New putative phenol oxidase in ascidian blood cells.

The phenol oxidase system is ancient and ubiquitously distributed in all living organisms. In various groups it serves for the biosynthesis of pigments and neurotransmitters (dopamine), defence reactions and tissue hardening. Ascidians belong to subphylum Tunicata, which is considered the closest living relative to Vertebrates. Two phenol oxidases previously described for ascidians are vertebrate-like and arthropod-like phenol oxidases. In our present study, we described a new ascidian protein, Tuphoxin, with putative phenol oxidase function, which bears no sequence similarity with two enzymes described previously. The closest related proteins to Tuphoxin are mollusc haemocyanins. Unlike haemocyanins, which are oxygen transporting plasma proteins, Tuphoxin is synthesised in ascidian blood cells and secreted in the extracellular matrix of the tunic-ascidian outer coverings. Single mature transcript coding for this phenol oxidase can give several protein products of different sizes. Thus limited proteolysis of the initial protein is suggested. A unique feature of Tuphoxins and their homologues among Tunicata is the presence of thrombospondin first type repeats (TSP1) domain in their sequence which is supposed to provide interaction with extracellular matrix. The finding of TSP1 in the structure of phenol oxidases is new and we consider this to be an innovation of Tunicata evolutionary lineage.

Nuclear organization by satellite DNA, SAF-A/hnRNPU and matrix attachment regions.

The need of large-scale chromatin organization in the nucleus has become more and more appreciated. The higher order nuclear organization ultimately regulate a plethora of biological processes including transcription, DNA replication, and DNA repair. In this context, it is of critical importance to understand the mechanisms that allow higher order nuclear organization. Scaffold Attachment Factor A (SAF-A/hnRNPU), which was originally identified as the component of nuclear matrix, has emerged as an important regulator of higher order nuclear organization. It is shown that SAF-A/hnRNPU binds to tandem repeats (TRs) and scaffold/matrix attachment regions (S/MAR) in a sequence-non-specific, but structure-specific manner (e.g. DNA curvature). Recent studies showed that SAF-A interacts with chromatin-associated RNAs (caRNAs) to regulate interphase chromatin structures in a transcription-dependent manner. It is proposed that SAF-A/hnRNPU and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes chromatin in a large scale. The common structural features of S/MAR and pericentromeric (periCEN) TR promotes SAF-A-mediated association with each other. Collectively a model is presented wherein SAF-A/hnRNPU and periCEN TR are the key players in large-scale nuclear organization that supports general transcription.

Human pericentromeric tandemly repeated DNA is transcribed at the end of oocyte maturation and is associated with membraneless mitochondria-associated structures.

Most of the human genome is non-coding. However, some of the non-coding part is transcriptionally active. In humans, the tandemly repeated (TR) pericentromeric non-coding DNA-human satellites 2 and 3 (HS2, HS3)-are transcribed in somatic cells. These transcripts are also found in pre- and post-implantation embryos. The aim of this study was to analyze HS2/HS3 transcription and cellular localization of transcripts in human maturating oocytes. The maternal HS2/HS3 TR transcripts transcribed from both strands were accumulated in the ooplasm in GV-MI oocytes as shown by DNA-RNA FISH (fluorescence in-situ hybridization). The transcripts' content was higher in GV oocytes than in somatic cumulus cells according to real-time PCR. Using bioinformatics analysis, we demonstrated the presence of polyadenylated HS2 and HS3 RNAs in datasets of GV and MII oocyte transcriptomes. The transcripts shared a high degree of homology with HS2, HS3 transcripts previously observed in cancer cells. The HS2/HS3 transcripts were revealed by a combination of FISH and immunocytochemical staining within membraneless RNP structures that contained DEAD-box helicases DDX5 and DDX4. The RNP structures were closely associated with mitochondria, and are therefore similar to membraneless bodies described previously only in oogonia. These membraneless structures may be a site for spatial sequestration of RNAs and proteins in both maturating oocytes and cancer cells.

Who Needs This Junk, or Genomic Dark Matter.

