The nucleus of pre-implantation mammalian embryos is characterized by peculiar structural organization. At the initial stages of cleavage, the nucleus of the embryo contains the so-called nucleolus precursor bodies (NPBs) or pronucleoli rather than functionally active nucleoli. The NPBs are fibrillar electron-dense structures inactive in RNA synthesis. The vast majority of NPBs are surrounded by a ring-shaped zone of transcriptionally inactive heterochromatin. Intriguingly, these zones contain not only tri-methylated histone Н3K9me3 as an epigenetic mark of repressed chromatin but also acetylated histone H4K5ac, a well-known marker of active chromatin. Immunocytochemical data suggest that the molecular composition of this ‘ring heterochromatin’ in mouse embryos changes during the realization of embryonic genome activation events, as well as during artificial suppression of transcription. In zygotes, some factors of mRNA biogenesis including splicing factor SC35 (SRSF2) and basal transcription factor TFIID are detectable in the ring chromatin. At later stages of development, other nuclear proteins such as Y14, a core component of the exon-exon junction complex (EJC), as well as the proteins involved in chromatin remodeling (ATRX, Daxx) are also detectable in this area. A typical component of the ‘ring heterochromatin’ is actin. Anti-actin immunocytochemical labeling is most expressed at the two-cell cleavage stage after activation of the embryonic genome. Indicatively, the molecular composition of the ‘ring heterochromatin’ associated with different NPBs may differ significantly even in the same nucleus. This seems to reflect the functional heterogeneity of morphologically similar NPBs according to their competence to the process of nucleologenesis. Here, we discuss briefly some peculiarities of the molecular composition and possible functions of the NPB-associated heterochromatin in mouse early embryos.

Rye (Secale) is among staple cereals along with other members of the Triticeae tribe: wheat and barley. The genus Secale includes perennial and annual, cross-pollinating and self-pollinating species, and they can be donors of valuable genes in wheat and rye breeding programs. Studies of the structure of the gene for centromeric histone H3 (CENH3), essential for centromere functions, are relevant to the breeding of agronomically important crops. We have investigated the nucleotide diversity of sequences of two variants of the rye CENH3 gene inside the N-terminal tail (NTT) and the conservative HFD (histone fold domain) domain in the genus Secale. The mean values of nucleotide diversity in the NTT and HFD of wild cross- and self-pollinating taxa are close in αCENH3: πtot = 0.0176–0.0090 and 0.0136–0. 0052, respectively. In the case of βCENH3, the mean values for NTT (πtot = 0.0168–0.0062) are lower than for HFD (πtot = 0.0259–0.084). The estimates of nucleotide and haplotype diversity per site for the CENH3 domains are considerably lower in taxa with narrow geographic ranges: S. cereale subsp. dighoricum and S. strictum subsp. kuprijanovii. Commercial breeding reduces the nucleotide sequence variability in αCENH3 and βCENH3. Cultivated rye varieties have π values within 0.0122–0.0014. The nucleotide and haplotype diversity values in αCENH3 and βCENH3 are close in S. sylvestre, which is believed to be the oldest rye species. The results of this study prove that the frequency of single nucleotide polymorphisms and nucleotide diversity of sequences in genes for CENH3 in Secale species are influenced by numerous factors, including reproduction habits, the geographic isolation of taxa, breeding, and the evolutionary age of species.

Drosophila melanogaster is one of the popular model organisms in DNA replication studies. Since the 1960s, DNA replication of polytene chromosomes has been extensively studied by cytological methods. In the recent two decades, the progress in our understanding of DNA replication was associated with new techniques. Use of fluorescent dyes increased the resolution of cytological methods significantly. High-throughput methods allowed analysis of DNA replication on a genome scale, as well as its correlation with chromatin structure and gene activi ty. Precise mapping of the cytological structures of polytene chromosomes to the genome assembly allowed comparison of replication between polytene chromosomes and chromosomes of diploid cells. New features of replication characteristic for D. melanogaster were described for both diploid and polytene chromosomes. Comparison of genomic replication profiles revealed a significant similarity between Drosophila and other well-studi ed eukaryotic species, such as human. Early replication is often confined to intensely transcribed gene-dense regions characterized by multiple replication initiation sites. Features of DNA replication in Drosophila might be explained by a compact genome. The organization of replication in polytene chromosomes has much in common with the organization of replication in chromosomes in diploid cells. The most important feature of replication in polytene chromosomes is its low rate and the dependence of S-phase duration on many factors: external and internal, local and global. The speed of replication forks in D. melanogaster polytene chromosomes is affected by SUUR and Rif1 proteins. It is not known yet how universal the mechanisms associated with these factors are, but their study is very promising.

