Genome organization of major tandem repeats and their specificity for heterochromatin of macro- and microchromosomes in Japanese quail.

Tandemly repeated DNAs form heterochromatic regions of chromosomes, including the vital centromeric chromatin. Despite the progress in new genomic technologies, tandem repeats remain poorly deciphered and need targeted analysis in the species of interest. The Japanese quail is one of the highest-producing poultry species as well as a model organism. Its genome differs by a noticeable accumulation of heterochromatin, which led to an increase by 1/7 compared to the chicken genome size. Prominent heterochromatin blocks occupy the short arms of acrocentric macrochromosomes and of microchromosomes. We have applied de novo repeat finder approach to unassembled raw reads of the Japanese quail genome. We identified the 20 most common tandem repeats with the abundance >1 Mb, which represent about 4.8% of the genome. We found that tandem repeat CjapSAT primarily contributes to the centromeric regions of the macrochromosomes CJA1-8. Cjap31B together with previously characterized BglII makes up centromere regions of microchromosomes and W chromosome. Other repeats populate heterochromatin of microchromosomal short arms in unequal proportions, as revealed by fluorescence in situ hybridization. The Cjap84A, Cjap408A, and CjapSAT repeat sequences show similarities to retrotransposon motifs. This suggests that retroelements may have played a crucial role in the distribution of repeats throughout the Japanese quail genome.

Germline-restricted chromosome (GRC) is widespread among songbirds.

An unusual supernumerary chromosome has been reported for two related avian species, the zebra and Bengalese finches. This large, germline-restricted chromosome (GRC) is eliminated from somatic cells and spermatids and transmitted via oocytes only. Its origin, distribution among avian lineages, and function were mostly unknown so far. Using immunolocalization of key meiotic proteins, we found that GRCs of varying size and genetic content are present in all 16 songbird species investigated and absent from germline genomes of all eight examined bird species from other avian orders. Results of fluorescent in situ hybridization of microdissected GRC probes and their sequencing indicate that GRCs show little homology between songbird species and contain a variety of repetitive elements and unique sequences with paralogs in the somatic genome. Our data suggest that the GRC evolved in the common ancestor of all songbirds and underwent significant changes in the extant descendant lineages.

On some structural and evolutionary aspects of rDNA amplification in oogenesis of Trachemys scripta turtles.

The features of rDNA amplification have been studied in oocytes of the red-eared slider Trachemys scripta using a number of specific histochemical and cytomolecular methods. A single nucleolus in early diplotene oocytes is associated with the nucleolus organizer region (NOR). With oocyte growth, the number of nucleoli increases dramatically and reaches hundreds by the lampbrush chromosome stage (pre-vitellogenesis). RNA-polymerase I, fibrillarin, and PCNA immunodetection in the amplified nucleoli and FISH of the 5'ETS probe to the oocyte nuclear content suggest pre-rRNA and rDNA synthesis in the nucleoli at all stages studied. This implies a continuous reproduction of the nucleoli during oocyte development from early diplotene up to vitellogenesis. The data obtained offer a different way for rDNA amplification and formation of extrachromosomal nucleoli in turtle oocytes compared with the amplified nucleoli formation in amphibian and fish oocytes. In the Sauropsida clade of Archelosauria, which includes turtles, crocodiles, and birds, rDNA function is known to be suppressed in avian oogenesis during the lampbrush stage (Gaginskaya et al. in Cytogenet Genome Res 124:251-267, 2009).

New high copy tandem repeat in the content of the chicken W chromosome.

The content of repetitive DNA in avian genomes is considerably less than in other investigated vertebrates. The first descriptions of tandem repeats were based on the results of routine biochemical and molecular biological experiments. Both satellite DNA and interspersed repetitive elements were annotated using library-based approach and de novo repeat identification in assembled genome. The development of deep-sequencing methods provides datasets of high quality without preassembly allowing one to annotate repetitive elements from unassembled part of genomes. In this work, we search the chicken assembly and annotate high copy number tandem repeats from unassembled short raw reads. Tandem repeat (GGAAA)n has been identified and found to be the second after telomeric repeat (TTAGGG)n most abundant in the chicken genome. Furthermore, (GGAAA)n repeat forms expanded arrays on the both arms of the chicken W chromosome. Our results highlight the complexity of repetitive sequences and update data about organization of sex W chromosome in chicken.