Centromeres (CEN), pericentromeric regions (periCEN), and subtelomeric regions (subTel) comprise the areas of constitutive heterochromatin (HChr). Tandem repeats (TRs or satellite DNA) are the main components of HChr forming no less than 10% of the mouse and human genome. HChr is assembled within distinct structures in the interphase nuclei of many species - chromocenters. In this review, the main classes of HChr repeat sequences are considered in the order of their number increase in the sequencing reads of the mouse chromocenters (ChrmC). TRs comprise ~70% of ChrmC occupying the first place. Non-LTR (-long terminal repeat) retroposons (mainly LINE, long interspersed nuclear element) are the next (~11%), and endogenous retroviruses (ERV; LTR-containing) are in the third position (~9%). HChr is not enriched with ERV in comparison with the whole genome, but there are differences in distribution of certain elements: while MaLR-like elements (ERV3) are dominant in the whole genome, intracisternal A-particles and corresponding LTR (ERV2) are prevalent in HChr. Most of LINE in ChrmC is represented by the 2-kb fragment at the end of the 2nd open reading frame and its flanking regions. Almost all tandem repeats classified as CEN or periCEN are contained in ChrmC. Our previous classification revealed 60 new mouse TR families with 29 of them being absent in ChrmC, which indicates their location on chromosome arms. TR transcription is necessary for maintenance of heterochromatic status of the HChr genome part. A burst of TR transcription is especially important in embryogenesis and other cases of radical changes in the cell program, including carcinogenesis. The recently discovered mechanism of epigenetic regulation with noncoding sequences transcripts, long noncoding RNA, and its role in embryogenesis and pluripotency maintenance is discussed.

Genomic diversity of cercarial clones of Himasthla elongata (Trematoda, Echinostomatidae) determined with AFLP technique.

The aim of this study was to reveal genomic diversity formed during parthenogenetic reproduction of rediae of the trematode Himasthla elongata in its molluskan host Littorina littorea. We applied amplification fragment length polymorphism (AFLP) to determine the genomic diversity of individual cercariae within the clone, that is, the infrapopulation of parthenogenetic progeny in a single molluskan host. The level of genomic diversity of particular cercariae isolates from a single clone, detected with EcoR1/Mse1 AFLP reaction, was significantly lower than the variability of cercariae from different clones. The presence of intraclonal genomic diversity indicates a nonsexual shuffle of alleles during parthenogenesis in the rediae of H. elongata. The obtained polymorphic AFLP fragments were long enough to detect the sequences that may be responsible for clonal genomic variability. Based on this, AFLP can be recommended as a tool for the study of genetic mechanisms of this variability.

EXTRA-CELLULAR DNA FOR THE UNSOLVED EVOLUTIONAL PROBLEMS.

The genome assembly and new sequencing methods shed light on extra-cellular DNA (ecDNA) content and uptake. The recent data are described. The asymmetry in repetitive sequences distribution in ecDNA are shown: 1) significant enrichment in pericentromeric tandem repeats (TR), but decrease of centromeric alpha-satellite; 2) enrichment in Alu (SINE) but decrease of LINEs. In the genomes, Alu repeats (SINE) are located mostly to the gene-rich regions, while LINEs enrich facultative heterochromatin as evidenced by bioinformatics and cytological data (FISH). Human shows a greater fraction of tissue-specific genes and a greater ratio of the total expression of tissue-specific genes to housekeeping genes in each tissue studied. A higher level of evolutionary cell differentiation (specialization) in human correlates with the substitution of 4 mouse SINEs with Alu (SINE) repeat. Sperm-mediated gene transfer became the ordinary methods of the veterinary. The overall substitution of ecDNA SINEs in the whole genome by recombination is possible due to Alu element and mouse B1 (SINE) sequence similarity. The orthologous genes are not altered initially but chromatin landscape change lifts up the transcription and provides the evolutionary advantage for the newly born species. So, the scheme suggested may remove two of the contradictions of the Modern evolutionary synthesis theory: 1) gene mutations are not necessary for the progressive evolution but repetitive elements substitution; 2) ancient mammalians could have litters with the number of offspring altered by ecDNA and inbred mating was possible for the descendant altered.

THE STRUCTURE OF DNA CHROMOCENTRES IN MOUSE IN SILICO AND IN SITU. LINE AND ERV FRAGMENTS ARE AN OBLIGATORY COMPONENTS OF DNA CHROMOCENTRES BESIDES TANDEM REPEATS.

