[Reason to split the genus Aurelia. Mesoglein from two populations differ].

The medusa, Aurelia aurita (Scyphozoa, Cnidaria), is considered to be a cosmopolitan species with a worldwide distribution in most seas from the poles to the tropics. Cnidarian is thought to possess two tissue layers: endoderm (gastroderm) and ectoderm, which are separated by huge mesoglea in medusa. The basic morphology of medusa is similar in different populations. Previously we have determined a new protein "mesoglein" as one of the main components of mesoglea. Deduced amino acid sequence of mesoglein contains Zona Pellucida (ZP) domain. In this paper, we have comparied of mesoglein and its gene in medusa from three habitats (White Sea (WsA), Black Sea (BsA), Japonic Sea (JsA)). The set of the mesoglea protein bands after SDS-PAGE is similar in all samples. Nevertheless, JsA mesogleins' M(r) is 53-55 kDa, while WsA and BsA mesogleins have M(r) of 47 kDa. Antibodies raised against WsA mesoglein recognize only mesogleins with M(r) of 47 kDa, but not 53-55 kDa, both on immunoblot and immunocytochemistry. Mesogleal cells and elastic fibrils are stained intensively in the mesoglea both from WsA and BsA but not from JsA. The possibility of gene divergency was checked by PCR with primers specific for WsA mesoglein gene. PCR products of expected length obtained on polyA-cDNA template from mesogleal cells of WsA and BsA medusa but not on cDNA of JsA medusa. Our results evidence that there are two different species in genus Aurelia: Aurelia aurita inhabits White and Black Seas while Aurelia sp. inhabits Japonic Sea. This is consistent with findings of other recept molecular biological studies.

[Line class retroposon is the component of the DNA polymorphic fragments pattern of trematode Himasthla elongata parthenitae].

We have determined that S-SAP method (Sequence specific amplification polymorphism) reveals clonal variability in the genomes of larvae of flatworm Himasthla elongata (Trematoda, Echinostomatidae). Being parthenogenetic the larvae were previously considered to be genetically homogeneous. Cloning and sequencing of a -500 bp conservative fragment (B1) from the fragments' pattern has been performed. Sequence analysis of B1 has shown that this fragment has maximum homology with LINE elements from CR1 family of Hydra and sparrow. In situ hybridization (FISH) has detected dispersed distribution of B1. Several other fragments cloned from the same lane of agarose electrophoresis correspond to conservative domain of reverse transcriptase (RT) from CR1 family. Thus, we have shown that 1) cercariae of trematode H. elongata have clonal variability; 2) the S-SAP method allows to obtaining patterns of fragment distribution characteristic of individual cercariae; 3) conservative domain of RT of CR1 family participates in the pattern of polymorphic fragments generation. Identification of the CR1 transcripts in cercariae of H. elongata transcriptome is the aim of the future work. Cloning of the variable fragments from the fragments' pattern is in progress.

[Morphodynamics of the contract plate in the course of oocyte maturation in the scyphozoan Aurelia aurita (Cnidaria: Semaeostomae)].

The structure forming in the area of contact between the oocyte and the germinal epithelium in the course of oocyte maturation of the scyphozoan Aurelia aurita is termed the contact plate. This study traces the successive stages of contact plate formation in the course of oocyte maturation at the light microscopic and ultrastructural levels. At early stages ofoocyte development, the appearance of granules is observed in the peripheral cytoplasm of the oocyte; these granules accumulate at the pole, which retains its connection with the germinal epithelium of the gonads. Two types of these granules are recognized: (1) granules with homogeneous content and (2) granules containing loose shapeless material in the form of thick cords. The transformation of type two granules into larger structures, as well as the consolidation of type one and type two granules at later stages of oocyte development, are probably the processes that lead to the formation of the characteristic structure and contact plate, visible in paraffin and semithin sections. It remains unclear where exactly the contact plate is localized at the moment of fertilization: inside or outside the oocyte. The content of granules and components of the plate specifically bind the antibodies (RA47) against mesoglein, the ZP domain-containing protein of the mesoglea of A. aurita. The contact plate, covering only the anomalous pole of the oocyte but detected by the presence of ZP domain-containing proteins, may prove to be the simplest egg membrane of the zona pellucida type.

