Painting Probes Research Articles

Karyotype relationships among selected deer species and cattle revealed by bovine FISH probes.

The Cervidae family comprises more than fifty species divided into three subfamilies: Capreolinae, Cervinae and Hydropotinae. A characteristic attribute for the species included in this family is the great karyotype diversity, with the chromosomal numbers ranging from 2n = 6 observed in female Muntiacus muntjak vaginalis to 2n = 70 found in Mazama gouazoubira as a result of numerous Robertsonian and tandem fusions. This work reports chromosomal homologies between cattle (Bos taurus, 2n = 60) and nine cervid species using a combination of whole chromosome and region-specific paints and BAC clones derived from cattle. We show that despite the great diversity of karyotypes in the studied species, the number of conserved chromosomal segments detected by 29 cattle whole chromosome painting probes was 35 for all Cervidae samples. The detailed analysis of the X chromosomes revealed two different morphological types within Cervidae. The first one, present in the Capreolinae is a sub/metacentric X with the structure more similar to the bovine X. The second type found in Cervini and Muntiacini is an acrocentric X which shows rearrangements in the proximal part that have not yet been identified within Ruminantia. Moreover, we characterised four repetitive sequences organized in heterochromatic blocks on sex chromosomes of the reindeer (Rangifer tarandus). We show that these repeats gave no hybridization signals to the chromosomes of the closely related moose (Alces alces) and are therefore specific to the reindeer.

A unique set of complex chromosomal abnormalities in an infant with myeloid leukemia associated with Down syndrome

BackgroundChildren with Down syndrome (DS) have an enhanced risk of developing acute leukemia, with the most common subtype being acute megakaryoblastic leukemia (AMKL). Myeloid leukemia in Down syndrome (ML-DS) is considered a disease with distinct clinical and biological features. There are few studies focusing on the clonal cytogenetic changes during evolution of ML-DS.Case presentationHere, we describe a complex karyotype involving a previously unreported set of chromosomal abnormalities acquired during progression of ML-DS in an infant boy: derivative der(1)t(1;15)(q24;q23), translocation t(4;5)(q26;q33) and derivative der(15)t(7;15)(p21;q23). Different molecular cytogenetic probes and probesets including whole chromosome painting (WCP) and locus specific probes, as well as, multicolor-FISH and multicolor chromosome banding (MCB) were performed in order to characterize the chromosomal abnormalities involved in this complex karyotype. The patient was treated according to the acute myeloid leukemia-Berlin-Frankfurt-Munich-2004 (AML-BFM 2004) treatment protocol for patients with Down syndrome; however, he experienced a poor clinical outcome.ConclusionThe molecular cytogenetic studies performed, allowed the characterization of novel chromosomal abnormalities in ML-DS and possible candidate genes involved in the leukemogenic process. Our findings suggest that the complex karyotype described here was associated with the poor prognosis.

Pooling strategy and chromosome painting characterize a living zebroid for the first time.

We have investigated the complex karyotype of a living zebra-donkey hybrid for the first time using chromosome-specific painting probes produced from flow-sorted chromosomes from a zebra (Equus burchelli) and horse (Equus caballus). As the chromosomes proved difficult to distinguish from one another, a successful new strategy was devised to resolve the difficulty and characterize each chromosome. This was based on selecting five panels of whole chromosome painting probes that could differentiate zebra and donkey chromosomes by labelling the probes with either FITC or Cy3 fluorochromes. Each panel was hybridized sequentially to the same G-Q-banded metaphases and the results combined so that every zebra and donkey chromosome in each suitable metaphase could be identified. A diploid number of 2n = 53, XY was found, containing haploid sets of 22 chromosomes from the zebra and 31 chromosomes from the donkey, without evidence of chromosome rearrangement. This new strategy, developed for the first time, may have several applications in the resolution of other complex hybrid karyotypes and chromosomal aberrations.

