Maria Alessandra Morescalchi

Assessment of environmental stress by the micronucleus test and the Comet assay on the genome of teleost populations from two natural environments.

The aim of the present paper was to assess the biological damage caused by exposure of the test organism (Gambusia holbrooki: Cyprinodontiformes, Poecilidae) to various mutagenic agents present in the polluted waters of the Sarno River. For this purpose, we performed a micronuclei (MN) test and single cell gel electrophoresis (the Comet assay), testing DNA migration in an electrophoretic field using erythrocytes of G. holbrooki specimens both from the Sarno River and from the waters of the crater of the Astroni natural reserve as negative controls. The results indicate statistically higher values for both MN and DNA migration in the samples from the Sarno River compared with those from Astroni and point to a strong genotoxic action of the mixture of pollutants present in the Sarno River. These data were compared with the values found in the G. holbrooki specimens from the Sarno River kept under laboratory conditions for 100 days in clean water.

Karyotype and genome characterization in four cartilaginous fishes.

Different approaches can be used to elucidate the unsolved questions concerning taxonomic evolution in cartilaginous fish. The study of the karyological characteristics of these vertebrates by combining molecular and traditional techniques of chromosome preparation and banding has been demonstrated to be a very effective method. In this paper we studied the localization and the composition of the constitutive heterochromatin by using C- and restriction endonuclease-banding in four selachian species, belonging to two of the four superorders. We also characterized two different types of repetitive genomic sequences in these species: satellite DNA and (TTAGGG)(n) telomeric sequences. Finally, we analysed the nuclear ribosomal gene to determine the number of the nucleolar organizers and their position on chromosomes by using silver staining, chromomycin A(3), and FISH (fluorescent in situ hybridization). The results showed a prevailingly telomeric localization of constitutive heterochromatin in the Galeomorphii, the presence of additional nucleolar organizer sites in Raja asterias, an exclusively telomeric localization of the (TTAGGG)(n) sequences in Scyliorhinus stellaris and both telomeric and interstitial in Taeniura lymma. These data, together with those concerning the conservation of the satellite DNA, seem to support the hypothesis that Chondrichthyes have an evolutionary history leading them to the acquisition of large genomes rich in highly repeated sequences and subjected to some selective pressures favoring the conservation of this DNA fraction.

Chromosomal and molecular analysis of some repeated families in Discoglossus Otth, 1837 (Anura, Discoglossidae): Taxonomic and phylogenetic implications

Abstract We conducted a cytogenetic and molecular analysis in six out of the eight Discoglossus taxa CD. pictus pictus, D. p. auritus, D. sardus, D. montalentii, D. galganoi galganoi and D. g. jean‐neae) All the taxa possessed 2n = 28 biarmed chromosomes, except D. pictus pictus where the 13th pair was acrocentric, and the NORs were located on the short arm of the 7th pair in all the taxa but D. galganoi, where they were on the short arm of the 13th pair. Heterochromatin was detected by banding methods (C‐band‐ing, base‐specific fluorochromes, replication patterns and digestions with restriction enzymes). The techniques used were able to discriminate among all the Discoglussus taxa studied, identifying several heterochromatin families with marked differences in chromosome location and/or in the behaviour of the banding techniques used. The differentiation of these heterochromatin families can have either a phylogenetic (as is the case with A+T rich cen‐tromeric heterochromatin) or an adaptive (G+C rich in...

Cytogenetic and molecular studies in a lungfish, Protopterus annectens (Osteichthyes, Dipnoi)

A neontological approach to the problem of the origin of tetrapods consists in the examination of the available cytological and molecular data on the genome of these vertebrates. Dipnoans are a group of osteichthyian fishes, the evolutionary relationships of which with tetrapods have been disputed since their discovery. In the past, they were variously considered as being related to actinistians, tetrapods, and lower actinopterygians, though nowadays they are considered a monophyletic group, the sister group of crossopterygians. Dipnoans first appeared in the geologic record in the Early Devonian with 50 extinct genera, surviving up to date, with only three genera: Lepidosiren, Neoceratodus and Protopterus, including only six recognized species. Nothing is known of the genome of the early tetrapods, except that they and the Choanoichthyes exhibited a remarkable interspecific variability of the karyotype and of DNA content. These characteristics are often found in dipnoans and in the extant lissamphibians. Very little is known about the evolutionary karyology in the four Protopterus species and in the dipnoan clade in general. In this paper, we karyotyped ten male and female specimens of P. annectens (2n=34) from Nigeria. Moreover, we localized heterochromatin and nucleolar organizer regions by using base-specific fluorochromes and detected the human telomeric (TTAGGG)(n) sequences on all the telomeric sites of P. annectens chromosomes. DNA was also extracted and digested with seven restriction enzymes, which revealed the probable presence of almost three different families of satellite DNA. Nuclear DNA content was identified from blood samples by flow cytometry. New genomic and karyological data were compared and discussed with those on closer genera and taxa available in literature.