Structure of the intergenic spacers in chicken ribosomal DNA.

BACKGROUND: Ribosomal DNA (rDNA) repeats are situated in the nucleolus organizer regions (NOR) of chromosomes and transcribed into rRNA for ribosome biogenesis. Thus, they are an essential component of eukaryotic genomes. rDNA repeat units consist of rRNA gene clusters that are transcribed into single pre-rRNA molecules, each separated by intergenic spacers (IGS) that contain regulatory elements for rRNA gene cluster transcription. Because of their high repeat content, rDNA sequences are usually absent from genome assemblies. In this work, we used the long-read sequencing technology to describe the chicken IGS and fill the knowledge gap on rDNA sequences of one of the key domesticated animals. METHODS: We used the long-read PacBio RSII technique to sequence the BAC clone WAG137G04 (Wageningen BAC library) known to contain chicken NOR elements and the HGAP workflow software suit to assemble the PacBio RSII reads. Whole-genome sequence contigs homologous to the chicken rDNA repetitive unit were identified based on the Gallus_gallus-5.0 assembly with BLAST. We used the Geneious 9.0.5 and Mega software, maximum likelihood method and Chickspress project for sequence evolution analysis, phylogenetic tree construction and analysis of the raw transcriptome data. RESULTS: Three complete IGS sequences in the White Leghorn chicken genome and one IGS sequence in the red junglefowl contig AADN04001305.1 (Gallus_gallus-5.0) were detected. They had various lengths and contained three groups of tandem repeats (some of them being very GC rich) that form highly organized arrays. Initiation and termination sites of rDNA transcription were located within small and large unique regions (SUR and LUR), respectively. No functionally significant sites were detected within the tandem repeat sequences. CONCLUSIONS: Due to the highly organized GC-rich repeats, the structure of the chicken IGS differs from that of IGS in human, apes, Xenopus or fish rDNA. However, the chicken IGS shares some molecular organization features with that of the turtles, which are other representatives of the Sauropsida clade that includes birds and reptiles. Our current results on the structure of chicken IGS together with the previously reported ribosomal gene cluster sequence provide sufficient data to consider that the complete chicken rDNA sequence is assembled with confidence in terms of molecular DNA organization.

Chicken Microchromosomes in the Lampbrush Phase: A Cytogenetic Description.

Lampbrush chromosomes are giant, transcriptionally active, meiotic chromosomes found in oocytes of all vertebrates with the exception of mammals. Lampbrush chromosomes offer a convenient tool for cytogenetic mapping and, in particular, have been instrumental in mapping genes and linkage groups on chicken (GGA) chromosomes. Whereas cytogenetic maps of macrochromosome GGA1-10 and microchromosome GGA11-16 lampbrush bivalents have been established, identification and description of smaller microchromosome bivalents are still missing. In this work, we used specific FISH probes for the identification of 12 chicken lampbrush chromosomes formed by GGA17-28. Our observations on chromomere and lateral loop arrangement and chiasma position allowed us to construct the respective cytogenetic maps for these microchromosomes. For the 10 smallest chicken microchromosomes, GGA29-38, no individual molecular tags are available, yet they can be collectively marked using the PO41 repeat. The reported results contribute to building of working cytogenetic maps of the chicken karyotype.

Evolution of ribosomal internal transcribed spacers in Deuterostomia.

