STRUCTURAL AND FUNCTIONAL GENOMICS

By the end of the course, students will have acquired knowledge and skills related to the structural and functional organization of the eukaryotic genome, its evolution, and the main methodological approaches used to study chromosomes, chromatin, and genomic sequences.

Knowledge and understanding
Students will be able to:

• describe the structural and functional organization of the eukaryotic genome and its organization in metaphase chromosomes and interphase nuclei;
• understand the main mechanisms underlying the origin and evolution of genes and genomes;
• describe the organization and properties of the main structural components of chromosomes and repetitive sequences;
• understand the compositional organization of genomes and the relationships among genomic composition, chromosome structure, gene density, DNA replication, and gene expression;
• understand the principles of three-dimensional chromatin organization and the relationships among nuclear architecture, regulation of gene expression, and human diseases;
• understand the principles and main applications of cytogenetic, cytogenomic, and genomic methodologies used to study chromosomes and genomes.

Applying knowledge and understanding
Students will be able to:

• apply the acquired knowledge to the interpretation of the structural and functional organization of eukaryotic genomes;
• identify human chromosomes based on the main chromosome banding systems and interpret their organization according to cytogenetic nomenclature;
• understand and interpret results obtained using classical and molecular cytogenetic techniques and methodologies for the analysis of genomes and three-dimensional chromatin organization;
• correlate alterations in chromosome and chromatin organization with their potential consequences for gene expression and phenotype.

Making judgements
Students will be able to:

• critically analyse data and models concerning genome organization, function, and evolution;
• evaluate the most appropriate methodological approach for studying specific chromosomal or genomic features;
• integrate information derived from different levels of analysis, from genomic sequences to chromosomal and nuclear organization.

Communication skills
Students will be able to:

• describe structures, processes, and methodologies of structural and functional genomics using appropriate scientific terminology;
• clearly and coherently present and discuss the relationships among genome organization, chromatin structure, gene expression, and evolution;
• communicate and interpret the results of cytogenetic and genomic analyses.

Learning skills
Students will be able to:

• independently explore topics in structural and functional genomics through the consultation of specialized textbooks and scientific literature;
• integrate acquired knowledge with new data arising from advances in genomic and cytogenomic technologies;
• use the acquired knowledge and skills as a basis for advanced courses and for experimental and professional activities in the biological, biomedical, and genomic fields.

Lectures and exercises in the classroom with ongoing tests.

Information for students with disabilities and/or specific learning disorders (SLD): In accordance with current legislation and to ensure equal opportunities, students requiring accommodations may request an individual meeting with the instructor to discuss appropriate compensatory and/or dispensatory measures, consistent with the learning objectives of the course and their specific needs. Students may also contact the departmental representative of CInAP (Centre for Active and Participatory Inclusion – Services for Students with Disabilities and Specific Learning Disorders) for further assistance.

Basic knowledge covered in the courses of Cytology, Zoology, Genetics, Biochemistry, and Molecular Biology within degree programmes of the L-13 class (Biological Sciences).

Mandatory attendance of lectures, in accordance with the provisions of the Degree Program’s academic regulations.

The Eukaryotic Genome. Overview of the size, gene content, and organization of the nuclear genome and the genomes of cytoplasmic organelles. Hypotheses on the origin of genomes, their evolution, and the formation of the eukaryotic genome. From the Human Genome Project to modern genomics and pangenomics projects. Sequencing and characterization of the human genome. Characteristics of eukaryotic genes: structure, size, and genomic and chromosomal organization. Gene density and alternative splicing. Single-copy genes, multiple-copy genes, and gene families. Mechanisms underlying the origin of new genes: gene duplication; gene fusion and fission; retrotransposition; de novo gene emergence; orphan genes; horizontal gene transfer; acquisition and/or domestication of viral genes. Structural and functional diversification of new genes: exon duplication/expansion, exon shuffling, and alternative splicing. Processed and non-processed pseudogenes. Fixation of new genes in populations. Functional innovations and evolutionary advantages of new genes. Human- and mammal-specific genes.

