Cytogenetic analyses of chromosomal rearrangements in Mus minutoides/musculoides from North-West Zambia through mapping of the telomeric sequence (TTAGGG)n and banding techniques

Three specimens of M. minutoides/musculoides from Zambia were cytogenetically studied through G- and C-banding, DAPI staining and fluorescence in-situ hybridization (FISH) with a (TTAGGG)_n telomeric sequence. Biarmed chromosomes were identified according to the current nomenclature as follows: Rb(2.7), Rb(3.12), Rb(4.5), Rb(6.8), Rb(9.16), and the sex chromosomes Rb(1.X), Rb(1.Y) and Rb(1.X_d), originated from the deleted X chromosome. One female showed the diploid number 2n=24; in the two other individuals, the Rb(9.16) occurred in a heteromorphic condition, and, accordingly, the diploid number was 2n=25. FISH showed the sites of telomeric sequences at telomeres of all the chromosomes, and in an interstitial position at the centromeres of all Robertsonian metacentrics, except one – the Rb(6.8), though the patterns of hybridization varied between chromosomes. Sex chromosome pairs, in the male and females, showed a similar C-banding pattern, but revealed clear differences after FISH. Traces of telomeric sequences were found dispersed in the whole-heterochromatic arm of the Rb(1.X_d). No visible bond between C-positive heterochromatin and telomeric sequences were detected in the other either bi- or uniarmed chromosomes, indicating that they may actually represent retained telomeres in the Robertsonian metacentrics. This revised version was published online in July 2006 with corrections to the Cover Date.     

Unusual distribution pattern of telomeric repeats in the shrews <Emphasis Type="Italic">Sorex araneus</Emphasis> and <Emphasis Type="Italic">Sorex granarius</Emphasis>

Sorex araneus and Sorex granarius are sibling species within the Sorex araneus group with karyotypes composed of almost identical chromosome arms. S. granarius has a largely acrocentric karyotype, while, in S. araneus, various of these acrocentrics have combined together by Robertsonian (Rb) fusions to form metacentrics, with the numbers and types of metacentrics differing between chromosomal races. Our studies on telomeric sequences in S. araneus and S. granarius revealed differences between chromosomes and between species. In S. araneus (the Novosibirsk race), hybridization signals were present on the telomeres of all the chromosomes after FISH with a PCR-generated telomeric probe. In addition, hybridization signals were observed at high frequencies in the pericentric regions of some but not all metacentrics formed by Rb fusion. There were fewer signals on those metacentrics formed earlier in the evolution of S. araneus. This suggests that S. araneus chromosomes retain at least some telomeric repeats during Rb fusion, but that these repeats are lost or modified over time. These results are critical for the interpretation of the well-studied hybrid zones between chromosomal races of S. araneus, given that Rb fission has been postulated in such hybrid zones and that the likelihood of Rb fission will relate to presence/absence of telomeric sequences at the centromeres of metacentrics. In S. granarius, there were strong signals at the proximal (centromeric) telomeres of the acrocentrics after FISH with a DNA telomeric probe. FISH with a PNA telomeric probe on S. granarius acrocentrics showed that the proximal telomeres were 213 kb on average, while the length of the distal telomeres was 3.8 kb on average. Two-colour FISH, using a telomeric DNA probe and a microdissected probe generated from the pericentric regions of the S. granarius chromosomes a and b, revealed regions on distinct chromatin fibres where telomeric and microdissected probes were colocalized or localized sequentially. The proximal telomeres of S. granarius are highly unusual both in their large size and their heterogeneous structure relative to the telomeres of other mammals.     

