Nadezhda V. Vorobieva

Ancient DNA Analysis Affirms the Canid from Altai as a Primitive Dog

The origin of domestic dogs remains controversial, with genetic data indicating a separation between modern dogs and wolves in the Late Pleistocene. However, only a few dog-like fossils are found prior to the Last Glacial Maximum, and it is widely accepted that the dog domestication predates the beginning of agriculture about 10,000 years ago. In order to evaluate the genetic relationship of one of the oldest dogs, we have isolated ancient DNA from the recently described putative 33,000-year old Pleistocene dog from Altai and analysed 413 nucleotides of the mitochondrial control region. Our analyses reveal that the unique haplotype of the Altai dog is more closely related to modern dogs and prehistoric New World canids than it is to contemporary wolves. Further genetic analyses of ancient canids may reveal a more exact date and centre of domestication.

The proto-oncogene C-KIT maps to canid B-chromosomes

Plant and animal karyotypes sometimes contain variable elements, that are referred to as additional or B-chromosomes. It is generally believed that B-chromosomes lack major genes and represent parasitic and selfish elements of a genome. Here we report, for the first time, the localization of a gene to B-chromosomes of mammals: red fox (Vulpes vulpes) and two subspecies of raccoon dog (Nyctereutes procyonoides). Identification of the proto-oncogene C-KIT on B-chromosomes of two Canidae species that diverged from a common ancestor more than 12.5 million years ago argues against the current view of B-chromosomes. Analyses of fox B-chromosomal C-KIT gene from a flow-sorted fox B-chromosome-specific library revealed the presence of intron–exon boundaries and high identity between sequenced regions of canine and fox B-chromosomal C-KIT copies. Identification of C-KIT gene on all B-chromosomes of two canid species provides new insight into the origin and evolution of supernumeraries and their potential role in the genome.

Mapping of KIT adjacent sequences on canid autosomes and B chromosomes

B chromosomes are often considered to be one of the most mysterious elements of karyotypes (Camacho, 2004). It is generally believed that mammalian B chromosomes do not contain any protein coding genes. The discovery of a conserved KIT gene in Canidae B chromosomes has changed this view. Here we performed analysis of sequences surrounding KIT in B chromosomes of the fox and raccoon dog. The presence of the RPL23A pseudogene was shown in canid B chromosomes. The 3′ end fragment of the KDR gene was found in raccoon dog B chromosomes. The size of the B-specific fragment homologous to the autosome fragment was estimated to be a minimum of 480 kbp in both species. The origin and evolution of B chromosomes in Canidae are discussed.

Human chromosome 3: high-resolution fluorescencein situ hybridization mapping of 40 uniqueNotl linking clones homologous to genes and cDNAs

Forty newNotl linking clones representing sequence tagged sites (STSs) were mapped by fluorescencein situ hybridization (FISH) to different regions of human chromosome 3 (HSA3). Clone NL1-245, containing human aminoacylase 1, was localized to 3p21.2–p21.1. Our previous localization of the CLC-2 chloride channel protein gene was refined to 3q27. Clone NL2-316 most likely contains a translocon-associated protein γ-subunit gene and was mapped to 3q23–q24. To our knowledge, this is the first time this gene has been mapped. OneNoti linking clone (NL1-229) probably contains a new protein phosphatase gene. This clone was mapped to 3p25. FiveNoti linking clones probably contain human expressed sequence tags (ESTs), as they possess sequences with a high level of identity (>90%) to cDNA clones. Other clones show 56–85% homology to known mammalian and human genes with various functions, including oncogenes and tumour-suppressor genes. These clones might represent new genes.

