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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 2.3×106 and 1.6×106 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. [ABSTRACT FROM PUBLISHER]

A new highly abundant satellite DNA from Discoglossus pictus (Dp-sat1) was isolated and characterized. The repetitive unit (0.51 kb) has 2 HindIII sites and only one SpeI site: digestion of genomic DNA with HindIII produces 3 fragments: HA (0.17 kb), HB (0.34 kb), and HC = HA + HB (0.51 kb), while digestion with SpeI produces the whole repetitive unit (0.51 kb) that contains both HindIII sites. Sequence analysis of cloned repeats indicates an average A + T content of 71%, with many A- and T-runs. Southern blot analysis shows an arrangement of multiple bands of the 0.51 kb monomer in SpeI-digested DNA, while HindIII-digested DNA shows a ladder composed of all the possible combinations of the 3 digested fragments. Quantitative dot-blot indicates that Dp-sat1 accounts for about 6% of the D. pictus genome: this value represents about 1.5 × 106 copies of repetitive units per nucleus. This satellite DNA is also a major repetitive DNA in 4 other Discoglossus species, in which the repetitive unit presents the same size and restriction sites except in D. montalentii where it contains a unique HindIII site. This satellite DNA was absent in all the other tested archaeo- and neo-bratrachian species, as well as non-amphibian species. Fluorescent in situ hybridization (FISH) analysis shows that Dp-sat1 is localized only in peri- and/or para-centromeric areas of the 7 small chromosome pairs, while no labeling was observed in the 7 large chromosome pairs. Remarkably, Dp-sat1 heterochromatin is found only at one pole of the nucleus, suggesting that during interphase all 7 small chromosome pairs are located in the same nuclear region. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Arginine-vasotocine (AVT) injected into male adult lizards, Podarcis s. sicula Raf., inhibits the last phases of spermatogenesis and the endocrine activity of gonads. In female lizards the substance induces an early interruption of egg deposition. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) constitute a considerable fraction of eukaryote genomes representing a major source of genetic variability. We describe two DNA sequences isolated in the lizard Zootoca vivipara , here named Zv516 and Zv817. Both sequences are single-copy nuclear sequences, including a truncation of two transposable elements (TEs), SINE Squam1 in Zv516 and a Tc1/Mariner-like DNA transposon in Zv817. FISH analyses with Zv516 showed the occurrence of interspersed signals of the SINE Squam1 sequence on all chromosomes of Z. vivipara and quantitative dot blot indicated that this TE is present with about 4700 copies in the Z. vivipara genome. FISH and dot blot with Zv817 did not produce clear hybridization signals. Bioinformatic analysis showed the presence of active SINE Squam 1 copies in the genome of different lacertids, in different mRNAs, and intronic and coding regions of various genes. The Tc1/Mariner-like DNA transposon occurs in all reptiles, excluding Sphenodon and Archosauria. Zv817 includes a trait of 284 bp, representing an amniote ultra-conserved element (UCE). Using amniote UCE homologous sequences from available whole genome sequences of major amniote taxonomic groups, we performed a phylogenetic analysis which retrieved Prototheria as the sister group of Metatheria and Eutheria. Within diapsids, Testudines are the sister group to Aves + Crocodylia (Archosauria), and Sphenodon is the sister group to Squamata. Furthermore, large trait regions flanking the UCE are conserved at family level.

We performed a molecular and a comparative cytogenetic analysis on different Helicoidea species and a review of all the available chromosome data on the superfamily to provide an updated assessment of its karyological diversity. Standard karyotyping, banding techniques, and Fluorescence in situ hybridization of Nucleolus Organizer Region loci (NOR-FISH) were performed on fifteen species of three families: two Geomitridae, four Hygromiidae and nine Helicidae. The karyotypes of the studied species varied from 2 n = 44 to 2 n = 60, highlighting a high karyological diversity. NORs were on a single chromosome pair in Cernuella virgata and on multiple pairs in four Helicidae, representing ancestral and derived conditions, respectively. Heterochromatic C-bands were found on pericentromeric regions of few chromosomes, being Q- and 4',6-diamidino-2-phenylindole (DAPI) negative. NOR-associated heterochromatin was C-banding and chromomycin A 3 (CMA 3 ) positive. Considering the available karyological evidence on Helicoidea and superimposing the chromosome data gathered from different sources on available phylogenetic inferences, we describe a karyotype of 2 n = 60 with all biarmed elements as the ancestral state in the superfamily. From this condition, an accumulation of chromosome translocations led to karyotypes with a lower chromosome number (2 n = 50-44). This process occurred independently in different lineages, while an augment of the chromosome number was detectable in Polygyridae. Chromosome inversions were also relevant chromosome rearrangements in Helicoidea, leading to the formation of telocentric elements in karyotypes with a relatively low chromosome count.

