Chromosomes, Nuclear Genes and the Phylogenetic Placement Within the Reptilia of Sphenodon (Tuatara)

<p>Chromosomes were examined from five populations of Sphenodon (tuatara) using giemsa, Ag-NOR, C-, G- and RE- banding. There were no differences between species, populations or sexes, although one animal had a structural heteromorphism. Chromosome morphology homology to Testudines (turtles), Aves (birds) and to a lesser extent Crocodylia (crocodiles) allowed reconstruction ofa Reptilian proto-karyotype, dated to 300 million years ago. DNA sequence was isolated from the WT1, AMH, DMRT1, FoxG1 and 28S. No variation was present in Sphenodon 28S, FoxG1 or AMH sequence. 28S could be dated to a common ancestor with Testudines, similar to the archaic karyotype. FoxG1 and AMH reflect an Oligocene divergence, WT1 divides north-eastern North Island and Cook Strait, and can be dated to the Pleistocene or the Pliocene, and DMRT1appears a recent post- pliocene divergence. FISH localised DIG-labelled probes of AMH to chromosome 11 and WT1 to chromosome 13 or 14. Human telomeric probes localised to Sphenodon telomeric regions demonstrating the highly conserved nature of telomeric sequences. Comparative genomic hybridisation with chicken chromosomes did not produce any regions of homology, implying significant chromosomal and DNA changes since the Orders shared a common ancestor, although macrochromosome morphology has remained similar. Sphenodon chromosomal and nuclear DNA analyses demonstrate evolutionary decoupling, supporting recent mtDNA work.</p>

Chromosomes, Nuclear Genes and the Phylogenetic Placement Within the Reptilia of Sphenodon (Tuatara)

<p>Chromosomes were examined from five populations of Sphenodon (tuatara) using giemsa, Ag-NOR, C-, G- and RE- banding. There were no differences between species, populations or sexes, although one animal had a structural heteromorphism. Chromosome morphology homology to Testudines (turtles), Aves (birds) and to a lesser extent Crocodylia (crocodiles) allowed reconstruction ofa Reptilian proto-karyotype, dated to 300 million years ago. DNA sequence was isolated from the WT1, AMH, DMRT1, FoxG1 and 28S. No variation was present in Sphenodon 28S, FoxG1 or AMH sequence. 28S could be dated to a common ancestor with Testudines, similar to the archaic karyotype. FoxG1 and AMH reflect an Oligocene divergence, WT1 divides north-eastern North Island and Cook Strait, and can be dated to the Pleistocene or the Pliocene, and DMRT1appears a recent post- pliocene divergence. FISH localised DIG-labelled probes of AMH to chromosome 11 and WT1 to chromosome 13 or 14. Human telomeric probes localised to Sphenodon telomeric regions demonstrating the highly conserved nature of telomeric sequences. Comparative genomic hybridisation with chicken chromosomes did not produce any regions of homology, implying significant chromosomal and DNA changes since the Orders shared a common ancestor, although macrochromosome morphology has remained similar. Sphenodon chromosomal and nuclear DNA analyses demonstrate evolutionary decoupling, supporting recent mtDNA work.</p>

Comparative Cytogenetics Analysis Among Peckoltia Species (Siluriformes, Loricariidae): Insights on Karyotype Evolution and Biogeography in the Amazon Region

Peckoltia is widely distributed genus in the Amazon and Orinoco basins and the Guiana Shield, containing 18 valid species, and distinct morphotypes still needing description in the scientific literature due to its great taxonomic complexity. This study performed a comparative chromosomal analysis of two undescribed Peckoltia species (Peckoltia sp. 3 Jarumã and Peckoltia sp. 4 Caripetuba) from the Brazilian Amazon using conventional chromosome bands methods and in situ localization of the repetitive DNA (5S and 18S rRNA and U1 snRNA genes and telomeric sequences). Both species presented 2n = 52 but differed in their karyotype formula, probably due to inversions or translocations. The nucleolus organizer regions (NORs) showed distal location on a probably homeologous submetacentric pair in both species, besides an extra signal in a subtelocentric chromosome in Peckoltia sp. 4 Caripetuba. Heterochromatin occurred in large blocks, with different distributions in the species. The mapping of the 18S and 5S rDNA, and U1 snDNA showed differences in locations and number of sites. No interstitial telomeric sites were detected using the (TTAGGG)n probes. Despite 2n conservationism in Peckoltia species, the results showed variation in karyotype formulas, chromosomal bands, and locations of repetitive sites, demonstrating great chromosomal diversity. A proposal for Peckoltia karyotype evolution was inferred in this study based on the diversity of location and number of chromosomal markers analyzed. A comparative analysis with other Peckoltia karyotypes described in the literature, their biogeography patterns, and molecular phylogeny led to the hypothesis that the derived karyotype was raised in the left bank of the Amazon River.

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.

Multiple hPOT1–TPP1 cooperatively unfold contiguous telomeric G-quadruplexes proceeding from 3′ toward 5′, a feature due to a 3′-end binding preference and to structuring of telomeric DNA

Telomeres are DNA repeated sequences that associate with shelterin proteins and protect the ends of eukaryotic chromosomes. Human telomeres are composed of 5′TTAGGG repeats and ends with a 3′ single-stranded tail, called G-overhang, that can be specifically bound by the shelterin protein hPOT1 (human Protection of Telomeres 1). In vitro studies have shown that the telomeric G-strand can fold into stable contiguous G-quadruplexes (G4). In the present study we investigated how hPOT1, in complex with its shelterin partner TPP1, binds to telomeric sequences structured into contiguous G4 in potassium solutions. We observed that binding of multiple hPOT1–TPP1 preferentially proceeds from 3′ toward 5′. We explain this directionality in terms of two factors: (i) the preference of hPOT1–TPP1 for the binding site situated at the 3′ end of a telomeric sequence and (ii) the cooperative binding displayed by hPOT1–TPP1 in potassium. By comparing binding in K+ and in Li+, we demonstrate that this cooperative behaviour does not stem from protein-protein interactions, but from structuring of the telomeric DNA substrate into contiguous G4 in potassium. Our study suggests that POT1-TPP1, in physiological conditions, might preferentially cover the telomeric G-overhang starting from the 3′-end and proceeding toward 5′.

