Tandem Repeat Diversity in Two Closely Related Hamster Species-The Chinese Hamster (Cricetulus griseus) and Striped Hamster (Cricetulus barabensis).

The Chinese hamster (Cricetulus griseus) and striped hamster (Cricetulus barabensis) are very closely related species with similar karyotypes. The karyotypes differ from each other by one Robertsonian rearrangement and X-chromosome morphology. The level of the tandem repeat (TR) sequences' evolutional variability is high. The aim of the current work was to trace the TR distribution on the chromosomes of two very closely related species. The striped hamster genome has not yet been sequenced. We classified the Chinese hamster TR in the assemblies available and then compared the mode of the TR distribution in closely related species. Chinese and striped hamsters are separate species due to the relative species specificity of Chinese hamster TR and prominent differences in the TR distribution in both species. The TR variation observed within homologous striped hamster chromosomes is caused by a lack of inbreeding in natural populations. The set of TR tested could be used to examine the CHO lines' instability that has been observed in heterochromatic regions.

The Long Linker Region of Telomere-Binding Protein TRF2 Is Responsible for Interactions with Lamins.

Telomere-binding factor 2 (TRF2) is part of the shelterin protein complex found at chromosome ends. Lamin A/C interacts with TRF2 and influences telomere position. TRF2 has an intrinsically disordered region between the ordered dimerization and DNA-binding domains. This domain is referred to as the long linker region of TRF2, or udTRF2. We suggest that udTRF2 might be involved in the interaction between TRF2 and lamins. The recombinant protein corresponding to the udTRF2 region along with polyclonal antibodies against this region were used in co-immunoprecipitation with purified lamina and nuclear extracts. Co-immunoprecipitation followed by Western blots and mass spectrometry indicated that udTRF2 interacts with lamins, preferably lamins A/C. The interaction did not involve any lamin-associated proteins, was not dependent on the post-translation modification of lamins, nor did it require their higher-order assembly. Besides lamins, a number of other udTRF2-interacting proteins were identified by mass spectrometry, including several heterogeneous nuclear ribonucleoproteins (hnRNP A2/B1, hnRNPA1, hnRNP A3, hnRNP K, hnRNP L, hnRNP M), splicing factors (SFPQ, NONO, SRSF1, and others), helicases (DDX5, DHX9, and Eif4a3l1), topoisomerase I, and heat shock protein 71, amongst others. Some of the identified interactors are known to be involved in telomere biology; the roles of the others remain to be investigated. Thus, the long linker region of TRF2 (udTRF2) is a regulatory domain responsible for the association between TRF2 and lamins and is involved in interactions with other proteins.

Amino acid sequence associated with bacteriophage recombination site helps to reveal genes potentially acquired through horizontal gene transfer.

BACKGROUND: Horizontal gene transfer, i.e. the acquisition of genetic material from nonparent organism, is considered an important force driving species evolution. Many cases of horizontal gene transfer from prokaryotes to eukaryotes have been registered, but no transfer mechanism has been deciphered so far, although viruses were proposed as possible vectors in several studies. In agreement with this idea, in our previous study we discovered that in two eukaryotic proteins bacteriophage recombination site (AttP) was adjacent to the regions originating via horizontal gene transfer. In one of those cases AttP site was present inside the introns of cysteine-rich repeats. In the present study we aimed to apply computational tools for finding multiple horizontal gene transfer events in large genome databases. For that purpose we used a sequence of cysteine-rich repeats to identify genes potentially acquired through horizontal transfer. RESULTS: HMMER remote similarity search significantly detected 382 proteins containing cysteine-rich repeats. All of them, except 8 sequences, belong to eukaryotes. In 124 proteins the presence of conserved structural domains was predicted. In spite of the fact that cysteine-rich repeats are found almost exclusively in eukaryotic proteins, many predicted domains are most common for prokaryotes or bacteriophages. Ninety-eight proteins out of 124 contain typical prokaryotic domains. In those cases proteins were considered as potentially originating via horizontal transfer. In addition, HHblits search revealed that two domains of the same fungal protein, Glycoside hydrolase and Peptidase M15, have high similarity with proteins of two different prokaryotic species, hinting at independent horizontal gene transfer events. CONCLUSIONS: Cysteine-rich repeats in eukaryotic proteins are usually accompanied by conserved domains typical for prokaryotes or bacteriophages. These proteins, containing both cysteine-rich repeats, and characteristic prokaryotic domains, might represent multiple independent horizontal gene transfer events from prokaryotes to eukaryotes. We believe that the presence of bacteriophage recombination site inside cysteine-rich repeat coding sequence may facilitate horizontal genes transfer. Thus computational approach, described in the present study, can help finding multiple sequences originated from horizontal transfer in eukaryotic genomes.

