Satellitome Analysis on Talpa aquitania Genome and Inferences about the satDNAs Evolution on Some Talpidae.

In the genus Talpa a new species, named Talpa aquitania, has been recently described. Only cytogenetic data are available for the nuclear genome of this species. In this work, we characterize the satellitome of the T. aquitania genome that presents 16 different families, including telomeric sequences, and they represent 1.24% of the genome. The first satellite DNA family (TaquSat1-183) represents 0.558%, and six more abundant families, including TaquSat1-183, comprise 1.13%, while the remaining 11 sat-DNAs represent only 0.11%. The average A + T content of the SatDNA families was 50.43% and the median monomer length was 289.24 bp. The analysis of these SatDNAs indicated that they have different grades of clusterization, homogenization, and degeneration. Most of the satDNA families are present in the genomes of the other Talpa species analyzed, while in the genomes of other more distant species of Talpidae, only some of them are present, in accordance with the library hypothesis. Moreover, chromosomal localization by FISH revealed that some satDNAs are localized preferentially on centromeric and non-centromeric heterochromatin in T. aquitania and also in the sister species T. occidentalis karyotype. The differences observed between T. aquitania and the close relative T. occidentalis and T. europaea suggested that the satellitome is a very dynamic component of the genomes and that the satDNAs could be responsible for chromosomal differences between the species. Finally, in a broad context, these data contribute to the understanding of the evolution of satellitomes on mammals.

Molecular Composition of Heterochromatin and Its Contribution to Chromosome Variation in the Microtus thomasi/Microtus atticus Species Complex.

The voles of the Microtus thomasi/M. atticus species complex demonstrate a remarkable variability in diploid chromosomal number (2n = 38-44 chromosomes) and sex chromosome morphology. In the current study, we examined by in situ hybridization the topology of four satellite DNA motifs (Msat-160, Mth-Alu900, Mth-Alu2.2, TTAGGG telomeric sequences) and two transposons (LINE, SINE) on the karyotypes of nine chromosome races (i.e., populations with unique cytogenetic traits) of Microtus thomasi, and two chromosomal races of M. atticus. According to the topology of the repetitive DNA motifs, we were able to identify six types of biarmed chromosomes formed from either Robertsonian or/and tandem fusions. In addition, we identified 14 X chromosome variants and 12 Y chromosome variants, and we were able to reconstruct their evolutionary relations, caused mainly by distinct mechanisms of amplification of repetitive DNA elements, including the telomeric sequences. Our study used the model of the Microtus thomasi/M. atticus species complex to explore how repetitive centromeric content can alter from chromosomal rearrangements and can shape the morphology of sex chromosomes, resulting in extensive inter-species cytogenetic variability.

Complete Mitochondrial Genome of Three Species of the Genus Microtus (Arvicolinae, Rodentia).

The 65 species of the genus Microtus have unusual sex-related genetic features and a high rate of karyotype variation. However, only nine complete mitogenomes for these species are currently available. We describe the complete mitogenome sequences of three Microtus, which vary in length from 16,295 bp to 16,331 bp, contain 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes and a control region. The length of the 13 PCGs and the coded proteins is the same in all three species, and the start and stop codons are conserved. The non-coding regions include the L-strand origin of replication, with the same sequence of 35 bp, and the control region, which varies between 896 bp and 930 bp in length. The control region includes three domains (Domains I, II and III) with extended termination-associated sequences (ETAS-1 and ETAS-2) in Domain I. Domain II and Domain III include five (CSB-B, C, D, E and F) and three (CSB-1, CSB-2, and CSB-3) conserved sequence blocks, respectively. Phylogenetic reconstructions using the mitochondrial genomes of all the available Microtus species and one representative species from another genus of the Arvicolinae subfamily reproduced the established phylogenetic relationships for all the Arvicolinae genera that were analyzed.

MCPH1 Lack of Function Enhances Mitotic Cell Sensitivity Caused by Catalytic Inhibitors of Topo II.

The capacity of Topoisomerase II (Topo II) to remove DNA catenations that arise after replication is essential to ensure faithful chromosome segregation. Topo II activity is monitored during G2 by a specific checkpoint pathway that delays entry into mitosis until the chromosomes are properly decatenated. Recently, we demonstrated that the mitotic defects that are characteristic of cells depleted of MCPH1 function, a protein mutated in primary microcephaly, are not a consequence of a weakened G2 decatenation checkpoint response. However, the mitotic defects could be accounted for by a minor defect in the activity of Topo II during G2/M. To test this hypothesis, we have tracked at live single cell resolution the dynamics of mitosis in MCPH1 depleted HeLa cells upon catalytic inhibition of Topo II. Our analyses demonstrate that neither chromosome alignment nor segregation are more susceptible to minor perturbation in decatenation in MCPH1 deficient cells, as compared with control cells. Interestingly, MCPH1 depleted cells were more prone to mitotic cell death when decatenation was perturbed. Furthermore, when the G2 arrest that was induced by catalytic inhibition of Topo II was abrogated by Chk1 inhibition, the incidence of mitotic cell death was also increased. Taken together, our data suggest that the MCPH1 lack of function increases mitotic cell hypersensitivity to the catalytic inhibition of Topo II.