Cytogenetics of a new cytotype of African Mus (subgenus Nannomys) minutoides (Rodentia, Muridae) from Kenya: C- and G- banding and distribution of (TTAGGG) n telomeric sequences

We present the results of a cytogenetic study on Mus (Nannomys) minutoides from Kenya by means of C- and G- banding and in-situ fluorescence hybridization (FISH) to localize the telomeric sequences. The karyotype is characterized by the occurrence of several Rb chromosomes Rb(1.X), Rb(1.Y). Rb(2.17), Rb(3.13), Rb(4.10), Rb(5.11), Rb(6.7), Rb(8.12), not previously described for this species. This finding suggests a high level of chromosomal diversification, which means it is possible to consider this cytotype as a new, well-differentiated, chromosomal lineage within the subgenus. The C-banding of the metaphases illustrated conspicuous blocks of centromeric heterochromatin at the paracentromeric regions of all telocentric chromosomes. Centromeric heterochromatin is not visible on all biarmed chromosomes. Following hybridization with telomeric probes, bright interstitial telomeric sequence (ITS) fluorescence signals are evident at the pericentromeric area of all Rb chromosomes, with the exception of Rb(2.17). Considering the localization of the C-positive heterochromatin and of the telomeric sequences, the events leading to the Kenyan cytotype from an all-telocentric condition probably included two steps: first, fusion without loss of heterochromatin and pericentromeric telomeric sequences; second, the reduction of the C-positive satellite DNA followed by the amplification of telomeric sequences in the C-negative paracentromeric region of Rb chromosomes. The presence of a single Rb(2.17) without ITS indicates possible variations of this mechanism.     

Intraspecific variation in the distribution of the interstitial telomeric (TTAGGG)n sequences in Micoureus demerarae (Marsupialia: Didelphidae)

The distribution of the telomeric sequence (TTAGGG)_n was studied in chromosomes of Micoureus demerarae (2n=14), a South American marsupial, by fluorescence in-situ hybridization (FISH). The telomeric repeat sequence was present at both ends of all chromosomes, but also various interstitial telomeric sequences (ITS) were detected in the pericentromeric heterochromatic regions. Intraspecific differences in the number of ITS (2 to 8) were observed without intraindividual variation. The presence of telomere-like sequences in the same regions of constitutive heterochromatin suggest that these segments are not necessarily remnants of true telomeres resulting from chromosome rearrangements but could be part of the satellite DNA.     

Spontaneous occurrence of a Robertsonian fusion involving chromosome 19 by single whole-arm reciprocal translocation (WART) in wild-derived house mice

Chromosomal races of the house mouse (Mus musculus domesticus) bear Robertsonian (Rb) fusions, which consist of centric translocations between two non-homologous acrocentric chromosomes. The high level of diversity of these fusions in house mice is generated by de-novo formation of Rb fusions and subsequent whole-arm reciprocal exchanges (WARTs). This paper describes the spontaneous occurrence of a new Rb fusion, Rb(4.19), in progeny of wild-derived house mice segregating for Rb(4.12). The chromosomal mutation was traced to a female which exhibited germline and somatic mosaicism indicating an early embryonic origin of the mutation. FISH analysis of centromerically-located ribosomal genes suggested that no modification was observed on chromosomes 12 and 19 prior to or following the occurrence of Rb(4.19). Distribution of telomeric sequences showed that both Rb fusions lacked telomeres in their centromeric regions. It is argued that this spontaneous mutation most likely originated by single whole-arm reciprocal translocation (WART) between Rb(4.12) and an acrocentric chromosome 19, resulting in Rb(4.19) and a neo-acrocentric chromosome 12. Sequences required for centromeric function and proximal telomeres would have been transferred to the neo-chromosome 12 from chromosome 19 during the translocation. The existence of such WARTs which generate derived acrocentric chromosomes has several implications for chromosomal evolution in house mice.     

Whole-arm reciprocal translocation (WART) between Robertsonian chromosomes: finding of a Robertsonian heterozygous mouse with karyotype derived through WARTs

The karyotype of a mouse trapped in a hybrid zone between a Robertsonian (Rb) population (2n=22) and a population with the standard karyotype (2n=40-all-telocentrics) shows two Rb chromosomes with new arm compositions. We suggest that whole-arm reciprocal translocations between Rb chromosomes gave rise to the new chromosome constitution and that such events can greatly help in understanding house mouse karyotype diversification and chromosomal speciation.     