Constitutive heterochromatin in higher eukaryotes is located in the primary chromosomal strangulation (centromeres and pericentromeres) and subtelomeric regions of chromosomes. In house mouse constitutive chromatin formes structures in the nuclei of cells, brightly colored with DAPI. It is known that the constitutive heterochromatin is enriched with large tandem repeats, however, its exact composition is unknown. In this paper chromocenters were studied using several approaches: cloning and sequencing by Sanger, as well as by high-throughput sequencing. We have shown that DNA components of chromocenters and constitutive chromatin are full-length endogenous retroviruses (ERV) and a 2 kbp fragment of the 3R-end of the LINE.

THE KARYOSPHERE CAPSULE IN OOCYTES OF HIBERNATING FROGS RANA TEMPORARIA CONTAINS ACTIN, LAMINS AND SMALL NUCLEAR RNP.

The nuclei of late vitellogenic oocytes of hibernating frogs Rana temporaria were studied. During the studied period of oogenesis, chromosomes are significantly inactivated and surrounded by a fibrillar karyosphere capsule. Formation of the karyosphere capsule in the grass frog oocytes was previously investigated in detail at the light and electron microscopic levels, but the molecular composition of the capsule still remains uncertain. Immunofluorescent staining of whole-mount preparations of oocyte nuclei revealed the following proteins in the karyosphere capsule: actin, lamins A, C and B, and the Sm proteins of snRNPs. A putative role of these proteins in formation of the karyosphere capsule is discussed.

[Nucleosome fracton of extracellular dna as the index of apoptosis].

Review is devoted to the analysis of changes of the extracellular DNA (excDNA) in pathological conditions involving the process of apoptosis, and the possibility of using of excDNA in the diagnosis and evaluation of course of various diseases. Apoptosis is the main mechanism of the appearance of the DNA in the circulation. ExcDNA found in the norm, its function is considered to be the part of the immune response. The excDNA content increases substantially during the induction of apoptosis. Dynamics of increase of excDNA content in stroke allows to diagnose the form of stroke and massiveness of destruction of brain tissue. Reduced content of excDNA is associated with the inhibition of apoptosis, it was shown that under such conditions there is a change of composition of excDNA. Investigation of excDNA character changes in the progression and treatment of cancer substantiates the possibility of early assessment the effectiveness of treatment. It is experimentally shown the immunosuppressive action of excDNA of tumor and its transforming effect on the cells. Ionizing radiation is an examples that demonstrated the association of induction of apoptosis and the release of excDNA. It is characterize some of the genome sequences of excDNA. Created on the basis of excDNA study tests of minimally invasive diagnostics are potentially useful in oncology and other areas of medicine. The study of tandem repeats, which are absent in the assembled genome, but there is a part of excDNA, will create tests for the diagnosis of cancer in the early stages.

[Large tandem repeats of mesocricetus a uratus in silico and in situ].

The class of tandemly repeated sequences exists only in eukaryotic genomes and absent in prokaryotes. The tens percent of eukaryotic genome are built up of the tandem repeats. The whole set of different tandem repeats is not revealed to any of the eukaryote species in spite of the half century history of its investigation by molecular biology methods. Previously we found the set of tandem repeats in the database of well assembled mouse genome with the bioinformatics methods. In the current work we applied the same methods to the poorly assembled hamster Mesocricetus auratus genome. 19 tandem repeats families have been found in hamster genome by bioinformatics (in silico). Only one of tandem repeats' families found have been cloned previously and exists in the Repbase, the database of all known repetitive fragments. The rest of the families are new and need the experimental verification by FISH (in situ). Oligo probes were designed at the base of in silico found sequences. Oligo probe for the known tandem repeat gives the same signal as the cloned probe, i.e., probes designed are suitable for oligo-FISH. All four oligo probes tested give signal at the heterochromatic centromeric region as expected, though with different intensities and at different number of chromosomes. The results show the power of the in silico methods for the mostly mysterious genome component, tandem repeats, investigation.

[Sequencing of low-molecular-weight DNA in blood plasma of irradiated rats].