Methods for Chromosome Analysis. In vitro cell cultures. Standard techniques for the preparation of metaphase and prometaphase chromosomes. Chromosome staining and its use in the analysis of unknown karyotypes and genotoxicity testing. Vertebrate karyotypes and microchromosomes in birds and reptiles (overview). Structural G-, R-, and T-banding: main techniques and analytical methods. The human karyotype and identification of individual chromosomes by G-banding. Karyotype resolution and standard nomenclature. The International System for Human Cytogenomic Nomenclature (ISCN). Dynamic chromosome banding. Use of bromodeoxyuridine, replication banding, and prometaphase chromosomes. Sister chromatid exchanges. Fluorescence in situ hybridization: general principles, probe-labelling methods, and signal detection. Hybridization on chromosomes, nuclei, and chromatin fibres. Classical and molecular cytogenetics in biomedical diagnostics. Chromosome-level genome analysis in the post-genomic era. From classical cytogenetics to cytogenomics: array-CGH, SNP arrays, and optical genome mapping.

Structural Organization of the Eukaryotic Genome. The centromere: centromeric proteins, centromeric DNA and chromatin, the pairing domain, and chromosomal passenger proteins. Centromeres in S. cerevisiae, S. pombe, C. elegans, and higher eukaryotes. Neocentromeres and neocentromerization. The telomere: telomeric sequences and chromosome shortening, telomerase and telomere length, ageing and oncogenesis. Origins of replication: comparison between prokaryotes and eukaryotes; the origin of replication in E. coli, yeast ARSs, and origins of replication in higher eukaryotes. The pre-replication complex. Replicons and replication timing in higher eukaryotes, replication foci and their sequential activation. Repetitive sequences and reassociation kinetics: highly and moderately repetitive sequences. Structure of repetitive sequences. Simple and complex repetitive sequences. Mobile elements: transposons and retrotransposons. LINE sequences (L1 family) and SINE sequences (Alu family). LTR sequences and their transposition mechanisms. Highly polymorphic simple repetitive sequences and their use in standard protocols for forensic genetic analysis.

Compositional Organization and Evolution of the Eukaryotic Genome. Isochores and CsCl density-gradient centrifugation for genome analysis. The genomic phenotype. Compositional features of the genomes of poikilothermic and homeothermic vertebrates, invertebrates, and unicellular eukaryotes. Compositional organization of plant genomes (overview). Compositional distribution of genes. Genome organization into isochores and the Bernardi model. Structural and functional features associated with isochores. The paleogenome and the neogenome. Compositional evolution of genomes. The major and minor compositional shifts in vertebrates. Hypotheses on the origin of GC-rich isochores. Chromosomal compositional mapping. Correlation between chromosome bands and genomic sequences. Isochores and chromosome bands: gene distribution, repetitive sequences, and replication timing. Housekeeping and tissue-specific genes: chromosomal distribution and expression levels in different chromosome bands.

Functional Organization of Chromatin in the Interphase Nucleus. Chromosome territories and interchromosomal domains, with reference to the Cremer model. Structure and function of nuclear speckles. Compositional organization of chromosome territories and its relationship with transcriptional activity, gene density, and replication timing. Transcriptionally active and inactive chromatin. Heterochromatinization as a mechanism of gene inactivation. Position effects in the modulation of gene expression and the consequences of chromosomal rearrangements, both neutral and disease-associated. DNA methylation and genomic imprinting. Imprinting control regions (ICRs): characteristics and mechanisms of gene activation/inactivation. Uniparental disomy syndromes and genetic disorders associated with genomic imprinting. Analysis of interactions between genomic sequences using chromatin conformation capture (3C) and derived methods (4C, 5C, Hi-C). Relationships among the compositional organization of the genome, chromatin compartments, TADs, and LADs. A/B chromatin compartments and their relationships with genomic composition, gene density, transcriptional activity, and replication timing. X-chromosome inactivation and the Xist gene.