From traveler to homebody: Which signaling mechanisms sponge larvae use to become adult sponges?

Cell-to-cell signaling is responsible for regulation of many developmental processes such as proliferation, cell migration, survival, cell fate specification and axis patterning. In this article we discussed the role of signaling in the metamorphosis of sponges with a focus on epithelial-mesenchymal transition (EMT) accompanying this event. Sponges (Porifera) are an ancient lineage of morphologically simple animals occupying a basal position on the tree of life. The study of these animals is necessary for understanding the origin of multicellularity and the evolution of developmental processes. Development of sponges is quite diverse. It finishes with the metamorphosis of a free-swimming larva into a young settled sponge. The outer surface of sponge larvae consists of a ciliated epithelial sheath, which ensures locomotion, while their internal structure varies from genus to genus. The fate of larval ciliated cells is the most intriguing aspect of metamorphosis. In this review we discuss the fate of larval ciliated cells, the processes going on in cells during metamorphosis at the molecular level and the regulation of this process. The review is based on information about several sponge species with a focus on Halisarca dujardini, Sycon ciliatum and Amphimedon queenslandica. In our model sponge, H. dujardini, ciliated cells leave the larval epithelium during metamorphosis and migrate to the internal cell mass as amoeboid cells to be differentiated into choanocytes of the juvenile sponge. Ciliated cells undergo EMT and internalize within minutes. As EMT involves the disappearance of adherens junctions and as cadherin, the main adherens junction protein, was identified in the transcriptome of several sponges, we suppose that EMT is regulated through cadherin-containing adherens junctions between ciliated cells. We failed to identify the master genes of EMT in the H. dujardini transcriptome, possibly because transcription was absent in the sequenced stages. They may be revealed by a search in the genome. The master genes themselves are controlled by various signaling pathways. Sponges have all the six signaling pathways conserved in Metazoa: Wnt, TGF-beta, Hedgehog, Notch, FGF and NO-dependent pathways. Summarizing the new data about intercellular communication in sponges, we can put forward two main questions regarding metamorphosis: (1) Which of the signaling pathways and in what hierarchical order are involved in metamorphosis? (2) How is the organization of a young sponge related to that of the larva or, in other words, is there a heredity of axes between the larva and the adult sponge?

Actin depolymerization disrupts karyosphere capsule integrity but not residual transcription in late oocytes of the grass frog Rana temporaria.

Late diplotene oocytes are characterized by an essential decrease in transcriptional activity. At this time, chromosomes condense and form a compact structure named a karyosphere. The karyosphere of grass frogs Rana temporaria is surrounded by a fibrillar karyosphere capsule (KC). One of the main protein constituents of R. temporaria KC is actin. In this study, we used antibodies against different actin epitopes to trace different forms of actin in the KC. We also investigated the effect of F-actin depolymerization on the oocyte nuclear structures and transcription of chromatin DNA and rDNA in the amplified nucleoli. It was determined that disruption of actin filaments leads to chromosome shrinkage, nucleoli fusion, and distortion of the KC structure, but does not inhibit residual transcription in both the karyosphere and the nucleoli.

Development and characterization of polyclonal antibodies against the linker region of the telomere-binding protein TRF2

