Tatyana V. Karamysheva

Comparative analysis of micro and macro B chromosomes in the Korean field mouse Apodemus peninsulae (Rodentia, Murinae) performed by chromosome microdissection and FISH

Comparative analysis of micro B and macro B chromosomes of the Korean field mouse Apodemus peninsulae, collected in populations from Siberia and the Russian Far East, was performed with Giemsa, DAPI, Ag-NOR staining and chromosome painting with whole and partial chromosome probes generated by microdissection and DOP-PCR. DNA composition of micro B chromosomes was different from that of macro B chromosomes. All analyzed micro B chromosomes contained clusters of DNA repeats associated with regions characterized by an uncondensed state in mitosis. Giemsa and DAPI staining did not reveal these regions. Their presence in micro B chromosomes led to their special morphology and underestimation in size. DNA repeat clusters homologous to DNA of micro B chromosome arms were also revealed in telomeric regions of some macro B chromosomes of specimens captured in Siberian regions. Neither active NORs nor clusters of ribosomal DNA were found in the uncondensed regions of micro B chromosomes. Possible evolutionary pathways for the origin of macro and micro B chromosomes are discussed.

Somatic Mosaicism in Cases with Small Supernumerary Marker Chromosomes

Somatic mosaicism is something that is observed in everyday lives of cytogeneticists. Chromosome instability is one of the leading causes of large-scale genome variation analyzable since the correct human chromosome number was established in 1956. Somatic mosaicism is also a well-known fact to be present in cases with small supernumerary marker chromosomes (sSMC), i.e. karyotypes of 47,+mar/46. In this study, the data available in the literature were collected concerning the frequency mosaicism in different subgroups of patients with sSMC. Of 3124 cases with sSMC 1626 (52%) present with somatic mosaicism. Some groups like patients with Emanuel-, cat-eye- or i(18p)- syndrome only tend rarely to develop mosaicism, while in Pallister-Killian syndrome every patient is mosaic. In general, acrocentric and non-acrocentric derived sSMCs are differently susceptible to mosaicism; non-acrocentric derived ones are hereby the less stable ones. Even though, in the overwhelming majority of the cases, somatic mosaicism does not have any detectable clinical effects, there are rare cases with altered clinical outcomes due to mosaicism. This is extremely important for prenatal genetic counseling. Overall, as mosaicism is something to be considered in at least every second sSMC case, array-CGH studies cannot be offered as a screening test to reliably detect this kind of chromosomal aberration, as low level mosaic cases and cryptic mosaics are missed by that.

Comparative FISH analysis of distribution ofB chromosome repetitive DNA in A an d B chromosomes in two subspecies of Podisma sapporensis (Orthoptera, Acrididae)

FISH analysis of B chromosome repetitive DNA distribution in A and B chromosomes of two subspecies of Podisma sapporensis (P. s. sapporensis and P. s. krylonensis) was performed. In the B chromosomes, C-positive regions contained homologous DNA repeats present also in some C-positive A chromosome regions. Most C-negative regions contained DNA repeats characteristic of A chromosome euchromatic regions. The two subspecies analyzed differed in the location of A chromosome regions enriched with repeats homologous to repeats of B chromosomes. The only common region enriched with these B chromosome repeats in both subspecies was the X chromosome pericentromeric region. The origin of B chromosomes in P. sapporensis is discussed.

A comparative study of genome organization and inferences for the systematics of two large bushcricket genera of the tribe Barbitistini (Orthoptera: Tettigoniidae: Phaneropterinae)

BackgroundPoecilimon and Isophya are the largest genera of the tribe Barbitistini and among the most systematically complicated and evolutionarily intriguing groups of Palearctic tettigoniids. We examined the genomic organization of 79 taxa with a stable chromosome number using classical (C–banding, silver and fluorochrome staining) and molecular (fluorescence in situ hybridization with 18S rDNA and (TTAGG)n telomeric probes) cytogenetic techniques. These tools were employed to establish genetic organization and differences or similarities between genera or species within the same genus and determine if cytogenetic markers can be used for identifying some taxonomic groups of species.ResultsDifferences between the karyotypes of the studied genera include some general changes in the morphology of the X chromosome in Isophya (in contrast to Poecilimon). The number of major rDNA clusters per haploid genome divided Poecilimon into two main almost equal groups (with either one or two clusters), while two rDNA clusters predominated in Isophya. In both genera, rDNA loci were preferentially located in the paracentromeric region of the autosomes and rarely in the sex chromosomes. Our results demonstrate a coincidence between the location of rDNA loci and active NORs and GC-rich heterochromatin regions. The C/DAPI/CMA3 bands observed in most Poecilimon chromosomes suggest the presence of more families of repetitive DNA sequences as compared to the heterochromatin patterns in Isophya.ConclusionsThe results show both differences and similarities in genome organization among species of the same genus and between genera. Previous views on the systematics and phylogenetic grouping of certain lineages are discussed in light of the present cytogenetic results. In some cases, variation of chromosome markers was observed to correspond with variation in other evolutionary traits, which is related to the processes of ongoing speciation and hybridization in zones of secondary contact. It was concluded that the physical mapping of rDNA sequences and heterochromatin may be used as an additional marker for understanding interspecific relationships in these groups and their routes of speciation.