Cross-species chromosome painting unveils cytogenetic signatures for the Eulipotyphla and evidence for the polyphyly of Insectivora

Insectivore-like animals are traditionally believed among the first eutherian mammals that have appeared on the earth. The modern insectivores are thus crucial for understanding the systematics and phylogeny of eutherian mammals as a whole. Here cross-species chromosome painting, with probes derived from flow-sorted chromosomes of human, was used to delimit the homologous chromosomal segments in two Soricidae species, the common shrew (Sorex araneus, 2n = 20/21), and Asiatic short-tailed shrew (Blarinella griselda, 2n = 44), and one Erinaceidae species, the shrew-hedgehog (Neotetracus sinensis, 2n = 32), and human. We report herewith the first comparative maps for the Asiatic short-tailed shrew and the shrew-hedgehog, in addition to a refined comparative map for the common shrew. In total, the 22 human autosomal paints detected 40, 51 and 58 evolutionarily conserved segments in the genomes of common shrew, Asiatic short-tailed shrew, and shrew-hedgehog, respectively, demonstrating that the common shrew has retained a conserved genome organization while the Asiatic short-tailed shrew and shrew-hedgehog have relatively rearranged genomes. In addition to confirming the existence of such ancestral human segmental combinations as HSA 3/21, 12/22, 14/15 and 7/16 that are shared by most eutherian mammals, our study reveals a shared human segmental combination, HSA 4/20, that could phylogenetically unite the Eulipotyphlan (i.e., the core insectivores) species. Our results provide cytogenetic evidence for the polyphyly of the order Insectivora and additional data for the eventual reconstruction of the ancestral eutherian karyotype.

Chromosomal localization of six repeated DNA sequences among species of Microtus (Rodentia)

C-banding and fluorescence in situ hybridization (FISH) document the distribution of constitutive heterochromatin and six highly repeated DNA families (MSAT2570, MSAT21, MSAT160, MS2, MS4 and STR47) in the chromosomes of nine species of Microtus (M. chrotorrhinus, M. rossiaemeridionalis, M. arvalis, M. ilaeus, M. transcaspicus, M. cabrerae, M. pennsylvanicus, M. miurus and M. ochrogaster). Autosomal heterochromatin is largely centromeric and contains different repeated families in different species. Similarly, large C-band positive blocks on the sex chromosomes of four species contain different repeated DNAs. This interspecific variation in the chromosomal distribution and copy number of the repeats suggests that a common ancestor to modern species contained most of the repetitive families, and that descendant species selectively amplified or deleted different repeats on different chromosomes.

Localization of human ARF2 and NCK genes and 13 other Notl-linking clones to chromosome 3 by fluorescence in situ hybridization

Two human genes containing NotI sites, ADP-ribosylation factor (ARF2) and melanoma NCK protein, were mapped by fluorescence in situ hybridization to 3p21.2-->p21.1 and 3q21, respectively. Thirteen other NotI-linking clones, representing sequence tagged sites, were also mapped to different regions of human chromosome 3. Two of these clones that contain sequences 80% homologous to the rat tropoelastin gene and brain Cl- channel protein CLC-2 gene probably represent new human genes closely related to the known rat genes.

Segmental paleotetraploidy revealed in sterlet (Acipenser ruthenus) genome by chromosome painting

BackgroundAcipenseriformes take a basal position among Actinopteri and demonstrate a striking ploidy variation among species. The sterlet (Acipenser ruthenus, Linnaeus, 1758; ARUT) is a diploid 120-chromosomal sturgeon distributed in Eurasian rivers from Danube to Enisey. Despite a high commercial value and a rapid population decline in the wild, many genomic characteristics of sterlet (as well as many other sturgeon species) have not been studied.ResultsCell lines from different tissues of 12 sterlet specimens from Siberian populations were established following an optimized protocol. Conventional cytogenetic studies supplemented with molecular cytogenetic investigations on obtained fibroblast cell lines allowed a detailed description of sterlet karyotype and a precise localization of 18S/28S and 5S ribosomal clusters. Localization of sturgeon specific HindIII repetitive elements revealed an increased concentration in the pericentromeric region of the acrocentric ARUT14, while the total sterlet repetitive DNA fraction (C0t30) produced bright signals on subtelomeric segments of small chromosomal elements. Chromosome and region specific probes ARUT1p, 5, 6, 7, 8 as well as 14 anonymous small sized chromosomes (probes A-N) generated by microdissection were applied in chromosome painting experiments. According to hybridization patterns all painting probes were classified into two major groups: the first group (ARUT5, 6, 8 as well as microchromosome specific probes C, E, F, G, H, and I) painted only a single region each on sterlet metaphases, while probes of the second group (ARUT1p, 7 as well as microchromosome derived probes A, B, D, J, K, M, and N) marked two genomic segments each on different chromosomes. Similar results were obtained on male and female metaphases.ConclusionsThe sterlet genome represents a complex mosaic structure and consists of diploid and tetraploid chromosome segments. This may be regarded as a transition stage from paleotetraploid (functional diploid) to diploid genome condition. Molecular cytogenetic and genomic studies of other 120- and 240-chromosomal sturgeons are needed to reconstruct genome evolution of this vertebrate group.