Polytene chromosomes of Drosophila melanogaster are a convenient model for studying interphase chromosomes of eukaryotes. They are giant in size in comparison with diploid cell chromosomes and have a pattern of cross stripes resulting from the ordered chromatid arrangement. Each region of polytene chromosomes has a unique banding pattern. Using the model of four chromatin types that reveals domains of varying compaction degrees, we were able to correlate the physical and cytological maps of some polytene chromosome regions and to show the main properties of genetic and molecular organization of bands and interbands, that we describe in this review. On the molecular map of the genome, the interbands correspond to decompacted aquamarine chromatin and 5’ ends of ubiquitously active genes. Gray bands contain lazurite and malachite chromatin, intermediate in the level of compaction, and, mainly, coding parts of genes. Dense black transcriptionally inactive bands are enriched in ruby chromatin. Localization of several dozens of interbands on the genome molecular map allowed us to study in detail their architecture according to the data of whole genome projects. The distribution of proteins and regulatory elements of the genome in the promoter regions of genes localized in the interbands shows that these parts of interbands are probably responsible for the formation of open chromatin that is visualized in polytene chromosomes as interbands. Thus, the permanent genetic activity of interbands and gray bands and the inactivity of genes in black bands are the basis of the universal banding pattern in the chromosomes of all Drosophila tissues. The smallest fourth chromosome of Drosophila with an atypical protein composition of chromatin is a special case.  Using the model of four chromatin states and fluorescent in situ hybridization, its cytological map was refined and the genomic coordinates of all bands and interbands were determined. It was shown that, in spite of the peculiarities of this chromosome, its band organization in general corresponds to the rest of the genome. Extremely long genes of different Drosophila chromosomes do not fit the common scheme, since they can occupy a series of alternating bands and interbands (up to nine chromosomal structures) formed by parts of these genes.

The formation of interphase chromosomes is a multi-level process in which DNA is compacted several thousandfold by association with histones and non-histone proteins. The first step of compaction includes the formation of nucleosomes – the basic repeating units of chromatin. Further packaging occurs due to DNA binding to histone H1 and non-histone proteins involved in enhancer-promoter and insulator interactions. Under these conditions, the genome retains its functionality due to the dynamic and uneven DNA compaction along the chromatin fiber. Since the DNA compaction level affects the transcription activity of a certain genomic region, it is important to understand the interplay between the factors acting at different levels of the packaging process. Drosophila polytene chromosomes are an excellent model system for studying the molecular mechanisms that determine DNA compaction degree. The unevenness of DNA packaging along the chromatin fiber is easily observed along these chromosomes due to their large size and specific banding pattern. The purpose of this study was to figure out the role of two non-histone regulatory proteins, ADF1 and BEAF-32, in the DNA packaging process from nucleosome positioning to the establishment of the final chromosome structure. We studied the impact of mutations that affect ADF1 and BEAF-32 binding sites on the formation of 61C7/C8 interband – one of the decompacted regions of Drosophila polytene chromosomes. We show that such mutations led to the collapse of an interband, which was accompanied with increased nucleosome stability. We also find that ADF1 and BEAF-32 binding sites are essential for the rescue of lethality caused by the null allele of bantam microRNA gene located in the region 61C7/C8.