Laser microdissection-based analysis of the Y sex chromosome of the Antarctic fish Chionodraco hamatus (Notothenioidei, Channichthyidae)

Abstract Microdissection, DOP-PCR amplification and microcloning were used to study the large Y chromosome of Chionodraco hamatus, an Antarctic fish belonging to the Notothenioidei, the dominant component of the Southern Ocean fauna. The species has evolved a multiple sex chromosome system with digametic males showing an X1YX2 karyotype and females an X1X1X2X2 karyotype. Fluorescence in situ hybridization, performed with a painting probe made from microdissected Y chromosomes, allowed a deeper insight on the chromosomal rearrangement, which underpinned the fusion event that generated the Y. Then, we used a DNA library established by microdissection and microcloning of the whole Y chromosome of Chionodraco hamatus for searching sex-linked sequences. One clone provided preliminary information on the presence on the Y chromosome of the CHD1 gene homologue, which is sex-linked in birds but in no other vertebrates. Several clones from the Y-chromosome mini-library contained microsatellites and transposable elements, one of which mapped to the q arm putative fusion region of the Y chromosome. The findings confirm that interspersed repetitive sequences might have fostered chromosome rearrangements and the emergence of the Y chromosome in Chionodraco hamatus. Detection of the CHD1 gene in the Y sex-determining region could be a classical example of convergent evolution in action.

A molecular approach to systematics of Polypteriformes among Osteichthyes

Abstract The Sarcopterygii are considered to be the living species most closely related to the ancestors of the tetrapods: they include the extinct rhipidistians, the coelacanths and the dipnoans. Furthermore, many Authors debate whether the Polypteriformes should be assigned a very peculiar place in the phylogeny of the bony fishes. To investigate the group of Polypteriformes and the Dipnoans and to provide new support for the classic morphological and molecular data and previous karyological evidence, we examined the DNA sequences of the mitochondrial genes 16S, 12S and cyt‐b in two polypterids (Polypterus palmas and Erpetoichthys calabaricus) and two lungfish (Protopterus annectens and P. aethiopicus). In all the trees, Polypteriformes and Dipnoi are grouped together, while coelacanths remain as a sister group of these two. This molecular evidence supports the earliest hypotheses in which Polypteriformes were grouped in the same subclass together with the coelacanths and Dipnoi.

Short interspersed DNA elements and miRNAs: a novel hidden gene regulation layer in zebrafish?

In this study, we investigated by in silico analysis the possible correlation between microRNAs (miRNAs) and Anamnia V-SINEs (a superfamily of short interspersed nuclear elements), which belong to those retroposon families that have been preserved in vertebrate genomes for millions of years and are actively transcribed because they are embedded in the 3′ untranslated region (UTR) of several genes. We report the results of the analysis of the genomic distribution of these mobile elements in zebrafish (Danio rerio) and discuss their involvement in generating miRNA gene loci. The computational study showed that the genes predicted to bear V-SINEs can be targeted by miRNAs with a very high hybridization E-value. Gene ontology analysis indicates that these genes are mainly involved in metabolic, membrane, and cytoplasmic signaling pathways. Nearly all the miRNAs that were predicted to target the V-SINEs of these genes, i.e., miR-338, miR-9, miR-181, miR-724, miR-735, and miR-204, have been validated in similar regulatory roles in mammals. The large number of genes bearing a V-SINE involved in metabolic and cellular processes suggests that V-SINEs may play a role in modulating cell responses to different stimuli and in preserving the metabolic balance during cell proliferation and differentiation. Although they need experimental validation, these preliminary results suggest that in the genome of D. rerio, as in other TE families in vertebrates, the preservation of V-SINE retroposons may also have been favored by their putative role in gene network modulation.

Polypteridae (Actinopterygii: Cladistia) and DANA-SINEs insertions

SINE sequences are interspersed throughout virtually all eukaryotic genomes and greatly outnumber the other repetitive elements. These sequences are of increasing interest for phylogenetic studies because of their diagnostic power for establishing common ancestry among taxa, once properly characterized. We identified and characterized a peculiar family of composite tRNA-derived short interspersed SINEs, DANA-SINEs, associated with mutational activities in Danio rerio, in a group of species belonging to one of the most basal bony fish families, the Polypteridae, in order to investigate their own inner specific phylogenetic relationships. DANA sequences were identified, sequenced and then localized, by means of fluorescent in situ hybridization (FISH), in six Polypteridae species (Polypterus delhezi, P. ornatipinnis, P. palmas, P. buettikoferi P. senegalus and Erpetoichthys calabaricus) After cloning, the sequences obtained were aligned for phylogenetic analysis, comparing them with three Dipnoan lungfish species (Protopterus annectens, P. aethiopicus, Lepidosiren paradoxa), and Lethenteron reissneri (Petromyzontidae)was used as outgroup. The obtained overlapping MP, ML and NJ tree clustered together the species belonging to the two taxonomically different Osteichthyans groups: the Polypteridae, by one side, and the Protopteridae by the other, with the monotypic genus Erpetoichthys more distantly related to the Polypterus genus comprising three distinct groups: P. palmas and P. buettikoferi, P. delhezi and P. ornatipinnis and P. senegalus. In situ hybridization with DANA probes marked along the whole chromosome arms in the metaphases of all the Polypteridae species examined.