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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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 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 weights300 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.

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 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.

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.

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.

Heterochromatin consists mainly of satellite DNAs (stDNA), the most rapidly evolving type of DNA sequences of the eucaryotic genome. On the other hand, stDNA is involved in the formation of the functionally conserved centromere structure. Centromeres and pericentromeric stDNA, are known to be in association with nuclear matrix or chromosome scaffold at all stages of the cell cycle. Several lines of evidence show that attachment of stDNA to the nuclear matrix is specific. The first defined parts of the genes found in association with nuclear matrix/scaffold, MAR/SAR, possess some common features with the stDNA. A number of different mechanisms have previously been implicated in heterochromatin formation and centromere conservation. The role of nuclear matrix proteins, which are able to recognize common structural features of MAR/SAR and stDNA, in constitutive heterochromatin organization is discussed in the current review.

DNA transcription and replication are organized by the nuclear matrix. It has been recently shown that the osteocalcin gene promoter binds specifically two proteins, NMP-1 and NMP-2 (nuclear matrix proteins 1 and 2), from cell line ROS 17/2.8 (Bidwell et al., 1993). Osteocalcin in one of the proteins taking part in extracellular matrix mineralization during osteogenic differentiation. Osteopontin gene is also active at the late stages of osteogenesis, so we supposed that its promoter could be bound to the nuclear matrix as well. Among the nuclear matrix proteins of ROS 17/2.8 we have found a protein factor which binds the promoter region of the osteopontin gene specifically with the help of gel shift assay. Two proteins with isoelectric points of pH8.0 and mol. masses of 38 and 43 kDa, respectively, have been purified by affinity chromatography with the promoter region of osteopontin attached to cellulose. The mol. masses of the revealed proteins are similar to those of already characterized NMP-2 (38 kDa) and one of subunits of heterodimer NMP-1 (43 kDa). Computer analysis shows that the binding sites for these proteins exist in the osteopontin gene promoter region. So, it is very likely that we dealt with the same proteins. A further investigation could lead to characterization of a group of nuclear matrix attached osteogenic-specific transcriptional factors.

Minisatellites are tandemly repeated DNA sequences with repeat units in the range 6-100 bp. They are preferentially located in subtelomeric euchromatin of chromosomes of higher eukaryotes. Minisatellite loci frequently show variability in allele length. This variability is a consequence of high frequency of mutations which change repeat copy number of locus. The most part of minisatellite mutations are germline specific and possibly meiotic. Examples of different mutations and involved genomic mechanisms are reviewed in this paper. A possible biological role of minisatellites is discussed.

The pattern of cell membrane collagen-binding proteins was analysed by affinity chromatography with collagen types I, III and IV columns. We have found that connective tissue cells have three polypeptides which are able to recognize the types of collagen. These proteins have molecular weights 130, 190, and 250 kDa (reduced) according to SDS-PAGE. Proteins with M. W. 130 and 250 kDa show high affinity to collagen type III and IV, but not to collagen type I. Protein with M. W. 190 kDa binds collagen type I only. The distribution of these proteins between cells from various tissues strongly correlates with the set of collagen types interacting with cells in the intact tissue.

A telomere-binding activity was found in the nuclear envelope of frog oocytes Rana temporaria by methods of binding on nitrocellulose filters and retardation. At present it is hardly possible to detect the proteins responsible for this activity. However, the results of this study allowed us to identify a specific mechanism of DNA-protein interaction, which provides the binding of the telomere to the envelope in meiotic cells.

Problem of three-dimensional structure of chromatin is reviewed. Data concerning ranged distribution of chromosomes in nucleoplasm are shown. Special attention being paid to the role of nuclear matrix in maintenance of nucleus topology. Possible role of satellite sequences and DNA-binding proteins is discussed.

Chinese hamster A238 TK- -cells were transformed with plasmids (derivatives of pBR325) containing thymidine kinase (TK) gene of Herpes simplex virus type 1 (HSV1). The results of dot- and blot-hybridization indicate the presence of pBR325 sequences in the chromosomal fractions of DNA in the transformant clones. These sequences are probably tandemly arranged, and each cluster contains 25--50 copies. SV40 sequences cloned in pBR325 were introduced into the Chinese hamster cells by co-transformation with TK-gene of HSV1-containing plasmid DNA, and all the co-transformant clones selected for TK+-phenotype were shown by hydridization to contain 3V40 DNA fragments. Isoelectrofocusing in polyacrylamide gel shows that thymidine kinase from TK+-transformant clones is of viral type (isoelectric point 7), in contrast to the cellular enzyme (coded by chromosomal gene) having alkaline isoelectric point (pH 9). The results suggest that the true TK+-transformant cells are selected by the procedure used in this study.

Nuclear proteins from HeLa cells specifically binding to the Alu-repeat cloned in the plasmid Blur8 have been studied. 0.35 M nuclear extract proteins have been separated on DEAE-cellulose. The presence of DNA-binding proteins has been found in all fractions by the technique of DNA-binding on nitrocellulose filters. The labelled restricted DNA of the plasmid Blur8 was incubated with the proteins of different fractions with the subsequent identification of specific Alu-protein complexes in polyacrylamide gel at low ionic strength. At least two proteins have been found to have the different affinity to Alu-repeat. Various functions of Alu-repeats and the possibility of their participation in the initiation of DNA replication are discussed.

