The chromosomal distribution of 41-bp repeats, known as CNM and PO41 repeats in the chicken genome and BglII repeats in the Japanese quail, was analyzed precisely using giant lampbrush chromosomes (LBC) from chicken, Japanese quail, and turkey growing oocytes. The PO41 repeat is conserved in all galliform species, whereas the other repeats are species specific. In chicken and quail, the centromere and subtelomere regions share homologous satellite sequences. RNA polymerase II transcribes the 41-bp repeats in both centromere and subtelomere regions. Ongoing transcription of these repeats was demonstrated by incorporation of BrUTP injected into oocytes at the lampbrush stage. RNA complementary to both strands of CNM and PO41 repeats is present on chicken LBC loops, whereas strand-specific G-rich transcripts are characteristic of BglII repeats in the Japanese quail. The RNA from 41-bp repeats does not undergo cotranscriptional U snRNP-dependent splicing. At the same time, the ribonucleoprotein matrix of transcription units with C-rich RNA of CNM and PO41 repeats was enriched with hnRNP protein K. Potential promoters for satellite transcription are discussed.

Lampbrush chromosomes (LBCs) are highly extended bivalents that function in the growing oocytes of many animals. Due to their distinctive chromomere-loop organization and intense transcriptional activity of lateral loops the LBCs, mainly amphibian ones, have served as a powerful system for exploring the general principles of chromosome organization and function. The exploitation of avian LBCs has considerably broadened the opportunities for comparative genome research and for cytogenetic analysis of domestic species. In this review we highlight the advantages of avian LBCs for research in different areas including integration of genome organization studies with studies on gene activity in vivo, analysis of co-transcriptional events occurring on nascent transcripts and investigation of chromosome-associated intranuclear domains. Recent findings concerning the organization of transcriptionally active and silent chromatin together with involvement of cohesin and condensin complexes into maintenance of structural integrity of LBCs are presented. The biological significance of the LBC phenomenon is discussed. The intensive transcription on LBCs shows some specific features: very long transcription units, deregulated termination, and transcription of non-coding satellite repeats. Here, based on the modern view on a role of RNA interference machinery in regulation of genome expression, we suggest a mechanism of initiation of satellite DNA transcription and offer a novel interpretation of the ‘classical’ hypothesis that sought to explain the significance of widespread transcription during oocyte growth.

BackgroundOver the past two decades, chromosome microdissection has been widely used in diagnostics and research enabling analysis of chromosomes and their regions through probe generation and establishing of chromosome- and chromosome region-specific DNA libraries. However, relatively small physical size of mitotic chromosomes limited the use of the conventional chromosome microdissection for investigation of tiny chromosomal regions.ResultsIn the present study, we developed a workflow for mechanical microdissection of giant transcriptionally active lampbrush chromosomes followed by the preparation of whole-chromosome and locus-specific fluorescent in situ hybridization (FISH)-probes and high-throughput sequencing. In particular, chicken (Gallus g. domesticus) lampbrush chromosome regions as small as single chromomeres, individual lateral loops and marker structures were successfully microdissected. The dissected fragments were mapped with high resolution to target regions of the corresponding lampbrush chromosomes. For investigation of RNA-content of lampbrush chromosome structures, samples retrieved by microdissection were subjected to reverse transcription. Using high-throughput sequencing, the isolated regions were successfully assigned to chicken genome coordinates. As a result, we defined precisely the loci for marker structures formation on chicken lampbrush chromosomes 2 and 3. Additionally, our data suggest that large DAPI-positive chromomeres of chicken lampbrush chromosome arms are characterized by low gene density and high repeat content.ConclusionsThe developed technical approach allows to obtain DNA and RNA samples from particular lampbrush chromosome loci, to define precisely the genomic position, extent and sequence content of the dissected regions. The data obtained demonstrate that lampbrush chromosome microdissection provides a unique opportunity to correlate a particular transcriptional domain or a cytological structure with a known DNA sequence. This approach offers great prospects for detailed exploration of functionally significant chromosomal regions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2437-4) contains supplementary material, which is available to authorized users.

In the oocyte nuclei (germinal vesicle or GV) of a variety of avian species, prominent spherical entities termed protein bodies (PBs) arise at the centromeric regions of the lampbrush chromosomes (LBCs). In spite of the obvious protein nature of PBs, nothing is known about their composition. We show that an antibody against DNA topoisomerase II (topo II), the DNA unwinding enzyme, recognizes PBs from chaffinch and pigeon oocytes. In later chaffinch oocytes, the PBs fuse to form a karyosphere, which is also labeled by the anti-topo II antibody. Furthermore, we show that proteins characteristic of Cajal bodies and B-snurposomes are not found in PBs, despite morphological similarities among these structures. Using immunoelectron microscopy and immunofluorescent laser scanning microscopy we demonstrated that topo II localizes predominantly in the dense material of PBs. Two antigens of approximately 170 kDa (which corresponds to topo II) and approximately 100 kDa were revealed with the antibody against topo II on immunoblots of avian GV proteins. We propose that the smaller protein results from oocyte specific topo II cleavage, since it was not detected in nuclei from testis cells. This represents the first report of a defined protein in the centromeric PBs on avian LBCs.

In diplotene oocyte nuclei of all vertebrate species, except mammals, chromosomes lack interchromosomal contacts and chromatin is linearly compartmentalized into distinct chromomere-loop complexes forming lampbrush chromosomes. However, the mechanisms underlying the formation of chromomere-loop complexes remain unexplored. Here we aimed to juxtapose somatic topologically associating domains (TADs), recently identified in chicken embryonic fibroblasts, with chromomere-loop complexes in lampbrush meiotic chromosomes.By measuring 3D-distances and colocalization between linear equidistantly located genomic loci, positioned within one TAD or separated by a TAD border, we confirmed the presence of predicted TADs in chicken embryonic fibroblast nuclei. Using three-colored FISH with BAC probes we mapped equidistant genomic regions included in several sequential somatic TADs on isolated chicken lampbrush chromosomes. Eight genomic regions, each comprising two or three somatic TADs, were mapped to non-overlapping neighboring lampbrush chromatin domainslateral loops, chromomeres or chromomere-loop complexes. Genomic loci from the neighboring somatic TADs could localize in one lampbrush chromomere-loop complex, while genomic loci belonging to the same somatic TAD could be localized in neighboring lampbrush chromomereloop domains. In addition, FISH-mapping of BAC probes to the nascent transcripts on the lateral loops indicates transcription of at least 17 protein-coding genes and 2 non-coding RNA genes during the lampbrush stage of chicken oogenesis, including genes involved in oocyte maturation and early embryo development.