Telomeric and interstitial telomeric sequences in holokinetic chromosomes of Lepidoptera: Telomeric DNA mediates association between postpachytene bivalents in achiasmatic meiosis of females

Telomeres, besides their main role in the protection and maintenance of chromosome ends, have several other vital functions in the cell cycle. We studied their role in the achiasmatic meiosis of female Lepidoptera, insects with holokinetic chromosomes. By fluorescence in-situ hybridization (FISH) with the insect telomeric probe, (TTAGG) _n , we mapped the distribution of telomeric and interstitial telomeric sequences (ITS) in female meiotic chromosomes of two species, Orgyia antiqua with a reduced chromosome number (2n=28) and Ephestia kuehniella mutants, possessing a radiation-induced chromosome fusion in the genome (2n=59). In addition to the strong typical telomeric signals, O. antiqua displayed weaker hybridization signals in interstitial sites of pachytene bivalents. The observed ITS most probably reflect remnants of chromosomal rearrangements and support the hypothesis that the Orgyia karyotype had arisen by multiple fusions of ancestral chromosomes. On the other hand, the absence of ITS in the chromosome fusion of Ephestia indicated the loss of telomeres before the two original chromosomes fused. When the telomeric probe was amplified by enzymatic reaction with tyramid, the number of ITS observed increased in Orgyia, and a few ITS were also observed in several chromosomes of Ephestia but not in the fused chromosome. This suggests that the genomes of both species also contain ITS other than those originating from chromosome fusions. The analysis of female meiotic prophase I revealed non-homologous associations of postpachytene bivalents mediated by telomeric DNA, which were not observed in the pachytene stage. Surprisingly, in early postpachytene nuclei the telomeric associations also involved ITS, whereas later postpachytene nuclei displayed chains of bivalents interconnected only by true telomeres. This finding favours a hypothesis that telomeric associations between bivalents play a role in chromosome segregation in the achiasmatic meiosis of female Lepidoptera. This revised version was published online in July 2006 with corrections to the Cover Date.     

A centromeric satellite DNA may be involved in heterochromatin compactness in gobiid fishes

Centromere and telomere composition and organization were studied in various gobiid species exhibiting and not exhibiting chromosome polymorphisms involving Robertsonian rearrangements. In Gobius cobitis, we isolated an AT-rich centromeric DNA satellite, designated pCOB, and found that several sequences contain adenine stretches, various CA/TG dinucleotide steps, and a sequence 76% homologous to the yeast CDE III centromeric sequence. All of these traits are generally considered important for centromeric function, and the hypothesis has been advanced that some are involved in the control of DNA curvature and thus in the degree of centromeric chromatin compactness. Based on these features, and on the fact that they are found only in the species not exhibiting Robertsonian biarmed chromosomes, a role for pCOB in preventing centric fusions has been hypothesized. Our data also suggest that, as in other species, the formation of Robertsonian biarmed chromosomes is accompanied by the loss of telomeric sequences. This revised version was published online in July 2006 with corrections to the Cover Date.     

Telomeric (TTAGGG)n Sequences Are Associated With Nucleolus Organizer Regions (NORs) in the Wood Lemming

The distribution of the (TTAGGG)_n telomeric sequence was studied in chromosomes of the wood lemming, Myopus schisticolor, by fluorescence in-situ hybridization. As expected, the hybridization signals were observed at telomeres of all chromosomes. However, quite a number of interstitial telomeric sites were present in the pericentric heterochromatic regions. Consistent strong hybridization signals were also seen at one terminus of chromosomes 5, 7 and 12–15. By post-hybridization G-banding and silver-staining, the large blocks of the telomeric sequences on chromosomes 5 and 12 were localized to nucleolus organizer regions (NORs). This revised version was published online in August 2006 with corrections to the Cover Date.     