Extracellular low-molecular-weight DNA in blood of irradiated rats was sequenced for the first time. The screening of sequences in the DDBJ database displayed homology of various parts of the rodent genome. Sequences of low-molecular-weight DNA in rat's plasma are enriched with G/C pairs and long interspersed elements relative to rat genome. DNA sequences in blood of rats irradiated at the doses of 8 and 100 Gy have marked distinctions. Data of sequencing of extracellular DNA from normal humans and with pathology were analyzed. DNA sequences of irradiated rats differ from the human ones by a wealth of long interspersed elements. This new knowledge lays the foundation for development of minimally invasive technologies of diagnosing the probability of pathology and controlling the adaptive resources of people in extreme environments.

SAF-A/hnRNP-U localization in interphase and metaphase.

SAF-A/hnRNP U is an abundant nuclear protein that interacts specifically with nuclear matrix attachment region DNA (MAR) and RNA as a component of hnRNPs. SAF-A/hnRNP U was also shown to specifically bind mouse major satellite DNA (satMa). Antibodies against SAF-A and GFP-fusion constructs were used in the current work in order to trace SAF-A localization. In accordance with its diverse nucleic acid binding specificity, SAF-A was found to be localized in three different domains: outside the chromosomes, on the surface of the chromosome arms (probably MARs), and in the centromere region where it apparently binds specifically to the satMa. GFP-fusion constructs with different SAF-A/hnRNP U domains confirms the functional significance of the protein's functional domains in interphase cells. In telophase cells, the anti-SAF-A antibody signal appeared as a kind of network covering unfolded chromosomes.

Human chromosome 1 satellite 3 DNA is decondensed, demethylated and transcribed in senescent cells and in A431 epithelial carcinoma cells.

Constitutive heterochromatin mainly consists of different classes of satellite DNAs and is defined as a transcriptionally inactive chromatin that remains compact throughout the cell cycle. The aim of this work was to investigate the level of condensation, methylation and transcriptional status of centromeric (alphoid DNA) and pericentromeric satellites (human satellite 3, HS3) in tissues (lymphocytes, placenta cells) and in cultured primary (MRC5, VH-10, AT2Sp) and malignant (A431) cells. We found that alphoid DNA remained condensed and heavily methylated in all the cell types. The HS3 of chromosome 1 (HS3-1) but not of chromosome 9 (HS3-9) was strongly decondensed and demethylated in A431 cells. The same observation was made for aged embryonic lung (MRC5) and juvenile foreskin (VH-10) fibroblasts obtained at late passages (32(nd) and 23(rd), respectively). Decondensation was also found in ataxia telangiectasia AT2Sp fibroblasts at the 16(th) passage. One of the manifestations of the disease is premature aging. The level of HS3-1 decondensation was higher in aged primary fibroblasts as compared to A431. The HS3-1 extended into the territory of neighbouring chromosomes. An RT-PCR product was detected in A431 and senescent MRC5 fibroblasts using primers specific for HS3-1. The RNA was polyadenylated and transcribed from the reverse chain. Our results demonstrate the involvement of satellite DNA in associations between human chromosomes and intermingling of chromosome territories. The invading satellite DNA can undergo decondensation to a certain level. This process is accompanied by demethylation and transcription.

A novel Aurelia aurita protein mesoglein contains DSL and ZP domains.

Body of the scyphoid jellyfish Aurelia aurita consists of 2 epithelia -- epidermis and gastroderm. The layers are separated by a thick layer of extracellular matrix -- mesoglea. A. aurita has a lot of cells in the mesoglea unlike many other Cnidarians. The major protein of the mesoglea with apparent molecular mass of 47 kDa was detected by SDS-PAGE. A partial mRNA of the protein 1421 bp long was cloned and sequenced. The search for homologous nucleotide and protein sequences shows that the mRNA sequence is novel. Deduced amino acid sequence of 416 aa contains zona pellucida (ZP) domain and Delta/Serrate/Lag-2 (DSL) domain. The protein was named mesoglein. According to reverse transcription PCR analysis it is expressed in the mature medusa exclusively in the mesogleal cells. Mesoglein belongs to the lowest phyla among ZP domain-containing proteins. The protein is supposed to be a structural element of the mesoglea extracellular matrix.