Chromatin assembly is a fundamental process essential for chromosome duplication subsequent to DNA replication. In addition, histone removal and incorporation take place constantly throughout the cell cycle in the course of DNA-utilizing processes, such as transcription, damage repair or recombination. In vitro studies have revealed that nucleosome assembly relies on the combined action of core histone chaperones and ATP-utilizing molecular motor proteins such as ACF or CHD1. Despite extensive biochemical characterization of ATP-dependent chromatin assembly and remodeling factors, it has remained unclear to what extent nucleosome assembly is an ATP-dependent process in vivo. Our original and published data about the functions of ATP-dependent chromatin assembly and remodeling factors clearly demonstrated that these proteins are important for nucleosome assembly and histone exchange in vivo. During male pronucleus reorganization after fertilization CHD1 has a critical role in the genomescale, replication-independent nucleosome assembly involving the histone variant H3.3. Thus, the molecular motor proteins, such as CHD1, function not only in the remodeling of existing nucleosomes but also in de novo nucleosome assembly from DNA and histones in vivo. ATP-dependent chromatin assembly and remodeling factors have been implicated in the process of histone exchange during transcription and DNA repair, in the maintenance of centromeric chromatin and in the loading and remodeling of nucleosomes behind a replication fork. Thus, chromatin remodeling factors are involved in the processes of both replication-dependent and replication-independent chromatin assembly. The role of these proteins is especially prominent in the processes of large-scale chromatin reorganization; for example, during male pronucleus formation or in DNA repair. Together, ATP-dependent chromatin assembly factors, histone chaperones and chromatin modifying enzymes form a “chromatin integrity network” to ensure proper maintenance and propagation of chromatin landscape.

The Su(Hw) protein was first identified as a DNA-binding component of an insulator complex in Drosophila. Insulators are regulatory elements that can block the enhancer-promoter communication and exhibit boundary activity. Some insulator complexes contribute to the higher-order organization of chromatin in topologically associated domains that are fundamental elements of the eukaryotic genomic structure. The Su(Hw)-dependent protein complex is a unique model for studying the insulator, since its basic structural components affecting its activity are already known. However, the mechanisms involving this complex in various regulatory processes and the precise interaction between the components of the Su(Hw) insulators remain poorly understood. Our recent studies reveal the fine mechanism of formation and function of the Su(Hw) insulator. Our results provide, for the first time, an example of a high complexity of interactions between the insulator proteins that are required to form the (Su(Hw)/Mod(mdg4)-67.2/CP190) complex. All interactions between the proteins are to a greater or lesser extent redundant, which increases the reliability of the complex formation. We conclude that both association with CP190 and Mod(mdg4)-67.2 partners and the proper organization of the DNA binding site are essential for the efficient recruitment of the Su(Hw) complex to chromatin insulators. In this review, we demonstrate the role of multiple interactions between the major components of the Su(Hw) insulator complex (Su(Hw)/Mod(mdg4)-67.2/CP190) in its activity. It was shown that Su(Hw) may regulate the enhancer–promoter communication via the newly described insulator neutralization mechanism. Moreover, Su(Hw) participates in direct regulation of activity of vicinity promoters. Finally, we demonstrate the mechanism of organization of “insulator bodies” and suggest a model describing their role in proper binding of the Su(Hw) complex to chromatin.

SAGA/TFTC, which is a histone acetyltransferase complex, plays an important role in the regulation of transcription. We have identified that the metazoan TFTC/STAGA complexes had histone H2A and H2B deubiquitinase activity that is carried out by a DUBm (deubiquitination module). We studied the DUBm of SAGA in Drosophila melanogaster and identified Drosophila homologs of yeast DUBm components. Two subunits of DUBm (Sus1/ENY2 and Sgf11) were shown to have functions separate from DUBm function. Thus, Sus1/ENY2 was shown to be present in several different complexes. Sgf11 was found to be associated with the cap-binding complex (CBC) and recruited onto growing messenger ribonucleic acid (mRNA). Also, we have shown that Sgf11 interacted with the TREX-2/AMEX mRNA export complex and was essential for mRNA export from the nucleus. Immunostaining of the polytene chromosomes of Drosophila larvae revealed that Sgf11 is present at the sites of localization of snRNA genes. It was also found in immunostaining experiments that dPbp45, the subunit of the PBP complex, the key player in the snRNA transcription process, is associated not only with the snRNA gene localization sites, but with other sites of active transcription by PolII. We also revealed that Sgf11 was present at many active transcription sites in interbands and puffs on polytene chromosomes, Sgf11 was localized at all Brf1 (the component of the RNA polymerase III basal transcription complex) sites. We concluded that SAGA coactivated transcription of both the PolII and PolIII-dependent snRNA genes.

