12946 - Molecular Biology

Learning outcomes

At the end of the course, the student is acquainted with qualitative and quantitative notions of the principal cellular processes and sports an in-depth understanding of riboregulation mechanisms in eukaryotes and prokaryotes. The successful student is able to properly frame the breadth of new discoveries in these fields, to evaluate their applicative potential and to critically integrate these informations in a biotech context. Specifically, the student has skills on i) sizes, concentrations, and rates that characterize the lives of cells ii) genetic regulation mechanisms mediated by non-coding RNAs and derived ablative strategies, iii) genome-editing methodologies.

Course contents

Introduction

The Path to Biological Numeracy. Rigorous Rules for sloppy calculations. Cell atlas.

Cell Biology by Numbers

Concentrations and absolute numbers. Rates and durations in the Central dogma, in Cellular dynamics and in the cellular Life cycle. Information and errors: genomes and mutations. Quantitative miscellany.

Riboregulation and RNAi

A major paradigm shift in biology. Quelling. Co-suppression in plant. Genetic bases of RNAi. Molecular dissection of RNAi, dicing and slicing. DICER. RISC assembly and maturation, guide strand selection, ARGONAUTES. endo-siRNA, pseudogenes.

microRNAs

Heterochronic gene regulation, miRNA biogenesis, DROSHA, microprocessor, mirtrons, miRNA trimming, nuclear export. mechanisms of miRNA-dependent regulation, mRNA decay, P-bodies. Network motifs and miRNA: a Systems Biology perspective.

ncRNAs and epigenetic regulation.

Silencing amplification, RdRPs, RNAi and heterochromatin, RITS, nuclearRNAi. piRNAs and PIWIs: guarding the germline, lncRNAs.

Bacterial regulatory ncRNAs

Riboswitches, sRNAs, 6S RNAs, CRISPRs

Genome editing methodologies

CRISPR applications, ZFN, TALENs, Cre-lox, Flp

 

WET-LAB COURSE

Functional analysis of the post-transcriptional regulation mediated by the RyhB sRNA with GFP reporter-fusions

Methods

• Site-specific mutagenesis
• Molecular cloning
• DNA miniprep
• Restriction analysis
• Plasmid incompatibility groups
• GFP reporter analysis
• Molecular microbiology techniques
• Multiplate reader measurements

Readings/Bibliography

Teaching material and articles will be made available through the Insegnamenti Online (Moodle) repository.

 

Additional readings

Watson. Molecular Biology of the Gene. 7th edition. Chapter 7

Milo & Phillips. Cell Biology by the Numbers. Freely available from bionumbers.org

  

Research Articles to be critically analyzed

RNAi discovery
Fire et al. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806-811.

21-23 nt dsRNA effectors; DICER
Zamore et al (2000). RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleoide Intervals. Cell 101, 25-33.
Berstein et al (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366.

RISC assembly and maturation
Schwarz et al. (2003). Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208.

microRNA regulation and RNAi share common genes and effector mechanisms
Grishok et al (2001). Genes and Mechanisms Related to RNA Interference Regulate Expression of the Small Temporal RNAs that Control C. elegans Developmental Timing. Cell 106, 23-34.

the MICROPROCESSOR complex
Han et al (2006). Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125, 887-901.

RITS and heterochromatin silencing
Buhler et al (2006). Tethering RITS to a nascent transcript initiates RNAi- and heterochromatin-dependent gene silencing. Cell 125, 873-886.

Bacterial non-coding regulatory RNAs
Pfeiffer et al (2009). Coding sequence targeting by MicC RNA reveals bacterial mRNA silencing downstream of translational initiation. Nature Struct. Mol. Biol., 16, 840-847.

Teaching methods

Introductory lessons to principal themes
Analysis and in class discussion of seminal research papers

Estimates and exercizes to train biological numeracy

Problem posing and solving sessions, student workgroups, flipped classroom approaches, active learning exercises
Summarizing powerpoint presentations and podcasts
Lab: post-transcriptional regulation mediated by the RhyB sRNA in Escherichia coli

Assessment methods

Oral exam; lab protocol. The exam aims at the assessment of the expected learning outcomes. The final grade arises from three questions on the subjects taught.

 

SERVICE FOR STUDENTS WITH DISABILITIES AND SLD

Teaching tools

.pdf files of seminal papers and scientific articles.
Powerpoint presentations of experimental approaches, results, and models.

Strip sequences
Wetlab course NOTE! The wetlab training activity requires that students take modules 1 and 2 in e-learning mode [https://www.unibo.it/it/servizi-e-opportunita/salute-e-assistenza/salute-e-sicurezza/sicurezza-e-salute-nei-luoghi-di-studio-e-tirocinio] and participate to module 3 of specific training on safety and health in the workplace. Information on dates and attendance of module 3 can be found in the appropriate section of the course website.

Registered students can download lesson presentations, articles and other teaching materials through the Insegnamenti Online site: https://virtuale.unibo.it
using their login credentials.

Office hours

See the website of Alberto Danielli

See the website of