Dynamics of satellite binding protein CENP-B and telomere binding protein TRF2/MTBP in the nuclei of mouse spermatogenic line.

The location of centromeric protein CENP-B and telomeric protein TRF2/MTBP in the mouse spermatogenic line has been studied using indirect immunofluorescent and immunoelectron microscopy. CENP-B localized to the heterochromatic parts of the nuclei at meiotic stages. A clearly distinct chromocenter forms in the nucleus at stages 3-4 of spermatid maturation; CENP-B localizes in it and in the area adjacent to the future acrosome. CENP-B localization in the subacrosomal area and in the chromocenters' periphery demonstrates that centromeres are organized in two groups in mouse spermatozoa, unlike human centromeres. TRF2/MTBP concentrates around the forming chromocenter at spermiogenesis early stages. The TRF2/MTBP main signal migrates into the area of acrosomal membrane at the course of spermatozoon maturation. TRF2/MTBP never localizes inside the synaptonemal complex but can be found in the areas where the synaptonemal complex attaches to the nuclear envelope. At the pachytene and diplotene stages when chromosomes separate from the nuclear envelope, some amount of the protein remains bound to the nuclear membrane while the other part reveals itself in chromosomes. TRF2/MTBP accumulates in the future acrosome from the very beginning of its formation. In the mature spermatozoon TRF2/MTBP decorates the acrosomal membrane as well as spreads in condensed chromatin.

Evidence for the existence of satellite DNA-containing connection between metaphase chromosomes.

Physical connections between mitotic chromosomes have been reported previously. It was assumed that the interchromosome connection was based on the DNA-protein thread. However, the data about DNA sequences and protein component in the thread is fragmentary. We demonstrated on the mouse cultured cell line and prematurely condensed chromosomes that: (a) all four mouse satellite DNA fragments (major and minor satellite, mouse satellite 3 (MS3) and mouse satellite 4 (MS4)) were involved in the thread formation; (b) MS4 was involved in the thread to the least extent among all the other fragments; (c) telomere was never a member of the thread; (d) the thread was synthesized at a late G(2) phase; (e) RNA helicase p68 and CENP-B were among the protein components of the interchromosome connection. It was shown by FACS analysis that in mouse and human cell lines: (1) the flow karyotype spectrums were never free from chromosome aggregates; (2) chromosome association did not depend on the chromosome length and each chromosome was free to associate with the other.

Telomere and TRF2/MTBP localization in respect to satellite DNA during the cell cycle of mouse cell line L929.

The mouse Mus musculus chromosomes are all acrocentric; each centromere (CEN) is adjacent to a telomere. The aim of the current work is to find out if at least half of the mouse telomeres (Tel) always follow satellite DNA sequences and if membrane telomere binding protein TRF2/MTBP is always in association with the Tel during the cell cycle. FISH, immunoFISH and confocal microscopy were used. During the cell cycle, Tel undergo extensive movement and rearrangement. Most Tel tend to aggregate into large conglomerates in G0/G1. Aggregates colocalize with major satellite (MaSat) and minor satellite (MiSat) to a lesser extent. Tel aggregates are embedded into the MaSat granules at G0/G1. A number of single Tel signals underline the nuclear envelope. In prometaphase, during the metaphase plate formation, half of the Tel, together with CEN, are arranged in a circle and half of the long arms form four clusters. Most of the Tel hybridization signals are colocalized with TRF2/MTBP throughout all stages of the cell cycle, although it is possible to find some telomeres that are not covered with the protein. A prominent shift of TRF2/MTBP signals in respect to the Tel signals is visible in the prophase. The biochemical features of TRF2/MTBP make it possible for the protein to be responsible for Tel clustering.

Telomere-binding TRF2/MTBP localization during mouse spermatogenesis and cell cycle of the mouse cells L929.