Comparative FISH analysis of C-positive blocks of centromeric chromosomal regions of pygmy wood mice Sylvaemus uralensis (Rodentia, Muridae)

The composition and homology of centromeric heterochromatin DNA has been compared in representatives of the Asian race and two chromosomal forms (Eastern European and Southern European) of the European race of the pygmy wood mouse Sylvaemus uralensis by means of in situ hybridization with metaphase chromosomes of microdissection DNA probes obtained from centromeric C-blocks of mice of the Southern European chromosomal form and the Asian race. Joint hybridization of both DNA probes yielded all possible variants of centromeric regions in terms of the presence of repetitive sequences homologous to those of some or another dissection region, which indicates a diversity of centromeric regions differing in DNA composition. However, most variations of the fluorescent in situ hybridization (FISH) patterns are apparently related to quantitative differences of repetitive elements of the genome. Experiments with the DNA probe obtained from the genome of the Southern European form of the pygmy wood mouse have shown that the number of intense FISH signals roughly corresponds to the number of large C-segments in representatives of the European race, which is characterized by a large amount of the centromeric C-heterochromatin in the karyotype. However, intense signals have been also detected in experiments on hybridization of this probe with chromosomes of representatives of the Asian race, which has no large C-blocks in the karyotype; thus, DNA sequences homologous to heterochromatic ones are also present in nonheterochromatic regions adjacent to C-segments. Despite the variations of the numbers of both intense and weak FISH signals, all chromosomal forms/races of S. uralensis significantly differ of the samples from one another in these characters. The number of intense FISH signals in DNA in pygmy wood mice of the samples from eastern Turkmenistan (the Kugitang ridge) and southern Omsk oblast (the vicinity of the Talapker railway station) was intermediate between those in the European and Asian races, which is apparently related to a hybrid origin of these populations (the hybridization having occurred long ago in the former case and recently in the latter case).

[Comparative FISH analysis of C-positive regions of chromosomes of wood mice (Rodentia, Muridae, Sylvaemus)].

The homology of DNA of C-positive centromeric regions of chromosomes in wood mice of the genus Sylvaemus (S. uralensis, S. fulvipectus, S. sylvaticus, S. flavicollis, and S. ponticus) was estimated for the first time. DNA probes were generated by microdissection from the centromeric regions of individual autosomes of each species, and their fluorescence in situ hybridization (FISH) with metaphase chromosomes of representatives of all studied wood mouse species was carried out. Unlike in the chromosomal forms and races of S. uralensis, changes in the DNA composition of the chromosomal centromeric regions in the wood mouse species of the genus Sylvaemus (including closely related S. flavicollis and S. ponticus) are both quantitative and qualitative. The patterns of FISH signals after in situ hybridization of the microdissection DNA probes with chromosomes of the species involved in the study demonstrate significant differences between C-positive regions of wood mouse chromosomes in the copy number and the level of homology of repetitive sequences as well as in the localization of homologous repetitive sequences. It was shown that C-positive regions of wood mouse chromosomes can contain both homologous and distinct sets of repetitive sequences. Regions enriched with homologous repeats were detected either directly in C-positive regions of individual chromosomes or only on the short arms of acrocentrics, or at the boundary of C-positive and C-negative regions.

How to narrow down chromosomal breakpoints in small and large derivative chromosomes – a new probe set

Here a new fluorescence in situ hybridization (FISH-) based probe set is presented and its possible applications are highlighted in 34 exemplary clinical cases. The so-called pericentric-ladder-FISH (PCL-FISH) probe set enables a characterization of chromosomal breakpoints especially in small supernumerary marker chromosomes (sSMC), but can also be applied successfully in large inborn or acquired derivative chromosomes. PCL-FISH was established as 24 different chromosome-specific probe sets and can be used in two- up multicolor-FISH approaches. PCL-FISH enables the determination of a chromosomal breakpoint with a resolution between 1 and ∼10 megabasepairs and is based on locus-specific bacterial artificial chromosome (BAC) probes. Results obtained on 29 sSMC cases and five larger derivative chromosomes are presented and discussed. To confirm the reliability of PCL-FISH, eight of the 29 sSMC cases were studied by array-comparative genomic hybridization (aCGH); the used sSMC-specific DNA was obtained by glass-needle based microdissection and DOP-PCR-amplification. Overall, PCL-FISH leads to a better resolution than most FISH-banding approaches and is a good tool to narrow down chromosomal breakpoints.