A novel mosaic complex supernumerary marker chromosome in a girl with seizures: systematic characterization of the complex marker

Small supernumerary marker chromosomes (sSMC) are a heterogeneous group of chromosomes which are reported in variable phenotypes. They are chromosomal fragments or markers whose origins often cannot be determined by conventional cytogenetic methods alone and require molecular approaches. In general sSMC are equal in size or smaller than chromosome 20. The complex sSMC (CsSMC) arise from two different chromosomes. The aim of the present study is to identify and characterize the chromosomal abnormality in an 11year old girl with seizures, intellectual disability and developmental delay. Different methods like GTG banding, C-banding and NOR staining, spectral karyotyping (SKY), fluorescence in-situ hybridization (FISH) using whole chromosome paint probes (WCP) and bacterial artificial chromosome (BAC) clones were used. Also array CGH was done to check the gains and losses in the genome. The chromosomal analysis on the metaphases revealed a karyotype of mos 47,XX,+mar/46,XX. The marker was identified by SKY and aCGH, as a 33Mb duplication of the 3q region and a 38Mb duplication of the chromosome 9p region. This was confirmed by WCP FISH. Further fine mapping was done with 8 BAC clones from the 3q and 9p regions to confirm the marker chromosome. The marker showed the centromeric 9 region which was also confirmed by the BAC clones. The application of the combined cytogenetic methods like aCGH and FISH helped in the systematic characterization of the CsSMC. The accurate characterization of the CsSMC helps in increasing the knowledge of chromosomal origin, gene content, UPD and other imbalance in the genome.

Extensive Sex Chromosome Polymorphism of Microtus thomasi/Microtus atticus Species Complex Associated with Cryptic Chromosomal Rearrangements and Independent Accumulation of Heterochromatin

The sibling species Microtus thomasi and M. atticus represent probably the highest karyotypic diversity within the genus Microtus and are an interesting model for chromosomal evolution studies. In addition to variation in autosomes, they show a high intraspecific variation in the size and morphology of both sex chromosomes. We analyzed individuals with different sex chromosome constitutions using 3 painting probes, 2 from Y chromosome variants and 1 from the small arm of the submetacentric X chromosome. Our comparative painting approach uncovered 12 variants of Y and 14 variants of X chromosomes, which demonstrates that the polymorphism of sex chromosomes is substantially larger than previously reported. We suggest that 2 main processes are responsible for this sex chromosome polymorphism: change of morphology from acrocentric to submetacentric or metacentric chromosomes and increase in size due to accumulation of repetitive DNA sequences, generating heterochromatic blocks. Strong genetic drift in small and fragmented populations of these 2 species could be related to the origin and maintenance of the large polymorphism of sex chromosomes. We proposed that a similar polymorphism variation combined with random drift fixing the biggest sex chromosomes could have occurred in the origin of some of the actual Microtus species with giant sex chromosomes.

Comparative Chromosomal Studies inRhinolophus formosaeandR. luctusfrom China and Vietnam: Elevation ofR. l. lanosusto Species Rank

For the majority of Rhinolophus species a karyotype consisting of either 58 or 62 chromosomes was reported. The members of the R. trifoliatus clade are therefore distinguished from other rhinolophids by low diploid chromosome numbers (2n) and a high rate of chromosomal changes. Recently, based on cytogenetic characters, a cryptic species, R. luctoides, was described and a subspecies, R. luctusmorio, was elevated to species rank. In the present work, the karyotypes of a male bat from Vietnam and a female one from China, both classified as R. luctus by morphological characters, were studied by G-banding and fluorescence in situ hybridization with whole chromosome painting probes derived from Aselliscus stoliczkanus. In the male specimen from Vietnam, the composition of the 15 autosomal bi-armed pairs (2n = 32, FN = 60) is identical to that of R. luctoides from Malaysia. For reason of differences in body size and baculum length as well as in skull morphology, this specimen is provisionally treated as R. cf. ...

Chromosome divergence during evolution of the tetraploid clawed frogs, Xenopus mellotropicalis and Xenopus epitropicalis as revealed by Zoo-FISH.