Background: TRF2 (telomeric repeat binding factor 2) is an essential component of the telomere-binding protein complex shelterin. TRF2 induces the formation of a special structure of telomeric DNA and counteracts activation of DNA damage-response pathways telomeres. TRF2 has a poorly characterized linker region (udTRF2) between its homodimerization and DNA-binding domains. Some lines of evidence have shown that this region could be involved in TRF2 interaction with nuclear lamina. Results: In this study, the fragment of the TERF2 gene encoding udTRF2 domain of telomere-binding protein TRF2 was produced by PCR and cloned into the pET32a vector. The resulting plasmid pET32a-udTRF2 was used for the expression of the recombinant udTRF2 in E. coli RosettaBlue (DE3). The protein was isolated and purified using ammonium sulfate precipitation followed by ion-exchange chromatography. The purified recombinant protein udTRF2 was injected into guinea pigs to generate polyclonal antibodies. The ability of anti-udTRF2 antibodies to bind endogenous TRF2 in human skin fibroblasts was tested by western blotting and immunofluorescent staining. Conclusions: In this study, the recombinant protein udTRF2 and antibodies to it were generated. Both protein and antibodies will provide a useful tool for investigation of the functions of the udTRF2 domain and its role in the interaction between TRF2 and nuclear lamina.

Mouse chromocenters DNA content: sequencing and in silico analysis.

BACKGROUND: Chromocenters are defined as a punctate condensed blocks of chromatin in the interphase cell nuclei of certain cell types with unknown biological significance. In recent years a progress in revealing of chromocenters protein content has been made although the details of DNA content within constitutive heterochromatin still remain unclear. It is known that these regions are enriched in tandem repeats (TR) and transposable elements. Quick improvement of genome sequencing does not help to assemble the heterochromatic regions due to lack of appropriate bioinformatics techniques. RESULTS: Chromocenters DNA have been isolated by a biochemical approach from mouse liver cells nuclei and sequenced on the Illumina MiSeq resulting in ChrmC dataset. Analysis of ChrmC dataset by the bioinformatics tools available revealed that the major component of chromocenter DNA are TRs: ~ 66% MaSat and ~ 4% MiSat. Other previously classified TR families constitute ~ 1% of ChrmC dataset. About 6% of chromocenters DNA are mostly unannotated sequences. In the contigs assembled with IDBA_UD there are many fragments of heterochromatic Y-chromosome, rDNA and other pseudo-genes and non-coding DNA. A protein coding sfi1 homolog gene fragment was also found in contigs. The Sfi1 homolog gene is located on the chromosome 11 in the reference genome very close to the Golden Pass Gap (a ~ 3 Mb empty region reserved to the pericentromeric region) and proves the purity of chromocenters isolation. The second major fraction are non-LTR retroposons (SINE and LINE) with overwhelming majority of LINE - ~ 11% of ChrmC. Most of the LINE fragments are from the ~ 2 kb region at the end of the 2nd ORF and its' flanking region. The precise LINEs' segment of ~ 2 kb is the necessary mouse constitutive heterohromatin component together with TR. The third most abundant fraction are ERVs. The ERV distribution in chromocenters differs from the whole genome: IAP (ERV2 class) is the most numerous in ChrmC while MaLR (ERV3 class) prevails in the reference genome. IAP and its LTR also prevail in TR containing contigs extracted from the WGS dataset. In silico prediction of IAP and LINE fragments in chromocenters was confirmed by direct fluorescent in situ hybridization (FISH). CONCLUSION: Our data of chromocenters' DNA (ChrmC) sequencing demonstrate that IAP with LTR and a precise ~ 2 kb fragment of LINE represent a substantial fraction of mouse chromocenters (constitutive heteroсhromatin) along with TRs.

The exon junction complex factor Y14 is dynamic in the nucleus of the beetle Tribolium castaneum during late oogenesis.

BACKGROUND: The oocyte chromosomes of the red flour beetle, Tribolium castaneum, are gathered into a knot, forming a karyosphere at the diplotene stage of meiotic prophase. Chromatin rearrangement, which is a characteristic feature of oocyte maturation, is well documented. The T. castaneum karyosphere is surrounded by a complex extrachromosomal structure termed the karyosphere capsule. The capsule contains the vast majority of oocyte RNA. We have previously shown using a BrUTP assay that oocyte chromosomes in T. castaneum maintain residual transcription up to the very end of oocyte maturation. Karyosphere transcription requires evidently not only transcription factors but also mRNA processing factors, including the components of the exon junction complex with its core component, the splicing factor Y14. We employed a gene engineering approach with injection of mRNA derived from the Myc-tagged Y14 plasmid-based construct in order to monitor the newly synthesized fusion protein in the oocyte nuclei. RESULTS: Our preliminary data have been presented as a brief correspondence elsewhere. Here, we provide a full-length article including immunoelectron-microscopy localization data on Y14-Myc distribution in the nucleus of previtellogenic and vitellogenic oocytes. The injections of the fusion protein Y14-Myc mRNA into the oocytes showed a dynamic pattern of the protein distribution. At the previtellogenic stage, there are two main locations for the protein: SC35 domains (the analogues of interchromatin granule clusters or nuclear speckles) and the karyosphere capsule. At the vitellogenic stage, SC35 domains were devoid of labels, and Y14-Myc was found in the perichromatin region of the karyosphere, presumably at the places of residual transcription. We show that karyosphere formation is accompanied by the movement of a nuclear protein while the residual transcription occurs during genome inactivation. CONCLUSIONS: Our data indicate that the karyosphere capsule, being a destination site for a protein involved in mRNA splicing and export, is not only a specializes part of nuclear matrix separating the karyosphere from the products of chromosome activity, as believed previously, but represents a special nuclear compartment involved in the processes of gene expression in the case the karyosphere retains residual transcription activity.