Observations of the organization and distribution of telomeres (Tel) in somatic tissues still remain controversial. The Tel topography revealed by modern microscopy shows them to be associated with the nuclear envelope (NE) in a wide variety of eukaryotic cells, although not at the Rabl orientation (peripheral position at one pole of the nucleus at prophase). We used two cell types that have different nuclear architectures. The cell line L929 shows lack of any rigid Tel architecture in the nucleus. In contrast, spermatozoa have a precise architecture established during spermiogenesis. We observed Tel and membrane Tel binding protein (MTBP/TRF2) position by immunoFISH in L929 cells and by immunofluorescence and immunogold electron microscopy, using antibodies against Membrane Tel Binding Protein (MTBP/TRF2), during different stages of spermiogenesis. At all stages of the L929 cell cycle, MTBP/TRF2 is co-localized with Tel. The only Tel order found in this cell type is similar to the Rabl-orientation, probably due to fast divisions. In the mouse pachytene spermatocytes, the membrane structures abut on the synaptonemal complex (SC) attachment sites contain MTBP/TRF2. In fully formed spermatozoa and during spermiogenesis, apart from the expected MTBP/TRF2 position at the nuclear periphery, MTBP/TRF2 unexpectedly localized at the acrosomal membrane that is adjacent to the nucleus. The difference in the MTBP/TRF2 distribution in the oocyte and spermatozoa leads to the suggestion that the MTBP/TRF2 location might reflect preparation for fertilization events. The Tel distribution is not static in cultured cells throughout the cell cycle or during spermatogenesis. When the Tel are attached to the NE, as during SC formation, MTBP/TRF2 is the member of the protein complex, which appears to be responsible for this attachment.

Structure-specific DNA-binding proteins as the foundation for three-dimensional chromatin organization.

Any functions of tandem repetitive sequences need proteins that specifically bind to them. Telomere-binding TRF2/MTBP attaches telomeres to the nuclear envelope in interphase due to its rod-domain-like motif. Interphase nuclei organized as a number of sponge-like ruffly round chromosome territories that could be rotated from outside. SAF-A/hnRNP-U and p68-helicase are proteins suitable to do that. Their location in the interchromosome territory space, ATPase domains, and the ability to be bound by satellite DNAs (satDNA) make them part of the wires used to help chromosome territory rotates. In case of active transcription p68-helicase can be involved in the formation of local "gene expression matrices" and due to its satDNA-binding specificity cause the rearrangement of the local chromosome territory. The marks of chromatin rearrangement, which have to be heritable, could be provided by SAF-A/hnRNP-U. During telophase unfolding the proper chromatin arrangement is restored according to these marks. The structural specificity of both proteins to the satDNAs provides a regulative but relatively stable mode of binding. The structural specificity of protein binding could help to find the "magic" centromeric sequence. With future investigations of proteins with the structural specificity of binding during early embryogenesis, when heterochromatin formation goes on, the molecular mechanisms of the "gene gating" hypothesis (Blobel, 1985) will be confirmed.

Specificity of SAF-A and lamin B binding in vitro correlates with the satellite DNA bending state.

There is evidence that Matrix Attachment Region (MAR)-binding proteins also bind satellite DNA (satDNA). The aim of the current work was to determine whether the major nuclear matrix (NM) MAR-binding proteins are able to recognize satDNAs of different locations and what DNA structural features are important for the recognition. In nuclei and NM, a number of the same polypeptides were recognized on a southwestern blot when MAR of immunoglobulin kappa gene (Ig kappa MAR) and pericentromeric (periCEN) satDNA fragments were used. However, the binding decreased dramatically when human and mouse CEN satDNA were used for the probes. After an NM extract was subjected to ion exchange chromatography, the main DNA-binding proteins were identified as SAF-A (scaffold attachment factor A) and lamin B. It was not possible to test the binding of lamin B by gel mobility shift assay (GMSA), but SAF-A showed an ability to distinguish CEN and periCEN satDNA fragments in GMSA. While periCEN fragments have an abnormally slow mobility on electrophoresis, which is a hallmark of bent DNA, CEN satDNA fragments have a normal mobility. A computer analysis was done using the wedge model (Ulanovsky and Trifonov [1987] Nature 326:720-722), which describes how the curved state depends on particular nucleotide sequences. The curved states of the fragments predicted by the model are in good agreement with their ability to be recognized by NM proteins. Thus SAF-A and lamin B are able to recognize conserved structural features of satDNA in the same way that MAR-binding proteins recognize MARs in spite of a lack of a consensus sequence. CEN and periCEN satDNAs are distinguished by proteins in correlation with the helical curvature of these fragments.