Sequences of ribosomal internal transcribed spacers (ITSs) are of great importance to molecular phylogenetics and DNA barcoding, but remain unstudied in some large taxa of Deuterostomia. We have analyzed complete ITS1 and ITS2 sequences in 62 species from 16 Deuterostomia classes, with ITS sequences in 24 species from 11 classes initially obtained using unannotated contigs and raw read sequences. A general tendency for both ITS length and GC-content increase from interior to superior Deuterostomia taxa, a uniform GC-content in both ITSs within the same species, thymine content decrease in sense DNA sequences of both ITSs are shown. A possible role of GC-based gene conversion in Deuterostomia ITS evolutionary changes is hypothesized. The first example of non-LTR retrotransposon insertion into ITS sequence in Deuterostomia is described in turtle Geochelone nigra. The roles of mobile genetic element insertions in the evolution of ITS sequences in some Sauropsida taxa are discussed as well.

Ribosomal RNA gene functioning in avian oogenesis.

Despite long-term exploration into ribosomal RNA gene functioning during the oogenesis of various organisms, many intriguing problems remain unsolved. In this review, we describe nucleolus organizer region (NOR) activity in avian oocytes. Whereas oocytes from an adult avian ovary never reveal the formation of the nucleolus in the germinal vesicle (GV), an ovary from juvenile birds possesses both nucleolus-containing and non-nucleolus-containing oocytes. The evolutionary diversity of oocyte NOR functioning and the potential non-rRNA-related functions of the nucleolus in oocytes are also discussed.

Homoploid hybrid speciation and genome evolution via chromosome sorting.

Genomes of numerous diploid plant and animal species possess traces of interspecific crosses, and many researches consider them as support for homoploid hybrid speciation (HHS), a process by which a new reproductively isolated species arises through hybridization and combination of parts of the parental genomes, but without an increase in ploidy. However, convincing evidence for a creative role of hybridization in the origin of reproductive isolation between hybrid and parental forms is extremely limited. Here, through studying Agrodiaetus butterflies, we provide proof of a previously unknown mode of HHS based on the formation of post-zygotic reproductive isolation via hybridization of chromosomally divergent parental species and subsequent fixation of a novel combination of chromosome fusions/fissions in hybrid descendants. We show that meiotic segregation, operating in the hybrid lineage, resulted in the formation of a new diploid genome, drastically rearranged in terms of chromosome number. We also demonstrate that during the heterozygous stage of the hybrid species formation, recombination was limited between rearranged chromosomes of different parental origin, representing evidence that the reproductive isolation was a direct consequence of hybridization.

Assessment of quadrivalent characteristics influencing chromosome segregation by analyzing human preimplantation embryos from reciprocal translocation carriers.

Patterns of meiotic chromosome segregation were analyzed in cleavage stage and blastocyst stage human embryos from couples with autosomal reciprocal translocations (ART). The influence of quadrivalent asymmetry degree, the presence of terminal breakpoints, and the involvement of acrocentric chromosomes in the rearrangement were analyzed to evaluate their contribution to the formation of non-viable embryos with significant chromosomal imbalance due to pathological segregation patterns and to assess the selection of human embryos by the blastocyst stage. A selection of viable embryos resulting from alternate and adjacent-1 segregation and a significant reduction in the detection frequency of the 3 : 1 segregation pattern were observed in human embryos at the blastocyst stage. The presence of terminal breakpoints increased the frequency of 3 : 1 segregation and was also associated with better survival of human embryos resulting from adjacent-1 mode, reflecting the process of natural selection of viable embryos to the blastocyst stage. The demonstrated patterns of chromosome segregation and inheritance of a balanced karyotype in humans will contribute to optimizing the prediction of the outcomes of in vitro fertilization programs and assessing the risks of the formation of unbalanced embryos for ART carriers.

Cytological maps of lampbrush chromosomes of European water frogs (Pelophylax esculentus complex) from the Eastern Ukraine.

