Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

Electric Field Effects on Brain Activity: Implications for Epilepsy and Burst Suppression.

Electric fields are now considered a major mechanism of epileptiform activity. However, it is not clear if another electrophysiological phenomenon, burst suppression, utilizes the same mechanism for its bursting phase. Thus, the purpose of this study was to compare the role of ephaptic coupling-the recruitment of neighboring cells via electric fields-in generating bursts in epilepsy and burst suppression. We used local injections of the GABA-antagonist picrotoxin to elicit epileptic activity and a general anesthetic, sevoflurane, to elicit burst suppression in rabbits. Then, we applied an established computational model of pyramidal cells to simulate neuronal activity in a 3-dimensional grid, with an additional parameter to trigger a suppression phase based on extra-cellular calcium dynamics. We discovered that coupling via electric fields was sufficient to produce bursting in scenarios where inhibitory control of excitatory neurons was sufficiently low. Under anesthesia conditions, bursting occurs with lower neuronal recruitment in comparison to seizures. Our model predicts that due to the effect of electric fields, the magnitude of bursts during seizures should be roughly 2-3 times the magnitude of bursts that occur during burst suppression, which is consistent with our in vivo experimental results. The resulting difference in magnitude between bursts during anesthesia and epileptiform bursts reflects the strength of the electric field effect, which suggests that burst suppression and epilepsy share the same ephaptic coupling mechanism.

Functional Deficiency of Interneurons and Negative BOLD fMRI Response.

The functional deficiency of the inhibitory system typically appears during development and can progress to psychiatric disorders or epilepsy, depending on its severity, in later years. It is known that interneurons, the major source of GABAergic inhibition in the cerebral cortex, can make direct connections with arterioles and participate in the regulation of vasomotion. The goal of this study was to mimic the functional deficiency of interneurons through the use of localized microinjections of the GABA antagonist, picrotoxin, in such a concentration that it did not elicit epileptiform neuronal activity. First, we recorded the dynamics of resting-state neuronal activity in response to picrotoxin injections in the somatosensory cortex of an awake rabbit; second, we assessed the altered neuronal and hemodynamic responses to whisker stimulation using BOLD fMRI and electrophysiology recordings; third, we evaluated brain tissue oxygen levels before and after picrotoxin injection. Our results showed that neuronal activity typically increased after picrotoxin administration, the BOLD responses to stimulation became negative, and the oxygen response was nearly abolished. Vasoconstriction during the resting baseline was not observed. These results indicate that picrotoxin provoked imbalanced hemodynamics either due to increased neuronal activity, decreased vascular response, or a combination of both.

Maps of Constitutive-Heterochromatin Distribution for Four Martes Species (Mustelidae, Carnivora, Mammalia) Show the Formative Role of Macrosatellite Repeats in Interspecific Variation of Chromosome Structure.

Constitutive-heterochromatin placement in the genome affects chromosome structure by occupying centromeric areas and forming large blocks. To investigate the basis for heterochromatin variation in the genome, we chose a group of species with a conserved euchromatin part: the genus Martes [stone marten (M. foina, 2n = 38), sable (M. zibellina, 2n = 38), pine marten (M. martes, 2n = 38), and yellow-throated marten (M. flavigula, 2n = 40)]. We mined the stone marten genome for the most abundant tandem repeats and selected the top 11 macrosatellite repetitive sequences. Fluorescent in situ hybridization revealed distributions of the tandemly repeated sequences (macrosatellites, telomeric repeats, and ribosomal DNA). We next characterized the AT/GC content of constitutive heterochromatin by CDAG (Chromomycin A3-DAPI-after G-banding). The euchromatin conservatism was shown by comparative chromosome painting with stone marten probes in newly built maps of the sable and pine marten. Thus, for the four Martes species, we mapped three different types of tandemly repeated sequences critical for chromosome structure. Most macrosatellites are shared by the four species with individual patterns of amplification. Some macrosatellites are specific to a species, autosomes, or the X chromosome. The variation of core macrosatellites and their prevalence in a genome are responsible for the species-specific variation of the heterochromatic blocks.

Brain tissue oxygen dynamics while mimicking the functional deficiency of interneurons.

