[Telomere Recombination in Normal Mammalian Cells].

Two mechanisms of telomere length maintenance are known to date. The first includes the use of a special enzymatic telomerase complex to solve the problems that arise during the replication of linear DNA in a normal diploid and part of tumor cells. Alternative lengthening of telomeres (ALT), which is based on the homologous recombination of telomere DNA, represents the second mechanism. Until recently, ALT was assumed to be expressed only in 15-20% of tumors lacking active telomerase and, together with telomerase reactivation represented one of two possibilities to overcome the replicative senescence observed in somatic mammalian cells due to aging or during cell culturing in vitro. Previously described sporadic cases of combinations of the two mechanisms of telomere length maintenance in several cell lines in vitro were attributed to the experimental design rather than to a real biological phenomenon, since active cellular division without active telomerase was considered to be the "gold standard" of ALT. The present review describes the morphological and functional reorganizations of mammalian telomeres observed with ALT activation, as well as recently observed,and well-documented cases of combinations between ALT-like and telomerase-dependent mechanisms in mammalian cells. The possible role of telomere recombination in telomerase-dependent cells is discussed.

Recombinogenic telomeres in diploid Sorex granarius (Soricidae, Eulipotyphla) fibroblast cells.

The telomere structure in the Iberian shrew Sorex granarius is characterized by unique, striking features, with short arms of acrocentric chromosomes carrying extremely long telomeres (up to 300 kb) with interspersed ribosomal DNA (rDNA) repeat blocks. In this work, we investigated the telomere physiology of S. granarius fibroblast cells and found that telomere repeats are transcribed on both strands and that there is no telomere-dependent senescence mechanism. Although telomerase activity is detectable throughout cell culture and appears to act on both short and long telomeres, we also discovered that signatures of a recombinogenic activity are omnipresent, including telomere-sister chromatid exchanges, formation of alternative lengthening of telomeres (ALT)-associated PML-like bodies, production of telomere circles, and a high frequency of telomeres carrying marks of a DNA damage response. Our results suggest that recombination participates in the maintenance of the very long telomeres in normal S. granarius fibroblasts. We discuss the possible interplay between the interspersed telomere and rDNA repeats in the stabilization of the very long telomeres in this organism.

[Mammalian telomere biology].

Review is devoted to detailed consideration of the functioning in normal and immortal cells of one of the main chromosomal regions, telomeres, being dynamic nucleoprotein structures that cap the ends of eukaryotic chromosomes, protecting them from degradation and end-to-end fusion. The role of telomeres in maintenance of genome stability and cell division was also analyzed. Telomere function depends on many interrelated parameters such as telomerase activity, status of the telomere safety complex shelterin and telomere associated proteins (factors of replication, recombination, and reparation of DNA breaks, and so on). We have focused on mechanisms of telomere length control in normal and immortal cells as well as in cells containing active telomerase and cells wherein it is absent. We have analyzed the features attributed to alternative telomere lengthening, namely in view of recently discovered additional mechanism of telomere shortening by trimming of t-cycles. We have viewed a possibility of expression in normal mammalian cells of both telomerase dependent and recombinational ways of telomere length control and the role of shelterin proteins in choice of the one of them as the dominant way. The role oftelomeres in spatial organization of nucleus, in mitosis and meiosis has been also considered. Diversity of telomere organization in mammalians including unusual telomeres in Iberian shrews has been discussed.

[Generation of rat induced pluripotent stem cells: the analysis of reprogramming and culturing media].

The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.

[The structure of long telomeres in chromosomes of the Iberian shrew].

It is shown that the size, localization, and structure of telomeres in the Iberian shrew (Sorex granarius) are not characteristic of mammals. In this species, long telomeres of an average size of 213 kb are localized on the short arms of all 32 acrocentrics; ribosomal blocks and active nucleolus-organizing regions (NORs) were also discovered there. At the remaining chromosome ends the average size of telomeres is 3.8 kb. However, in a closely related species, Sorex araneus, all telomeres have size similar to that of human telomeres, i.e., 6.8-15.2 kb. Despite the fact that some long telomeres contain ribosomal repeats in addition to telomeric ones, the long telomeres have preserved asymmetry of G- and C-rich strands as in functional telomeres. It is probable that long telomeres were formed in meiosis at the stage of chromosome bouquet as a result of global reorganization of the chromosome ends. The provoking factors for such reorganization might be the fission of several metacentrics and the necessity of telomerization of the resulting acrocentrics.

