Chromosome Painting in Cercopithecus petaurista (Schreber, 1774) Compared to Other Monkeys of the Cercopithecini Tribe (Catarrhini, Primates).

The Cercopithecini tribe includes terrestrial and arboreal clades whose relationships are controversial, with a high level of chromosome rearrangements. In order to provide new insights on the tribe's phylogeny, chromosome painting, using the complete set of human syntenic probes, was performed in Cercopithecus petaurista, a representative species of the Cercopithecini tribe. The results show C. petaurista with a highly rearranged karyotype characterized by the fission of human chromosomes 1, 2, 3, 5, 6, 8, 11, and 12. These results compared with the literature data permit us to confirm the monophyly of the Cercopithecini tribe (fissions of chromosomes 5 and 6), as previously proposed by chromosomal and molecular data. Furthermore, we support the monophyly of the strictly arboreal Cercopithecus clade, previously proposed by the molecular approach, identifying chromosomal synapomorphies (fissions of chromosomes 1, 2, 3, 11, 12). We also add additional markers that can be useful for deciphering arboreal Cercopithecini phylogeny. For example, the fission of chromosome 8 is synapomorphy linking C. petaurista, C. erythrogaster, and C. nictitans among the arboreal species. Finally, a telomeric sequence probe was mapped on C. petaurista, showing only classic telomeric signals and giving no support to a previous hypothesis regarding a link between interspersed telomeric sequences in high rearranged genomes.

Ciona robusta macrophage migration inhibitory factor (Mif1 and Mif2) genes are differentially regulated in the lipopolysaccharide-challenged pharynx.

The effects of lipopolysaccharide (LPS) on Mif (macrophage migration inhibitory factor) gene expression in the pharynx (haemapoetic tissue) of Ciona robusta were investigated using quantitative reverse-transcription PCR (qRT-PCR) and in situ hybridisation (ISH). To verify the induction of an inflammatory response in the pharynx, a qRT-PCR analysis was performed to evaluate the change in the expression of proinflammatory marker genes such as Mbl, Ptx-like, Tnf-α and Nf-kb, which were shown to be upregulated 1 h post LPS challenge. The change in the expression of the two Mif paralogs in the pharynx was assessed before and after stimulation, and qRT-PCR and ISH results showed that, although Mif2 and Mif2 were expressed in clusters of haemocytes in pharynx vessels, only Mif1 expression increased after LPS stimulation. This indicates that the Mif genes are differently regulated and respond to different ambient inputs that need further analysis.

Transcriptomic and Bioinformatic Analyses Identifying a Central Mif-Cop9-Nf-kB Signaling Network in Innate Immunity Response of Ciona robusta.

The Ascidian C. robusta is a powerful model for studying innate immunity. LPS induction activates inflammatory-like reactions in the pharynx and the expression of several innate immune genes in granulocyte hemocytes such as cytokines, for instance, macrophage migration inhibitory factors (CrMifs). This leads to intracellular signaling involving the Nf-kB signaling cascade that triggers downstream pro-inflammatory gene expression. In mammals, the COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) complex also results in the activation of the NF-kB pathway. It is a highly conserved complex in vertebrates, mainly engaged in proteasome degradation which is essential for maintaining processes such as cell cycle, DNA repair, and differentiation. In the present study, we used bioinformatics and in-silico analyses combined with an in-vivo LPS exposure strategy, next-generation sequencing (NGS), and qRT-PCR to elucidate molecules and the temporal dynamics of Mif cytokines, Csn signaling components, and the Nf-κB signaling pathway in C. robusta. A qRT-PCR analysis of immune genes selected from transcriptome data revealed a biphasic activation of the inflammatory response. A phylogenetic and STRING analysis indicated an evolutionarily conserved functional link between the Mif-Csn-Nf-kB axis in ascidian C. robusta during LPS-mediated inflammation response, finely regulated by non-coding molecules such as microRNAs (miRNAs).

Mapping Retrotransposon LINE-1 Sequences into Two Cebidae Species and Homo sapiens Genomes and a Short Review on Primates.

