Nikolay B. Rubtsov

DNA content of the B chromosomes in grasshopper Podisma kanoi Storozh. (Orthoptera, Acrididae)

A DNA library derived from the B chromosome of Podisma kanoi was obtained by chromosome microdissection. A total of 153 DNA clones were isolated from the microdissected DNA library. Twenty of them were sequenced. A comparison of B chromosome DNA sequences with sequences of other species from the DDBJ/GenBank/EMBL database (http://www.ddbj.nig.ac.jp/) was performed. Different patterns of signals were observed after FISH with labeled cloned DNA fragments. FISH signals with cloned DNA fragments painted either whole Bs or their different regions. Some clones also gave signals in pericentromeric regions of A chromosomes. Other cloned DNA fragments gave only background-like signals on A and B chromosomes. Comparative FISH analysis of B chromosomes in Podisma kanoi and P. sapporensis with DNA probes derived from the Bs of these species revealed homologous DNA that was confined within pericentromeric and telometric regions of the B chromosome in P. kanoi. In contrast to the B chromosomes in P. sapporensis containing large regions enriched with rDNA, only a small cluster of rDNA was detected in one of the examined B chromosomes in P. kanoi. The data strongly suggest an independent origin of B chromosomes in two closely related Podisma species.

Comparative cytogenetics of opisthorchid species (Trematoda, Opisthorchiidae)

In the present study karyotypes and chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus (Rivolta, 1884), O. viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), M. bilis (Braun, 1893), and Clonorchis sinensis (Cobbold, 1875)) were compared. Karyotypes of O. felineus, M. xanthosomus, M. bilis and C. sinensis consist of two pairs of large meta- and submetacentrics and five pairs of small chromosomes (2n = 14). The karyotype of O. viverrini is 2n = 12, which indicates a fusion of two chromosomes of opisthorchid ancestral karyotype. Analysis of mitotic and meiotic chromosomes was performed by heterologous in situ hybridization of microdissected DNA probes obtained from chromosomes 1 and 2 of O. felineus and chromosomes 1 and 2 of M. xanthosomus. Results of chromosome staining (C- and AgNOR-banding) and FISH of telomeric probes and ribosomal DNA probe on opisthorchid chromosomes were used for chromosome comparison. Data on chromosome number in opisthorchid species were also discussed.

Microdissection and sequence analysis of pericentric heterochromatin from the Drosophila melanogaster mutant Suppressor of Underreplication

Abstract. In the Suppressor of Underreplication (SuUR) mutant strain of Drosophila melanogaster, the heterochromatin of polytene chromosomes is not underreplicated and, as a consequence, a number of β-heterochromatic regions acquire a banded structure. The chromocenter does not form in these polytene chromosomes, and heterochromatic regions, normally part of the chromocenter, become accessible to cytological analysis. We generated four genomic DNA libraries from specific heterochromatic regions by microdissection of polytene chromosomes. In situ hybridization of individual libraries onto SuUR polytene chromosomes shows that repetitive DNA sequences spread into the neighboring euchromatic regions. This observation allows the localization of eu-heterochromatin transition zones on polytene chromosomes. We find that genomic scaffolds from the eu-heterochromatin transition zones are enriched in repetitive DNA sequences homologous to those flanking the suppressor of forked gene [su(f) repeat]. We isolated and sequenced about 300 clones from the heterochromatic DNA libraries obtained. Most of the clones contain repetitive DNA sequences; however, some of the clones have unique DNA sequences shared with parts of unmapped genomic scaffolds. Hybridization of these clones onto SuUR polytene chromosomes allowed us to assign the cytological localizations of the corresponding genomic scaffolds within heterochromatin. Our results demonstrate that the SuUR mutant renders possible the mapping of heterochromatic scaffolds on polytene chromosomes.