The dynamic interaction between excitatory and inhibitory activity in the brain is known as excitatory-inhibitory balance (EIB). A significant shift in EIB toward excitation has been observed in numerous pathological states and diseases, such as autism or epilepsy, where interneurons may be dysfunctional. The consequences of this on neurovascular interactions remains to be elucidated. Specifically, it is not known if there is an elevated metabolic consumption of oxygen due to increased excitatory activity. To investigate this, we administered microinjections of picrotoxin, a gamma aminobutyric acid (GABA) antagonist, to the rabbit cortex in the awake state to mimic the functional deficiency of GABAergic interneurons. This caused an observable shift in EIB toward excitation without the induction of seizures. We used chronically implanted electrodes to measure both neuronal activity and brain tissue oxygen concentrations (PO2) simultaneously and in the same location. Using a high-frequency recording rate for PO2, we were able to detect two important phenomena, (1) the shift in EIB led to a change in the power spectra of PO2 fluctuations, such that higher frequencies (8-15 cycles per minute) were suppressed and (2) there were brief periods (dips with a duration of less than 100 ms associated with neuronal bursts) when PO2 dropped below 10 mmHg, which we defined as the threshold for hypoxia. The dips were followed by an overshoot, which indicates either a rapid vascular response or decrease in oxygen consumption. Our results point to the essential role of interneurons in brain tissue oxygen regulation in the resting state.

Early Development of the GABAergic System and the Associated Risks of Neonatal Anesthesia.

Human and animal studies have elucidated the apparent neurodevelopmental effects resulting from neonatal anesthesia. Observations of learning and behavioral deficits in children, who were exposed to anesthesia early in development, have instigated a flurry of studies that have predominantly utilized animal models to further interrogate the mechanisms of neonatal anesthesia-induced neurotoxicity. Specifically, while neonatal anesthesia has demonstrated its propensity to affect multiple cell types in the brain, it has shown to have a particularly detrimental effect on the gamma aminobutyric acid (GABA)ergic system, which contributes to the observed learning and behavioral deficits. The damage to GABAergic neurons, resulting from neonatal anesthesia, seems to involve structure-specific changes in excitatory-inhibitory balance and neurovascular coupling, which manifest following a significant interval after neonatal anesthesia exposure. Thus, to better understand how neonatal anesthesia affects the GABAergic system, we first review the early development of the GABAergic system in various structures that have been the focus of neonatal anesthesia research. This is followed by an explanation that, due to the prolonged developmental curve of the GABAergic system, the entirety of the negative effects of neonatal anesthesia on learning and behavior in children are not immediately evident, but instead take a substantial amount of time (years) to fully develop. In order to address these concerns going forward, we subsequently offer a variety of in vivo methods which can be used to record these delayed effects.

Karyotype Evolution in 10 Pinniped Species: Variability of Heterochromatin versus High Conservatism of Euchromatin as Revealed by Comparative Molecular Cytogenetics.

Pinnipedia karyotype evolution was studied here using human, domestic dog, and stone marten whole-chromosome painting probes to obtain comparative chromosome maps among species of Odobenidae (Odobenus rosmarus), Phocidae (Phoca vitulina, Phoca largha, Phoca hispida, Pusa sibirica, Erignathus barbatus), and Otariidae (Eumetopias jubatus, Callorhinus ursinus, Phocarctos hookeri, and Arctocephalus forsteri). Structural and functional chromosomal features were assessed with telomere repeat and ribosomal-DNA probes and by CBG (C-bands revealed by barium hydroxide treatment followed by Giemsa staining) and CDAG (Chromomycin A3-DAPI after G-banding) methods. We demonstrated diversity of heterochromatin among pinniped karyotypes in terms of localization, size, and nucleotide composition. For the first time, an intrachromosomal rearrangement common for Otariidae and Odobenidae was revealed. We postulate that the order of evolutionarily conserved segments in the analyzed pinnipeds is the same as the order proposed for the ancestral Carnivora karyotype (2n = 38). The evolution of conserved genomes of pinnipeds has been accompanied by few fusion events (less than one rearrangement per 10 million years) and by novel intrachromosomal changes including the emergence of new centromeres and pericentric inversion/centromere repositioning. The observed interspecific diversity of pinniped karyotypes driven by constitutive heterochromatin variation likely has played an important role in karyotype evolution of pinnipeds, thereby contributing to the differences of pinnipeds' chromosome sets.

Evolutionary rearrangements of X chromosomes in voles (Arvicolinae, Rodentia).

