Search

Sexual differentiation (SD) during development results in anatomical, metabolic, and physiological differences that involve not only the gonads, but also a variety of other biological structures, such as the brain, determining differences in morphology, behavior, and response in the breeding season. In many reptiles, whose sex is determined by egg incubation temperature, such as the leopard gecko, Eublepharis macularius, embryos incubated at different temperatures clearly differ in the volume of brain nuclei that modulate behavior. Based on the premise that "the developmental decision of gender does not flow through a single gene", we performed an analysis on E. macularius using three approaches to gain insights into the genes that may be involved in brain SD during the thermosensitive period. Using quantitative RT-PCR, we studied the expression of genes known to be involved in gonadal SD such as WNT4, SOX9, DMRT1, Erα, Erβ, GnRH, P450 aromatase, PRL, and PRL-R. Then, further genes putatively involved in sex dimorphic brain differentiation were sought by differential display (DDRT-PCR) and PCR array. Our findings indicate that embryo exposure to different sex determining temperatures induces differential expression of several genes that are involved not only in gonadal differentiation (PRL-R, Wnt4, Erα, Erβ, p450 aromatase, and DMRT1), but also in neural differentiation (TN-R, Adora2A, and ASCL1) and metabolic pathways (GP1, RPS15, and NADH12). These data suggest that the brains of SDT reptiles might be dimorphic at birth, thus behavioral experiences in postnatal development would act on a structure already committed to male or female.

The common lizard (Zootoca vivipara) displays characteristic cytogenetic, reproductive, molecular, and biogeographic variability. This species comprises oviparous and viviparous populations with disjunct distribution and sex chromosome polymorphisms, from simple ZZ/ZW to complex Z1Z1Z2Z2/Z1Z2W systems with different morphologies of the W chromosome. In this study, we used the primers SINE A and SINE B and a newly designed primer pair to (1) obtain information on the presence and distribution of transposable elements (TEs) in 8 squamate families and (2) assess the chromosomal location of SINE Squam elements in Z. vivipara. PCR amplification with SINE A and SINE B produced single or multiple products in different Z. vivipara populations, subsequently used to design the SINE-Zv primers. Using the newly designed SINE-Zv primers, we identified 2 sequences of about 700 and 300 bp (SINE-Zv 700 and SINE-Zv 300) in all the investigated populations of Z. vivipara. Fluorescence in situ hybridizations showed a preferential localization of SINE-Zv sequences in the peritelomeric regions of almost all chromosomes, with the exception of the W. Both sequences contained a distinct segment of SINE Squam2. SINE-Zv 700 appeared to be restricted to Z. vivipara, while SINE-Zv 300 contained a partial Gypsy sequence that is highly conserved among Squamata and showed high identity values (72-93%) with several transcripts from different species. Using the same primers, we also highlighted the presence of another highly conserved Gypsy-like fragment in snakes which displayed significant similarity with the stomatin-like protein 2 of colubrids. Our results suggest that SINEs and the Gypsy-like elements are widely distributed among squamates and may have played an active role in their genomic evolution and differentiation.

Due to their particular phylogenetic position and biological characteristics, squamate reptiles and, in particular, snakes are becoming an increasingly important model for fields such as evolutionary biology, molecular ecology and adaptation. Recently, during a study to analyze the evolutionary history of European whip snakes, we found a LINE1 (L1)-like sequence (GenBank accession no. LM644476), herein called TRL1L, and while there are data on the abundance of L1 in snakes, their genomic and chromosome localization is still largely unexplored. We therefore performed a study to obtain information on TRL1L abundance, distribution and conservation in snake species, belonging to the Colubridae, Lamprophiidae and Viperidae families, using quantitative dot-blot and fluorescence in situ hybridization (FISH). TRL1L showed a high identity with homologous segments of L1s of lizards the Anolis carolinensis and Lacerta agilis and the zebrafish Danio rerio. The discovered sequences are truncated L1 elements which occur with a low copy number, about 0.1% of the genome of the species studied. This evidence suggests that L1 retroposons have a similar landscape in lizard and snake genomes, probably because similar processes limited L1 distribution in their genomes. TRL1L showed a non-random chromosome distribution pattern. It was scarcely located on autosomes and on the euchromatic W chromosome of Cerastes vipera, while mostly found on the heterochromatic W chromosome of Hierophis carbonarius and Elaphe quatuorlineata. Our data support the hypothesis that a ‘purifying selection’ against the accumulation of L1 elements takes place in recombining regions and highlight the possible role of these elements in the differentiation processes of the snake heterochromatic W chromosome. Interestingly, the preferential distribution of TRL1L on the heterochromatic W chromosomes of the studied snakes appears to be similar to that observed in mammals for L1 accumulation on differentiated Y chromosomes. This finding suggests that a convergent process may have taken place in the differentiation of vertebrate heterochromatic sex chromosomes

Within the scope of a project on the characterization of satellite DNAs in polar mollusks, the Antarctic chiton Nuttallochitonmirandus (Thiele, 1906) was analyzed. Two novel families of tandemly repeated DNAs, namely NmH and NmP, are described in their structure and chromosomal localization, and, furthermore, their presence was analyzed in related species. Data reported here display a particular variability in the structural organization of DNA satellites within this species. Processes driving satellite evolution, which are likely responsible for the intriguing variability of the identified satellite DNAs, are discussed.

