Telomeric-Like Repeats Flanked by Sequences Retrotranscribed from the Telomerase RNA Inserted at DNA Double-Strand Break Sites during Vertebrate Genome Evolution

Interstitial telomeric sequences (ITSs) are stretches of telomeric-like repeats located at internal chromosomal sites. We previously demonstrated that ITSs have been inserted during the repair of DNA double-strand breaks in the course of evolution and that some rodent ITSs, called TERC-ITSs, are flanked by fragments retrotranscribed from the telomerase RNA component (TERC). In this work, we carried out an extensive search of TERC-ITSs in 30 vertebrate genomes and identified 41 such loci in 22 species, including in humans and other primates. The fragment retrotranscribed from the TERC RNA varies in different lineages and its sequence seems to be related to the organization of TERC. Through comparative analysis of TERC-ITSs with orthologous empty loci, we demonstrated that, at each locus, the TERC-like sequence and the ITS have been inserted in one step in the course of evolution. Our findings suggest that telomerase participated in a peculiar pathway of DNA double-strand break repair involving retrotranscription of its RNA component and that this mechanism may be active in all vertebrate species. These results add new evidence to the hypothesis that RNA-templated DNA repair mechanisms are active in vertebrate cells.

Cytogenetically Elusive Sex Chromosomes in Scincoidean Lizards

The lizards of the species-rich clade Scincoidea including cordylids, gerrhosaurids, skinks, and xantusiids, show an almost cosmopolitan geographical distribution and a remarkable ecological and morphological divergence. However, previous studies revealed limited variability in cytogenetic traits. The sex determination mode was revealed only in a handful of gerrhosaurid, skink, and xantusiid species, which demonstrated either ZZ/ZW or XX/XY sex chromosomes. In this study, we explored the karyotypes of six species of skinks, two species of cordylids, and one gerrhosaurid. We applied conventional and molecular cytogenetic methods, including C-banding, fluorescence in situ hybridization with probes specific for telomeric motifs and rDNA loci, and comparative genomic hybridization. The diploid chromosome numbers are rather conserved among these species, but the chromosome morphology, the presence of interstitial telomeric sequences, and the topology of rDNA loci vary significantly. Notably, XX/XY sex chromosomes were identified only in Tiliqua scincoides, where, in contrast to the X chromosome, the Y chromosome lacks accumulations of rDNA loci. We confirm that within the lizards of the scincoidean clade, sex chromosomes remained in a generally poor stage of differentiation.

High heritability of telomere length, but low evolvability, and no significant heritability of telomere shortening in wild jackdaws

Telomere length (TL) and shortening rate predict survival in many organisms. Evolutionary dynamics of TL in response to survival selection depend on the presence of genetic variation that selection can act upon. However, the amount of standing genetic variation is poorly known for both TL and TL shortening rate, and has not been studied for both traits in combination in a wild vertebrate. We used experimental (cross-fostering) and statistical (animal models) means to disentangle and estimate genetic and environmental contributions to TL variation in pedigreed free-living jackdaws (Corvus monedula). TL was measured twice early in life (age- and interval-standardized), when shortening is highest, using the high-precision TRF technique, adapted to exclude interstitial telomeric sequences. TL shortened significantly during the nestling phase (10.4 bp/day), was highly repeatable within individuals (R=0.97) and genetically correlated between the two ages (rG>0.99). Additive genetic effects explained the major part of TL variation between individuals, with heritability on average estimated at h2=0.74. Parent- offspring regressions yielded similar estimates for the sexes when accounting for changes in paternal TL over life. Cohort effects explained a small but significant part of TL variation. Heritable variation for telomere shortening was negligible. Despite the high heritability of TL, its evolvability, which scales the additive genetic variance by mean TL, was close to zero. Hence evolutionary change of TL is likely to be slow despite significant selection.