Text-1: S. Saccone - C. Federico. IL GENOMA DEGLI EUCARIOTI: organizzazione ed evoluzione. EdiSES Edizioni S.r.l., Napoli. [EBOOK], 2024. ISBN: 9788836231829.

Text-2: Saccone, S.; Brancato, D.; Bruno, F.; Coniglio, E.; Sturiale, V.; Federico, C.  ORIGIN AND EVOLUTION OF GENES IN EUKARYOTES: MECHANISMS, DYNAMICS, AND FUNCTIONAL IMPLICATIONS. Genes 2025, 16, 702. https://doi.org/10.3390/genes16060702

FINAL EXAMINATION

The final examination consists of a preliminary test comprising a written component, with two open-ended questions, and a practical component involving the identification of human chromosomes. Both components contribute to the overall assessment of the preliminary test. The examination is completed by an oral test, which students may take after obtaining a passing grade in the preliminary test. During the course, two in itinere assessments will be administered, structured in the same way as the preliminary test of the final examination, with a written and a practical component.

The examination is designed to assess:

1. knowledge and understanding of the course contents;
2. the ability to apply acquired knowledge to the analysis of issues related to the structural and functional organization of the genome;
3. the ability to critically analyse and integrate the different levels of genome organization, from DNA sequence to chromosomal and nuclear organization;
4. the ability to interpret data and results obtained through cytogenetic, cytogenomic, and genomic methodologies;
5. the ability to present and discuss topics clearly and coherently, using appropriate scientific terminology.

All assessments are graded on a 30-point scale, with 18/30 as the minimum passing grade.

IN ITINERE ASSESSMENTS

The in itinere assessments contribute to the final grade. Each assessment includes a written component consisting of two open-ended questions, evaluated according to the following criteria: 1) relevance and completeness of the answers; 2) accuracy of concepts and definitions; 3) ability to analyse and establish connections among topics; 4) level of detail and depth of knowledge; 5) appropriate use of scientific terminology.

Each in itinere assessment also includes a practical component involving the identification of human chromosomes with G-banding. The practical component contributes to the overall grade of the in itinere assessment according to the number of chromosomes correctly identified.

The two open-ended questions of the first in itinere assessment cover topics from the first part of the course programme, whereas those of the second and final in itinere assessment cover the entire course programme.

A positive evaluation in the in itinere assessments allows students, during the first examination session, to take only the oral examination, with the grade obtained in the in itinere assessments replacing the grade of the preliminary test of the final examination.

Students who receive a negative evaluation in the in itinere assessments, or who consider the grade obtained unsatisfactory, must take the complete final examination, including the preliminary test, with both its written and practical components, and the oral examination. In this case, the grade obtained in the in itinere assessments will not be taken into account.

Information for students with disabilities and/or specific learning disorders (SLD): In accordance with current legislation and to ensure equal opportunities, students requiring accommodations may request an individual meeting with the instructor to discuss appropriate compensatory and/or dispensatory measures, consistent with the learning objectives of the course and their specific needs. Students may also contact the departmental representative of CInAP (Centre for Active and Participatory Inclusion – Services for Students with Disabilities and Specific Learning Disorders) for further assistance.

Examples of open-ended questions:

Correlation between genome size and organismal complexity.
Dynamic chromosome banding.
Karyotype resolution and the ISCN system.
Describe the organization of the centromere in S. cerevisiae.
Neocentromerization.
Telomeres and cellular ageing.
Origin and evolution of Alu sequences.
Single-locus probes in genotypic diagnosis: characteristics and applications.
Compositional features of the human genome: describe the profile obtained by CsCl density-gradient centrifugation.
GC-rich isochores: characteristics and hypotheses on their origin.
Organization of chromosomes in the interphase nucleus.
Organization of a nuclear speckle.
Mechanisms of allelic inactivation: chromatin structure and the role of ICRs.
The Xist gene: characteristics and functions.

Example of a practical question:

Identify chromosomes 3, 6, 13, 15, 19, and 21 in a G-banded human metaphase spread.