Sex chromosome evolution in fish. II. Second occurrence of an X1X2Y sex chromosome system in Gymnotiformes

A multiple sex chromosome system of the X₁X₁X₂X₂:X₁X₂Y type is reported to occur in the fish species Brachyhypopomus pinnicaudatus (Gymnotiformes, Hypopomidae), being the second occurrence of this sex chromosome system in Gymnotiformes and the fifth among Neotropical freshwater fish. The possible origin of this system was hypothesized to be a centric fusion, which occurred in an ancestral form, of two medium-sized acrocentrics, giving origin to the metacentric neo-Y. Heterochromatic DAPI-positive regions were visualized in the pericentromeric region of all the chromosomes, including the Y-chromosome. In-situ hybridization with (TTAGGG) _n (all-human-telomeres probe) did not detect any telomeric interstitial regions (ITS), indicating a possible loss of terminal segments of the chromosomes involved in the neo-Y formation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Intrachromosomal distribution of telomeric repeats in Eumops glaucinus and Eumops perotis (Molossidae, Chiroptera)

The location of chromosomal telomeric repeats (TTAGGG)_n was investigated in two species of the Molossidae family, Eumops glaucinus and Eumops perotis. The diploid chromosome number (2n) is 40 in E. glaucinus and 48 in E. perotis and the fundamental numbers (FN) are 64 and 58, respectively. It has been suggested that the E. glaucinus karyotype has evolved from the E. perotis karyotype through Robertsonian fusion events. In the present study, the telomeric sequences were detected at the termini of chromosomes in both species. In addition, E. glaucinus also displayed telomeric repeats in centromeric and pericentromeric regions in almost all biarmed chromosomes. Conversely, in E. perotis pericentromeric signals were only observed in two biarmed chromosomes. In both E. glaucinus and E. perotis, such telomeric sequences were observed as part of the heterochromatin. The interstitial sites of telomeric sequences suggest that they are remnants of telomeres of ancestral chromosomes that participated in the fusion event.     

Exceptional minute sex-specific region in the X0 mammal,Ryukyu spiny rat

The Ryukyu spiny rats (genus Tokudaia) inhabit only three islands in the Nansei Shoto archipelago in Japan, and have the variations of karyotype among the islands. The chromosome number of T. osimensis in Amami-Oshima Island is 2n = 25, and T. tokunoshimensis in Tokunoshima Island is 2n = 45, and the two species have X0 sex chromosome constitution with no cytogenetically visible Y chromosome in both sexes. We constructed the standard ideograms for these species at the 100 and 200 band levels. Comparing the banding patterns between these species, it was suggested that at least 10 times the number of Robertsonian fusions occurred in T. osimensis chromosomes. However, no karyotypic differences were observed between sexes in each species. To detect the sex-specific chromosomal region of these X0 species we applied the comparative genomic hybridization (CGH) method. Although the male- and female-derived gains and losses were detected in several chromosome regions, all of them were located in the heterochromatic and/or telomeric regions. This result suggested that the differences detected by CGH might be caused by the polymorphism on the copy numbers of repeated sequences in the heterochromatic and telomeric regions. Our result indicated that the sex-specific region, where the key to sex determination lies, is very minute in X0 species of Tokudaia.     

Cytogenetics of the bleak (Alburnus alburnus), with special emphasis on the B chromosomes

Some of the largest B chromosomes so far discovered in vertebrates are present in the cyprinid fish Alburnus alburnus. Previous cytogenetic analyses revealed a diploid chromosome number of 2n = 50. In addition, in some individuals one or two unusually large B chromosomes are present. Two morphologically different types of B chromosomes were observed. The frequency of animals bearing a supernumerary chromosome was found to vary considerably between different populations. A more detailed analysis of the A and B chromosomes of A. alburnus by conventional banding techniques, as well as fluorescence in-situ hybridization (FISH) with the telomeric DNA repeats (GGGTTA)₇/(TAACCC)₇, 18S + 28S rDNA and 5S rDNA were performed in the present study. Furthermore, a B chromosome-specific DNA probe obtained by amplified length polymorphism (AFLP) was hybridized on metaphases of A. alburnus carrying supernumerary B chromosomes. The banding analyses showed that the B chromosomes are completely heterochromatic, consist of GC-rich DNA sequences, replicate their DNA in the very late S-phase of the cell cycle and are composed mainly of a specific retrotransposable DNA element. Finally, blood probes from A. alburnus were collected for DNA-flow cytometric measurements. It could be shown that the huge supernumerary chromosomes represent nearly 10% of the total genome size of A. alburnus.     