Heterochromatic Tandem Repeats in the Extracellular DNA.

Only limited sequencing data of the normal extracellular DNA (ecDNA) are currently available. The uptake of the ecDNA by cultured cells and its integration into the host chromatin have been demonstrated. A number of membrane-bearing vesicles in plasma and serum have been shown to carry nucleic acids. The presence of Tandem Repeat (TR) in both apoptotic DNA of HUVEC culture medium and membrane-associated DNA is shown. The existence and successful application of CREST serum also show the presence of fragments of the centromeric heterochromatin together with their TR and specific proteins in blood. Apparently, pericentromeric and centromeric DNA (TR) should be part of ecDNA in all cases.

Nucleolus-like body of mouse oocytes contains lamin A and B and TRF2 but not actin and topo II.

BACKGROUND: During the final stages of oocyte development, all chromosomes join in a limited nuclear volume for the final formation of a single complex chromatin structure - the karyosphere. In the majority of mammalian species, the chromosomes surround a round protein/fibrillar body known as the central body, or nucleolus-like body (NLB). Nothing seems to unite the inner portion of the karyosphere with the nucleolus except position at its remnants. Nevertheless, in this study we will use term NLB as the conventional one for karyosphere with the central body. At the morphological level, NLBs consist of tightly-packed fibres of 6-10 nm. The biochemical structure of this dense, compact NLB fibre centre remains uncertain. RESULTS: The aim of this study was to determine which proteins represent the NLB components at final stages of karyosphere formation in mouse oogenesis. To determine this, three antibodies (ABs) have been examined against different actin epitopes. Examination of both ABs against the actin N-end provided similar results: spots inside the nucleus. Double staining with AB against SC35 and actin revealed the colocalization of these proteins in IGCs (interchromatin granule clusters/nuclear speckles/SC35 domains). In contrast, examination of polyclonal AB against peptide at the C-end reveals a different result: actin is localized exclusively in connection with the chromatin. Surprisingly, no forms of actin or topoisomerase II are present as components of the NLB. It was discovered that: (1) lamin B is an NLB component from the beginning of NLB formation, and a major portion of it resides in the NLB at the end of oocyte development; (2) lamin A undergoes rapid movement into the NLB, and a majority of it remains in the NLB; (3) the telomere-binding protein TRF2 resides in the IGCs/nuclear speckles until the end of oocyte development, when significant part of it transfers to the NLB. CONCLUSIONS: NLBs do not contain actin or topo II. Lamin B is involved from the beginning of NLB formation. Both Lamin A and TRF2 exhibit rapid movement to the NLB at the end of oogenesis. This dynamic distribution of proteins may reflect the NLB's role in future chromatin organization post-fertilisation.

Karyotype features of trematode Himasthla elongata.