[Satellite DNA as a phylogenetic marker: case study of three genera of the Murinae subfamily].

Satellite DNA (satDNA) represent tens percent of any of the vertebrate genome. Still, a complete set of sat-DNA fragments is not determined for either species. It is known that some genus with species-specific modifications possess a satDNA characteristic for the genus. So, satDNA was used as a phylogenetic marker in some cases when precise satDNA fragment was cloned. We used the probe of the whole pericentromeric region and 4 cloned satDNA fragments of Mus musculus in order to consider probes value for phylogenesis of 3 Murinae genera. Fluorescent in situ hybridization (FISH) revealed similar pattern on metaphase spreads inside genus Mus, though some difference was noted. None of the satDNA fragment gave signal in the centromeric region on chromosomes from genera Sylvaemus and Apodemus. These data are in agreement with those on satDNA fragments in the genome determined by dot-blot hybridization: M musculus satDNA fragments are absent in the genomes of both remote genera while they are present in the genomes of the genera Mus, though in different amounts. SatDNA of each genera should be cloned for the phylogenetic purposes.

[Immuno- and histochemistry characteristics of morula and test cells in three ascidian species].

One of the hypotheses suggests that test cells play a part in a larval tunic formation like morula cells in adult ascidians. It was shown that the antibodies against morula cell proteins of 26 and 48 kDa of the ascidian Styela rustica react on the paraffin sections with both the granules of morula cells and test cells of ascidians S. rustica and Boltenia echinata. Among the test cell proteins of S. rustica SDS-electrophoresis revealed at least 5 major proteins but no one with the molecular mass of 26 and 48 kDa and none of them react with the antibodies. At the same time AB26 bind the proteins with similar molecular masses in blood cells and in the probe containing test cells--27 and 28 kDa, correspondingly,--of ascidian Molgula citrina. Comparative histochemical analysis of morula and test cells of these three ascidian species was carried out. There are a lot of acid polysaccharides combined with proteins in test cells whereas morula cells contain mainly positively charged proteins. Thus it could be supposed that degree of manifestation of antigens might be different in the conditions of immunoblot and immunohistochemical analysis. The hypothesis of the similarity in morula and test cells functions and their interrelationship is discussed.

[Mouse centromeric tandem repeats in silico and in situ].

The search for all sequences containing centromeric (CEN) minor satellite (MiSat) or pericentromeric (peri-CEN) mouse major satellite (MaSat) was conducted in the whole genome shotgun (WGS) database. The sequences were checked for the presence of the known dispersed repeats using the Censor software. The presence of tandem repeats was tested using Tandem Repeat Finder (TRF). Monotonous MiSat and MaSat arrays and MaSat to MiSat array transitions were detected. Moreover, two other types of contacts were revealed: (1) MiSat transition to fragments of retroelements LINE and IAP (ERV family, intracisternal A-type particles), mainly to ORF2 and 5'-LTR containing elements; (2) MaSat transition to two tandem repeats with monomers 21 bp and 31 bp in size. The presence of the MiSat/IAP transition could be checked experimentally. The common DNA motif among the IAP fragments close to MiSat was isolated. IAP-specific primers were constructed and the fragments obtained in PCR with LAP and MiSat primers compiled the plasmid vector library. Clone n51 with the maximum length of the possible insertion (approximately no. 800 bp) was selected from the library. FISH on extended chromatin fibers (fiberFISH) carried out on the n51 clone demonstrated that the main signal definitely belonged to CEN. However, the signals on the chromosome arms were also detected that could be due to the partial homology of n51 to the dispersed repeats. The duplicated fiberFISH with MiSat and n51 allowed to measure the distances between the fragments. The previously obtained MS3 sequence has some homology to IAP and CEN localization. Accordingly, the regular associations of MiSat with IAP retroelements were shown in silico and in situ. Together with the published data, the present findings suggest that retroelements or their fragments may be essential components of the normal centromere of higher eukaryotes.