[Chromosomal instability of in vitro cultured mouse embryonic stem cells and induced pluripotent stem cells].

A perspective of using embryonic stem (ES) and induced pluripotent stem (iPS) cells in clinical medicine makes karyological analysis of these cells an important issue. Using methods of classical and molecular cytogenetics chromosomal analysis, we have carried out karyological study of two mouse ES and two iPS cell lines derived de novo. We have found monosomy of X chromosome in all studied ES and iPS cell lines, thus making a modal number of chromosomes in these cell lines 39. A chromosomal instability (aneuploidy) was revealed in both studied iPS cell lines. Moreover, we have detected chromosomal rearrangements and chromosomal fragments in one of iPS cell line. Our findings underline the importance of careful cytogenetic evaluation of pluripotent cell lines, especially iPS cell lines, which should be carried out prior to any clinical use of these cells.

[Telomeric DNA allocation in chromosomes of common shrew Sorex araneus, Eulipotyphla].

Recently we have displayed shrew species, Iberian shrew S. granarius, with telomeres of unusual for mammals structure, including long telomeres on the short acrocentrics arms containing 213 kb on average and short telomeres (3.8 kb) on the other chromosomal ends (Zhdanova et al., 2005, 2007). However, it is not clear if such telomeres are characteristic of all shrew species or only of S. granarius. S. granarius and common shrew Sorex araneus are the sibling species. In this investigation by using modified Q-FISH, we demonstrated that telomeres in S. araneus from different chromosomal races differing in the numbers of metacentrics contain 6.8-15.2 kb of telomeric tracts. Thus, the S. araneus telomere lengths appeared to correspond with telomere lengths both in shrews and majority wild mammalian species, and S. granarius has telomeres with unique or scarce structure. Furthermore, using DNA and RNA modified with probe high specificity to telomeric repeats (PNA and LNA) we showed that interstitial telomeric sites in S. araneus chromosomes contained mainly telomeric DNA and their localization coincided with some evolutionary breakpoints. Interstitial telomeric DNA in S. granarius chromosomes was not revealed. Thus, distribution of telomeric DNA can greatly differ even in closely related species whose chromosomes are composed from almost identical chromosomal arms.

[3D organization of interphase fibroblast nuclei in two closely related shrew species, Sorex granarius and S. araneus, differed by the structure of chromosome termini].

To study 3D organization of fibroblast interphase nuclei in two sibling shrew species, Sorex araneus from Cordon race and S. granarius, FISH with probe to telomeric and rDNA repeats, and immunofluorescence with ANA CREST and antibodies to nucleolus protein B23 were used. Karyotypes of studied species are composed of near identical chromosomal arms and differ by the number of metacentrics and the structure of terminal chromosome regions. The large telomeres containing on the average 218 kbp of telomere repeats characterize the short arms in all of 32 S. granarius acrocentrics. Telomere repeats in them alternate with nbosomal repeats. These regions also contain active NORs. In contrast, active NORs in S. araneus are localized at the terminal regions of 8 chromosomal arms (Zhdanova et al., 2005, 2007b). We have shown that telomere associations of chromosomes and contacts of a part of telomere clusters with inner nuclear membrane and nucleolus characterize interphase nuclei of both S. granarius and S. araneus. Moreover, the partial colocalization of telomere and ribosomal clusters, and spatial nearness of centomeric and telomeric regions were revealed in the interphase nuclei of S. granarius. Evidently, only those ribosomal clusters that contain a number of active ribosomal genes display connection with nucleolus. The stripping of nucleolus materials during transition of fibroblasts to mitosis and the role of B23 protein in this process has been studied.