Contrasting origin of B chromosomes in two cervids (Siberian roe deer and grey brocket deer) unravelled by chromosome-specific DNA sequencing

BackgroundB chromosomes are dispensable and variable karyotypic elements found in some species of animals, plants and fungi. They often originate from duplications and translocations of host genomic regions or result from hybridization. In most species, little is known about their DNA content. Here we perform high-throughput sequencing and analysis of B chromosomes of roe deer and brocket deer, the only representatives of Cetartiodactyla known to have B chromosomes.ResultsIn this study we developed an approach to identify genomic regions present on chromosomes by high-throughput sequencing of DNA generated from flow-sorted chromosomes using degenerate-oligonucleotide-primed PCR. Application of this method on small cattle autosomes revealed a previously described KIT gene region translocation associated with colour sidedness. Implementing this approach to B chromosomes from two cervid species, Siberian roe deer (Capreolus pygargus) and grey brocket deer (Mazama gouazoubira), revealed dramatically different genetic content: roe deer B chromosomes consisted of two duplicated genomic regions (a total of 1.42-1.98 Mbp) involving three genes, while grey brocket deer B chromosomes contained 26 duplicated regions (a total of 8.28-9.31 Mbp) with 34 complete and 21 partial genes, including KIT and RET protooncogenes, previously found on supernumerary chromosomes in canids. Sequence variation analysis of roe deer B chromosomes revealed a high frequency of mutations and increased heterozygosity due to either amplification within B chromosomes or divergence between different Bs. In contrast, grey brocket deer B chromosomes were found to be more homogeneous and resembled autosomes in patterns of sequence variation. Similar tendencies were observed in repetitive DNA composition.ConclusionsOur data demonstrate independent origins of B chromosomes in the grey brocket and roe deer. We hypothesize that the B chromosomes of these two cervid species represent different stages of B chromosome sequences evolution: probably nascent and similar to autosomal copies in brocket deer, highly derived in roe deer. Based on the presence of the same orthologous protooncogenes in canids and brocket deer Bs we argue that genomic regions involved in B chromosome formation are not random. In addition, our approach is also applicable to the characterization of other evolutionary and clinical rearrangements.

DNA Double-Strand Breaks Coupled with PARP1 and HNRNPA2B1 Binding Sites Flank Coordinately Expressed Domains in Human Chromosomes

Genome instability plays a key role in multiple biological processes and diseases, including cancer. Genome-wide mapping of DNA double-strand breaks (DSBs) is important for understanding both chromosomal architecture and specific chromosomal regions at DSBs. We developed a method for precise genome-wide mapping of blunt-ended DSBs in human chromosomes, and observed non-random fragmentation and DSB hot spots. These hot spots are scattered along chromosomes and delimit protected 50–250 kb DNA domains. We found that about 30% of the domains (denoted forum domains) possess coordinately expressed genes and that PARP1 and HNRNPA2B1 specifically bind DNA sequences at the forum domain termini. Thus, our data suggest a novel type of gene regulation: a coordinated transcription or silencing of gene clusters delimited by DSB hot spots as well as PARP1 and HNRNPa2B1 binding sites.