Rapid chromosomal evolution in the bush-cricket Gonatoxia helleri Hemp, 2016 (Orthoptera, Phaneropterinae)

Gonatoxia helleri Hemp, 2016 is one of the most widespread bush-crickets of the genus Gonatoxia Karsch, 1889 in East Africa. This species with seven large chromosomes (2n♂ = 7) differs from other representatives of the genus Gonatoxia drastically by its reduced chromosome number, the asymmetrical karyotype including karyomorphs rarely found in tettigoniids, as well as in irregularities in the course of meiosis. To better understand the origin of such an exceptional karyotype, chromosomes of 29 specimens from four populations/localities were studied using classical techniques, such as C-banding, silver impregnation, fluorochrome double staining and fluorescence in situ hybridization (FISH) technique with 18S rDNA and (TTAGG)n telomeric probes. FISH showed many 18S rDNA loci as well as interstitial telomeric sequences, where chromosome morphology varied in these components in terms of quantity and distribution. The 18S rDNA loci coincided with active NORs and C-banding patterns. We suggest that a combination of Robertsonian rearrangements and/or multiple common tandem fusions involving the same chromosomes contributed to the formation of this karyotype/karyomorphs. The results are the first step towards a better understanding of chromosomal reorganization and evolution within the genus Gonatoxia. Low chromosome number, together with the incidence of chromosomal polymorphism that is higher in G. helleri than previously reported in bush-crickets, implies that this species can be a valuable new model for cytogenetic and speciation studies. Our findings suggest that chromosomal translocations lead to diversification and speciation in this species and could be the driving force of adaptive radiation.

Insertion of Telomeric Repeats in the Human and Horse Genomes: An Evolutionary Perspective

Interstitial telomeric sequences (ITSs) are short stretches of telomeric-like repeats (TTAGGG)n at nonterminal chromosomal sites. We previously demonstrated that, in the genomes of primates and rodents, ITSs were inserted during the repair of DNA double-strand breaks. These conclusions were derived from sequence comparisons of ITS-containing loci and ITS-less orthologous loci in different species. To our knowledge, insertion polymorphism of ITSs, i.e., the presence of an ITS-containing allele and an ITS-less allele in the same species, has not been described. In this work, we carried out a genome-wide analysis of 2504 human genomic sequences retrieved from the 1000 Genomes Project and a PCR-based analysis of 209 human DNA samples. In spite of the large number of individual genomes analyzed we did not find any evidence of insertion polymorphism in the human population. On the contrary, the analysis of ITS loci in the genome of a single horse individual, the reference genome, allowed us to identify five heterozygous ITS loci, suggesting that insertion polymorphism of ITSs is an important source of genetic variability in this species. Finally, following a comparative sequence analysis of horse ITSs and of their orthologous empty loci in other Perissodactyla, we propose models for the mechanism of ITS insertion during the evolution of this order.

Interspecific cytogenetic relationships in three Acestrohynchus species (Acestrohynchinae, Characiformes) reveal the existence of possible cryptic species

The karyotypes and chromosomal characteristics of three Acestrorhynchus Eigenmann et Kennedy, 1903 species were examined using conventional and molecular protocols. These species had invariably a diploid chromosome number 2n = 50. Acestrorhynchus falcatus (Block, 1794) and Acestrorhynchus falcirostris (Cuvier, 1819) had the karyotype composed of 16 metacentric (m) + 28 submetacentric (sm) + 6 subtelocentric (st) chromosomes while Acestrorhynchus microlepis (Schomburgk, 1841) had the karyotype composed of 14m+30sm+6st elements. In this species, differences of the conventional and molecular markers between the populations of Catalão Lake (AM) and of Apeu Stream (PA) were found. Thus the individuals of Pará (Apeu) were named Acestrorhynchus prope microlepis. The distribution of the constitutive heterochromatin blocks was species-specific, with C-positive bands in the centromeric and telomeric regions of a number of different chromosomes, as well as in interstitial sites and completely heterochromatic arms. The phenotypes of nucleolus organizer region (NOR) were simple, i. e. in a terminal position on the p arm of pair No. 23 except in A. microlepis, in which it was located on the q arm. Fluorescence in situ hybridization (FISH) revealed 18S rDNA sites on one chromosome pair in karyotype of A. falcirostris and A. prope microlepis (pair No. 23) and three pairs (Nos. 12, 23, 24) in A. falcatus and (Nos. 8, 23, 24) in A. microlepis; 5S rDNA sites were detected in one chromosome pair in all three species. The mapping of the telomeric sequences revealed terminal sequences in all the chromosomes, as well as the presence of interstitial telomeric sequences (ITSs) in a number of chromosome pairs. The cytogenetic data recorded in the present study indicate that A. prope microlepis may be an unnamed species.