Whole genome duplication (WGD) generates new species and genomic redundancy. In African clawed frogs of the genus Xenopus, this phenomenon has been especially important in that (i) all but one extant species are polyploid and (ii) whole genome sequences of some species provide an evidence for genomic rearrangements prior to or after WGD. Within Xenopus in the subgenus Silurana, at least one allotetraploidization event gave rise to three extant tetraploid (2n = 4x = 40) species–Xenopus mellotropicalis, X. epitropicalis, and X. calcaratus–but it is not yet clear the degree to which these tetraploid genomes experienced rearrangements prior to or after allotetraploidization. To explore genome evolution during diversification of these species, we performed cytogenetic analyses of X. mellotropicalis, including assessment of the localization of nucleolar organizer region, chromosome banding, and determination of the p/q arm ratios for each chromosome pair. We compared these data to a previously characterized karyotype of X. epitropicalis. Morphometric, C-banding and Zoo-FISH data support a previously hypothesized common allotetraploid predecessor of these species. Zoo-FISH with whole chromosome painting (WCP) probes derived from the closely related diploid species X. tropicalis confirmed the existence of ten chromosomal quartets in X. mellotropicalis somatic cells, as expected by its ploidy level and tetraploid ancestry. The p/q arm ratio of chromosome 2a was found to be substantially different between X. mellotropicalis (0.81) and X. epitropicalis (0.67), but no substantial difference between these two species was detected in this ratio for the homoeologous chromosome pair 2b, or for other chromosome pairs. Additionally, we identified variation between these two species in the locations of a heterochromatic block on chromosome pair 2a. These results are consistent with a dynamic history of genomic rearrangements before and/or after genome duplication, a surprising finding given the otherwise relatively conserved genomic structure of most frogs.

Chromosomal Aberrations in Large Japanese Field Mice (Apodemus speciosus) Captured near Fukushima Dai-ichi Nuclear Power Plant.

Since the Fukushima Dai-ichi Nuclear Power Plant accident, radiation effects on nonhuman biota in the contaminated areas have been a major concern. Here, we analyzed the frequencies of chromosomal aberrations (translocations and dicentrics) in the splenic lymphocytes of large Japanese field mice (Apodemus speciosus) inhabiting Fukushima Prefecture. A. speciosus chromosomes 1, 2, and 5 were flow-sorted in order to develop A. speciosus chromosome-specific painting probes, and FISH (fluorescence in situ hybridization) was performed using these painting probes to detect the translocations and dicentrics. The average frequency of the translocations and dicentrics per cell in the heavily contaminated area was significantly higher than the frequencies in the case of the noncontaminated control area and the slightly and moderately contaminated areas, and this aberration frequency in individual mice tended to roughly increase with the estimated dose rates and accumulated doses. In all four sampling areas, the proportion of aberrations occurring in chromosome 2 was approximately >3 times higher than that in chromosomes 1 and 5, which suggests that A. speciosus chromosome 2 harbors a fragile site that is highly sensitive to chromosome breaks induced by cellular stress such as DNA replication. The elevated frequency of chromosomal aberrations in A. speciosus potentially resulting from the presence of a fragile site in chromosome 2 might make it challenging to observe the mild effect of chronic low-dose-rate irradiation on the induction of chromosomal aberrations in A. speciosus inhabiting the contaminated areas of Fukushima.

Euchromatic variants of 8q21.2 in twins

ObjectiveEuchromatic variants (EVs) of 8q21.2 are extremely rare chromosomal abnormalities. So, far there have only been two reports on EVs of 8q21.2. Here, we report an 8q21.2 EV detected in cultured amniotic-fluid cells of twins. It was later found to be inherited from the mother, who did not present with abnormal phenotypes. Case ReportA pregnant woman underwent amniocentesis at 16 weeks of gestation because of advanced maternal age. This pregnancy was monozygotic twins conceived naturally. A cytogenetic analysis of cultured amniocytes revealed 46,XY,?dup(8)(q21.2). Chromosomal microarray revealed no abnormalities. C-banding and fluorescent in situ hybridization using chromosome 8 painting probe suggested euchromatic nature of the extra chromosomal band. Karyotyping of the parents showed that the EV was inherited from the mother. ConclusionMany, but not all, EVs are clinically innocuous. This is the first case of 8q21.2 EV reported in the ethnic Han. More cases are needed to clarify whether 8q21.2 duplication as a bona fide EV.

