High BrdU Sensitivity of Passeriformes Chromosomes: Conservation of BrdU-Sensitive Fragile Sites on Their Z Chromosomes during Evolution.

Amongst 15 bird species, representative of 7 orders, recurrent breakages evocating the presence of fragile sites were detected in the chromosomes of the 5 species belonging to Passeriformes. These breaks appeared when 5-bromodeoxyuridine (BrdU) was added to the cell culture medium at a dose inefficient for inducing chromosome structure alterations in other birds and mammals. They involved, similarly in male and female, 3 loci on the Z chromosome of 3 Turdus species (Turdidae). Labeling by BrdU antibody confirmed the correlation between BrdU incorporation into DNA and breakage, especially around and in the sites of breakage. Thus, 3 BrdU-sensitive fragile sites were present in the Z chromosomes of these birds. Three fragile sites were also detected at different locations in the Z chromosomes of the European robin (Erithacus rubecula, Muscicapidae), suggesting that a structural rearrangement occurred during the evolution of Turdidae and Muscicapidae. Chromosome banding confirmed this interpretation. Finally, in the more distantly related species Parus major (Paridae), the almost acrocentric Z chromosome displayed a single BrdU-sensitive fragile site in its short arm, and the W appeared to be pulverized by BrdU incorporation. Although it cannot be excluded that the BrdU-sensitive fragile sites may be involved in rearrangements, their conservation in many species, and possibly all Passeriformes, provides evidence that they do not constitute a pejorative character during evolution.

The Relationship between the (In-)Stability of NORs and Their Chromosomal Location: The Example of Cercopithecidae and a Short Review of Other Primates.

Amongst Cercopithecidae, the species of the Cercopithecini tribe underwent a very active chromosome evolution, principally by fissions, which increased their chromosome number up to 72. In contrast, all the species of Papionini have fairly similar karyotypes with 42 chromosomes. In animals, nucleolus organizer regions (NORs) are generally considered as instable structures, which frequently vary in size, number, and location at both infra- and interspecific levels. Although in Cercopithecinae the NORs, involved in breaks, exchanges, and translocations, behave like fragile sites in somatic cells, their number and location appear to be very stable between species. Fluorescence in situ hybridization of a 28S rDNA probe on metaphase chromosomes displayed a unique interstitial location in either an acrocentric pair (in 12 species of Cercopithecini) or a metacentric pair (in 6 species of Papionini). A non-exhaustive survey of literature data on NOR location in other primates shows that numerical variations of the NORs principally depend on their location: most multiple NORs are in terminal positions, while almost all unique NORs are in interstitial positions. We propose that this correlation is the consequence of the selection against gametic imbalances involving the chromosomal material distal to the NORs, which is effective when they are interstitially, but not terminally, located. Thus, the consequences of the interstitial NOR instability for reproduction are essentially limited to their size variations, as observed in Cercopithecidae.

Karyotype diversity suggests that Laonastes aenigmamus (Laotian rock rat) (Rodentia, Diatomyidae) is a multi-specific genus.

Laonastes aenigmamus (Khanyou) is a recently described rodent species living in geographically separated limestone formations of the Khammuan Province in Lao PDR. Chromosomes of 21 specimens of L. aenigmamus were studied using chromosome banding as well as fluorescent in situ hybridization (FISH) techniques using human painting, telomere repeats, and 28S rDNA probes. Four different karyotypes were established. Study with human chromosome paints and FISH revealed that four large chromosomes were formed by multiple common tandem fusions, with persistence of some interstitial telomeres. The rearrangements separating the different karyotypes (I to IV) were also reconstructed. Various combinations of Robertsonian translocations or tandem fusions involving the same chromosomes differentiate these karyotypes. These rearrangements create a strong gametic barrier, which isolates specimens with karyotype II from the others. C-banding and FISH with telomere repeats also exhibit large and systematized differences between karyotype II and others. These data indicate an ancient reproductive separation and suggest that Laonastes is not a mono-specific genus.

Diversity and distribution of alpha satellite DNA in the genome of an Old World monkey: Cercopithecus solatus.

BACKGROUND: Alpha satellite is the major repeated DNA element of primate centromeres. Evolution of these tandemly repeated sequences has led to the existence of numerous families of monomers exhibiting specific organizational patterns. The limited amount of information available in non-human primates is a restriction to the understanding of the evolutionary dynamics of alpha satellite DNA. RESULTS: We carried out the targeted high-throughput sequencing of alpha satellite monomers and dimers from the Cercopithecus solatus genome, an Old World monkey from the Cercopithecini tribe. Computational approaches were used to infer the existence of sequence families and to study how these families are organized with respect to each other. While previous studies had suggested that alpha satellites in Old World monkeys were poorly diversified, our analysis provides evidence for the existence of at least four distinct families of sequences within the studied species and of higher order organizational patterns. Fluorescence in situ hybridization using oligonucleotide probes that are able to target each family in a specific way showed that the different families had distinct distributions on chromosomes and were not homogeneously distributed between chromosomes. CONCLUSIONS: Our new approach provides an unprecedented and comprehensive view of the diversity and organization of alpha satellites in a species outside the hominoid group. We consider these data with respect to previously known alpha satellite families and to potential mechanisms for satellite DNA evolution. Applying this approach to other species will open new perspectives regarding the integration of satellite DNA into comparative genomic and cytogenetic studies.