A Case of Trisomy 13 Mosaicism Presenting with a Severe Aortic Root Dilatation and Marfanoid Habitus due to an Unpredictable Cytogenetic Mechanism

In this report, we present a new case of mosaic trisomy 13 with prolonged survival, firstly detected by array-CGH analysis which was carried out because of moderate intellectual disability with postaxial hexadactyly, dermatologic features, ventricular septal defect, bicuspid aortic valve, and aortic dystrophy in a 19-year-old male patient. In a subset of 15% of the cells, the patient carried a derivative chromosome 10 generated by a nonreciprocal (10;13) translocation inherited from his healthy mother who carried the translocation in a balanced and homogeneous state. FISH analyses showed interstitial telomeric sequences at the breakpoints. To our knowledge, this is the second report of a patient with trisomy 13 mosaicism displaying a severe aortic root dilatation. We also discuss the mechanisms which could explain the mosaic state, the most likely one being related to the instability of the interstitial telomere.

Chromosome spreading of the (TTAGGG)n repeats in the Pipa carvalhoi Miranda-Ribeiro, 1937 (Pipidae, Anura) karyotype

Pipidae is a clade of Anura that diverged relatively early from other frogs in the phylogeny of the group. Pipids have a unique combination of morphological features, some of which appear to represent a mix of adaptations to aquatic life and plesiomorphic characters of Anura. The present study describes the karyotype of Pipa carvalhoi Miranda-Ribeiro, 1937, including morphology, heterochromatin distribution, and location of the NOR site. The diploid number of P. carvalhoi is 2n=20, including three metacentric pairs (1, 4, 8), two submetacentric (2 and 7), three subtelocentric (3, 5, 6), and two telocentric pairs (9 and 10). C-banding detected centromeric blocks of heterochromatin in all chromosome pairs and the NOR detected in chromosome pair 9, as confirmed by FISH using the rDNA 28S probe. The telomeric probes indicated the presence of interstitial telomeric sequences (ITSs), primarily in the centromeric region of the chromosomes, frequently associated with heterochromatin, suggesting that these repeats are a significant component of this region. The findings of the present study provide important insights for the understanding of the mechanisms of chromosomal evolution in the genus Pipa, and the diversification of the Pipidae as a whole.

Assessing the Epigenetic Status of Human Telomeres

The epigenetic modifications of human telomeres play a relevant role in telomere functions and cell proliferation. Therefore, their study is becoming an issue of major interest. These epigenetic modifications are usually analyzed by microscopy or by chromatin immunoprecipitation (ChIP). However, these analyses could be challenged by subtelomeres and/or interstitial telomeric sequences (ITSs). Whereas telomeres and subtelomeres cannot be differentiated by microscopy techniques, telomeres and ITSs might not be differentiated in ChIP analyses. In addition, ChIP analyses of telomeres should be properly controlled. Hence, studies focusing on the epigenetic features of human telomeres have to be carefully designed and interpreted. Here, we present a comprehensive discussion on how subtelomeres and ITSs might influence studies of human telomere epigenetics. We specially focus on the influence of ITSs and some experimental aspects of the ChIP technique on ChIP analyses. In addition, we propose a specific pipeline to accurately perform these studies. This pipeline is very simple and can be applied to a wide variety of cells, including cancer cells. Since the epigenetic status of telomeres could influence cancer cells proliferation, this pipeline might help design precise epigenetic treatments for specific cancer types.