Transcription-dependent radial distribution of TCF7L2 regulated genes in chromosome territories.

Human chromosomes occupy distinct territories in the interphase nucleus. Such chromosome territories (CTs) are positioned according to gene density. Gene-rich CTs are generally located in the center of the nucleus, while gene-poor CTs are positioned more towards the nuclear periphery. However, the association between gene expression levels and the radial positioning of genes within the CT is still under debate. In the present study, we performed three-dimensional fluorescence in situ hybridization experiments in the colorectal cancer cell lines DLD-1 and LoVo using whole chromosome painting probes for chromosomes 8 and 11 and BAC clones targeting four genes with different expression levels assessed by gene expression arrays and RT-PCR. Our results confirmed that the two over-expressed genes, MYC on chromosome 8 and CCND1 on chromosome 11, are located significantly further away from the center of the CT compared to under-expressed genes on the same chromosomes, i.e., DLC1 and SCN3B. When CCND1 expression was reduced after silencing the major transcription factor of the WNT/β-catenin signaling pathway, TCF7L2, the gene was repositioned and mostly detected in the interior of the CT. Thus, we suggest a non-random distribution in which over-expressed genes are located more towards the periphery of the respective CTs.

Tissue-specific features of the X chromosome and nucleolus spatial dynamics in a malaria mosquito, Anopheles atroparvus.

Spatial organization of chromosome territories is important for maintenance of genomic stability and regulation of gene expression. Recent studies have shown tissue-specific features of chromosome attachments to the nuclear envelope in various organisms including malaria mosquitoes. However, other spatial characteristics of nucleus organization, like volume and shape of chromosome territories, have not been studied in Anopheles. We conducted a thorough analysis of tissue-specific features of the X chromosome and nucleolus volume and shape in follicular epithelium and nurse cells of the Anopheles atroparvus ovaries using a modern open-source software. DNA of the polytene X chromosome from ovarian nurse cells was obtained by microdissection and was used as a template for amplification with degenerate oligo primers. A fluorescently labeled X chromosome painting probe was hybridized with formaldehyde-fixed ovaries of mosquitoes using a 3D-FISH method. The nucleolus was stained by immunostaining with an anti-fibrillarin antibody. The analysis was conducted with TANGO—a software for a chromosome spatial organization analysis. We show that the volume and position of the X chromosome have tissue-specific characteristics. Unlike nurse cell nuclei, the growth of follicular epithelium nuclei is not accompanied with the proportional growth of the X chromosome. However, the shape of the X chromosome does not differ between the tissues. The dynamics of the X chromosome attachment regions location is tissue-specific and it is correlated with the process of nucleus growth in follicular epithelium and nurse cells.

Rapid Karyotype Evolution in Lasiopodomys Involved at Least Two Autosome - Sex Chromosome Translocations.

The generic status of Lasiopodomys and its division into subgenera Lasiopodomys (L. mandarinus, L. brandtii) and Stenocranius (L. gregalis, L. raddei) are not generally accepted because of contradictions between the morphological and molecular data. To obtain cytogenetic evidence for the Lasiopodomys genus and its subgenera and to test the autosome to sex chromosome translocation hypothesis of sex chromosome complex origin in L. mandarinus proposed previously, we hybridized chromosome painting probes from the field vole (Microtus agrestis, MAG) and the Arctic lemming (Dicrostonyx torquatus, DTO) onto the metaphases of a female Mandarin vole (L. mandarinus, 2n = 47) and a male Brandt's vole (L. brandtii, 2n = 34). In addition, we hybridized Arctic lemming painting probes onto chromosomes of a female narrow-headed vole (L. gregalis, 2n = 36). Cross-species painting revealed three cytogenetic signatures (MAG12/18, 17a/19, and 22/24) that could validate the genus Lasiopodomys and indicate the evolutionary affinity of L. gregalis to the genus. Moreover, all three species retained the associations MAG1bc/17b and 2/8a detected previously in karyotypes of all arvicolins studied. The associations MAG2a/8a/19b, 8b/21, 9b/23, 11/13b, 12b/18, 17a/19a, and 5 fissions of ancestral segments appear to be characteristic for the subgenus Lasiopodomys. We also validated the autosome to sex chromosome translocation hypothesis on the origin of complex sex chromosomes in L. mandarinus. Two translocations of autosomes onto the ancestral X chromosome in L. mandarinus led to a complex of neo-X1, neo-X2, and neo-X3 elements. Our results demonstrate that genus Lasiopodomys represents a striking example of rapid chromosome evolution involving both autosomes and sex chromosomes. Multiple reshuffling events including Robertsonian fusions, chromosomal fissions, inversions and heterochromatin expansion have led to the formation of modern species karyotypes in a very short time, about 2.4 MY.