The proteins with the BTB domain play an important role in the processes of activation and repression of transcription. Interestingly, BTB-containing proteins are widely distributed only among higher eukaryotes. Many BTB-containing proteins are transcriptional factors involved in a wide range of developmental processes. One of the key regulators of early development is the BTB-containing protein Ttk (tramtrack), which is able to interact with the Drosophila nucleosome remodeling and histone deacetylation (dNuRD) complex. Ttk69 directly interacts with two protein components of the dNuRD complex, dMi-2 and MEP1. It can be assumed that Ttk69 represses some target genes by remodeling chromatin structure through the recruitment of the dNuRD complex. However, it is still unknown what provides for specific recruitment of Ttk to chromatin in the process of negative/positive regulation of a target gene expression. Although Ttk69 has DNA-binding activity, no extended specific motif has been identified. The purpose of this study was to find proteins that can participate in the recruitment of Ttk to regulatory elements. To identify Ttk partner proteins, screening in the yeast two-hybrid system was performed against a collection of proteins with clusters of C2H2 domains, which bind effectively and specifically to sites on chromatin. As a results, the CG10321 and CG1792 proteins were identified as potential DNA-binding partners of Ttk. We suppose that the CG10321 and CG1792 proteins provide specificity for the recruitment of Ttk and, as a result, of the NuRD-complex to the genome regulatory elements. We found that the Ttk protein is able to interact with the MEP1 and ZnF proteins at once.

Chromatin remodelling multiprotein complexes play an important role in regulation of gene expression in embryogenesis and in the adult organism. Mutations in the subunits of the complexes are often lethal or lead to developmental defects. Complexes consist of core subunits and a specific module. The core consists of ATPase and structure subunits, specific subunits of the module are necessary for chromatin binding. PHF10 (PHD finger protein 10) is a subunit of the PBAF (polybromo-associated BAF) chromatin remodelling complex subfamily. Conserved and highly regulated PHF10 is ubiquitously expressed in mammals as four different isoforms. The isoforms of PHF10 differ by domain structures and posttranslational modifications. All isoforms are highly regulated and included in the PBAF complex in a mutually exclusive manner. Two of the PHF10 isoforms (PHF10-P) are expressed at a high level in neuronal and myeloid progenitors and are necessary for cell proliferation. These isoforms contain PHD (plant homeodomain) fingers for nucleosome binding and recruit RNA polymerase II on the promoters of cell cycle genes. Two other isoforms (PHF10-S) instead of PHD have PDSM (phosphorylation-dependent sumoylation motif), the motif for SUMO1 conjugation. PHF10 is the most unstable subunit of the PBAF complex. Stability can alter the turnover rate of the subunits of the PBAF complex. All PHF10 isoforms are degraded by β-TrCP ubiquitin ligase but PHF10-S isoforms contain a cluster of serins (X-cluster) for multiple phosphorylation by casein kinase I. This phosphorylation protects the β-TrCP degron from β-TrCP recognition and subsequently stabilizes the PHF10-S isoforms. Thus, the incorporation of PHF10 isoforms with different phosphorylation patterns and different stability into the PBAF complexes alters the functions of the entire PBAF complex and determines the range of genes undergoing remodelling.