The chromosomal distribution and organization of sheep satellite I and II centromeric DNA using characterized sheep-hamster somatic cell hybrids

A panel of sheep-hamster somatic cell hybrids containing single sheep chromosomes was used to study the chromosomal distribution and organization of two families of sheep centromeric satellite DNA. This study shows that the centromeres of the sheep metacentric chromosomes 1,2 and 3 differ in their organization and relative quantities of sheep satellite I DNA. The results, when correlated with the proposed formation of these metacentric chromosomes by ancient Robertsonian translocations, suggest a loss or replacement of satellite I centromeric DNA from the centromeres of these sheep chromosomes. Using Southern blot analysis and fluorescencein situ hybridization, this study shows that the recent centric fusion chromosome t2 (rob 9;10) contains little satellite II DNA. Together these resuults suggest the possibility of substantial reorganization of sheep centromeric DNA families after Robertsonian translocations.accepted for publication by R. J. Baker     

Chromosome healing by addition of telomeric repeats in wheat occurs during the first mitotic divisions of the sporophyte and is a gradual process

Alien gametocidal chromosomes cause extensive chromosome breakage prior to S-phase in the first mitotic division of gametophytes lacking the alien chromosome. The broken chromosomes may be healed either by addition of telomeric repeats in the gametophyte or undergo fusions to form dicentric or translocation chromosomes. We show that dicentric chromosomes undergo breakage–fusion–bridge (BFB) cycles in the first few mitotic divisions of the sporophyte, are partially healed before the germ line differentiation regimen, and are healed completely in the ensuing gametophytic stage. The gametocidal factor on chromosome 4M_g of Aegilops geniculata was used to induce dicentrics involving the satellite chromosomes1B and 6B of wheat, Triticum aestivum. The dicentrics 1BS·1BL-2AL·2AS and 6BS·6BL-4BL·4BS initiated BFB cycles that ceased 2 to 4 weeks after seed germination. At the end of the BFB cycles, we observed deficient 1B and 6B chromosomes with breakpoints in proximal regions of the 1BL and 6BL arms. The process of chromosome healing was analyzed in root tip meristems, at meiotic metaphase I, and in the derived progenies by fluorescence in-situ hybridization analysis using a telomeric probe pAtT4. The results show that chromosome healing in wheat occurs during very early mitotic divisions in the sporophyte by de-novo addition of telomeric repeats and is a gradual process. Broken chromosome ends have to pass through several cell divisions in the sporophyte to acquire the full telomeric repeat length.     

Characterization of the atypical karyotype of the black-winged kite Elanus caeruleus (Falconiformes: Accipitridae) by means of classical and molecular cytogenetic techniques

The karyotype of the black-winged kite (Elanus caeruleus), a small diurnal raptor living in Africa, Asia and southern Europe, was studied with classical (G-, C-, R-banding, and Ag-NOR staining) and molecular cytogenetic methods, including primed in-situ labelling (PRINS) and fluorescence in-situ hybridization (FISH) with telomeric (TTAGGG) and centromeric DNA repeats. The study revealed that the genome size, measured by flow cytometry (3.1pg), is in the normal avian range. However, the black-winged kite karyotype is particularly unusual among birds in having a moderate diploid number of 68 chromosomes, and containing only one pair of dot-shaped microchromosomes. Moreover, the macrochromosomes are medium-sized, with the Z and W gonosomes being clearly the largest in the set. C-banding shows that constitutive heterochromatin is located at the centromeric regions of all chromosomes, and that two pairs of small acrocentrics and the pair of microchromosomes are almost entirely heterochromatic and G-band negative. The distribution pattern of a centromeric repeated DNA sequence, as demonstrated by PRINS, follows that of C-heterochromatin. The localization of telomeric sequences by FISH and PRINS reveals many strong telomeric signals but no extratelomeric signal was observed. The atypical organization of the karyotype of the black-winged kite is considered in the context of the modes of karyotypic evolution in birds.     