A Comparative Study of Pygmy Hippopotamus (Choeropsis liberiensis) Karyotype by Cross-Species Chromosome Painting

The family Hippopotamidae is comprised of two genera with two living species, the common hippo (Hippopotamus amphibius) and the pygmy hippo (Choeropsis liberiensis). Unlike the common hippo, the karyotype of C. liberiensis has not yet been investigated via cross-species chromosome painting methods. We established chromosomal homologies between the pygmy hippo, pig, and cattle by fluorescence in situ hybridization using whole chromosome, arm-specific, region specific, and bacterial artificial chromosome (BAC) probes. Probes from the 18 pig autosomes painted 45 conserved chromosomal segments in the pygmy hippo genome. The pygmy hippo and cattle homology map was deduced from our hybridization results of painting probes to pygmy hippo chromosomes with a combination of previously published dromedary hybridization data. On the pygmy hippo and cattle homology map, 29 cattle autosomes revealed 39 conservative segments on pygmy hippo chromosomes. For a more detailed structural analysis of genome rearrangements and X chromosome structure, we used cattle region specific and BAC probes. Our report demonstrates that cattle probes are useful not only in comparative studies within Ruminantia, but also in more phylogenetically distant Artiodactyla species.

Chromosome Painting in Triatomine Insects Reveals Shared Sequences Between X Chromosomes and Autosomes.

In order to provide a broad picture on the origin and evolution of holocentric X chromosomes in heteropteran species, we prepared a sex chromosome painting probe by microdissection of the X1 and X2 chromosomes from a kissing bug Mepraia spinolai (Hemiptera: Reduviidae: Triatominae). Fluorescence in situ hybridization on four species of the Triatomini having different amounts of autosomal heterochromatin and sex chromosome systems show that the Xs probe hybridizes on the euchromatin, located both on autosomes and X chromosomes. The heterochromatic Y chromosome and autosomal heterochromatic regions always appear free of hybridization signals. The hybridization results of the Xs probe on Rhodnius prolixus (Rhodniini) is completely different to that observed in Triatomini species. The hybridization signals are small and scattered on all euchromatin, without specific regions including the X chromosome. These results are in accordance with previous data obtained by genomic in situ hybridization and fluorescent banding, suggesting a clear differentiation in the repeat sequence composition of both sex chromosomes between Triatomini and Rhodniini tribes. These results also support that each sex chromosome in Triatomini has evolved independently from different autosomal pairs of a common ancestor, as described in other insect orders.

Sorting of chromosomes on FACSAria(TM) SORP for the preparation of painting probes.