[The plate in the zone of oocyte and germinal epithelium contact in scyphomedusa Aurelia aurita binds antibodies to ZP-domain-containing protein mesoglein].

Cnidaria are lower multicellular animals with the body consisting of two epithelial layers. An extracellular substance--mesoglea--is situated between epidermal and gastrodermal layers of these animals. Mesoglein is one of the major mesogleal proteins of adult medusa of Scyphozoan jellyfish Aurelia aurita. Search for the known domains in mesoglein amino acid sequence reveals prominent zona pellucida (ZP) domain (which was found at first in the mammal oocyte zona pellucida proteins), so the protein belongs to ZP family of extracellular matrix proteins and it is an early metazoan member of ZP-domain-containing protein family. However, nothing is known about oogenesis related ZP-domain proteins in the lower multicellular animals. Oogenesis in Scyphozoa is described poorly. In this work morphological features of the zone in contact area between the oocyte and the germinal epithelium were investigated in semi-fine sections: To make it more convenient we identified seven stages according to the oocyte size and the structure found in this area was named the plate. It was shown that the components of the plate bound specifically the antibodies against mesoglein. So it seems the plate material contains ZP-domain proteins. Electrophoresis and immunoblot results give evidence that the proteins immunologically related to mesoglein have a higher molecular mass. It might be due to either the posttranslational modifications of the precursors or that they represent other proteins of ZP-domain family in Cnidaria.

[Localization of satellite DNA and associated protein in respect to nucleolar precursor bodies in one- and two-cell mouse embryos].

Nucleolar precursor bodies (NPB) are characteristic structures in the nuclei of one- and two cell mouse embryos. The alignment of centromeric (CEN) and pericentromeric (periCEN) chromosome regions to the chromatin layer surrounding NPB is known. Mus musculus 4 satellite DNA (satDNA) types are known to be located in CEN region--mouse minor satellite (MiSat) and mouse satellite 3 (MS3); and periCEN region--mouse major satellite (MaSat) and mouse satellite (MS4). We determined the localization of 4 types of mouse satDNA CEN and periCEN regions and associated proteins: RNA-helicase p68, SMC3, Rad21 subunits of the cohesin complex and SYCP3 subunit of the synaptonemal complex (SC). Partially flattened nuclei of the one- and two-cell embryos and embryos treated with ocadaic acids (OA) were used. Different satDNA fragments revealed distinct domains at the surface of NPB: periCEN MaSat was always localized in NPB more internally covering almost entire surface of NPB while CEN MiSat, MS3 and periCEN MS4 showed more peripheral localization. All 4 satDNA did not cover the entire areas of the NPB, indicating the presence of other DNA sequence involved in its formation. RNA-helicase p68 and components of multiprotein cohesin and synaptonemal complexes are the necessary components of NPB. Our results support the opinion that NPB serve as a precursor of chromocenters.

[Heterochromatin and centromere structure paradox].

Centromere (CEN) is the structure responsible for the chromatid association, chromosome attachment to the spindle, and correct position in the plate. The only DNA found in the mammalian CEN belongs to the satellite DNA--high repeated tandem repeats. Mounting evidence indicates that both types of chromatin (CEN and peri-CEN) are required for proper centromere function. CEN, peri-CEN and peritelomeric regions remain white spots at the chromosome maps appeared after reading genomes of human, mouse, and rat. SatDNA is considered to be species-specific. Library hypothesis regards heterochromatin as the library of different satDNA one fragments of which became spread and fixed in species fixation. We have analyzed database Chromosome Unknown (ChrUn) and found several new classes of mouse tandem repeats. The features of these classes are similar with the ones from rat ChrUn, as well as their distributions according to GC-richness. We believe that similar fragments' structure, i. e. intermingling of fragments with different curvature rather than their primary sequence will help to solve the paradox, when CEN or peri-CEN fragments from different animals have nothing in common, but bind the same sets of proteins.