BACKGROUND: Hybridogenesis (hemiclonal inheritance) is a kind of clonal reproduction in which hybrids between parental species are reproduced by crossing with one of the parental species. European water frogs (Pelophylax esculentus complex) represent an appropriate model for studying interspecies hybridization, processes of hemiclonal inheritance and polyploidization. P. esculentus complex consists of two parental species, P. ridibundus (the lake frog) and P. lessonae (the pool frog), and their hybridogenetic hybrid - P. esculentus (the edible frog). Parental and hybrid frogs can reproduce syntopically and form hemiclonal population systems. For studying mechanisms underlying the maintenance of water frog population systems it is required to characterize the karyotypes transmitted in gametes of parental and different hybrid animals of both sexes. RESULTS: In order to obtain an instrument for characterization of oocyte karyotypes in hybrid female frogs, we constructed cytological maps of lampbrush chromosomes from oocytes of both parental species originating in Eastern Ukraine. We further identified certain molecular components of chromosomal marker structures and mapped coilin-rich spheres and granules, chromosome associated nucleoli and special loops accumulating splicing factors. We recorded the dissimilarities between P. ridibundus and P. lessonae lampbrush chromosomes in the length of orthologous chromosomes, number and location of marker structures and interstitial (TTAGGG)n-repeat sites as well as activity of nucleolus organizer. Satellite repeat RrS1 was mapped in centromere regions of lampbrush chromosomes of the both species. Additionally, we discovered transcripts of RrS1 repeat in oocytes of P. ridibundus and P. lessonae. Moreover, G-rich transcripts of telomere repeat were revealed in association with terminal regions of P. ridibundus and P. lessonae lampbrush chromosomes. CONCLUSIONS: The constructed cytological maps of lampbrush chromosomes of P. ridibundus and P. lessonae provide basis to define the type of genome transmitted within individual oocytes of P. esculentus females with different ploidy and from various population systems.

Amplified Fragments of an Autosome-Borne Gene Constitute a Significant Component of the W Sex Chromosome of Eremias velox (Reptilia, Lacertidae).

Heteromorphic W and Y sex chromosomes often experience gene loss and heterochromatinization, which is frequently viewed as their "degeneration". However, the evolutionary trajectories of the heterochromosomes are in fact more complex since they may not only lose but also acquire new sequences. Previously, we found that the heterochromatic W chromosome of a lizard Eremias velox (Lacertidae) is decondensed and thus transcriptionally active during the lampbrush stage. To determine possible sources of this transcription, we sequenced DNA from a microdissected W chromosome sample and a total female DNA sample and analyzed the results of reference-based and de novo assembly. We found a new repetitive sequence, consisting of fragments of an autosomal protein-coding gene ATF7IP2, several SINE elements, and sequences of unknown origin. This repetitive element is distributed across the whole length of the W chromosome, except the centromeric region. Since it retained only 3 out of 10 original ATF7IP2 exons, it remains unclear whether it is able to produce a protein product. Subsequent studies are required to test the presence of this element in other species of Lacertidae and possible functionality. Our results provide further evidence for the view of W and Y chromosomes as not just "degraded" copies of Z and X chromosomes but independent genomic segments in which novel genetic elements may arise.

[NSI (+)]: a novel non-Mendelian nonsense suppressor determinant in Saccharomyces cerevisiae.

Non-Mendelian determinants that control heritable traits in yeast are subdivided into two major groups-one that includes DNA- or RNA-based elements and another that comprises protein-based factors that are analogous to mammalian prion. All yeast non-Mendelian determinants show dominant inheritance, and some of them demonstrate cytoplasmic infectivity. Only prions, however, harbor-specific features, such as high frequency of induction following overproduction of prion-encoding protein, loss of the protein's normal function, and reversible curability. Here, we describe a novel nonchromosomal determinant that, in addition to [PSI (+)] and [ISP (+)], is involved in epigenetic control of nonsense suppression. This determinant, which we have designated [NSI (+)], causes nonsense suppression in the strains bearing the N-terminal-deleted or -modified SUP35 gene, but has no manifestation in the strains with the intact copy of SUP35. [NSI (+)] shows dominant non-Mendelian inheritance, reversible curability and may be transmitted by cytoduction, albeit with low frequency. Similar to yeast prions, this determinant can be cured by deletion or mutational inactivation of Hsp104. We have shown that [NSI (+)] does not correspond to the already identified yeast prions. Based on the data obtained, we hypothesize that [NSI (+)] is a novel prion factor involved in epigenetic control of nonsense suppression.

