[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.

[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.