Draft genome of the lowland anoa (Bubalus depressicornis) and comparison with buffalo genome assemblies (Bovidae, Bubalina).

Genomic data for wild species of the genus Bubalus (Asian buffaloes) are still lacking while several whole genomes are currently available for domestic water buffaloes. To address this, we sequenced the genome of a wild endangered dwarf buffalo, the lowland anoa (Bubalus depressicornis), produced a draft genome assembly and made comparison to published buffalo genomes. The lowland anoa genome assembly was 2.56 Gbp long and contained 103,135 contigs, the longest contig being 337.39 kbp long. N50 and L50 values were 38.73 and 19.83 kbp, respectively, mean coverage was 44× and GC content was 41.74%. Two strategies were adopted to evaluate genome completeness: (1) determination of genomic features with de novo and homology-based predictions using annotations of chromosome-level genome assembly of the river buffalo and (2) employment of benchmarking against universal single-copy orthologs (BUSCO). Homology-based predictions identified 94.51% complete and 3.65% partial genomic features. De novo gene predictions identified 32,393 genes, representing 97.14% of the reference's annotated genes, whilst BUSCO search against the mammalian orthologs database identified 71.1% complete, 11.7% fragmented, and 17.2% missing orthologs, indicating a good level of completeness for downstream analyses. Repeat analyses indicated that the lowland anoa genome contains 42.12% of repetitive regions. The genome assembly of the lowland anoa is expected to contribute to comparative genome analyses among bovid species.

A paradox revealed: karyotype evolution in the four-horned antelope occurs by tandem fusion (Mammalia, Bovidae, Tetracerus quadricornis).

The four-horned antelope, Tetracerus quadricornis, is a karyotypic novelty in Bovidae since chromosomal evolution in this species is driven by tandem fusions in contradiction to the overwhelming influence of Robertsonian fusions in other species within the family. Using a combination of differential staining and molecular cytogenetic techniques, we provide the first description of the species' karyotype, draw phylogenetic inferences from the cytogenetic data and discuss possible mechanisms underlying the formation of the tandem fusions in this species. We show (a) that pairs 1-6 of Tetracerus correspond to a combination of Bos taurus orthologous chromosomes that are tandemly fused head to tail, (b) the presence of interstitial centromeric satellite DNA at the junctions of orthologous blocks defined by the cross-species painting data and (c) that in some instances, residual telomeric sequences persist at these sites. We conclude that the attendant result of each fusion is an enlarged acrocentric fusion element comprising a single functional centromere and two terminal telomeres that, collectively, led to a reduction of the 2n = 58 bovid ancestral acrocentric chromosomal complement to the 2n = 38 detected in the four-horned antelope.

Chromosome evolution in the subtribe Bovina (Mammalia, Bovidae): the karyotype of the Cambodian banteng (Bos javanicus birmanicus) suggests that Robertsonian translocations are related to interspecific hybridization.

Three subspecies of banteng (Bos javanicus) have been described: B. j. javanicus in Java, B. j. lowi in Borneo, and B. j. birmanicus in Cambodia, Lao PDR, Myanmar, Thailand and Vietnam. In this paper we provide the first description of the karyotype of the Cambodian banteng. The chromosomal complement of B. j. birmanicus differs from that of B. j. javanicus, which was previously found to be similar to that of cattle, Bos taurus (2n = 60). The Cambodian banteng karyotype has a diploid number of 2n = 56 (FN = 62) and the karyotype consists of 26 pairs of acrocentric chromosomes and two pairs of submetacentric chromosomes. Comparisons with other species of the subtribe Bovina show that the two pairs of bi-armed chromosomes resulted from two centric fusions involving the equivalent of cattle chromosomes 1 and 29, and 2 and 28, respectively. Cross-species fluorescence in-situ hybridization (FISH) with B. taurus whole chromosome paints and satellite DNA I probes was used to identify the chromosomes involved in the translocations, and their orientation. We suggest that Robertsonian translocations (1;29) and (2;28) have been fixed in the common ancestor of Cambodian banteng as a consequence of hybridization with the kouprey (Bos sauveli) during the Pleistocene epoch.

The Targeted Sequencing of Alpha Satellite DNA in Cercopithecus pogonias Provides New Insight Into the Diversity and Dynamics of Centromeric Repeats in Old World Monkeys.