Complete mitochondrial genome of the Iberian Mole Talpa occidentalis (Talpidae, Insectivora) and comparison with Talpa europaea.

The complete mitogenome of Talpa occidentalis, the Iberian mole, was sequenced using a combination of the Illumina and Sanger methods. The 16,962 bp genome obtained contains 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a control region. Thirty-seven identical repetitions of a 10-nucleotide (CACACGTACG) repeat element were identified in the non-coding control region (D-loop). The number, order, and orientation of the mitochondrial genes are the same as in T. europaea, the only mitogenome published so far for this genus. These two mitogenomes differ only at the repeat element included in the control region. The phylogeny obtained for the Talpidae species using the protein-coding genes of these mitogenomes agrees with the current classification of this family.

Structural and Evolutionary Relationships in the Giant Sex Chromosomes of Three Microtus Species.

The genus Microtus has high karyotypic diversity. The existence of notable differences in the length of its sex chromosomes contributes to this variation. Variations in size are attributed to the enlargement of their heterochromatin content, which is of such magnitude in some species that they are referred to as "giant sex chromosomes". Here, we perform an intra- and interspecific analysis of the molecular composition of the heterochromatic blocks in three species with giant sex chromosomes (Microtus chrotorrhinus, M. cabrerae and M. agrestis). Our results show that the heterochromatic content is very similar in both the X and Y chromosomes of M. chrotorrhinus, and that their molecular composition is more closely related to the heterochromatic blocks of M. agrestis than to the sex heterochromatin of M. cabrerae; however, species-specific differences do clearly exist. Interestingly, the euchromatic regions of the X chromosome of all three of these species share a homologous region composed of heterochromatic-related sequences. Our results therefore reinforce the idea that certain similarities in the original organization of these X chromosomes could have facilitated their later enlargement.

Correction to: Whole chromosome painting reveals independent origin of sex chromosomes in closely related forms of a fish species.

ere, we report that a paragraph from the "Discussion" section of Cioffi et al. (2011; p. 1070, 4th paragraph of column 1) was transcribed (with only minor edits) from an introductory paragraph previously published in Chromosome Research by O'Meally et al.

Intragenomic distribution of RTE retroelements suggests intrachromosomal movement.

Much is known about the abundance of transposable elements (TEs) in eukaryotic genomes, but much is still unknown on their behaviour within cells. We employ here a combination of cytological, molecular and genomic approaches providing information on the intragenomic distribution and behaviour of non-long terminal repeat (LTR) retrotransposon-like elements (RTE). We microdissected every chromosome in a single first meiotic metaphase cell of the grasshopper Eyprepocnemis plorans and polymerase chain reaction (PCR) amplified a fragment of the RTE reverse transcriptase gene with specific primers. PCR products were cloned and 139 clones were sequenced. Analysis of molecular variance (AMOVA) showed significant intragenomic structure for these elements, with 4.6 % of molecular variance being found between chromosomes. A maximum likelihood tree built with the RTE sequences revealed the frequent presence of two or more elements showing very high similarity and being located on the same chromosome, thus suggesting intrachromosome movement. The 454 pyrosequencing of genomic DNA gave strong support to the microdissection results and provided evidence for the existence of 5' truncated elements. Our results thus indicate a tendency of RTE elements to reinsert into the same chromosome from where they were transcribed, which could be achieved if retrotranscription and insertion takes place immediately after transcription.

A synaptonemal complex-derived mechanism for meiotic segregation precedes the evolutionary loss of homology between sex chromosomes in arvicolid mammals.

Synapsis and reciprocal recombination between sex chromosomes are restricted to the pseudoautosomal region. In some animal species, sex chromosomes do not present this region, although they utilize alternative mechanisms that ensure meiotic pairing and segregation. The subfamily Arvicolinae (Rodentia, Cricetidae) includes numerous species with achiasmate sex chromosomes. In order to know whether the mechanism involved in achiasmate segregation is an ancient feature in arvicolid species, we have compared the sex chromosomes of both the Mediterranean vole (Microtus duodecimcostatus) and the water vole (Arvicola terrestris). By means of immunofluorescence, we have found that sex chromosomes in M. duodecimcostatus are asynaptic and develop a synaptonemal complex-derived structure that mediates pairing and facilitates segregation. In A. terrestris, sex chromosomes are synaptic and chiasmate but also exhibit a synaptonemal complex-derived filament during anaphase I. Since phylogenetic relationships indicate that the synaptic condition is ancestral in arvicolids, this finding indicates that the mechanism for achiasmate sex chromosome segregation precedes the switching to the asynaptic condition. We discuss the origin of this synaptonemal complex-derived mechanism that, in turn, could counterbalance the disruption of homology in the sex chromosomes of those species.