[Characterization of transposon mariner from the Himasthla elongata genome].

A novel transposable element Hemar 1 was identified and characterized in the genome of Himasthla elongata flatworm. This element is a member of mariner family; it belongs to capitata subfamily and highly homologues to turbellarian worm Dugesia tigrina mariner transposon. Hemar 1 is a dispersed repeat and accounts for about 0.01% of H. elongata genome. Identified element Hemar 1 represents useful tool for investigation of functions of the mobile elements in H. elongata genome.

[DNA of some regions of constitutive heterochromatin is demethylated and decondensed in MRC5 and A431 cells].

It is believed that satellite DNA is compact and transcriptionally inert during interphase. We determined localization, range of compactization and methylation state of the centromeric and pericentromeric satellite DNA using the method of fluorescence hybridization in situ (FISH) combined with the antibody immunostaining against the methylated DNA. We investigated the tissue cells (the cells of placenta and lymphocytes), primary (MRC5 fibroblasts) and malignant (A431) cell cultures. Centromeric satellite DNA was condensed and stained with antibodies against 5-methylcytosine in all the cases. Pericentromeric satellite 3 of the chromosome 1 was condensed in lymphocytes, placenta cells and young culture of fibroblasts. The unwrapping of satellite 3 of the chromosome 1 has been observed in the senescent MRC5 fibroblasts and in the malignant cell line A431. The compact areas of pericentromeric satellites were stained with antibodies against the methylated DNA, white the decondensed areas were'nt stained. Thus, we observed pericentromeric satellite 3 decondensation in senescent fibroblasts culture MRC5 and in cell line A431. The decondensation was accompanied by the partial demethylation of the satellite DNA, which is believed to belong to constitutive heterochromatin.

[Multifunctional nuclear protein NAP57 specifically interacts with dead RNA-helicase p68].

NAP57 has been found as a component of nuclear matrix protein complex with ability to specifically bind alphoid DNA. Polyclonal antibodies against NAP57 were raised in order to investigate intranuclear localization and interactions of the protein. Two types of localization were observed: a) nucleoplasmic and b) nucleolar. A bulk of nucleoplasmic fraction is present in splicing factors compartments (SFC). The type of localization pattern does not depend on the cell cycle phase, but we revealed changes in NAP57 localization pattern during S phase. According to immunoprecipitation and immunofluorescence assays, NAP57 specifically interacts with DEAD RNA helicase p68 in vitro and co-localizes with helicase p68 in the nucleus of cultured cells. We suppose participation of both proteins in processing of small nuclear RNA on the SFC periphery, and positioning of the nucleolus according to centromere regions of chromosomes.

[Larval cells of sponge Halisarca dujardini (Demospongiae, Halisarcida). I. Separation of the cells and their aggregation properties]. / Osobennosti lichinochnykh kletok gubki Halisarca dujardini (Demospongiae, Halisarcida). I. Rasdelenie kletok i sposobnost' ikh k agregatsii.

The majority of Demospongia members have a parenchymula larva, whose inner cells are similar to definite sponge cells. There are some contradictory opinions about the structure of larva in the marine sponge Halisarca dujardini: some authors deny the presence of inner cells, while other investigators consider this larva as a real parenchymula. We defined the larval cell types by cell separation in the percoll gradient and analysed their morphology and behaviour. The results showed the existence of 6 cell types in the parenchymula larva of H. dujardini, including 2 types of external flagellated cells. Each cell category differs in its morphology and aggregation ability.

