Universitą degli studi di Pavia


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Raimondi research activity


The research activity carried on in the Laboratory of Molecular Cytogenetics is focused on the analysis of genome plasticity, on the investigation of the mechanisms responsible for karyotypes evolution and on the study of the physical organization of the centromere in mammals.
We make use of a biological model system which is represented by species belonging to the genus Equus and our researches are performed in collaboration with the Laboratory of Molecular and Cellular Biology of the Department of Biology and Biotechnology of the University of Pavia, headed by Prof. Elena Giulotto.
Equids belong to the order Perissodactila which includes three families: Equidae, Tapiridae and Rinocerotidae. All the Equid extant species belong to the genus Equus. In the different branches of the Perissodactila phylogenetic tree, the evolutionary rate is comparable to that observed in other mammals. This figure is radically different when the genus Equus is concerned; the radiation of this genus occurred 2-3 millions years ago and it has been observed an increase of 80 times in the evolutionary rate with respect to that observed in ancient Ceratomorpha. Perissodactila are especially interesting also from a cytogenetic point of view due to the wide variability in the diploid chromosome number (2n) with 2n = 32-66 in Equidae, 2n = 52-80 in Tapiridae and 2n = 82-84 in Rinocerotidae. Worthy of note is that Equids, despite their recent divergence, the morphological similarity and the possibility of cross between different species, show very different karyotypes for both chromosome number and structure. In this scenario, Equid genomes seem particularly suitable to obtain molecular data on the evolutionary dynamics into and among species.

1. Karyotype evolution
Comparative analyses of Perissodactila chromosomes have been performed by means of multidirectional interspecific "chromosome painting" and allowed researchers to create whole genome comparative maps of ten Perissodactila species thus reconstructing an hypothetical ancestral Perissodactila karyotype.
We used a high resolution molecular-cytogenetic approach to compare the localization of single-copy DNA sequences on metaphase chromosomes from the horse, from the donkey and from two zebras. Our preliminary results strongly suggest that some chromosomes, so far considered orthologues, have been indeed originated by independent chromosomal rearrangements. These data will contribute to clarify some evolutionary mechanisms and will eventually allow us to shed light on debated nodes of the radiation of Equine related species.

2. Study of the organization of the centromere
The segregation of chromosomes is the basis of heredity, essential for normal development but also, when this function is compromised, playing a role in disease, most notably cancer and fertility or developmental disorders. The chromosomal element that directs segregation is the centromere, the primary constriction of mitotic chromosomes. The centromere is a deeply enigmatic structure whose fundamental features remain key challenges of biology. Unlike other genetic loci, centromeres are not determined by their underlying DNA sequences, but rather by the proteins that associate with DNA domains which show exceptional plasticity in evolution.
In a previous work, performed in collaboration with the laboratory headed by Prof. Elena Giulotto, we have demonstrated that two highly conserved satellite DNA families are localized in the centromeric region of all the horse chromosomes, while they show a predominantly telomeric localization in in the donkey as well in two zebra species. The results of these researches allowed us to hypothesize that in the Equids, contrary to what observed in all the other vertebrates, the centromeric function is not coupled to the presence of highly repeated DNA. We set up a high resolution fluorescence in situ hybridization technique on extended chromatin fibers, coupled with immunofluorescence with antibodies against the proteins that constitute the kinetochore (immuno-FISH). This kind of experiments are suitable to analyze the physical organization of different satellite DNA families and allow the ascertainment of the protein-DNA interactions. Our aim is to define which and how many equine satellite DNA families are directly involved in centromere function. Moreover, in the contest of a work aimed at sequencing the whole horse genome, was determined the DNA sequence of the evolutionary new centromere on horse chromosome 11 which demonstrated that this centromere lacks any satellite DNA sequences; this is the only Equid centromere completely characterised so far. By means of immuno-FISH experiments, performed using single copy DNA sequences located in the functional horse chromosome 11 centromere as probes, we plan to identify the functional domains responsible for centromeric function.

3. Insertion polymorphism
A large fraction of the genome of mammals is occupied by interspersed repeats that were generated during evolution by the propagation of transposable elements. Short INterspersed Elements (SINEs) are non-autonomous retrotransposons that make use of a transposition process in which an RNA intermediate is reverse transcribed and the resulting cDNA is inserted into a new genomic location. Depending on the age of its integration, an insertion sequence can be present in all the individuals of a species (fixed) or alternatively can be present only in some individuals (insertion polymorphism). Although transposable elements are considered "junk DNA", in a number of examples they have acquired a functional role, a process termed 'exaptation', and provide an origin for at least some of the many highly conserved vertebrate-specific genomic sequences. The insertion of transposable elements inside genes or in their proximity may alter gene structure or expression through gene interruption, introduction of promoter sequences or splice sites. In some rare cases, transposons are implicated in genetic disease or cancer.
We are carrying out a genome wide analysis of the perissodactyl-specific SINE family of Equine Repetitive Elements (ERE) focusing our attention on insertion polymorphism in relation to sequence conservation. Moreover, in order to investigate if ERE elements may play a role in modulating gene expression and in the evolution of gene function, we searched for the presence of ERE insertion elements within introns or in the 5’ regulatory region. Our preliminary results demonstrate that a fraction of ERE elements, significantly higher than expected, is located inside intronic sequences, moreover we have observed that some ERE sequences are positioned next to the promoter of expressed genes.

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