High purity chromosome sorting can be performed on instruments such as MoFlo MLS and BD influx, which are stream-in-air sorters equipped with water-cooled high power lasers. The FACSAria is a true fixed alignment, low laser powered instrument with a quartz flow cell gel-coupled to the collection optics. However, whether high purity mouse and human chromosomes can be obtained by sorting on the BD FACSAria(TM) Special Order Research Product (FACSAria SORP) remains to be determined. Here, we report that the high resolution flow karyotype of mouse lymphocytes and normal male human peripheral blood mononuclear cells (hPBMCs) can be obtained on the FACSAria SORP using laser power settings of 50mW for 355nm and 20mW for 444nm excitation. Furthermore, the use of Fluorescence in situ hybridization (FISH) confirmed that chromosome paints prepared from the sorted chromosomes demonstrated high purity and signal specificity. Notably, human chromosome 12 was separated from the chromosome 9-12 cluster in the flow karyotype, and its identity was confirmed using FISH in trisomy 12 human ES cell lines B2-C7 and B2-B8. In addition, multicolor FISH (mFISH) with human chromosome painting probes to 13,18, 21, and sex chromosomes X and Y showed high signal specificity in hPBMCs. Taken together, our findings demonstrated that high resolution flow karyotype can be obtained using FACSAria SORP. Moreover, a FISH analysis confirmed high purity of the sorted chromosomes. Additionally, in contrast to centromeric satellite probes, chromosome painting probes with high specificity are more suitable for detection of chromosome aberrations, such as deletions and translocations, in prenatal diagnosis. © 2016 International Society for Advancement of Cytometry.

Comparative analysis of individual chromosome involvement in micronuclei induced by mitomycin C and bleomycin in human leukocytes.

BackgroundMicronucleus (MN) assay is a well standardized approach for evaluation of clastogenic/aneugenic effects of mutagens. Fluorescence in situ hybridization (FISH) is successfully used to characterize the chromosomal content of MN. However, the relationships between nuclear positioning, length, and gene density of individual chromosomes and their involvement in MN induced by different mutagens have not been clearly defined.ResultsChromosomal content of MN was characterized in human leukocytes treated with mitomycin C (MMC) and bleomycin (BLM) by FISH using centromeric (cep) and whole-chromosome painting (wcp) probes. Involvement of chromosomes 8, 15 and 20 in MMC-induced and chromosomes 1, 9 and 16 in BLM-induced MN was studied, and correlated with chromosome size, gene density and interphase position. The results obtained were analyzed together with previous own data on the frequencies of inclusion of chromosomes 3, 4, 6, 7, 9, 16, 17, 18, and X in MMC-induced MN. It could be shown that MMC- and BLM-induced MN could contain material derived from all chromosomes investigated. Involvement of whole chromosomes 8, 15 and 20 in MMC-induced MN negatively correlated with gene density; however, analysis together with earlier studied chromosomes did not confirm this correlation. Inclusion of chromosomes 8, 15 and 20 in MMC-induced MN does not depend on their size and interphase position; the same result was found for the twelve overall analyzed chromosomes. In BLM-treated cells significant correlation between frequencies of involvement of chromosomes 1, 9 and 16 in MN and their size was found.ConclusionsOur results clearly revealed that BLM differs from MMC with respect to the distribution of induced chromosome damage and MN formation. Thus, DNA-damaging agents with diverse mechanism of action induce qualitatively different MN with regard to their chromosomal composition. Also this study demonstrates the utility of combined sequential application of cep and wcp probes for efficient detection of MN chromosomal content in terms of centric and acentric fragments.

Karyotypic Evolution in Malagasy Flying Foxes (Pteropodidae, Chiroptera) and Their Hipposiderid Relatives as Determined by Comparative Chromosome Painting

Pteropodidae and Hipposideridae are 2 of the 9 chiropteran families that occur on Madagascar. Despite major advancements in the systematic study of the island's bat fauna, few karyotypic data exist for endemic species. We utilized G- and C-banding in combination with chromosome painting with Myotismyotis probes to establish a genome-wide homology among Malagasy species belonging to the families Pteropodidae (Pteropus rufus 2n = 38; Rousettus madagascariensis, 2n = 36), Hipposideridae (Hipposideros commersoni s.s., 2n = 52), and a single South African representative of the Rhinolophidae (Rhinolophus clivosus, 2n = 58). Painting probes of M. myotis detected 26, 28, 28, and 29 regions of homology in R. madagascariensis, P. rufus, H. commersoni s.s, and R. clivosus, respectively. Translocations, pericentric inversions, and heterochromatin additions were responsible for karyotypic differences amongst the Malagasy pteropodids. Comparative chromosome painting revealed a novel pericentric inversion on P. rufus chromosome 4. Chromosomal characters suggest a close evolutionary relationship between Rousettus and Pteropus. H. commersoni s.s. shared several chromosomal characters with extralimital congeners but did not exhibit 2 chromosomal synapomorphies proposed for Hipposideridae. This study provides further insight into the ancestral karyotypes of pteropodid and hipposiderid bats and corroborates certain molecular phylogenetic hypotheses.