A karyological analysis on six Italian populations the slow worm (Anguis veronensis Pollini, 1818) was performed and their genetic differentiation at the mitochondrial 16S rRNA gene fragment from a Spanish sample has been assessed. The Italian populations were karyologically uniform, all showing 2n= 44 elements, of which 20 were macrochromosomes and 24 microchromosomes. Comparison with literature data on Central European populations showed a difference on the morphology of the 10th chromosome pair: submetacentric in Italian populations and telocentric in the Central European ones. Our analysis showed the presence of a fragile site on chromosomes of this pair, suggesting its propensity for structural rearrangements. Analysis of the 16S rRNA gene fragment showed uniformity among Italian populations (uncorrected genetic distance of 0.4%), and their genetic distinctness from the Spanish individual (uncorrected genetic distance of 4.2%). Our results confirm the existence of two different Anguis fragilis Linnaeus, 1758 lineages, each one characterized by a different cytotype.

A karyological study conducted by both conventional and banding staining methods (Ag-NOR-, CMA3, and C-banding) evidenced a peculiar heterochromatin pattern on chromosomes of Moldovan specimens of the agile frog, Rana dalmatina. As is normal for all agile frogs, the Moldovan population presented 2n = 26 chromosomes, with NOR loci on the short arms of the third chromosome pair, but differed in showing heavy centromeric, CMA3 positive C-bands on the seventh chromosome pair, and light centromeric, DAPI positive C-bands on five chromosome pairs. In contrast, Moldovan specimens showed no significant difference in two segments of the 16S mitochondrial rRNA gene and in the S1 satellite DNA sequence and organization, in comparison with the GenBank deposited 16S rDNA and S1 DNA satellite sequences of R. dalmatina. Molecular similarity and chromosomal differences between agile frogs from Moldovan and extra-Moldovan populations are discussed.

The aim of this work is to investigate the sequence conservation and the evolution of repeated DNA in related species. Satellite DNA is a component of eukaryotic genomes and is made up of tandemly repeated sequences. These sequences are affected by high rates of mutation that lead to the occurrence of species-specific satellite DNAs, which are different in terms of both quantity and quality. In this work, a novel repetitive DNA family, named PjHhaI sat, is described in Pecten jacobaeus. The quantitative analyses revealed a different abundance of this element in the molluscan species investigated in agreement with the "library hypothesis" even if, in this case, at a high taxonomic level. In addition, the qualitative analysis demonstrated an astonishing sequence conservation not only among scallops but also in six other molluscan species belonging to three classes. These findings suggest that the PjHhaI sat may be considered as the most ancients of DNA described so far, which remained "frozen" during molluscan evolution. The widespread distribution of this sat DNA in molluscs as well as its long evolutionary preservation open up questions on the functional role of this element. A future challenge might be the identification of proteins or molecules which interact with the PjHhaI sat.

Two new high repetitive DNAs were isolated from Discoglossus pictus genomic DNA digested with StuI. Sequence analysis of clones indicated two types of repetitive units 143–148 bp and 170–177 bp long, respectively. Southern blot analyses show ladders of bands indicating that both repetitive units are organized in long tandem arrays and belong to two different satellite DNAs that were named Dp-sat2 and Dp-sat3. A similar ladder of bands was also produced when genomic DNA was digested with ClaI in the case of Dp-sat2, or with ScaI in the case of Dp-sat3. Quantitative dot-blot analyses indicate that Dp-sat2 and Dp-sat3 account for about 2.6% and 2.2%, respectively, of the D. pictus genome. Considering that D. pictus has a genomic DNA content of 10.5 pg/N, our data indicate that Dp-sat2 and Dp-sat3 repetitive units are present in about 2.3 millions and 1.6 millions copies per diploid genome, respectively. Dp-sat2 and Dp-sat3 probes produced the same patterns of hybrid bands in Southern blots and the same genomic content in dot-blots in all the other Discoglossus species, except D. montalentii which showed a different band pattern and genomic content. The chromosomal distribution of the two StuI satellites shows some similarities: both Dp-sat2 and Dp-sat3 probes mainly labelled the pericentromeric regions of the large chromosome pairs 1–5 and 7. Moreover, probes from both satellite DNAs also produced faint fluorescent mini-spots along almost all chromosome pairs. These data suggest that both satellite DNAs have two types of organization, one in long arrays localized in pericentromeric positions, and the other in short arrays dispersed in the genome.

The present paper shows the results of chromosome banding and rDNA-FISH study performed on several specimens of different populations of Patella caerulea, Patella rustica and Patella ulyssiponensis. The taxonomic attribution of specimens was ascertained by the molecular phylogenetic analysis of the mitochondrial 16S rRNA gene. P. caerulea and P. rustica had 2n = 18 chromosomes with first seven of biarmed pairs and the remaining two uniarmed pairs. P. ulyssiponensis had 2n = 16 with all biarmed chromosomes. Ag-NOR loci were on the short arms of the first metacentric pair in the three studied limpets, whereas they showed a different pattern of heterochromatin distribution and composition. A chromosome mosaicism was observed in several P. caerulea specimens, which exhibited an unpaired metacentric element and loss of a telocentric pair. The obtained results suggest that in the genus Patella specific diversification was accompanied by variations in heterochromatin distribution and composition and reduction of chromosome number by Robertsonian centric fusion.