[Larval cells of sponge Halisarca dujardini (Demospongiae, Halisarcida). II. Some cell types marked by polyclonal antibodies]. / Osobennosti lichinochnykh kletok gubki Halisarca dujardini (Demospongiae, Halisarcida). II. Markirovanie nekotorykh tipov kletok lichinok gubki s pomoshch'iu poliklonal'nykh antitel.

The recent morphological and experimental data concerning the involvement of flagellated cells in sponge larvae are contradictory and testify to or against the germinal layers inversion. A study of morphogenetic processes in sponges, in particular larval metamorphosis, is complicated by difficulties in identification and succession of certain cell types. It is possible to trace the destiny of flagellated and other larval cells by marking them with antibodies (AB) specified for each cell type. We separated larval and adult sponge cells of Halisarca dujardini in percoll density gradient and obtained polyclonal AB for the majority of these cell types. The protein pattern of larval flagellated cells differed significantly from that of other cell types. The major proteins of flagellated, collencyte-like and spherulous cells were used to raise the corresponding AB. Immunoblot showed all AB to be specific for certain proteins and suitable for immunofluorescence. The AB for flagellated cells reacted with the apical cytoplasm, but not with the flagellum, the AB for major protein of collencyte-like cells stained cytoplasm granules. The AB for spherulous cells of the adult sponge reacted with larval spherulous cells supposed to be of maternal origin. So, the method of cell marking with specific polyclonal AB can facilitate analysis of the layers inversion problem, as well as elucidate the degree of cell differentiation in larvae, their conformity to cells of the adult sponge or their provisional destiny.

[The mammalian centromere organization]. / Organizatsiia tsentromera mlekopitaiushchikh.

Routine and recently obtained data on the pattern and functions of the mammalian centromeres and kinetochores have been reviewed. Several problems of kinetochore formation (centromere recognition, anaphase checkpoint) are specially discussed, in addition to the role played by centromere DNA in the interphase nucleus consideration.

[Protein composition of mesoglea and mesogloeal cells of medusa Aurelia aurita]. / Belkovyi sostav mezoglei i mezogleal'nykh kletok meduzy Aurelia aurita.

Protein composition of mesoglea of the scyphomedusa Aurelia aurita was revealed in SDS-PAGE. Some major bands are visible in mesoglea of a mature medusa: 30, 45-47, 85 kDa, three bands between 100-200 kDa, and several bands with molecular weights > 300 kDa. Polyclonal antisera RA45/47 against protein 45 kDa were raised. RA45/47 react with 45-47 kDa protein in mesogleal sample and protein 120 kDa in mesogleal cells on immunoblot. Immunohistochemical analysis of A. aurita histological sections of young and mature medusae showed antigen localization in mesogleal cell granules and in the apical part of ectodermal cells. In mature medusae, the antigen was localized also in elastic fibers. We can conclude that in A. aurita mesogleal cells, along with ectodermal cells, take part in the formation of extracellular matrix of mesoglea.

[Localization of proteins, specifically binding highly-repetitive sequences of DNA in human spermatozoids]. / Lokalizatsiia belkov, spetsificheski sviazyvaiushchikh vysokopovtoriaiushchiesia posledovatel'nosti DNK, v spermatozoide cheloveka.