Identification of sex chromosomes in Eremiasvelox (Lacertidae, Reptilia) using lampbrush chromosome analysis.

Reptiles are good objects for studying the evolution of sex determination, since they have different sex determination systems in different lineages. Lacertid lizards have been long-known for possessing ZZ/ZW type sex chromosomes. However, due to morphological uniformity of lacertid chromosomes, the Z chromosome has been only putatively cytologically identified. We used lampbrush chromosome (LBC) analysis and FISH with a W-specific probe in Eremiasvelox (Pallas, 1771) to unequivocally identify the ZW bivalent and investigate its meiotic behavior. The heterochromatic W chromosome is decondensed at the lampbrush stage, indicating active transcription, contrast with the highly condensed condition of the lampbrush W chromosomes in birds. We identified the Z chromosome by its chiasmatic association with the W chromosome as chromosome XIII of the 19 chromosomes in the LBC karyotype. Our findings agree with previous genetic and genomic studies, which suggested that the lacertid Z chromosome should be one of the smaller macrochromosomes.

Karyosystematics and molecular taxonomy of the anomalous blue butterflies (Lepidoptera, Lycaenidae) from the Balkan Peninsula.

The Balkan Peninsula represents one of the hottest biodiversity spots in Europe. However, the invertebrate fauna of this region is still insufficiently investigated, even in respect of such well-studied organisms as Lepidoptera. Here we use a combination of chromosomal, molecular and morphological markers to rearrange the group of so-called anomalous blue butterflies (also known as 'brown complex' of the subgenus Agrodiaetus Hübner, [1822] and as the Polyommatus (Agrodiaetus) admetus (Esper, 1783) species group) and to reveal its cryptic taxonomic structure. We demonstrate that Polyommatus aroaniensis (Brown, 1976) is not as widespread in the Balkans as was previously thought. In fact, it has a dot-like distribution range restricted to the Peloponnese Peninsula in South Greece. Polyommatus orphicus Kolev, 2005 is not as closely related to the Turkish species Polyommatus dantchenkoi (Lukhtanov & Wiemers, 2003) as was supposed earlier. Instead, it is a Balkan endemic represented by two subspecies: Polyommatus orphicus orphicus (Bulgaria) and Polyommatus orphicus eleniae Coutsis & De Prins, 2005 (Northern Greece). Polyommatus ripartii (Freyer, 1830) is represented in the Balkans by an endemic subspecies Polyommatus ripartii pelopi. The traditionally recognized Polyommatus admetus (Esper, 1783) is shown to be a heterogeneous complex and is divided into Polyommatus admetus sensu stricto (the Balkans and west Turkey) and Polyommatus yeranyani (Dantchenko & Lukhtanov, 2005) (east Turkey, Armenia, Azerbaijan and Iran). Polyommatus nephohiptamenos (Brown & Coutsis, 1978) is confirmed to be a species with a dot-like distribution range in Northern Greece. Finally, from Central Greece (Timfristos and Parnassos mountains) we describe Polyommatus timfristos Lukhtanov, Vishnevskaya & Shapoval, sp. n. which differs by its haploid chromosome number (n=38) from the closely related and morphologically similar Polyommatus aroaniensis (n=47-48) and Polyommatus orphicus (n=41-42). We provide chromosomal evidence for three separate south Balkan Pleistocene refugia (Peloponnesse, Central Greece and Northern Greece/South Bulgaria) and stress the biogeographic importance of Central Greece as a place of diversification. Then we argue that the data obtained have direct implications for butterfly karyology, taxonomy, biogeography and conservation.

Chicken rRNA Gene Cluster Structure.