Oligo-based aCGH analysis reveals cryptic unbalanced der(6)t(X;6) in pediatric t(12;21)-positive acute lymphoblastic leukemia

Secondary chromosomal aberrations are necessary for development of overt leukemia in t(12;21)/ETV6-RUNX1-positive acute lymphoblastic leukemia (ALL). Conventional cytogenetic analysis supplemented with locus-specific FISH analyses is gold standard to detect important clonal aberrations in this disease group. However, adequate chromosome banding analysis may often be hampered by poor chromosome morphology and banding patterns in pediatric ALL cases, which may hinder identification of possible clinical important additional chromosomal aberrations.We used oligo-based high-resolution aCGH (oaCGH) analysis as an adjunct tool to enhance conventional cytogenetic analysis in pediatric acute B-cell lymphoblastic leukemia in a prospective single center study during a 4-year period (2012–2015).In a consecutive series of 45 pediatric B-ALLs, we identified eight patients with t(12;21)/ETV6-RUNX1 fusion by FISH analysis. In three of the patients, oaCGH analysis revealed concurrent Xq duplication and 6q deletion, which was cryptic by G-banded analysis. FISH analyses with whole chromosome painting probes confirmed the imbalances and showed an unbalanced translocation der(6)t(X;6) in all three patients. A search in the literature revealed two additional pediatric patients with cryptic der(6)t(X;6) in t(12;21)-positive ALLs. No common break points on Xq or 6q could be determined between the five patients.This study highlights the importance of oaCGH analysis as an adjunct cytogenetic tool to detect cryptic chromosomal aberrations. Further, the study adds to understanding the full spectrum of secondary chromosomal aberrations in the very common t(12;21)-positive pediatric ALL disease group. We suggest that the unbalanced der(6)t(X;6), which is cryptic to conventional cytogenetics, is a non-random secondary event in this disease group. It might be that the specific combination of concurrent Xq duplication and 6q-deletion results in gain of possible oncogenes on Xq and loss of possible tumor suppressor genes on 6q that are important for the leukemic propagation of t(12;21)-positive hematopoietic cells in a subset of ALLs.

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.

Origin of B chromosomes in the genus Astyanax (Characiformes, Characidae) and the limits of chromosome painting.

Eukaryote genomes are frequently burdened with the presence of supernumerary (B) chromosomes. Their origin is frequently investigated by chromosome painting, under the hypothesis that sharing the repetitive DNA sequences contained in the painting probes is a sign of common descent. However, the intragenomic mobility of many anonymous DNA sequences contained in these probes (e.g., transposable elements) adds high uncertainty to this conclusion. Here we test the validity of chromosome painting to investigate B chromosome origin by comparing its results for seven B chromosome types in two fish species genus Astyanax, with those obtained (1) by means of the physical mapping of 18S ribosomal DNA (rDNA), H1 histone genes, the As51 satellite DNA and the (AC)15 microsatellite, and (2) by comparing the nucleotide sequence of one of these families (ITS regions from ribosomal DNA) between genomic DNA from B-lacking individuals in both species and the microdissected DNA from two metacentric B chromosomes found in these same species. Intra- and inter-specific painting suggested that all B chromosomes that were assayed shared homologous DNA sequences among them, as well as with a variable number of A chromosomes in each species. This finding would be consistent with a common origin for all seven B chromosomes analyzed. By contrast, the physical mapping of repetitive DNA sequences failed to give support to this hypothesis, as no more than two B-types shared a given repetitive DNA. Finally, sequence analysis of the ITS regions suggested that at least some of the B chromosomes could have had a common origin.