classes. Splicing mechanism. Autosplicing mechanism of group I and II introns. Molecular mechanism of tRNA splicing.

Module 4 (laboratory, 2 cfu, 30 hr)

Laboratory practical course module

PCR goals and applications: amplification and modification of a DNA template DNA and protein electrophoresis Generation of recombinant plasmids: restriction and ligation reactions. Expression of eukaryotic recombinant proteins in a heterologous prokaryotic system: the T7 system.

Readings/Bibliography

Module 1: Il Gene, Seconda Edizione Compatta, B. LEWIN, Zanichelli. Chapters: 1-3, 7-9, 11-15, 18-20

The slides presented in class do not replace the reference text but is a guide for the selection and understanding of the topics to be studied.

Module 2 and 3: Capranico G, Martegani E, Musci G, Raugei G, Russo T, Zambrano N, Zappavigna V. Biologia molecolare, EdiSES (ultima edizione)

Teaching methods

Class lectures

The course consists of three modules (10 CFU) of lectures aimed to the comprehension of the fundamental principles and basic mechanisms of cell functioning.

The laboratory course consists of 2 CFU, corresponding to 30 hours of practical work. Each student will performs the experimental protocol individually, thought working in groups of 4-5 students. During the course, exercises will be be given to students in order to strengthen the theory underneath lab techniques.

Assessment methods

Module1: Written test

The exam takes place only through the final exam, which ensures the acquisition of knowledge and expected skills by conducting a written test for 1.5 hours without the aid of notes or books.

The written test consists of 40 multiple choice questions and one open question. Each multiple choice question achieves 0.5 points, while the open question allows for a maximum of 10 points. The final grade of the module is calculated as the sum of the obtained points.

Module 2 and 3:

The maximum score is 30/30. For this module, registration on AlmaEsami is needed.

The exam consists of a written and a oral test. The written one consist of multiple answer-quizes (five answers, only one correct), which will be considered passed if >80% of the answers are correct. Passing the written test is compulsory for the admission to the verbal test, which consists of 3-4 questions on the course program. Immediately after the end of the course, and only for the students who attended, there will be the possibility to sit for a only-written exam, consisting of quizes, short answer-questions and a discussion of a general question. The max score for each question is indicated on the test. The total score is >31. The test is passed if the score is ≥18.

Laboratory practical course module: the assessment method consists of a written report on the laboratory experience. This musts be short (one page max) and include each different step performed in the lab and its relevance with respect to the general result. The report must be provided in three days from the course end, after this term, a oral examination must be arranged with the teacher. The final score will consider also the outcomes of the exercises provided during the course and the student behavior and involvement.

The total course score is the weighed mean of the scores obtained in the three modules.

Teaching tools

PC, videoprojection and board.

In the lab all the instruments and reagents necessary for the experimental procedures are provided to students.

Office hours

See the website of Vincenzo Scarlato

See the website of Jessica Marinello

See the website of Jessica Marinello