Evidence for Karyotype Polymorphism in the Free-Living Flatworm, Macrostomum lignano, a Model Organism for Evolutionary and Developmental Biology

Over the past decade, the free-living flatworm Macrostomum lignano has been successfully used in many areas of biology, including embryology, stem cells, sexual selection, bioadhesion and aging. The increased use of this powerful laboratory model, including the establishment of genomic resources and tools, makes it essential to have a detailed description of the chromosome organization of this species, previously suggested to have a karyotype with 2n = 8 and one pair of large and three pairs of small metacentric chromosomes. We performed cytogenetic analyses for chromosomes of one commonly used inbred line of M. lignano (called DV1) and uncovered unexpected chromosome number variation in the form of aneuploidies of the largest chromosomes. These results prompted us to perform karyotypic studies in individual specimens of this and other lines of M. lignano reared under laboratory conditions, as well as in freshly field-collected specimens from different natural populations. Our analyses revealed a high frequency of aneuploids and in some cases other numerical and structural chromosome abnormalities in laboratory-reared lines of M. lignano, and some cases of aneuploidy were also found in freshly field-collected specimens. Moreover, karyological analyses were performed in specimens of three further species: Macrostomum sp. 8 (a close relative of M. lignano), M. spirale and M. hystrix. Macrostomum sp. 8 showed a karyotype that was similar to that of M. lignano, with tetrasomy for its largest chromosome being the most common karyotype, while the other two species showed a simpler karyotype that is more typical of the genus Macrostomum. These findings suggest that M. lignano and Macrostomum sp. 8 can be used as new models for studying processes of partial genome duplication in genome evolution.

A comparative study of cell-free apoptotic and genomic DNA using FISH and massive parallel sequencing

Objective : Study of circulating DNA (cirDNA) generation mechanisms with respect to their influence on the content of cirDNA is very important since it could indicate the best molecular targets for diagnostic applications. Since apoptosis was shown to be one of the main sources of cirDNA, we performed in vitro comparative study of cell-free apoptotic and genomic DNA (gDNA). Methods : DNA isolated from culture medium of apoptotic human umbilical vein endothelial cells (cm-apoDNA) and the gDNA from the same living cells was analyzed using FISH and sequenced on SOLiD 3 platform. Results/conclusions : FISH demonstrates overrepresentation of C-positive chromosome regions in cm-apoDNA. SOLiD 3 data show enrichment of cm-apoDNA for Alu repeats: the content of AluJ, AluS and AluY repeats was, respectively, 2.47-fold (standard deviation (SD) 3.6%), 2.45-fold (SD 5.5%) and 2.79-fold (SD 6.1%) higher in cm-apoDNA. By contrast, some of L1 elements were underrepresented in cm-apoDNA: the content of L1MA and L1ME was, respectively, 1.4-fold (SD 22%) and 1.45-fold (SD 9%) lower in cm-apoDNA. In contrast to FISH, these data and the predominant location of Alu repeats in euchromatic regions evidence the non-uniform gDNA degradation during apoptosis leading to the enrichment of cm-apoDNA with coding sequences.

Telomeric DNA in chromosomes of five opisthorchid species.

The analysis of telomere repeat distribution in chromosomes of five opisthorchid species (Opisthorchis felineus (Rivolta, 1884), Opisthorchis viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), Metorchis bilis (Braun, 1890), Clonorchis sinensis (Cobbold, 1875)) was performed with fluorescent in situ hybridization (FISH) of labeled (TTAGGG)n DNA-probe and PNA telomere probe on mitotic and meiotic chromosomes of these species. It was shown that chromosome telomeres of all studied species contain large clusters of (TTAGGG)n telomeric repeats. Interstitial clusters of the (TTAGGG)n repeats have not been revealed in the chromosomes of any studied species even when FISH of PNA telomere probe on pachytene chromosomes was performed. Furthermore interstitial clusters of the (TTAGGG)n repeats have not been detected in the chromosomes of O. viverrini, one of chromosomes of this species is the result of a fusion of two ancestral opisthorchid 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.