Chameleons display high karyological diversity in chromosome number (from 2n = 20 to 62), morphology, heterochromatin distribution and location of specific chromosomal markers, making them unique study models in evolutionary cytogenetics. However, most available cytogenetic data are limited to the description of the chromosome number and morphology. Concerning sex chromosomes, our knowledge is limited to ZZ/ZW and Z1Z1Z2Z2/Z1Z2W systems in the genus Furcifer and the isolation of sex-linked, male-specific, sequences in Chamaeleo calyptratus, but the putative XY chromosomes have still to be identified in Chamaeleo and the conservation of male heterogamety in the genus needs confirmation from other species. In this study we performed a molecular and a cytogenetic analysis on C. chamaeleon, using standard, banding methods and molecular cytogenetics to provide a throughout karyological characterization of the species and to identify and locate the putative XY chromosomes. We confirm that the chromosome formula of the species is 2n = 24, with 12 metacentric macrochromosomes, 12 microchromosomes and NORs on the second chromosome pair. Heterochromatin was detected as weak C-bands on centromeric regions, differently from what was previously reported for C. calyptratus. Fluorescence in situ hybridization (FISH) showed the occurrence of interspersed telomeric signals on most macrochromosomes, suggesting that ancient chromosome fusions may have led to a reduction of the chromosome number. Using a combination of molecular and FISH analyses, we proved that male specific Restriction site-Associated DNA sequences (RADseq) isolated in C. calyptratus are conserved in C. chamaeleon and located the putative XY chromosomes on the second chromosome pair. We also identified different transposable elements in the focal taxa, which are highly interspersed on most chromosome pairs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The data presented in this paper stand as supplementary information of the associated article "Karyological characterization of the common chameleon ( Chamaeleo chamaeleon ) provides insights on the evolution and diversification of sex chromosomes in Chamaeleonidae" [1]. This work provides (i) raw experimental data on the karyology of the common chameleon Chamaeleo chamaeleon and (ii) the results of bioinformatic analysis on sex-specific and repeated DNA sequences found in the same species. The karyological information here presented includes traditional staining method (Giemsa staining) and sequential C-banding + fluorochromes performed on Tunisian samples of the species. The sequence data include the alignments of the isolated DNA sequences with homologous sequences found in squamate Short Read Archives (SRAs) and the results of searches in public nucleic acid databases., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2020 Published by Elsevier Inc.)

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., (© 2019 S. Karger AG, Basel.)

S1 satellite DNA from Palearctic brown frogs has a species-specific structure in all European species. We characterized S1 satellite DNA from the Anatolian brown frogs Rana macrocnemis, R. camerani, and R. holtzi in order to define their taxonomic rank and the structure of this satellite in this frog lineage. Southern blots of genomic DNA digested with KpnI, EcoRV, NdeI, NheI, or StuI produced the same pattern of satellite DNA bands. Moreover, quantitative dot blots showed that this satellite DNA accounts for 0.1 % of the genome in all taxa. Analysis of the overall genomic variability of the S1a repeat sequence in specimens from various populations demonstrated that this repetitive unit also has the same size (476 bp), the same most common sequence (MCS) and the same overall variability in all three taxa, and also in R. macrocnemis tavasensis. The S1a repetitive unit presents three deletions of 9, 8 and 1 bp compared to the 494-bp S1a repeat from European frogs. The S1a MCS has three variable positions (sequence WWTK in positions 183-186), due to the presence of two repeat subpopulations with motifs AATG and WWTT in all taxa. Unlike previously analyzed mitochondrial and nuclear sequences that show considerable variations among these taxa, no difference could be detected in the structure and variability of the S1 satellite repetitive units. This suggests that these taxa should belong to a single species. Our results indicate that this satellite DNA variety probably formed when the Anatolian lineage radiated from common ancestor about 4 mya, and since then has maintained its structure in all four taxa examined.