The nucleolus is a dynamic non-membrane-bound nuclear organelle, which plays key roles not only in ribosome biogenesis but also in many other cellular processes. Consistent with its multiple functions, the nucleolus has been implicated in many human diseases, including cancer and degenerative pathologies of the nervous system and heart. Here, we report the characterization of the Drosophila Non3 (Novel nucleolar protein 3) gene, which encodes a protein homologous to the human Brix domain-containing Rpf2 that has been shown to control ribosomal RNA (rRNA) processing. We used imprecise P-element excision to generate four new mutant alleles in the Non3 gene. Complementation and phenotypic analyses showed that these Non3 mutations can be arranged in an allelic series that includes both viable and lethal alleles. The strongest lethal allele (Non3∆600) is a genetically null allele that carries a large deletion of the gene and exhibits early lethality when homozygous. Flies heterozygous for Non3∆600 occasionally exhibit a mild reduction in the bristle size, but develop normally and are fertile. However, heteroallelic combinations of viable Non3 mutations (Non3197, Non3310 and Non3259) display a Minute-like phenotype, consisting in delayed development and short and thin bristles, suggesting that they are defective in ribosome biogenesis. We also demonstrate that the Non3 protein localizes to the nucleolus of larval brain cells and it is required for proper nucleolar localization of Fibrillarin, a protein important for post-translational modification and processing of rRNAs. In summary, we generated a number of genetic and biochemical tools that were exploited for an initial characterization of Non3, and will be instrumental for future functional studies on this gene and its protein product.

The Notch gene plays a key role in the development of organs and tissues of neuroectodermic origin, including the nervous system. In eukaryotic organisms, the Notch pathway is involved in cell fate determination. The Notch gene was first discovered in Drosophila melanogaster. In mammals, the family of Notch receptors includes four homologues. In humans, mutations in the Notch gene cause several hereditary diseases and carcinogenesis. Studies of the regulatory zone of the Notch gene in D. melanogaster have been conducted for several decades. We review their results and methods. The regulatory zone of the Notch gene is in the region of open chromatin state that corresponds to the 3C6/3C7 interband on the cytological map of polytene chromosomes of D. melanogaster salivary glands. The development of new methods for directed genome editing made it possible to create a system for introducing directed changes into the regulatory zone of the gene. Using the CRISPR/Cas9 system, we obtained a directed 4-kilobase deletion including the 5’-regulatory zone, promoter, and the first exon of the Notch gene and introduced the attP site into the first intron of the Notch gene. This approach enabled targeted changes of the sequence of the regulatory and promoter regions of the gene. Thus, it provided a new powerful tool for studies of Notch gene regulation and the organization of the open chromatin state.

The discovery of the position effect variegation phenomenon and the subsequent comprehensive analysis of its molecular mechanisms led to understanding that the local chromatin composition has a dramatic effect on gene activity. To study this effect in a high-throughput mode and at the genome-wide level, the Thousands of Reporters Integrated in Parallel (TRIP) approach based on the usage of barcoded reporter gene constructs was recently developed. Here we describe the construction and quality checks of high-diversity barcoded plasmid libraries supposed to be used for high-throughput analysis of chromatin position effects in Drosophila cells. First, we highlight the critical parameters that should be considered in the generation of barcoded plasmid libraries and introduce a simple method to assess the diversity of random sequences (barcodes) of synthetic oligonucleotides using PCR amplification followed by Sanger sequencing. Second, we compare the conventional restriction-ligation method with the Gibson assembly approach for cloning barcodes into the same plasmid vector. Third, we provide optimized parameters for the construction of barcoded plasmid libraries, such as the vector : insert ratio in the Gibson assembly reaction and the voltage used for electroporation of bacterial cells with ligation products. We also compare different approaches to check the quality of barcoded plasmid libraries. Finally, we briefly describe alternative approaches that can be used for the generation of such libraries. Importantly, all improvements and modifications of the techniques described here can be applied to a wide range of experiments involving barcoded plasmid libraries.