PHA-stimulated human lymphocytes contain the protein (SBP) which has selectivity in binding of 1.8 kb fragment of human satellite DNA III (HS3) as compared to other DNA sequences. It is shown that the binding site is localized within 1kb Sau3A-EcoR I fragment of HS3. SBP-binding activity is increased after treatment of cells with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). The essential increase in a number of metaphases with chromosome endoreduplications in TPA-treated lymphocytes indicates that SBP may be involved in initiation of chromosome replication or in alteration of the mitotic spindle function.

As demonstrated by dot-hybridization, the cells of HT-subclones isolated from the cells of transformant clones cultured on a non-selective medium differ significantly in the number of copies of thymidine kinase gene (tk-gene) of Herpes simplex virus (HSV1). Since the cells of transformant clones lose thymidine kinase-positive (TK+) phenotype during cultivation, this data are indicative of high frequence rearrangements in the region of transforming DNA as responsible for the transformant phenotype nonstability. These rearrangements, among other things, induce alterations in the number of copies of tk gene of HSV1. The analysis of cells of subclones isolated on a medium containing 5-bromodeoxyuridine (BrdU) shows that the number of copies of tk gene of HSV1 decreases as compared to the cells of parental clones. The decrease in the number of copies of tk gene of HSV1 in a row of BrdU-resistant subclones is accompanied by simultaneous increase in the number of sequences of pBR325 plasmide DNA to which tk gene of HSV1 is linked covalently in the pST826 plasmide introduced into cells of transformant clones. This evidence implies a most complex nature of transforming DNA rearrangements reducing the number of copies of tk gene of HSV1 due possibly to a genetic correction. The analysis of results permits a hypothesis that instability of cells in transformant phenotype may be determined by the genetic instability of insertion type. The rate of the loss of transformant phenotype depends on the frequency of rearrangements in the transforming DNA locus.

Extrachromosomal DNAs from TK+ transformant clones of A238 Chinese hamster cells isolated after the treatment with plasmid pST826 containing thymidine kinase gene (TK-gene) of Herpes simplex virus (HSV1) and 1.8 kb insert of human satellite III DNA (HSIII) were studied by hybridization technique. In two TK+-clones (2T301 and 2T16) large quantities of rearranged plasmid DNA molecules were found. Electron microscopy show in clone 2T301 the presence of circular DNAs with average length being 4.64 +/- 0.27 kb. These molecules were rescued by retransformation into E. coli and analysed by restriction mapping and hybridization. All of them contain deletions spanning the entire TK gene of HSV1 and pBR325 sequences situated just downstream from the ORI of replication. The origin of extra-replicating circular DNA in 2T301 clone is discussed.

Introduction of the plasmid containing the methotrexate-resistant (Mtx-r) bacterial gene of dihydrofolate reductase (DHFR) under the control of the early promoter of SV 40 into the donor bone cells of the mouse with subsequent transplantation of the cells into lethally irradiated mice results in the increase in the life span of mice under conditions of methotrexate selection. It is due to the stable transformation of the bone marrow colony-forming cells with the plasmic DNA and the synthesis of the bacterial Mtx-r DHFR in the spleen and bone marrow of the recipient mouse.

Metaphase chromosomes (MCh) isolated at pH 2.1 and 6.5 slightly differ in their composition and contain in average: 17% of DNA, 9% of RNA and 74% of protein. The three-fold increase of protein content in MCh was not accompanied by the change in nonhistone protein composition. The behaviour of chromosomes isolated at pH 2.1 (acid) and 6.5 (neutral) in two-phase polymer system is shown to be very similar. The function of certain chromosome components in the determination of MCh behaviour in two-phase systems and possible usefulness of acid and neutral MCh for fractionation of total MCh are discussed.

Protein solubilized in large quantities in solutions of low ionic strength, when myofibril-like preparations were isolated from ambulacral tube, was shown to be actin. When gelation of sarcoplasmic proteins occurs a gel consisting of actin microfilaments bundles is formed. The factor which inhibits actin polymerization is liberated during gel formation. The aggregate substance of sarcoplasmic actin under cell ionic conditions may be determined by binding with this factor.

Two proteins with molecular weights of 40 and 80 kDa which are able to bind human Alu-repeat in a sequence-specific manner were found in HeLa nuclear extracts. The proteins were partially purified by column chromatography on DEAE-cellulose, phosphocellulose and FPLC MonoQ sorbent. One of the Alu-binding proteins (ABP2 with m. w. of 80 kDa) was found to bind the sequence within the Alu-repeat that has a homology with the T-antigen binding site of SV40, suggesting that ABP2 is the cellular analog of SV40 T-antigen.

In gelatinated extracts of starfish ambulacral podia the actin polymerized during gel formation exists as oligomers incapable of binding to rabbit myosin. Actin is also the major protein extractable from the cells by glycerol- and Triton-containing solutions, which do not destroy the contractile mechanism of the cells. The weakly bound gel proteins washed from gel by low ionic strength solutions comprise a factor hampering the polymerization of rabbit actin. The data obtained suggest that the ambulacral sarcoplasmic proteins contain actin (profilament form) coupled to the factor preventing its polymerization.

A method combining advantages of both, charge electrophoresis and isoelectric focusing (IEF) was developed for analysing actins of different tissues. This method was used in investigations of actins from invertebrate muscle sources. Each type of actin has one isoform, pI of which is close to that of the smooth muscle beta-isoactin. Actins from different tissues of the mollusc Spisula s. were identical according to the IEF data.