[Comparative mapping of mink (Mustela vison) chromosome 8p: localization of three human BAC clones].

Using fluorescent in situ hybridization (FISH), three human BAC clones, localized in the terminal region of human chromosome 17p (HSA17p13; 1.44--3.68 Mp), were mapped to chromosome 8p of American mink (MVI8p). It was demonstrated that in MVI8p the region, homeologous to HSA17p13, was split into three fragments, which were detected within terminal, pericentric, and probably nucleolus-organizing regions. Using human BAC clones as heterologous markers for mapping of the gene sequences to the chromosomes of American mink, regional localization of eight sequences (PRPF8, SLC43A2, and RILP in MVI8p25; C17orf31 in MVI8p21-22; and CTNS, TAX1BP3, and P2RX5 in MVI8p11) was deduced.

[Terminal regions of mammal chromosomes: plasticity and role in evolution].

The review considers data on the composition, organization, and functional significance of terminal regions in mammalian chromosomes, including telomeres and subtelomeric regions. Because of specific structure, features of DNA replication, and characteristic localization in somatic and meiotic cells, these regions are hot spots for many events associated with genome functioning in mammals. Instability of these regions is of special interest. Evidence suggesting that instability of chromosomal regions containing telomeric DNA is a factor of chromosome evolution is discussed. The association of size and structure of telomeric regions with replicative aging and cell immortalization is considered. The review deals in detail with classical and alternative mechanisms of telomere size control, the significance of changes in telomeric region length in ontogeny, oncotransformation, and evolution. The issues related to telomere destabilization and the role of this process in chromosome rearrangement formation and chromosome evolution are discussed. The origin of telomere repeats in interstitial chromosome sites, including regions of evolutionary fusions-fissions is given special consideration. The possible role of ribosomal repeats and mechanisms similar to ALT (alternative lengthening of telomeres) in telomere reorganization in some taxa are discussed.

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.

Comparative mapping of cattle chromosome 19: cytogenetic localization of 19 BAC clones.

Here we present the results of fluorescent in situ hybridization (FISH) mapping of a set of cattle BAC clones preselected for assignment on cattle chromosome 19 (BTA19). The BAC clones were anchored to human chromosome 17 (HSA17) sequences by BLASTn similarity search of cattle BAC-ends against the human genome sequence (NCBI build 33). Five blocks of homologous synteny were defined in the comparative map of BTA19 and HSA17 built with FISH data and the human genome coordinates. The positions for four evolutionary breakpoints in the bovine and human chromosomes were identified. Comparison of the FISH comparative map with previously published comparative RH, physical, and cytogenetic maps of BTA19 did not reveal major conflicts and allowed for the extension of the boundaries of homology between BTA19 and HSA17. Comparative analysis of HSA17, BTA19, and mouse chromosome 11 (MMU11) demonstrates that most likely mice retain the ancestral organization of the synteny group, and both cattle and human chromosomes underwent several major internal rearrangements after the divergence of Primates, Rodentia, and Cetartiodactyla.

[The structure of a conserved region of porcine genome, represented in human genome by chromosome 17]. / Struktura v genome svin'i konservativnoso raiona, predstavlennogo v genome cheloveka khromosomnoi 17.

Radiation mapping of nine genes (H3F3B, HLR1, MYL4, STAT5B, THRA1, TOP2A, MCP1, NF1, and MPO) to porcine chromosome 12 was carried out. Also, subchromosomal location of the NF1 gene along with the two loci containing the DNA sequences homologous to the DNA of the two human BAC clones was determined. The NF1 position was ascertained via microdissection of chromosome 12 with subsequent PCR amplification of the gene fragment with specific primers. BAC clones were mapped using FISH. Comparative analysis of the gene order in porcine chromosome 12 and in the homologous human chromosome 17 was performed. It was demonstrated that the gene orders in these chromosomes differed relative to the position of the MPO gene.

[Genome radiation hybrid mapping: summary and future directions]. / Radiatsionnoe kartirovanie genomov. Nastoiashchee i budushchee.