New insights into the karyotype evolution of the free-living flatworm Macrostomum lignano (Platyhelminthes, Turbellaria)

The free-living flatworm Macrostomum lignano is a model organism for evolutionary and developmental biology studies. Recently, an unusual karyotypic diversity was revealed in this species. Specifically, worms are either ‘normal’ 2n = 8, or they are aneuploid with one or two additional large chromosome(s) (i.e. 2n = 9 or 2n = 10, respectively). Aneuploid worms did not show visible behavioral or morphological abnormalities and were successful in reproduction. In this study, we generated microdissected DNA probes from chromosome 1 (further called MLI1), chromosome 2 (MLI2), and a pair of similar-sized smaller chromosomes (MLI3, MLI4). FISH using these probes revealed that MLI1 consists of contiguous regions homologous to MLI2-MLI4, suggesting that MLI1 arose due to the whole genome duplication and subsequent fusion of one full chromosome set into one large metacentric chromosome. Therefore, one presumably full haploid genome was packed into MLI1, leading to hidden tetraploidy in the M. lignano genome. The study of Macrostomum sp. 8 — a sibling species of M. lignano — revealed that it usually has one additional pair of large chromosomes (2n = 10) showing a high homology to MLI1, thus suggesting hidden hexaploidy in its genome. Possible evolutionary scenarios for the emergence of the M. lignano and Macrostomum sp. 8 genomes are discussed.

Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and Apodemus B chromosomes

Supernumerary chromosomes sporadically arise in many eukaryotic species as a result of genomic rearrangements. If present in a substantial part of species population, those are called B chromosomes, or Bs. This is the case for 70 mammalian species, most of which are rodents. In humans, the most common types of extra chromosomes, sSMCs (small supernumerary marker chromosomes), are diagnosed in approximately 1 of 2000 postnatal cases. Due to low frequency in population, human sSMCs are not considered B chromosomes. Genetic content of both B-chromosomes and sSMCs in most cases remains understudied. Here, we apply microdissection of single chromosomes with subsequent low-pass sequencing on Ion Torrent PGM and Illumina MiSeq to identify unique and repetitive DNA sequences present in a single human sSMC and several B chromosomes in mice Apodemus flavicollis and Apodemus peninsulae. The pipeline for sequencing data analysis was made available in Galaxy interface as an addition to previously published command-line version. Human sSMC was attributed to the proximal part of chromosome 15 long arm, and breakpoints leading to its formation were located into satellite DNA arrays. Genetic content of Apodemus B chromosomes was species-specific, and minor alterations were observed in both species. Common features of Bs in these Apodemus species were satellite DNA and ERV enrichment, as well as the presence of the vaccinia-related kinase gene Vrk1. Understanding of the non-essential genome elements content provides important insights into genome evolution in general.

Chromosome Evolution in the Free-Living Flatworms: First Evidence of Intrachromosomal Rearrangements in Karyotype Evolution of Macrostomum lignano (Platyhelminthes, Macrostomida)

The free-living flatworm Macrostomum lignano is a hidden tetraploid. Its genome was formed by a recent whole genome duplication followed by chromosome fusions. Its karyotype (2n = 8) consists of a pair of large chromosomes (MLI1), which contain regions of all other chromosomes, and three pairs of small metacentric chromosomes. Comparison of MLI1 with metacentrics was performed by painting with microdissected DNA probes and fluorescent in situ hybridization of unique DNA fragments. Regions of MLI1 homologous to small metacentrics appeared to be contiguous. Besides the loss of DNA repeat clusters (pericentromeric and telomeric repeats and the 5S rDNA cluster) from MLI1, the difference between small metacentrics MLI2 and MLI4 and regions homologous to them in MLI1 were revealed. Abnormal karyotypes found in the inbred DV1/10 subline were analyzed, and structurally rearranged chromosomes were described with the painting technique, suggesting the mechanism of their origin. The revealed chromosomal rearrangements generate additional diversity, opening the way toward massive loss of duplicated genes from a duplicated genome. Our findings suggest that the karyotype of M. lignano is in the early stage of genome diploidization after whole genome duplication, and further studies on M. lignano and closely related species can address many questions about karyotype evolution in animals.