This work focuses on the distribution of LINE-1 (a Long Interspersed Nuclear Element) in primates and its role during evolution and as a constituent of the architecture of primate genomes. To pinpoint the LINE-1 repeat distribution and its role among primates, LINE-1 probes were mapped onto chromosomes of Homo sapiens (Hominidae, Catarrhini), Sapajus apella, and Cebus capucinus (Cebidae, Platyrrhini) using fluorescence in situ hybridisation (FISH). The choice of platyrrhine species are due to the fact they are taxa characterised by a high level of rearrangements; for this reason, they could be a useful model for the study of LINE-1 and chromosome evolution. LINE-1 accumulation was found in the two Cebidae at the centromere of almost all acrocentric chromosomes 16-22 and on some bi-armed chromosomes. LINE-1 pattern was similar in the two species but only for chromosomes 6, 8, 10, and 18, due to intrachromosomal rearrangements in agreement with what was previously hypothesised as through g banding. LINE-1 interstitial accumulation was found in humans on the 1, 8, 9, 13-15, and X chromosomes; on chromosomes 8, 9, and 13-15, the signal was also at the centromeric position. This is in agreement with recent and complete molecular sequence analysis of human chromosomes 8 and some acrocentric ones. Thus, the hypothesis regarding a link between LINE-1 and centromeres as well as a link with rearrangements are discussed. Indeed, data analysis leads us to support a link between LINE-1 and inter- and intrachromosomal rearrangements, as well as a link between LINE-1 and structural functions at centromeres in primates.

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.

Evolutionary and transcriptional analyses of a pentraxin-like component family involved in the LPS inflammatory response of Ciona robusta.

Pentraxins (PTXs) are a superfamily of conserved proteins which are components of the humoral arm of innate immunity. They are considered to be functional ancestors of antibodies and are classified into short and long types. In this study, we show that a pentraxin-like component (Ptx-like) with a C-terminal PTX domain, highly homologous to the short PTX of H. sapiens CRP, and a long N-terminal domain typical of long PTXs, is involved in the inflammatory response of Ciona robusta under LPS exposure in vivo. Analyses of protein domains as well as 3D modelling and phylogenetic tree supported the close relationship of Ptx-like with mammalian CRP, suggesting that C. robusta Ptx-like shares a common ancestor in the chordate lineages. qRT-PCR analysis showed that Ptx-like was transcriptionally upregulated during the inflammatory process induced by LPS inoculation and that it is involved in the initial phase as well as the secondary phase of the inflammatory response in which matrix remodelling and the achievement of homeostasis occur. In situ hybridisation assays revealed that gene transcription was upregulated in the pharynx post-LPS challenge in vivo, and that Ptx-like was expressed by clusters of haemocytes, mainly granulocytes, inside the pharynx vessels. We also found transcript-expressing granulocytes flowing in the musculature and in the lacunae of the circulatory system. These data supported that Ptx-like is a potential molecule of the acute-phase response in C. robusta immune defence systems against bacterial infection.

Molecular Cytogenetic Characterization of the Sicilian Endemic Pond Turtle Emys trinacris and the Yellow-Bellied Slider Trachemys scripta scripta (Testudines, Emydidae).

Turtles, a speciose group consisting of more than 300 species, demonstrate karyotypes with diploid chromosome numbers ranging from 2n = 26 to 2n = 68. However, cytogenetic analyses have been conducted only to 1/3rd of the turtle species, often limited to conventional staining methods. In order to expand our knowledge of the karyotype evolution in turtles, we examined the topology of the (TTAGGG)n telomeric repeats and the rDNA loci by fluorescence in situ hybridization (FISH) on the karyotypes of two emydids: the Sicilian pond turtle, Emys trinacris, and the yellow-bellied slider, Trachemys scripta scripta (family Emydidae). Furthermore, AT-rich and GC-rich chromosome regions were detected by DAPI and CMA3 stains, respectively. The cytogenetic analysis revealed that telomeric sequences are restricted to the terminal ends of all chromosomes and the rDNA loci are localized in one pair of microchromosomes in both species. The karyotype of the Sicilian endemic E. trinacris with diploid number 2n = 50, consisting of 13 pairs of macrochromosomes and 12 pairs of microchromosomes, is presented here for first time. Our comparative examination revealed similar cytogenetic features in Emys trinacris and the closely related E. orbicularis, as well as to other previously studied emydid species, demonstrating a low rate of karyotype evolution, as chromosomal rearrangements are rather infrequent in this group of turtles.

Evolution of the Human Chromosome 13 Synteny: Evolutionary Rearrangements, Plasticity, Human Disease Genes and Cancer Breakpoints.