Evolutionary dynamics of pseudoautosomal region 1 in humans and great apes

The pseudoautosomal region 1 (PAR1) is a 2.7 Mb telomeric region of human sex chromosomes. As the largest point of contact between the X and Y, PAR1 has a crucial role in ensuring proper segregation of sex chromosomes during male meiosis, exposing it to extreme recombination and associated mutational processes. We investigate PAR1 evolution using population genomic datasets of extant humans, eight populations of great apes and two archaic human genome sequences. We find that the PAR1 sequence is closer to nucleotide equilibrium than autosomal telomeric sequences. We detect a difference between long-term substitution patterns and extant diversity in PAR1 that is mainly driven by the conflict between strong mutation and recombination-associated fixation bias at CpG sites. Additionally, we detect excess C→G mutations in PAR1 of all great ape species, specific to the mutagenic effect of male recombination. Analysis of differences between frequencies of alleles segregating in females and males provided no evidence for sexually antagonistic selection in this region. Furthermore, despite recent evidence for Y chromosome introgression from humans into Neanderthals, we find that the Neanderthal PAR1 retained similarity to the Denisovan sequence, as is the case for the X chromosome and the autosomes. Lastly, we study repeat content and double-strand break hotspot regions in PAR1 and find that they may play roles in ensuring the obligate X-Y recombination event during male meiosis. Our study provides an unprecedented quantification of population genetic forces and insight into evolutionary processes governing PAR1 biology.

Repetitive Sequence Distribution on Saguinus, Leontocebus and Leontopithecus Tamarins (Platyrrhine, Primates) by Mapping Telomeric (TTAGGG) Motifs and rDNA Loci

Tamarins are a distinct group of small sized New World monkeys with complex phylogenetic relationships and poorly studied cytogenetic traits. In this study, we applied molecular cytogenetic analyses by fluorescence in situ hybridization with probes specific for telomeric sequences and ribosomal DNA loci after DAPI/CMA3 staining on metaphases from five tamarin species, namely Leontocebus fuscicollis, Leontopithecus rosalia, Saguinus geoffroyi, Saguinus mystax and Saguinus oedipus, with the aim to investigate the distribution of repetitive sequences and their possible role in genome evolution. Our analyses revealed that all five examined species show similar karyotypes, 2n = 46, which differ mainly in the morphology of chromosome pairs 16–17 and 19–22, due to the diverse distribution of rDNA loci, the amplification of telomeric-like sequences, the presence of heterochromatic blocks and/or putative chromosomal rearrangements, such as inversions. The differences in cytogenetic traits between species of tamarins are discussed in a comparative phylogenetic framework, and in addition to data from previous studies, we underline synapomorphies and apomorphisms that appeared during the diversification of this group of New World monkeys.

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.

The immediate early protein 1 of the human herpesvirus 6B counteracts NBS1 and prevents homologous recombination repair pathways

Integration of viral DNA in the genome of host cells triggers host-pathogens interaction that are consequential for the virus and the infected cells. In cells semi-permissive for viral replication, the human herpesvirus 6B (HHV-6B) integrates its genome into the host telomeric sequences. Interestingly, HHV-6B integration in gametes leads to a condition called inherited chromosomally integrated HHV-6B (iciHHV-6B), where the newborn carries a copy of HHV-6B in every cell of its body and is associated with health issues such as spontaneous abortion rates, pre-eclampsia and angina pectoris when transmitted to its offspring. Unlike retroviruses, the mechanism that leads to viral integration of DNA viruses and the consequences of these events on host cells are not well characterized. Here, we report that HHV-6B infection induce genomic instability by suppressing the ability of the host cell to sense DNA double-strand break (DSB). We discovered that this phenotype is mediated by the ability of the immediate-early HHV-6B protein IE1 to bind, delocalize, and inhibit the functions of the DNA damage sensor NBS1. These results raise the possibility that the genomic instability induced by the expression of IE1 from integrated genomes contributes to the development of iciHHV-6B-associated disease. As reported for other types of viruses, the inhibition of DSB sensing and signaling promotes viral replication. However, HHV-6B integration is not affected when this pathway is inhibited, supporting models where integration of the viral genome at telomeric sequence is dictated by mechanisms that promote telomere-elongation in a given infected cell and not solely DNA repair mechanisms.

Telomeric and Sub-Telomeric Structure and Implications in Fungal Opportunistic Pathogens

Telomeres are long non-coding regions found at the ends of eukaryotic linear chromosomes. Although they have traditionally been associated with the protection of linear DNA ends to avoid gene losses during each round of DNA replication, recent studies have demonstrated that the role of these sequences and their adjacent regions go beyond just protecting chromosomal ends. Regions nearby to telomeric sequences have now been identified as having increased variability in the form of duplications and rearrangements that result in new functional abilities and biodiversity. Furthermore, unique fungal telomeric and chromatin structures have now extended clinical capabilities and understanding of pathogenicity levels. In this review, telomere structure, as well as functional implications, will be examined in opportunistic fungal pathogens, including Aspergillus fumigatus, Candida albicans, Candida glabrata, and Pneumocystis jirovecii.