LAMPBRUSH CHROMOSOMES AS A MODEL FOR EXPLORATION INTO LOCI OF NUCLEAR DOMAINS FORMATION.

Nuclear domains can be divided into two major groups: those arising freely in nucleoplasm and those forming at specific chromosomal loci as a result of their activity. The advantages of giant transcriptionally active lampbrush chromosomes for the investigation of nuclear bodies formed in particular chromosomal regions have been demonstrated in a series of studies. We propose to use two strategies to analyze the loci of nuclear domains formation on lampbrush chromosomes typical for avian and amphibian oocytes. The first approach implies consecutive mapping of BAC-clones, containing the fragments of DNA assigned to genomic coordinates, in close proximity to the nuclear domains. The second approach is based on mechanical microdissection of chromosomal regions adjacent to a particular nuclear structure. DNA of dissected material can be amplified by PCR with degenerate primers and mapped by fluorescent in situ hybridization (FISH) on chromosomal spreads. Utilization of high-throughput sequencing (next generation sequencing, NGS) technologies also proves to be prospective for subsequent deciphering of regions underlying nuclear structures formation. Deciphered fragments can be aligned against reference genome assembly to define precisely the loci responsible for nuclear domains assembly. In this review, the possibilities of using two complementary strategies for investigation of nuclear domains associated with lampbrush chromosomes are demonstrated.

[Cajal bodies and histone locus bodies: molecular structure and function].

The review provides modern classification of evolutionarily conserved coilin-containing nuclear bodies of somatic and germ cells that is based on the characteristic features of their molecular composition and the nature of their functions. The main differences between Cajal bodies and histone locus bodies, which are involved in the biogenesis of small nuclear spliceosomal and nucleolar RNAs and in the 3'-end processing of histone precursor messenger RNA, respectively, are considered. It is shown that a significant contribution to the investigation of the diversity of coilin-containing bodies was made by the studies on the architecture of the RNA processing machinery in oocyte nuclei in a number of model organisms. The characteristics features of the molecular composition of coilin-containing bodies in the nuclei of growing oocytes (the so-called germinal vesicles) of vertebrates, including amphibians and birds, are described.

[Spatial arrangement of macro-, midi- and microchromosomes in transcriptionally active nuclei of growing oocytes in birds of the order galliformes].

Three-dimensional genome organization in the cell nucleus reflects its functional state and is one of the regulatory levels of gene expression. Thus, a detailed exploration of the interrelations between the genome spatial organization and functioning is essential. In this work, three-dimensional genome organization in growing oocytes of Galliform birds, with giant transcriptionally active nucleus characterized by nearly absolute lack of structural constraints on chromosome decondensation, is analyzed in detail. Radial distribution of three groups of chromosomes with different size and gene density in the nuclei of chicken and Japanese quail oocytes was analyzed using confocal laser scanning microscopy followed by the 3D-reconstruction. The chromosome position relative to the nuclear center was estimated by analyzing its localization in the certain radial zones of the nucleus and direct distance measurements from the centre of the nucleus to the terminal regions and the center of gravity of the chromosome. It has been shown that, in the transcriptionally active nuclei of avian oocytes, chromosomes are located at a significant distance from the nuclear envelope and the gene-rich microchromosomes have no preferential location close to the center of the nucleus and are localized mainly at the periphery of the region occupied by the whole chromosome set. Therefore the radial distribution of lampbrush chromosomes in the oocyte nucleus does not obey the regularity of the spatial arrangement of chromosomes in the interphase nucleus according to which the gene-rich chromosome territories are located at the nuclear center and the gene-poor ones are at the nuclear periphery. With the help of visualization of 3D-preserved lampbrush chromosomes in the intact nucleus, we have confirmed the presence of the repulsion forces between the lateral loops of lampbrush half-bivalents and the lack of interactions between the heterochromatic segments of different bivalents at the lampbrush stage of oogenesis.

[Organization of centromere regions of chromosomes in the lampbrush phase].

Centromeres have a pivotal role in the complex of structural elements that are required for precise segregation of eukaryotic chromosomes during two types of cellular divisions--mitosis and meiosis. Data of ultrastructural and cytomolecular analysis indicate significant changes in molecular composition and functional morphology of centromeres during preparation for the first meiotic division. The review is devoted to modern concepts of morpho-functional organization of chromosomal centromere regions in growing oocytes in birds and amphibians. Structure, molecular composition as well as domain organization of centromeres in the lampbrush phase are characterized; data of cytogenetic analysis are presented. Special attention is given to the significance and regulation of satellite DNA transcription in the nuclei of developing oocytes. Possible functions of centromere "protein bodies" formed at the primary constriction of meiotic bivalents are discussed.

[Chicken lampbrush chromosomes: transcription of tandemly repetitive DNA sequences].

The transcribed part of the genome includes both protein-coding sequences and a variety of sequences with unknown functions. Amphibian and avian lampbrush chromosomes represent a convenient experimental system for studying cell functions and the regulation of transcription of protein-noncoding DNA. Taking lumpy loops formed on chicken (Gallusgallus domesticus) chromosome 2 at the lampbrush stage as an example, we applied an approach allowing RNA sources to be identified in the lateral loops of lampbrush chromosomes. This approach involves a bioinformatic analysis of data from the chicken genome sequencing project and a high-resolution mapping of transcripts on microsurgically isolated bivalents. As a result, a novel tandemly repetitive DNA sequence, LL2R (lumpy loop 2 repeat), of approximately 440 bp in size was identified in the chicken genome, its transcripts taking part in the formation of lumpy loops with a massive RNP matrix on chromosome 2 in growing oocytes.

[Lampbrush chromosomes of the Japanese quail (Coturnix coturnix japonica): a new version of cytogenetic maps].

Avian oocyte chromosomes are transfomed into giant transcriptionally active lampbrush chromosomes (LBCs) at meiosis 1 diplotene. These chromosomes are a convenient tool for high-resulution cytogenetic analysis. Using differential staining with fluorochromes DAPI and CMA3, we have constructed detailed cytological maps for lampbrush macrochromosomes 1-5 and ZW of the Japanese quail Coturnix coturnix japonica. We also performed a comparative analysis ofmitotic chromosomes and LBCs corresponding to them. We estimated the decondensation coefficient during LBC formation and determined the centromere indices for mitotic and diplotene chromosomes and thus found that different chromosomes and chromosomal regions demonstrate unequal degrees of decondensation.