BACKGROUND: Trematodes have a complex life cycle with animal host changes and alternation of parthenogenetic and hermaphrodite generations. The parthenogenetic generation of the worm (rediae) from the first intermediate host Littorina littorea was used for chromosome spreads production. Karyotype description of parasitic flatworm Himasthla elongata Mehlis, 1831 (Digenea: Himasthlidae) based on fluorochrome banding and 18S rDNA mapping. RESULTS: Chromosome spreads were obtained from cercariae embryos and redial tissue suspensions with high pressure squash method.74.4 % of the analysed spreads contained 12 chromosome pairs (2n = 24). Chromosome classification was performed according to the morphometry and nomenclature published. H. elongata spread chromosomes had a rather bead-like structure. Ideograms of DAPI-banded chromosomes contained 130 individual bands. According to flow cytometry data, the H. elongata genome contains 1.25 pg of DNA, so one band contains, on average, 9.4 Mb of DNA. Image bank captures of individual high-resolution DAPI-banded chromosomes were provided. Differential DAPI- and CMA3-staining revealed the chromatin areas that differed in AT- or GC-content. Both dyes stained chromosomes all along but with varying intensities in different areas. FISH revealed that vast majority (95.0 %) of interphase nuclei contained one signal for 18S rDNA. This corresponded to the number of nucleoli per cell detected by observations in vivo. The rDNA signal was observed on one or two homologs of chromosome 10 in 72.2 % of analysed chromosome spreads, therefore chromosome 10 possessed the main rDNA cluster and minor ones on chromosomes 3 and 6, that corresponds with AgNOR results. CONCLUSIONS: Himasthla elongata chromosomes variations presented as image bank. Differential chromosome staining with fluorochromes and FISH used for 18S rDNA mapping let us to conclude: (1) Himasthla elongata karyotype is 2n = 24; (2) chromosome number deviates from the previously studied echinostomatids (2n = 14-22); (3). Chromosome 10 possesses the main rDNA cluster with the minor ones existing on chromosomes 3 and 6.

LINE-related component of mouse heterochromatin and complex chromocenters' composition.

Chromocenters are interphase nuclear landmark structures of constitutive heterochromatin. The tandem repeat (TR)-enriched parts of different chromosomes cluster together in chromocenters. There has been progress in recent years in determining the protein content of chromocenters, although it is not clear which DNA sequences underly constitutive heterochromatin apart from the TRs. The aim of the current work was to find out which DNA sequences besides TRs are involved in chromocenters' formation. Biochemically isolated chromocenters and microdissected centromeric regions were amplified by DOP-PCR, then cloned and sequenced. Alignment to Repbase, the mouse reference genome and WGS databases separated the sequences from both libraries into three groups: (1) sequences with similarity to pericentromere mouse major satellite; (2) sequences without similarity to any repetitive sequences; (3) sequences with similarity to long interspersed nuclear elements (LINEs). LINE-related sequences have a disperse pattern distribution on chromosomes predicted in silico. Selected clones were used for fluorescent in situ hybridization (FISH). The 10 clones tested hybridized to chromocenters and centromeric regions of metaphase chromosomes. These clones were used for double FISH with four known cloned TRs (satDNA, satellite DNA) and a probe specific for the sex chromosomes. The probes bind various chromocenters' regions without overlapping; so, FISH results reveal a complex chromocenter composition. We mapped 18 LINE-derived clones to the RepBase L1 records. Most of them grouped in a ∼2-kb region at the end of the second ORF and 3' untranslated region (UTR). So, even the limited number of the clones allows us to determine the region of the L1 element that is specific for heterochromatic regions. Although the L1 full-length probe did not hybridize at detectable levels to the heterochromatic region on any chromosome, the 2-kb fragment found is definitely a part of these regions. The precise LINE ∼2-kb fragment is the component of mouse and human constitutive heterochromatin enriched with TRs. The method used for amplification of the probes from two sources of the heterochromatic material uncovered the enrichment of a precise fragment of LINE within chromocenters.

Telomere Repeat-Binding Factor 2 Is Responsible for the Telomere Attachment to the Nuclear Membrane.

Telomeres are nucleoprotein structures that specify ends of eukaryotic chromosomes. They enable complete DNA replication, protect chromosomes from end-to-end fusions, and help organize chromatin structure. These functions are mediated by special telomeric proteins. TRF2 (telomeric repeat-binding factor 2) is an essential component of shelterin, a telomere-binding protein complex. TRF2 induces formation of a special structure of telomeric DNA, counteracts activation of double-strand break response pathway and ataxia telangiectasia mutated kinase pathway at telomeres. Some line of evidence implicates TRF2 in interactions with the nuclear envelope (NE). TRF2 is tightly bound to the nuclear membrane in frog oocytes nucleus, and it was found colocalized with NE or its remnants in mouse cells. Computer analysis of TRF2 amino acid sequence has shown that TRF2 possesses motifs, which resemble rod domain characteristic of intermediate filament proteins. These observations suggest that TRF2 is a good candidate for the attachment of telomeres to the NE in somatic cells.