We have characterized the S1 satellite from eight European populations of Rana dalmatina by Southern blot, cloning and a new method that determines the sequence variability of repetitive units in the genome. This report completes our previous studies on this satellite DNA family, thus providing the first characterization of the overall variability of the structure and genomic organization of a satellite DNA within a species and among related species. The S1 satellite from R. dalmatina has a pericentromeric location on ten chromosome pairs and presents two homologous repeats S1a (494 bp) and S1b (332 bp), mostly organized as composite S1a-S1b repetitive units. In other brown frog species, both repeats have different sequences and locations, and are usually organized as separate arrays, although composite S1a-S1b repeats represent a minor, widely variable component in Rana italica. The average genomic sequences indicate that the species contains an enormous number of variants of each repeat derived from a unique, species-specific common sequence. The repeat variability is restricted to specific base changes in specific sequence positions in all population samples. Our data show that the structure and evolution of S1 satellite family is not due to crossing-over and gene conversion, but to a mechanism that maintains the ability of the satellite DNA to assemble in constitutive heterochromatin by replacing altered satellite segments with new arrays generated by rolling circle amplification. The mode of action of this repair process not only directly explains the intra- and inter-specific variability of the structure and organization of the S1 satellite repeats from European brown frogs, but also accounts for all general features of satellite DNA in eukaryotes, including its discontinuous evolution. This repair mechanism can maintain the satellite structure in a species indefinitely, but also promote a rapid generation of new variants or types of satellite DNA when environmental conditions favor the formation of new species.

We investigated the overall variability of the S1a satellite DNA repeats in ten European populations of Rana temporaria by a new procedure that determines the average sequence of the repeats in a genome. The average genomic sequences show that only 17% of the S1a repeat sequence (494 bp) is variable. The variable positions contain the same major and minor bases in all or many of the population samples tested, but the percentages of these bases can greatly vary among populations. This indicates the presence in the species of an enormous number of repeats having a different distribution of bases in these variable positions. Individual genomes contain thousands of repeat variants, but these mixtures have very similar characteristics in all populations because they present the same type of restricted and species-specific variability. Southern blots analyses and sequences of cloned S1a repeats fully support this conclusion. The S1 satellite DNA of other European brown frog species also presents properties indicating the same type of variability. This first characterisation of the overall repeat variability of a satellite DNA in a species has revealed features that cannot be determined by gene conversion and crossing over. Our results suggest that a specific directional process based on rolling circle amplification should play a relevant role in the evolution of satellite DNA.

The brown frog Rana graeca was believed to be present in two areas, the Balkan Peninsula and the Italian Apennines. We have characterised the S1 satellite DNA family from Rana graeca graeca and compared it with that of Rana graeca italica. On Southern blots, the patterns of S1 satellite DNA bands are very different between Italian and Greek specimens, but homogeneous among various populations of the same taxon. The satellite DNA from the Greek taxon contains two repetitive units (S1a (494 bp) and S1b (363 bp)) that could be sequenced after amplification from genomic DNA to directly yield their consensus sequences in each genome. These consensus sequences were very similar among the Greek populations, but differed either in sequence (in S1a) or in both size and sequence (in S1b) from the corresponding repeats of the Italian taxon. A mechanism of concerted evolution is likely responsible for the high homogeneity of S1a and S1b repeat sequences within each genome and species. The genomic content of S1 satellite DNA was lower in the Greek than in the Italian populations (0.5 vs. 1.9%) and fluorescence in situ hybridization (FISH) analysis showed the S1 satellite on only 4 chromosome pairs in the Greek taxon and on all 13 chromosome pairs in the Italian taxon. The completely different structure and genomic organization of the S1 satellite DNA indicate that the Greek and Italian taxa are distinct species: R. graeca and R. italica.

1. Liver esterases of the green frog Rana esculenta have been fractionated on disc electrophoresis, thin-layer electrofocusing and column electrofocusing. 2. The enzyme resolves in several molecular forms some of which result sex dependent, since they disappear in castrated animals and can be induced by sex hormone administration. 3. The enzyme molecular forms which depend on female hormones might be involved in cellular modifications of hepatocytes related to yolk protein synthesis. induced by sex hormone administration. 3. The enzyme molecular forms which depend on female hormones might be involved in cellular modifications of hepatocytes related to yolk protein synthesis.