The mechanisms of ecdysone-dependent expression have been studied for many decades. Initially, the activation of individual genes under the influence of ecdysone was studied on the model of polythene chromosomes from salivary glands of Drosophila melanogaster. These works helped to investigate the many aspects of the Drosophila development. They also revealed plenty of valuable information regarding the fundamental mechanisms controlling the genes’ work. Many years ago, a model describing the process of gene activation by ecdysone, named after the author – Ashburner model – was proposed. This model is still considered an excellent description of the ecdysone cascade, which is implemented in the salivary glands during the formation of the Drosophila pupa. However, these days there is an opinion that the response of cells to the hormone ecdysone can develop with significant differences, depending on the type of cells. The same genes can be activated or repressed under the influence of ecdysone in different tissues. Likely, certain DNA-binding transcription factors that are involved in the ecdysonedependent response together with the EcR/Usp heterodimer are responsible for cell-type specificity. A number of transcriptional regulators involved in the ecdysone response have been described. Among them are several complexes responsible for chromatin remodeling and modification. It has been shown by various methods that ecdysone-dependent activation/repression of gene transcription develops with significant structural changes of chromatin on regulatory elements. The description of the molecular mechanism of this process, in particular, the role of individual proteins in it, as well as structural interactions between various regulatory elements is a matter of the future. This review is aimed to discuss the available information regarding the main regulators that interact with the ecdysone receptor. We provide a brief description of the regulator’s participation in the ecdysone response and links to the corresponding study. We also discuss general aspects of the mechanism of ecdysone-dependent regulation and highlight the most promising points for further research.

Despite the rapid development of approaches aimed to precisely control transcription of exogenous genes in time and space, design of systems providing similar tight regulation of endogenous gene expression is much more challenging. However, finding ways to control the activity of endogenous genes is absolutely necessary for further progress in safe and effective gene therapies and regenerative medicine. In addition, such systems are of particular interest for genetics, molecular and cell biology. An ideal system should ensure tunable and reversible spatio-temporal control over transcriptional activity of a gene of interest. Although there are drug-inducible systems for transcriptional regulation of endogenous genes, optogenetic approaches seem to be the most promising for the gene therapy applications, as they are noninvasive and do not exhibit toxicity in comparison with druginducible systems. Moreover, they are not dependent on chemical inducer diffusion rate or pharmacokinetics and exhibit fast activation-deactivation switching. Among optogenetic tools, long-wavelength light-controlled systems are more preferable for use in mammalian tissues in comparison with tools utilizing shorter wavelengths, since far-red/near-infrared light has the maximum penetration depth due to lower light scattering caused by lipids and reduced tissue autofluorescence at wavelengths above 700 nm. Here, we review such light-inducible systems, which are based on synthetic factors that can be targeted to any desired DNA sequence and provide activation or repression of a gene of interest. The factors include zinc finger proteins, transcription activator-like effectors (TALEs), and the CRISPR/Cas9 technology. We also discuss the advantages and disadvantages of these DNA targeting tools in the context of the light-inducible gene regulation systems.

The black kite Milvus migrans is a common bird of prey demonstrating remarkable ecological plasticity. It inhabits a variety of habitats and is an increasingly synanthropic species. The black kite is widespread in Eurasia, Africa, Australia and adjacent islands. Palearctic kites migrate to Africa, India and China in winter, but kites of Africa and Australia are partly sedentary and partly seasonal migrants. The wide range and high mobility are the reasons of a complex population structure of the black kite. Commonly five to seven M. migrans subspecies are distinguished, each of which is widespread over extensive areas and has more or less an apparent phenotype. Recently, studies of genetic differences between black kite populations started to emerge. On the grounds of earlier studies of mitochondrial and nuclear genes of this species, we check whether there is a genetic support for separation of the black kite subspecies. Recent studies of some mitochondrial loci substantiate the recognition of at least the European (M. m. migrans), Asian (M. m. lineatus and M. m. govinda), African (M. m. aegyptius and M. m. parasitus), and Australian (M. m. affinis) black kite subspecies. Furthermore, the mitochondrial haplotype difference suggests that the African yellow-billed kite, including M. m. aegyptius and M. m. parasitus, should be a separate species as already proposed, or even two separate species.