Genome mapping by means of radiation-induced interspecific cell hybrids is a direct way to localize both high- and low-polymorphic nucleotide sequences, including gene sequences, on animal chromosomes. Using radiation hybrid panels either individual chromosomes and loci or entire genome can be mapped. This efficient approach makes it possible to reach high resolution of markers (up to 100 bp) as well as unify the mapping language. Due to electronic means of communication, the same experimental material can be used in numerous laboratories to provide high-resolution extended genomic maps saturated with markers. Radiation hybrid mapping is a powerful tool for analysis of complex genome structure. Using radiation hybrid maps permitted verification of regions of chromosome homeology in various species and detection of regions with conserved sequence and conserved gene order. Identification of these regions is extremely important for understanding evolution of species karyotypes and for making use of positional cloning to isolate genes responsible for commercial traits as well as genes involved in hereditary human diseases.

[Use of PCR markers for mapping swine chromosome 12]. / Ispol'zovanie PTsR-markerov dlia kartirovaniia khromosomy 12 svin'i.

Using PCR analysis of pig-mink and pig-Chinese hamster hybrid cell lines and heterologous and homologous primers of various types, chromosomal and subchromosomal mapping of genes TOP2A, THRA, BRCA1, GAS, HLR1, MYL4, LIS1, MCP1, ENO3, CRYB1, P4HB, STAT5B, and H3F3B to pig chromosome 12 was carried out. The efficiency of using different types of heterologous primers for pig chromosome mapping was compared.

[Radiation mapping of the short arm of swine chromosome 2]. / Radiatsionnaia karta korotkogo plecha khromosomy 2 svin'i.

In recent years, maps of mammalian genomes have been acquiring increasingly higher resolution. Integration of maps of different types has become possible. As a tool in integrating maps of mammalian genomes of different types, high-resolution mapping with radiation-induced hybrids (RH) is used. Here, we present an RH6000 map of the short arm of porcine chromosome 2. The map contains 15 microsatellites and five genes (for parathyroid hormone, lactate dehydrogenase A, myogenic factor, follicle-stimulating hormone beta, and calpain I). The RH panel was obtained on the basis of a hybrid cell line bearing the single porcine chromosome 2 against the background of mink chromosomes. The mean frequency of preserving markers examined in the panel was 18.3%. Integration of four genes in the panel and a comparison of gene order in homologous regions of human and porcine chromosomes are presented.

Zoo-FISH with region-specific paints for mink chromosome 5q: delineation of inter- and intrachromosomal rearrangements in human, pig, and fox.

Comparison of evolutionarily conserved mammalian chromosomes homologous to human chromosome 17, performed with microdissected painting probes, revealed rearrangements inside these chromosomes in mink and pig and a disruption of this conserved region in the fox. Detection of a homologous region on an Iberian shrew chromosome showed the efficiency of microdissected painting probes for delineation of homologous chromosome regions in species belonging to orders that diverged at least 100 million years ago.

Dog chromosome-specific paints reveal evolutionary inter- and intrachromosomal rearrangements in the American mink and human.

Forty chromosome-specific paint probes of the domestic dog (Canis familiaris, 2n = 78) were used to delineate conserved segments on metaphase chromosomes of the American mink (Mustela vison, 2n = 30) by fluorescence in situ hybridisation. Half of the 38 canine autosomal probes each painted one pair of homologous segments in a diploid mink metaphase, whereas the other 19 dog probes each painted from two to five pairs of discrete segments. In total, 38 canine autosomal paints highlighted 71 pairs of conserved segments in the mink. These painting results allow us to establish a complete comparative chromosome map between the American mink and domestic dog. This map demonstrates that extensive chromosome rearrangements differentiate the karyotypes of the dog and American mink. The 38 dog autosomes could be reconstructed from the 14 autosomes of the American mink through at least 47 fissions, 25 chromosome fusions, and six inversions. Furthermore, comparison of the current dog/mink map with the published human/dog map discloses 23 cryptic intrachromosomal rearrangements in 10 regions of conserved synteny in the human and American mink genomes and thus further refined the human/mink comparative genome map.