STRUCTURAL AND FUNCTIONAL GENOMICS

Students deepen the knowledges on the organization of the genome into the metaphase chromosomes and interphase nuclei, also considering the evolutionary aspect. They will understand the correlation between architecture of chromatin, gene expression and the onset of human genetic diseases. From the application point of view, the students will acquire the methodological skills on the analysis of human chromosomes and of genomic sequences that can be used in various fields of work, publics and privates.

Lectures and exercises in the classroom with ongoing tests.

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

The Eukaryotic Genome
General overview of genome size, gene content, and the 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. Genome Projects and Human Genome Sequencing. Characteristics of eukaryotic genes: structure, size, and genomic and chromosomal organization. Gene density and alternative splicing. Single-copy genes, multicopy genes, and gene families. Mechanisms underlying the origin of new genes: gene duplication; gene fusion and fission; retrotransposition; emergence of new genes; orphan genes; horizontal gene transfer; acquisition and/or domestication of viral genes. Structural and functional diversification of new genes: exon duplication/expansion, exon shuffling, alternative splicing. Processed and unprocessed pseudogenes. Fixation of new genes in populations. Functional innovations and evolutionary advantages of new genes. Human- and mammal-specific genes. The epigenomes of multicellular eukaryotes.

Methods for the study of chromosomes
In vitro cell culture. Standard techniques for the preparation of metaphase and prometaphase chromosomes. Chromosome staining and its use in the analysis of unknown karyotypes and in genotoxicity testing. Vertebrate karyotypes and the microchromosomes of birds and reptiles (overview). Structural bandings G, R, T: main techniques and methods of analysis. The human karyotype and identification of individual chromosomes using G-banding. Karyotype resolution and standard nomenclature. The International System for Human Cytogenetic Nomenclature (ISCN). Dynamic bandings. Use of bromodeoxyuridine, replication banding, and prometaphase chromosomes. Sister chromatid exchanges. Fluorescence in situ hybridization (FISH): general principles, probe labeling and signal detection methods. Hybridization on chromosomes, nuclei, and chromatin fibers. Classical and molecular cytogenetics in biomedical diagnostics. Chromosome-level genome studies in the post-genomic era. Array-CGH, SNP arrays, and optical mapping.

Structural organization of the eukaryotic genome
The centromere: centromeric proteins, centromeric DNA and chromatin, the pairing domain, and chromosomal passenger proteins. The centromere of S. cerevisiae, S. pombe, C. elegans, and higher eukaryotes. Neocentromeres and neocentromerization. The telomere: telomeric sequences and chromosome shortening, telomerase and telomere length, aging and oncogenesis. The origin of replication: general features of ARS elements, the E. coli origin of replication, ARS in yeast and higher eukaryotes. ARS and the pre-replication complex. Replicons and replication timing in higher eukaryotes, replication foci and their sequential activation. Repeated sequences and reassociation kinetics: highly and moderately repeated sequences. Structure of repeated sequences. Simple versus complex repeats. Mobile elements: transposons and retrotransposons. LINE sequences (L1 family) and SINE sequences (Alu family). LTR sequences and their transposition mechanism. Highly polymorphic simple repeats and their application in standard forensic genetic protocols.

Compositional organization and evolution of the eukaryotic genome
Isochores and the CsCl gradient in genome studies. The genomic phenotype. Compositional features of the genomes of heterothermic and homeothermic vertebrates, invertebrates, and unicellular eukaryotes. Compositional genome organization in plants (overview). Gene distribution across compositional domains. Genome organization into isochores and the Bernardi model. Structural and functional features associated with isochores. Paleogenome and neogenome. Compositional genome evolution. Major and minor compositional shifts in vertebrates. Hypotheses on the origin of GC-rich isochores. Chromosomal compositional mapping. Correlation between chromosomal bands and genomic sequence. Isochores and chromosomal bands: gene distribution, repeated sequences, replication timing. Constitutive versus tissue-specific genes: chromosomal distribution and expression levels across different chromosomal 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 correlation with transcriptional activity, gene density, and replication timing. Transcriptionally active and inactive chromatin. Heterochromatinization as a mechanism of gene silencing. Position effect in the modulation of gene expression and consequences of chromosomal rearrangements, both neutral and disease-associated. DNA methylation and genomic imprinting. Imprinting control regions (ICRs): features and mechanisms of gene activation/silencing. Uniparental disomy syndromes and imprinting-related genetic disorders. Analysis of genomic sequence interactions through chromatin conformation capture (3C) and derived methods (4C, 5C, Hi-C). Chromatin loops and the mechanism of TAD formation. TADs, LADs, and isochores. 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

Examination Methods

The final examination consists of a preliminary written test with two open-ended questions and one question concerning the identification of human chromosomes. The examination concludes with an oral test, which can only be taken after successfully passing the preliminary written test with a sufficient grade. During the course, two written/practical mid-term tests will be administered, both identical in format to the preliminary written exam.

The purpose of the examination is to assess:

1. the level of learning and understanding,

2. the ability to analyze the topics covered during the course,

3. the depth of knowledge of the subject matter,

4. the use of supplementary teaching material,

5. the ability to apply methodological skills acquired during lectures and practical sessions.

All tests are graded on a 30-point scale, with a minimum passing grade of 18/30.

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Mid-term Tests

Mid-term tests are written and contribute to the final evaluation. Each test, consisting of two open-ended questions, is assessed according to the following criteria:

1. relevance of the answers,

2. completeness of the answers,

3. use of teaching material and depth of knowledge,

4. accuracy of concepts and definitions,

5. appropriateness of scientific terminology.

In addition, a practical exercise is included: identification of selected human chromosomes in a G-banded metaphase. The exercise is considered passed if at least half plus one of the required chromosomes are correctly identified. A positive result contributes to the final evaluation.

The first mid-term test is based on topics from the first part of the program. The second (final) mid-term test covers the entire course content.

A positive evaluation of the mid-term tests allows the student, in the first exam session, to take only the oral test, with the grades obtained in the mid-term tests serving as a substitute for the written exam.

Students who receive a negative evaluation in the mid-term tests, or who consider their evaluation unsatisfactory, must take both the written and the oral parts of the final exam. In this case, the negative mid-term results will not be taken into account.

Examples of Open-Ended Questions:

• Correlation between genome size and organismal complexity.

• Dynamic banding techniques.

• Karyotype resolution and the ISCN system.

• Describe the organization of the centromere in S. cerevisiae.

• Neocentromerization.

• Telomeres and cellular aging.

• Origin and evolution of Alu sequences.

• Single-locus probes in genotypic diagnosis: features and applicability.

• Compositional characteristics of the human genome: describe the profile obtained by CsCl gradient analysis.

• GC-rich isochores: features and hypotheses on their origin.

• Chromosome organization in the interphase nucleus.

• Organization of a “nuclear speckle.”

• Mechanism of allelic inactivation: chromatin structure and role of ICRs.

• The Xist gene: characteristics and functions.

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Example of Practical Question:

• Identify, in a metaphase spread of human chromosomes with G-banding, chromosomes 3, 6, 13, 15, 19, and 21.