The history of each human chromosome can be studied through comparative cytogenetic approaches in mammals which permit the identification of human chromosomal homologies and rearrangements between species. Comparative banding, chromosome painting, Bacterial Artificial Chromosome (BAC) mapping and genome data permit researchers to formulate hypotheses about ancestral chromosome forms. Human chromosome 13 has been previously shown to be conserved as a single syntenic element in the Ancestral Primate Karyotype; in this context, in order to study and verify the conservation of primate chromosomes homologous to human chromosome 13, we mapped a selected set of BAC probes in three platyrrhine species, characterised by a high level of rearrangements, using fluorescence in situ hybridisation (FISH). Our mapping data on Saguinus oedipus, Callithrix argentata and Alouatta belzebul provide insight into synteny of human chromosome 13 evolution in a comparative perspective among primate species, showing rearrangements across taxa. Furthermore, in a wider perspective, we have revised previous cytogenomic literature data on chromosome 13 evolution in eutherian mammals, showing a complex origin of the eutherian mammal ancestral karyotype which has still not been completely clarified. Moreover, we analysed biomedical aspects (the OMIM and Mitelman databases) regarding human chromosome 13, showing that this autosome is characterised by a certain level of plasticity that has been implicated in many human cancers and diseases.

Chromosomal Localization of 18S-28S rDNA and (TTAGGG)n Sequences in Two South African Dormice of the Genus Graphiurus (Rodentia: Gliridae).

Classical cytogenetics and mapping of 18S-28S rDNA and (TTAGGG)n sequences by fluorescence in situ hybridization (FISH) was performed on Graphiurus platyops (GPL) and Graphiurus ocularis (GOC) metaphases with the aim to characterize the genomes. In both species, inverted DAPI karyotypes showed the same diploid number, 2n = 46, and hybridization of the (TTAGGG)n probe revealed interstitial telomeric sequences (ITSs) at the centromeres of almost all bi-armed chromosomes. FISH with the rDNA probe localized nucleolus organizer regions (NORs), at the terminal ends of the p arms of the subtelocentric pairs 16 and 17 in both species and detected additional signals on GPL8 and GOC18, 19, and 22. The species have similar karyotypes, but their chromosome pairs 18-22 differ in morphology; these are acrocentric in G. platyops, as also confirmed by C-banding, and subtelocentric in G. ocularis. These differences in pairs 18-22 were also highlighted by hybridization of the telomeric probe (TTAGGG)n, which showed the small p arms in G. ocularis enriched with ITSs. FISH of rDNA probes detected multiple NOR loci in G. ocularis, underlining the intense evolutionary dynamics related to these genes. Although the Graphiurus species analyzed have similar karyotypes, the results on the repetitive sequences indicate a complex pattern of genomic reorganization and evolution occurring in these phylogenetically close species.

Evolutionary insight on localization of 18S, 28S rDNA genes on homologous chromosomes in Primates genomes.

We explored the topology of 18S and 28S rDNA units by fluorescence in situ hybridization (FISH) in the karyotypes of thirteen species representatives from major groups of Primates and Tupaia minor (Günther, 1876) (Scandentia), in order to expand our knowledge of Primate genome reshuffling and to identify the possible dispersion mechanisms of rDNA sequences. We documented that rDNA probe signals were identified on one to six pairs of chromosomes, both acrocentric and metacentric ones. In addition, we examined the potential homology of chromosomes bearing rDNA genes across different species and in a wide phylogenetic perspective, based on the DAPI-inverted pattern and their synteny to human. Our analysis revealed an extensive variability in the topology of the rDNA signals across studied species. In some cases, closely related species show signals on homologous chromosomes, thus representing synapomorphies, while in other cases, signal was detected on distinct chromosomes, leading to species specific patterns. These results led us to support the hypothesis that different mechanisms are responsible for the distribution of the ribosomal DNA cluster in Primates.

Distribution of Interstitial Telomeric Sequences in Primates and the Pygmy Tree Shrew (Scandentia).

