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.

[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.

[Tandem repeats in rodents genome and their mapping].

Tandemly-repeated sequences represent a unique class of eukaryotic DNA. Their content in the genome of higher eukaryotes mounts to tens of percents. However, the evolution of this class of sequences is poorly-studied. In our paper, 62 families of Mus musculus tandem repeats are analyzed by bioinformatic methods, and 7 of them are analyzed by fluorescence in situ hybridization. It is shown that the same tandem repeat sets co-occure only in closely related species of mice. But even in such species we observe differences in localization on the chromosomes and the number of individual tandem repeats. With increasing evolutionary distance only some of the tandem repeat families remain common for different species. It is shown, that the use of a combination of bioinformatics and molecular biology techniques is very perspective for further studies of the evolution of tandem repeats.

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

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

[Heterochromatin and centromere structure paradox].

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