Natural hybridization increases a lot phenotypic and genetic diversity and shapes intra-species patterns, which is a subject of phylogeography. We studied mitochondrial and complete genome variation in the bird family Corvidae, genera Corvus, Pica, Cyanopica, Perisoreus and Nucifraga. In the classic case of natural hybridization between carrion and hooded crows in Siberia, we found no decreased fitness of hybrids, but instead positive assortative mating which should restrict hybrid zone width. Several genetic markers were unable to discriminate between pure carrion and hooded crows. Mitochondrial DNA sequences revealed no difference between carrion and hooded crows, but instead two diverged haplogroups within the eastern part of the distribution range of the carrion crow. NGS resulted in a clear pattern of diversification of pure forms and hybrids (by using SNPs), and showed genomic regions of increased variability, the so-called “speciation islands”. Comparing European and Siberian crow hybrid zones, differences in genome regions bearing genes of melanogenesis supposedly under divergent selection were found. Comparative phylogeographic analysis of 10 widely distributed Palearctic species revealed two kinds of patterns: one with a division into two haplogroups, western and eastern, and another one without such a division. These two phylogeographic patterns might be explained by different habitat preferences: mainly open fields for the first group and forests for the second one. One glacial refuge was assigned to the latter group, while west-east group species might have survived in several refuges. One of such species, the Eurasian magpie (Pica pica) has a gap in its range in Transbaikalia, which is currently shrinking before our eyes. The two subspecies divided by this gap differ in phenotype, mtDNA and vocalization. In their young contact zone, some hybridization occurs with small introgression limited by certain post-zygotic isolation.

Chromosomal rearrangements can lead to the formation of new stable karyotypes, nevertheless changing the architectonics of the nucleus. The differences in locations might promote Robertsonian (Rb) translocations and encourage meiotic drive in favour of changed chromosomes or against them. We hypothesized that hybridization and meiotic drive may produce new chromosomal forms in Ellobius tancrei. We crossed two forms with 2n = 50, and two pairs of different Rb metacentrics with partial (monobrachial) homology. In 10 years of inbred crossings (sister – brother), we got 9 generations of hybrids (262 litters, 578 animals). In the first hybrid generation, two trivalents, a tetravalent and 20 bivalents were revealed at meiotic prophase I. Hybrids of the first generation had lower fertility, fertility increased starting from the third generation. Instead of returning to parental karyotypes, starting from the second generation, hybrids obtained new chromosome sets, with different 2n (48, 49, 51, 52) and combinations of Rb metacentrics. Analysis of F4, F7 and F9 hybrids revealed that synapsis of homologous parts take place despite the presence of heterozygotes and monobrachial homology of Rb metacentrics. The most common meiotic disturbance was delayed synapsis, which resumed later compared to the homologous crossings. The late synaptic adjustments nevertheless provide a proper segregation of chromosomes and normal sets in the gametes. Therefore, some cells pass through meiosis successfully and promote viable gametes. We proved the hypothesis that origin of monobrachially homologous Rb translocations may lead to divergence in several generations, due to meiotic drive.

Recurrent pregnancy loss (RPL) is a severe reproductive pathology with a significant component of unexplained etiology. Extended homozygous regions as a possible etiological factor for RPL were sought in the genomes of embryos. Twenty-two paired first-trimester spontaneously aborted embryos from eleven women with recurrent miscarriage were analyzed. All embryos had normal karyotypes according to metaphase karyotyping and conventional comparative genomic hybridization. SurePrint G3 Human CGH + SNP 4 × 180K microarrays (Agilent Technologies) were used to search for homozygous regions. As a result, 39 runs of homozygosity (ROH) were identified in extraembryonic tissues of 15 abortuses. Verification of recurrent homozygous regions was performed by Sanger sequencing. The presence of occasional heterozygous SNPs was shown in 25 extended ROHs, which may indicate that they did not arise de novo but were inherited from parents. In the course of inheritance in a series of generations, they may accumulate mutations, leading to heterozygosity for several sites in the initially homozygous population-specific regions. Homozygotization of recessive mutations is one of the putative mechanisms of the influence of such inherited ROHs on RPL development. The high frequency of extended ROHs detected in the present study may point to a role of inbreeding in RPL etiology. Homozygous regions may also occur due to uniparental disomy, and abnormalities of genomic imprinting may be another mechanism responsible for the pathological manifestation of ROHs in embryogenesis. Indeed, five predicted imprinted genes were identified within ROHs according to the Geneimprint database: OBSCN, HIST3H2BB, LMX1B, CELF4, and FAM59A. This work reports the first finding of a high frequency of extended ROHs in spontaneously aborted embryos with normal karyotypes from families with RPL.