Ribosomal RNA (rRNA) genes, whose activity results in nucleolus formation, constitute an extremely important part of genome. Despite the extensive exploration into avian genomes, no complete description of avian rRNA gene primary structure has been offered so far. We publish a complete chicken rRNA gene cluster sequence here, including 5'ETS (1836 bp), 18S rRNA gene (1823 bp), ITS1 (2530 bp), 5.8S rRNA gene (157 bp), ITS2 (733 bp), 28S rRNA gene (4441 bp) and 3'ETS (343 bp). The rRNA gene cluster sequence of 11863 bp was assembled from raw reads and deposited to GenBank under KT445934 accession number. The assembly was validated through in situ fluorescent hybridization analysis on chicken metaphase chromosomes using computed and synthesized specific probes, as well as through the reference assembly against de novo assembled rRNA gene cluster sequence using sequenced fragments of BAC-clone containing chicken NOR (nucleolus organizer region). The results have confirmed the chicken rRNA gene cluster validity.

Optional Endoreplication and Selective Elimination of Parental Genomes during Oogenesis in Diploid and Triploid Hybrid European Water Frogs.

Incompatibilities between parental genomes decrease viability of interspecific hybrids; however, deviations from canonical gametogenesis such as genome endoreplication and elimination can rescue hybrid organisms. To evaluate frequency and regularity of genome elimination and endoreplication during gametogenesis in hybrid animals with different ploidy, we examined genome composition in oocytes of di- and triploid hybrid frogs of the Pelophylax esculentus complex. Obtained results allowed us to suggest that during oogenesis the endoreplication involves all genomes occurring before the selective genome elimination. We accepted the hypothesis that only elimination of one copied genome occurs premeiotically in most of triploid hybrid females. At the same time, we rejected the hypothesis stating that the genome of parental species hybrid frogs co-exist with is always eliminated during oogenesis in diploid hybrids. Diploid hybrid frogs demonstrate an enlarged frequency of deviations in oogenesis comparatively to triploid hybrids. Typical for hybrid frogs deviations in gametogenesis increase variability of produced gametes and provide a mechanism for appearance of different forms of hybrids.

Yeast red pigment modifies Amyloid beta growth in Alzheimer disease models in both Saccharomyces cerevisiae and Drosophila melanogaster.

The effect of yeast red pigment on amyloid-ß (Aß) aggregation and fibril growth was studied in yeasts, fruit flies and in vitro. Yeast strains accumulating red pigment (red strains) contained less amyloid and had better survival rates compared to isogenic strains without red pigment accumulation (white strains). Confocal and fluorescent microscopy was used to visualise fluorescent Aß-GFP aggregates. Yeast cells containing less red pigment had more Aß-GFP aggregates despite the lower level of overall GFP fluorescence. Western blot analysis with anti-GFP, anti-Aß and A11 antibodies also revealed that red cells contained a considerably lower amount of Aß GFP aggregates as compared to white cells. Similar results were obtained with exogenous red pigment that was able to penetrate yeast cells. In vitro experiments with thioflavine and TEM showed that red pigment effectively decreased Aß fibril growth. Transgenic flies expressing Aß were cultivated on medium containing red and white isogenic yeast strains. Flies cultivated on red strains had a significant decrease in Aß accumulation levels and brain neurodegeneration. They also demonstrated better memory and learning indexes and higher locomotor ability.

Lampbrush chromosomes of the chaffinch (Fringilla coelebs L.).

The seven macrochromosomes of the chaffinch (Fringilla coelebs L.) are described in their lampbrush form. The relative lengths of bivalents, the positions and arrangements of chromosomal regions with lateral loops of similar length and appearance, as well as the positions of protein bodies and loops of peculiar morphology have been defined and mapped, so that each of the seven lampbrush macrobivalents may be identified in oocytes from every individual of the species. This morphological analysis has been supplemented by determining the positions of certain loci and objects that are specifically and consistently labelled after immunostaining or fluorescence in-situ hybridization with defined molecular probes.

Centromeric protein bodies on avian lampbrush chromosomes contain a protein detectable with an antibody against DNA topoisomerase II.

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.