It has been hypothesized that interstitial telomeric sequences (ITSs), i.e., repeated telomeric DNA sequences found at intrachromosomal sites in many vertebrates, could be correlated to chromosomal rearrangements and plasticity. To test this hypothesis, we hybridized a telomeric PNA probe through FISH on representative species of 2 primate infraorders, Strepsirrhini (Lemur catta, Otolemur garnettii, Nycticebus coucang) and Catarrhini (Erythrocebus patas, Cercopithecus petaurista, Chlorocebus aethiops, Colobus guereza), as well as on 1 species of the order Scandentia, Tupaia minor, used as an outgroup for primates in phylogenetic reconstructions. In almost all primate species analyzed, we found a telomeric pattern only. In Tupaia, the hybridization revealed many bright ITSs on at least 11 chromosome pairs, both biarmed and acrocentric. These ITS signals in Tupaia correspond to fusion points of ancestral human syntenic associations, but are also present in other chromosomes showing synteny to only a single human chromosome. This distribution pattern was compared to that of the heterochromatin regions detected through sequential C-banding performed after FISH. Our results in the analyzed species, compared with literature data on ITSs in primates, allowed us to discuss different mechanisms responsible for the origin and distribution of ITSs, supporting the correlation between rearrangements and ITSs.

Exploring evolution in Ceboidea (Platyrrhini, primates) by Williams-Beuren probe (HSA 7q11.23) chromosome mapping.

The ancestral platyrrhine karyotype was characterised by a syntenic association of human 5 and a small segment of human 7 orthologues. This large syntenic association has undergone numerous rearrangements in various phylogenetic lines. We used a locus-specific molecular cytogenetic approach to study the chromosomal evolution of the human 7q11.23 orthologous sequences (William-Beuren syndrome, WS) in various Ceboidea (Platyrrhini) species. The fluorescent in situ hybridisation of the WS probe revealed a two-way pattern of chromosomal organisation that suggests various evolutionary scenarios. The first pattern (seen in Callimico and Saimiri) includes a fairly simple disruption of the 7/5 syntenic association by a chromosome fission. The second pattern (seen in Atelinae, Alouattinae and in Callicebus) is characterised by an increasing complexity in the 7/5 association as a consequence of a series of inversions and translocations resulting in different syntenic associations. These data support recent proposals for phylogenomic groupings of New World monkeys. The study also illustrates how single-locus probe hybridisations can reveal intrachromosomal rearrangements.

Phylogenomics of species from four genera of New World monkeys by flow sorting and reciprocal chromosome painting.

BACKGROUND: The taxonomic and phylogenetic relationships of New World monkeys (Platyrrhini) are difficult to distinguish on the basis of morphology and because diagnostic fossils are rare. Recently, molecular data have led to a radical revision of the traditional taxonomy and phylogeny of these primates. Here we examine new hypotheses of platyrrhine evolutionary relationships by reciprocal chromosome painting after chromosome flow sorting of species belonging to four genera of platyrrhines included in the Cebidae family: Callithrix argentata (silvered-marmoset), Cebuella pygmaea (pygmy marmoset), Callimico goeldii (Goeldi's marmoset) and Saimiri sciureus (squirrel monkey). This is the first report of reciprocal painting in marmosets. RESULTS: The paints made from chromosome flow sorting of the four platyrrhine monkeys provided from 42 to 45 hybridization signals on human metaphases. The reciprocal painting of monkey probes on human chromosomes revealed that 21 breakpoints are common to all four studied species. There are only three additional breakpoints. A breakpoint on human chromosome 13 was found in Callithrix argentata, Cebuella pygmaea and Callimico goeldii, but not in Saimiri sciureus. There are two additional breakpoints on human chromosome 5: one is specific to squirrel monkeys, and the other to Goeldi's marmoset. CONCLUSION: The reciprocal painting results support the molecular genomic assemblage of Cebidae. We demonstrated that the five chromosome associations previously hypothesized to phylogenetically link tamarins and marmosets are homologous and represent derived chromosome rearrangements. Four of these derived homologous associations tightly nest Callimico goeldii with marmosets. One derived association 2/15 may place squirrel monkeys within the Cebidae assemblage. An apparently common breakpoint on chromosome 5q33 found in both Saimiri and Aotus nancymae could be evidence of a phylogenetic link between these species. Comparison with previous reports shows that many syntenic associations found in platyrrhines have the same breakpoints and are homologous, derived rearrangements showing that the New World monkeys are a closely related group of species. Our data support the hypothesis that the ancestral karyotype of the Platyrrhini has a diploid number of 2n = 54 and is almost identical to that found today in capuchin monkeys; congruent with a basal position of the Cebidae among platyrrhine families.