Aloe spp. – plants with vertebrate-like telomeric sequences

Chromosome termini of most eukaryotes end in tracks of short tandemly repeated GC-rich sequences, the composition of which varies among different groups of organisms. Plant species predominantly contain (TTTAGGG)_n repeats at their telomeres. However, a few plant species, including members of Alliaceae and Aloe spp. (Asphodelaceae) were found to lack such Arabidopsis-type (T₃AG₃)_n telomeric repeats. Recently, it has been proposed that the lack of T₃AG₃ telomeric repeat sequences extends to all species forming the Asparagales clade. Here, we analysed the composition of Aloe telomeres by single-primer PCR and fluorescence in-situ hybridization (FISH) with directly labelled Arabidopsis-type (TTTAGGG)_28–43 DNA probe, and with vertebrate-type (TTAGGG)_33–50 DNA and a (C₃TA₂)₃ peptide nucleic acid (PNA) probe. It was found that Nicotiana tabacum contained Arabidopsis-type telomeric repeats, while Aloe telomeres lacked the corresponding FISH signals. Surprisingly, FISH with the highly specific vertebrate-type (C₃TA₂)₃ PNA probe resulted in strong T₂AG₃-specific FISH signals at the ends of chromosomes of both Aloe and Nicotiana tabacum, suggesting the presence of T₂AG₃ telomeric repeats in these species. FISH with a long (TTAGGG)_33–50 DNA probe also highlighted Aloe chromosome ends, while this probe failed to reveal FISH signals on tobacco chromosomes. These results indicate the presence of vertebrate-like telomeric sequences at the telomeres of Aloe spp. chromosomes. However, single-primer PCR with (T₂AG₃)₅ primers failed to amplify such sequences in Aloe, which could indicate a low copy number of T₂AG₃ repeats at the chromosome ends and/or their co-orientation and interspersion with other repeat types. Our results suggest that telomeres of plant species, which were thought to lack GC-rich repeats, may in fact contain variant repeat types. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cloning of DNA libraries from mouse Y chromosomes purified by flow cytometry

To purify mouse Y chromosomes by flow cytometry, a male cell line containing the Robertsonian translocation Rb(9.19)163H has been established by SV40 transformation. Flow karyotypes obtained from these cells exhibit a well-isolated peak of fluorescence corresponding to the single Y chromosome, clearly distinct from that of chromosome 19. From this peak, 650,000 chromosomes were sorted, and two restriction fragment libraries were constructed from the DNA of the sorted chromosomes. The characterization of several Y-specific fragments has shown that the Y DNA was enriched at least 36-fold. Furthermore, given that there are likely homologies between the X and Y chromosomes, we can assume that this calculated value of the purification factor is an underestimation and that the Y DNA was more highly purified by flow sorting.     

Rapid,independent, and extensive amplification of telomeric repeats in pericentromeric regions in karyotypes of arvicoline rodents

The distribution of telomeric repeats was analyzed by fluorescence in situ hybridization in 15 species of arvicoline rodents, included in three different genera: Chionomys, Arvicola, and Microtus. The results demonstrated that in most or the analyzed species, telomeric sequences are present, in addition to normal telomeres localization, as large blocks in pericentromeric regions. The number, localization, and degree of amplification of telomeric sequences blocks varied with the karyotype and the morphology of the chromosomes. Also, in some cases telomeric amplification at non-pericentromeric regions is described. The interstitial telomeric sequences are evolutionary modern and have rapidly colonized and spread in pericentromeric regions of chromosomes by different mechanisms and probably independently in each species. Additionally, we colocalized telomeric repeats and the satellite DNA Msat-160 (also located in pericentromeric regions) in three species and cloned telomeric repeats in one of them. Finally, we discuss about the possible origin and implication of telomeric repeats in the high rate of karyotypic evolution reported for this rodent group.