Trematode Himasthla elongata mariner element (Hemar): structure and applications.

We cloned and analyzed Hemar1-the full-length mariner of Himasthla elongata. Hemar1 amount and distribution in the genome is typical for the transposable elements. Hemar1 closest relatives found in databases are the mariner-like element (MLE) of Girardia tigrina with 88% similarity in the most conserved transposase domain and Cemar1 of Caenorhabditis elegans with the most similar inverted terminal repeats. Hydra's (Cnidaria) MLE are the next in similarity to Hemar1. We checked whether sequences similar to Hemar1 exist in intermediate and definitive hosts of the parasitic trematode and did not find obvious similarity. This fact, together with the data of Hemar1 evolutionary position, argues against recent MLE-mediated horizontal transfer in this parasite-host model. Our results demonstrate that H. elongata generates genomic variability in asexual parthenogenetic generations within the snail. Transposon insertional display based on full-length sequence showed that Hemar1 could be located in the regions involved in generating clonal diversity in rediae and cercariae, that is, trematode parthenitae.

Aurelia aurita-Cnidarian with a prominent medusiod stage.

Aurelia aurita has a complex life cycle that consists of several stages including alternating generations of medusa and polyps, huge sexual, and tiny asexual stages. Cnidarian is thought to possess two tissue layers: endoderm (gastroderm) and ectoderm, which are separated by mesoglea in medusa. The determination of the composition of the A. aurita jellyfish mesoglea was performed. New protein "mesoglein" was determined as one of the main components of mesoglea. Mesoglein is synthesized by mesogleal cells (Mc), which are populated A. aurita mesoglea as a high molecular mass precursor. Mc are involved in the formation of noncollagenous "elastic" fibers. Deduced amino acid sequence of mesoglein contains Zona Pellucida (ZP) domain and Delta/Serrate/Lag-2 domain. According to reverse transcription PCR, mesoglein is expressed in the mature medusa exclusively in the Mc. The sperm binding to the ZP is particularly important for successful fertilization. Antibodies against mesoglein stain the plate in the place of contact of germinal epithelium and oocyte. The structure found was named the "contact plate." The contact plate could be the precursor of the ZP. All our data suggest that Mc and, probably, the whole mesoglea originate from the epidermis (ectoderm). Computer search for mesoglein relatives reveals Nematostella and Trichoplax proteins as predicted ORFs, indicating that ZP proteins are quite ancient purchase in the evolution.

Tandemly repeated DNA families in the mouse genome.

BACKGROUND: Functional and morphological studies of tandem DNA repeats, that combine high portion of most genomes, are mostly limited due to the incomplete characterization of these genome elements. We report here a genome wide analysis of the large tandem repeats (TR) found in the mouse genome assemblies. RESULTS: Using a bioinformatics approach, we identified large TR with array size more than 3 kb in two mouse whole genome shotgun (WGS) assemblies. Large TR were classified based on sequence similarity, chromosome position, monomer length, array variability, and GC content; we identified four superfamilies, eight families, and 62 subfamilies - including 60 not previously described. 1) The superfamily of centromeric minor satellite is only found in the unassembled part of the reference genome. 2) The pericentromeric major satellite is the most abundant superfamily and reveals high order repeat structure. 3) Transposable elements related superfamily contains two families. 4) The superfamily of heterogeneous tandem repeats includes four families. One family is found only in the WGS, while two families represent tandem repeats with either single or multi locus location. Despite multi locus location, TRPC-21A-MM is placed into a separated family due to its abundance, strictly pericentromeric location, and resemblance to big human satellites. To confirm our data, we next performed in situ hybridization with three repeats from distinct families. TRPC-21A-MM probe hybridized to chromosomes 3 and 17, multi locus TR-22A-MM probe hybridized to ten chromosomes, and single locus TR-54B-MM probe hybridized with the long loops that emerge from chromosome ends. In addition to in silico predicted several extra-chromosomes were positive for TR by in situ analysis, potentially indicating inaccurate genome assembly of the heterochromatic genome regions. CONCLUSIONS: Chromosome-specific TR had been predicted for mouse but no reliable cytogenetic probes were available before. We report new analysis that identified in silico and confirmed in situ 3/17 chromosome-specific probe TRPC-21-MM. Thus, the new classification had proven to be useful tool for continuation of genome study, while annotated TR can be the valuable source of cytogenetic probes for chromosome recognition.