Chromosomal Localization of Ribosomal and Telomeric DNA Provides New Insights on the Evolution of Gomphocerinae Grasshoppers

Chromosome location of ribosomal DNA (rDNA) and telomeric repeats was analysed in mitotic chromosomes of 15 species of Gomphocerinae grasshoppers belonging to the tribes Arcypterini, Gomphocerini, Stenobothrini, and Chrysochraontini. Two types of rDNA distribution were found in the Gomphocerini tribe. Type 1, found in 9 species, was characterized by the presence of rDNA in the short arm of the long biarmed chromosomes 2 and 3 and, in some species, also in the X chromosome. Type 2 was found only in Aeropus sibiricus and Stauroderus scalaris and consisted in the presence of pericentromeric rDNA blocks in all chromosomes. A comparison of rDNA distribution in Gomphocerini species with 2n♂ = 23, 2n♂ = 21, and 2n♂ = 17 suggested the possible involvement of chromosome 6 in the ancestral karyotype (2n♂ = 23) in 1 of the 3 centric fusions that decreased the chromosome number in these species. In the tribe Stenobothrini, Stenobothrus eurasius carried a single rDNA cluster in the X chromosome, likewise 2 Spanish species previously analysed, but Omocestus viridulus unusually showed a single rDNA cluster in the longest autosome. Telomeric repeats were located primarily on the ends of chromosome arms. In 2 species, however, we observed the presence of interstitial clusters outside telomeric regions. The first one, Aeropus sibiricus, exhibited a polymorphic interstitial site of telomeric repeats in chromosome 6 as a consequence of a paracentric inversion. Most remarkably, Chorthippus jacobsoni showed the presence of telomeric repeats in the pericentric regions of the 3 biarmed chromosome pairs originated by centric fusion, thus suggesting that these rearrangements were not of the Robertsonian type but true centric fusion with a probable generation of dicentric chromosomes.

Distribution of repetitive DNA sequences in chromosomes of five opisthorchid species (Trematoda, Opisthorchiidae).

Genomes of opisthorchid species are characterized by small size, suggesting a reduced amount of repetitive DNA in their genomes. Distribution of repetitive DNA sequences in the chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus 2n = 14 (Rivolta, 1884), Opisthorchis viverrini 2n = 12 (Poirier, 1886), Metorchis xanthosomus 2n = 14 (Creplin, 1846), Metorchis bilis 2n = 14 (Braun, 1890), Clonorchis sinensis 2n = 14 (Cobbold, 1875)) was studied with C- and AgNOR-banding, generation of microdissected DNA probes from individual chromosomes and fluorescent in situ hybridization on mitotic and meiotic chromosomes. Small-sized C-bands were discovered in pericentric regions of chromosomes. Ag-NOR staining of opisthorchid chromosomes and FISH with ribosomal DNA probe showed that karyotypes of all studied species were characterized by the only nucleolus organizer region in one of small chromosomes. The generation of DNA probes from chromosomes 1 and 2 of O. felineus and M. xanthosomus was performed with chromosome microdissection followed by DOP-PCR. FISH of obtained microdissected DNA probes on chromosomes of these species revealed chromosome specific DNA repeats in pericentric C-bands. It was also shown that microdissected DNA probes generated from chromosomes could be used as the Whole Chromosome Painting Probes without suppression of repetitive DNA hybridization. Chromosome painting using microdissected chromosome specific DNA probes showed the overall repeat distribution in opisthorchid chromosomes.

Identification of all pachytene bivalents in the common shrew using DAPI-staining of synaptonemal complex spreads

A major problem in studies of synaptonemal complexes (SC) is the difficulty in distinguishing individual chromosomes. This problem can be solved combining SC immunostaining with FISH of chromosome-specific sequences. However, this procedure is expensive, time-consuming and applicable only to a very limited number of species. In this paper we show how a combination of SC immunostaining and DAPI staining can allow identification of all chromosome arms in surface-spreads of the SC of the common shrew (Sorex araneus L.). Enhancement of brightness and contrast of the images with photo editing software allowed us to reveal clear DAPI-positive and negative bands with relative sizes and positions similar to DAPI landmarks on mitotic metaphase chromosomes. Using FISH with DNA probes prepared from chromosome arms m and n we demonstrated correct recognition of the chromosomes mp and hn on the basis of their DAPI pattern. We show that the approach we describe here may be applied to other species and can provide an important tool for identification of individual bivalents in pachytene surface-spreads.