Immunoblot revealed in spermatozoa alpha-satellite (sat) DNA-specific centromere protein B (CENP-B) and p70 (Enukashvily et al., 2000), a membrane telomere binding protein (MTBP/TRF2) (Podgornaya et al., 2000), and Alu-binding protein p68 (Lukyanov et al., 2000). The localization of some of these proteins in spermatozoa was defined using indirect immunofluorescence. Spermatozoa were fixed in methanol/acetic acid 3:1, or prior to fixation were treated with 5 mM heparin and 10 mM DTT. The heparin/DTT treatment causes the nuclear membrane destruction and a partial chromatin decondensation. In non-treated spermatozoa fluorescent signals from all ABs are registered near the membrane, with MTBP/TRF2 being localized closer to the acrosome than sat-DNA-specific proteins. In the treated spermatozoa MTBP/TRF2 was partially lost, whereas part of CENP-B and sat-p70 remained in contact with membrane. Another part of sat-binding proteins reveals a dot-like staining pattern, with dots confined to the DAPI-stained chromatin area, inside a nuclei. This is in partial agreement with the pattern of telomere and CEN position revealed by FISH. Commonly MTBP has a near membrane localization, being lost when the nuclear membrane is destroyed. Centromere-binding proteins are arranged in the order from the nuclear membrane towards the nuclear center, with CENP-B being situated more peripherally but not in the middle of the nucleus. This discrepancy may be explained by the fact, that some proteins are not associated with the appropriate sequences in a spermatozoon. Possibly, such a distribution of proteins may reflect their role in unpacking the paternal genetic material in a zygote.

[The murine nuclear matrix protein specificcaly binds to centromeric satellite DNA]. / Belok iadernogo matriksa myshi, spetsificheski vzaimodeistvuiushchii s tsentromernoi satellitnoi DNK.

The nuclear matrix (NM) of mouse contains a protein (miSat BP) that can specifically bind to mouse centromeric minor satellite DNA as shown by gel shift assay. The ion-exchange chromatography on DEAE-Sepharose was used as the first miSat BP purification. MiSat BT was eluted by 0.2 M NaCl. Antibodies against p70, a human NM protein of 70 kDa described earlier as a protein recognizing human alphoid DNA, produce hypershift effect when added to the retardation incubation mix. Immunoblotting of NM and an active NM fraction (0.2 M NaCl) with these antibodies revealed a protein with 70 kDa in both preparations. This antigen retained in NM in situ being associated with residual DNA as shown by indirect immunofluorescent staining. In the untreated interphase nucleus most of miSat BP granules were shown to be colocalized with prekinetochores. We suggest that miSat BP is capable of recognizing the minor satellite DNA due to its structural features, but it does not belong to a group of constitutive centromeric proteins.

[The Chi-like sites free minisatellite insertion in plasmids of pSV2neo family directs conversion of reporter neo gene in LM cell line]. / Mini-satellitnye posledovatel'nosti bez Chi-podobnykh saitov v plazmidakh semeistva pSV2neo napravliaiut gennuiu konversiiu testernogo gena neo v kletkakh linii LM.

Minisatellite loci show variability in copy number of repeat units probably due to their recombinogenic activity. In the presence of minisatellites with Chi-similar sites in plasmids the enhanced frequency of homologous recombination of defective plasmids copies of selectable gene was shown. In this work we have estimated recombinogenic activity of minisatellite DNAs without Chi-similar sites. The restoration frequency of a neo gene and the ratio of restored and not restored copies of this gene in genomic DNA of transformed clones was quantitatively estimated. We conclude that the presence of minisatellite insertion without Chi-similar sites stimulates events of gene conversion in adjacent DNA of plasmids. Plasmids with minisatellites act as acceptors of genetic information.

[Differentiation of osteogenic cells in culture]. / Differentsirovka osteogennykh kletok v kul'ture.

The regulatory factors and cell responses during osteogenic differentiation have been reviewed. Evidence has been provided on hormone and mechanical regulation and on the extracellular matrix (ECM). The data on responses of primary cell culture and constant cell lines of osteogenic origin to these regulations are represented in the number of tables. It looks likely that the relationship with the ECM may mediate the rest of regulations, and therefore the most attention was paid to the cell-ECM interactions and, namely, to collagen-integrin interplay. A comparison of the reviewed data leads to an assumption that the ECM influence is passed through integrin receptors to the nuclear matrix connected transcriptional factors causing the next step of osteogenic differentiation.