Whole chromosome painting reveals independent origin of sex chromosomes in closely related forms of a fish species.

The wolf fish Hoplias malabaricus includes well differentiated sex systems (XY and X(1)X(2)Y in karyomorphs B and D, respectively), a nascent XY pair (karyomorph C) and not recognized sex chromosomes (karyomorph A). We performed the evolutionary analysis of these sex chromosomes, using two X chromosome-specific probes derived by microdissection from the XY and X(1)X(2)Y sex systems. A putative-sex pair in karyomorph A was identified, from which the differentiated XY system was evolved, as well as the clearly evolutionary relationship between the nascent XY system and the origin of the multiple X(1)X(2)Y chromosomes. The lack of recognizable signals on the sex chromosomes after the reciprocal cross-FISH experiments highlighted that they evolved independently from non-homologous autosomal pairs. It is noteworthy that these distinct pathways occur inside the same nominal species, thus exposing the high plasticity of sex chromosome evolution in lower vertebrates. Possible mechanisms underlying this sex determination liability are also discussed.

Misregulation of mitotic chromosome segregation in a new type of autosomal recessive primary microcephaly.

Primary autosomal recessive microcephaly (MCPH) is a congenital disorder characterized by a pronounced reduction of brain size and mental retardation. We present here a consanguineous Turkish family clinically diagnosed with MCPH and without linkage to any of the known loci (MCPH1-MCPH7). Autozygosity mapping identified a homozygous region of 15.8 Mb on chromosome 10q11.23-21.3, most likely representing a new locus for MCPH. Although we were unable to identify the underlying genetic defect after extensive molecular screening, we could delineate a possible molecular function in chromosome segregation by the characterization of mitosis in the patients' cells. Analyses of chromosome nondisjunction in T-lymphocytes and fibroblasts revealed a significantly elevated rate of nondisjunction in the patients' cells as compared to controls. Mitotic progression was further explored by immunofluorescence analyses of several chromosome and spindle associated proteins. We detected a remarkable alteration in the anaphase distribution of Aurora B and INCENP, which are key regulators of chromosome segregation. In particular, a fraction of both proteins remained abnormally loaded on chromosomes during anaphase in MCPH patients' cells while in cells of normal control subjects both proteins are completely transferred to the spindle midzone. We did not observe any other alterations regarding cell cycle progression, chromosome structure, or response to DNA damage. Our observations point towards a molecular role of the underlying gene product in the regulation of anaphase/telophase progression possibly through interaction with chromosomal passenger proteins. In addition, our findings represent further evidence for the proposed role of MCPH genes in the regulation of mitotic progression.

MCPH1 patient cells exhibit delayed release from DNA damage-induced G2/M checkpoint arrest.

Mutations in the MCPH1 gene cause primary microcephaly associated with a unique cellular phenotype of misregulated chromosome condensation. The encoded protein contains three BRCT domains, and accumulating data show that MCPH1 is involved in the DNA damage response. However, most of this evidence has been generated by experiments using RNA interference (RNAi) and cells from non-human model organisms. Here, we demonstrate that patient-derived cell lines display a proficient G2/M checkpoint following ionizing irradiation (IR) despite homozygous truncating mutations in MCPH1. Moreover, chromosomal breakage rates and the relocation to DNA repair foci of several proteins functioning putatively in an MCPH1-dependent manner are normal in these cells. However, the MCPH1-deficient cells exhibit a slight delay in re-entering mitosis and delayed resolution of γH2AX foci following IR. Analysis of chromosome condensation behavior following IR suggests that these latter observations may be related to hypercondensation of the chromatin in cells with MCPH1 mutations. Our results indicate that the DNA damage response in human cells with truncating MCPH1 mutations differs significantly from the damage responses in cells of certain model organisms and in cells depleted of MCPH1 by RNAi. These subtle effects of human MCPH1 deficiency on the cellular DNA damage response may explain the absence of cancer predisposition in patients with biallelic MCPH1 mutations.

The karyotype and 5S rRNA genes from Spanish individuals of the bat species Rhinolophus hipposideros (Rhinolophidae; Chiroptera).

The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair.

Mapping the SRY gene in Microtus cabrerae: a vole species with multiple SRY copies in males and females.

The SRY gene is a single-copy, male-specific gene, located on the Y chromosome in most mammals. However, recently we have described the presence of multiple polymorphic copies of this gene in both males and females of the vole species Microtus cabrerae. Here, we present the chromosomal localization of SRY gene copies in this species by fluorescent in situ hybridization (FISH). This technique localized these gene copies in the short arm, and hence in the euchromatic region, of the Y chromosome. Furthermore, several copies of the SRY gene are located on the X chromosome. These copies are spread along the entire heterochromatic region of the X chromosome, occupying the whole short arm, the centromeric region, and the pericentromeric region of the long arm.