The fate of larval flagellated cells during metamorphosis of the sponge Halisarca dujardini.

Sponge larval flagellated cells have been known to form the external layer of larva, but their subsequent fate and morphogenetic role are still unclear. It is actually impossible to follow flagellated cell developmental fate unless a specific marker is found. We used percoll density gradient fractionation to separate different larval cell types of Halisarca dujardini (Demospongiae, Halisarcida). A total of 5 fractions were obtained which together contained all cell types. Fraction 1 contained about 100% FC and its polypeptide composition was very different to that of the other fractions. Of all larval cell types, flagellated cells displayed the lowest in vitro aggregation capacity. We raised a polyclonal antibody against a 68 kDa protein expressed by larval flagellated cells. Its specificity was tested on total protein extract from adult sponges by Western blotting and proved to be suitable for immunofluorescence. By means of double immunofluorescence using both this polyclonal antibody and commercial anti-tubulin antibodies, we studied the distribution of the 68 kDa protein in larval flagellated cells and its fate at successive stages of metamorphosis. In juvenile sponges just after metamorphosis the choanocytes and the upper pinacoderm were labelled with both antibodies. In larval flagellated cells, the 68 kDa protein was found all over the cytoplasm appearing as granules, while in adult sponges, it was present in the apical part of choanocytes in the vicinity of collars. Direct participation of the larval flagellated cells in the development of definitive structures was demonstrated.

Identification of the proteins specifically binding to the rat LINE1 promoter.

The initial step of LINE1 retrotransposons dissemination requires transcription from species-specific promoter located within 5'-untranslated region of LINE1. Although the 5'-untranslated region of the rat LINE1 element shows promoter activity, no promoter-binding proteins have been discovered so far. Using an EMSA and Southwestern blotting methods, we identified Sp1 and Sp3 proteins, which specifically bind to the rat LINE1 promoter in vitro. The Sp1/Sp3-binding motif within rat LINE1 promoter is located downstream of the major predicted transcription initiation site.

Analysis of polymorphic sites in the promoter of the nitric oxide synthase 2 gene.

A point mutation (G --> C) in the gene promoter for the human nitric oxide synthase (NOS) 2 at position -954 is associated with protection against severe Plasmodium falciparum malaria in Gabon. Carriers of this mutation show higher basal levels of nitric oxide production than wild type individuals. To obtain information about the possible binding transcription factors, nucleic proteins from the lung carcinoma cell line were enriched by affinity chromatography using DEAE-Sepharose and immobilized oligonucleotides derived from the promoter sequence. A mutational analysis was performed on 30 samples to detect polymorphisms in the NOS2 promoter region that contains important NF-kappaB sites. Three point mutations were identified in this region. In vitro studies with promoter constructs showed an altered expression of the marker gene depending on the promoter variant used.

New types of mouse centromeric satellite DNAs.

Genomic databases do not contain complete sequences of the centromeric regions. We created a pUC19-based library of DNA fragments from isolated chromocentres of interphase nuclei. In this library we have found major satellite (MaSat) and two new satellite sequences - MS3 and MS4. The computer analysis of MS3 and MS4 sequences by alignment, fragment curved state and search for MAR motifs in comparison with the mouse major and minor satellite (MiSat) DNA has shown them to be new satellite fragments. Southern blot of MS3 and MS4 with total DNA digested by restriction enzymes shows the ladder characteristic of satellite DNA. 2.2% of the total DNA consists of MS3, the monomer of which is 150 bp long. The MS4 monomer is 300 bp long and accounts for 1.6% of the total DNA. On metaphase chromosomes MS3 and MS4 are located at the centromeric region. FISH analysis of L929 nuclei during the cell cycle showed relative positions of MaSat, MiSat, MS3, and MS4. All mapped satDNA fragments except MaSat belong to the outer layer of the chromocentres in the G0/G1 phase. MS3 is likely to be involved in the centromere formation. The mouse genome contains at least four satDNA types: AT-rich (MaSat and MiSat), and CG-rich (MS3 and MS4).