Alpha satellite is the major repeated DNA element of primate centromeres. Specific evolutionary mechanisms have led to a great diversity of sequence families with peculiar genomic organization and distribution, which have till now been studied mostly in great apes. Using high throughput sequencing of alpha satellite monomers obtained by enzymatic digestion followed by computational and cytogenetic analysis, we compare here the diversity and genomic distribution of alpha satellite DNA in two related Old World monkey species, Cercopithecus pogonias and Cercopithecus solatus, which are known to have diverged about 7 Ma. Two main families of monomers, called C1 and C2, are found in both species. A detailed analysis of our data sets revealed the existence of numerous subfamilies within the centromeric C1 family. Although the most abundant subfamily is conserved between both species, our fluorescence in situ hybridization (FISH) experiments clearly show that some subfamilies are specific for each species and that their distribution is restricted to a subset of chromosomes, thereby pointing to the existence of recurrent amplification/homogenization events. The pericentromeric C2 family is very abundant on the short arm of all acrocentric chromosomes in both species, pointing to specific mechanisms that lead to this distribution. Results obtained using two different restriction enzymes are fully consistent with a predominant monomeric organization of alpha satellite DNA that coexists with higher order organization patterns in the C. pogonias genome. Our study suggests a high dynamics of alpha satellite DNA in Cercopithecini, with recurrent apparition of new sequence variants and interchromosomal sequence transfer.

Telomere regulation during ageing and tumorigenesis of the grey mouse lemur.

Telomere erosion leading to replicative senescence has been well documented in human and anthropoid primates, and provides a clue against tumorigenesis. In contrast, other mammals, such as laboratory mice, with short lifespan and low body weight mass have different telomere biology without replicative senescence. We analyzed telomere biology in the grey mouse lemur, a small prosimian model with a relative long lifespan currently used in ageing research. We report an average telomere length by telomere restriction fragment (TRF) among the longest reported so far for a primate species (25-30 kb), but without detectable overall telomere shortening with ageing on blood samples. However, we demonstrate using universal STELA (Single Telomere Length Amplification) the existence of short telomeres, the increase of which, while correlating with ageing might be related to another mechanism than replicative senescence. We also found a low stringency of telomerase restriction in tissues and an ease to immortalize fibroblasts in vitro upon spontaneous telomerase activation. Finally, we describe the first grey mouse lemur cancer cell line showing a dramatic telomere shortening and high telomerase activity associated with polyploidy. Our overall results suggest that telomere biology in grey mouse lemur is an exception among primates, with at best a physiologically limited replicative telomere ageing and closest to that observed in small rodents.

Phylogenomics of African guenons.

The karyotypes of 28 specimens belonging to 26 species of Cercopithecinae have been compared with each other and with human karyotype by chromosome banding and, for some of them, by Zoo-FISH (human painting probes) techniques. The study includes the first description of the karyotypes of four species and a synonym of Cercopithecus nictitans. The chromosomal homologies obtained provide us with new data on a large number of rearrangements. This allows us to code chromosomal characters to draw Cercopithecini phylogenetic trees, which are compared to phylogenetic data based on DNA sequences. Our findings show that some of the superspecies proposed by Kingdon (1997 The Kingdon Field Guide to African Mammals, Academic Press.) and Groves (2001 Primates Taxonomy, Smithsonian Institution Press) do not form homogeneous groups and that the genus Cercopithecus is paraphyletic, in agreement with previous molecular analyses. The evolution of Cercopithecini karyotypes is mainly due to non-centromeric chromosome fissions and centromeric shifts or inversions. Non-Robertsonian translocations occurred in C. hamlyni and C. neglectus. The position of chromosomal rearrangements in the phylogenetic tree leads us to propose that the Cercopithecini evolution proceeded by either repeated fission events facilitated by peculiar genomic structures or successive reticulate phases, in which heterozygous populations for few rearranged chromosomes were present, allowing the spreading of chromosomal forms in various combinations, before the speciation process.

Ductal breast carcinoma develops through different patterns of chromosomal evolution.

In a previous study that used comparative genomic hybridization (CGH) to analyze 43 ductal breast carcinomas selected for hyperdiploidy, we proposed the existence of two distinct pathways of chromosomal evolution. In the present study, in which we reassessed our cytogenetic findings on 158 ductal breast carcinomas selected for having a modal number of chromosomes of fewer than 60, we confirmed the existence of two subtypes of tumors. Along with the great majority of tumors (142 of 158) that evolved through structural rearrangements with no or very few whole-chromosome gains, we found that a minor subset (16 of 158) evolved through progressive gains of whole chromosomes with no or only a few associated rearrangements. In this article, we describe the karyotypes of these 16 tumors together with data from CGH, which was performed for 10 of them. Chromosomes 5, 7, 8, and 20 were the most frequently gained. Our findings support the evidence of a new pathway of chromosomal evolution in a small subset of ductal breast carcinomas characterized by numerical chromosome aberrations.