Euchromatic segments of the X chromosomes of placental mammals are the most conservative elements of the karyotype, only rarely subjected to either inter- or intrachromosomal rearrangements. Here, using microdissection-derived set of region-specific probes of Terricola savii we detailed the evolutionary rearrangements found in X chromosomes in 20 vole species (Arvicolinae, Rodentia). We show that the evolution of X chromosomes in this taxon was accompanied by multiple para- and pericentric inversions and centromere shifts. The contribution of intrachromosomal rearrangements to the karyotype evolution of Arvicolinae species was approximately equivalent in both the separate autosomal conserved segments and the X chromosomes. Intrachromosmal rearrangements and structural reorganization of the X chromosomes was likely accompanied by an accumulation, distribution, and evolution of repeated sequences.

Multiple intrasyntenic rearrangements and rapid speciation in voles.

Remarkably stable genomic chromosome elements (evolutionary conserved segments or syntenies) are the basis of large-scale chromosome architecture in vertebrate species. However, these syntenic elements harbour evolutionary important changes through intrachromosomal rearrangements such as inversions and centromere repositioning. Here, using FISH with a set of 20 region-specific probes on a wide array of 28 species, we analyzed evolution of three conserved syntenic regions of the Arvicolinae ancestral karyotype. Inside these syntenies we uncovered multiple, previously cryptic intrachromosomal rearrangements. Although in each of the three conserved blocks we found inversions and centromere repositions, the blocks experienced different types of rearrangements. In two syntenies centromere repositioning predominated, while in the third region, paracentric inversions were more frequent, whereas pericentric inversions were not detected. We found that some of the intrachromosomal rearrangements, mainly paracentric inversions, were synapomorphic for whole arvicoline genera or tribes: genera Alexandromys and Microtus, tribes Ellobini and Myodini. We hypothesize that intrachromosomal rearrangements within conserved syntenic blocks are a major evolutionary force modulating genome architecture in species-rich and rapidly-evolving rodent taxa. Inversions and centromere repositioning may impact speciation and provide a potential link between genome evolution, speciation, and biogeography.

Author Correction: Red fox genome assembly identifies genomic regions associated with tame and aggressive behaviours.

In the version of this Article originally published, there were some errors in the affiliations: Stephen J. O'Brien's affiliations were incorrectly listed as 8,9; they should have been 7,9. Affiliation 3 was incorrectly named the Institute of Cytology and Genetics of the Russian Academy of Sciences; it should have read Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences. Affiliation 4 was incorrectly named the Institute of Molecular and Cell Biology of the Russian Academy of Sciences; it should have read Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences. These have now been corrected.

Red fox genome assembly identifies genomic regions associated with tame and aggressive behaviours.

Strains of red fox (Vulpes vulpes) with markedly different behavioural phenotypes have been developed in the famous long-term selective breeding programme known as the Russian farm-fox experiment. Here we sequenced and assembled the red fox genome and re-sequenced a subset of foxes from the tame, aggressive and conventional farm-bred populations to identify genomic regions associated with the response to selection for behaviour. Analysis of the re-sequenced genomes identified 103 regions with either significantly decreased heterozygosity in one of the three populations or increased divergence between the populations. A strong positional candidate gene for tame behaviour was highlighted: SorCS1, which encodes the main trafficking protein for AMPA glutamate receptors and neurexins and suggests a role for synaptic plasticity in fox domestication. Other regions identified as likely to have been under selection in foxes include genes implicated in human neurological disorders, mouse behaviour and dog domestication. The fox represents a powerful model for the genetic analysis of affiliative and aggressive behaviours that can benefit genetic studies of behaviour in dogs and other mammals, including humans.

Chromosome Synapsis and Recombination in Male-Sterile and Female-Fertile Interspecies Hybrids of the Dwarf Hamsters (Phodopus, Cricetidae).

Hybrid sterility is an important step in the speciation process. Hybrids between dwarf hamsters Phodopus sungorus and P.campbelli provide a good model for studies in cytological and genetic mechanisms of hybrid sterility. Previous studies in hybrids detected multiple abnormalities of spermatogenesis and a high frequency of dissociation between the X and Y chromosomes at the meiotic prophase. In this study, we found that the autosomes of the hybrid males and females underwent paring and recombination as normally as their parental forms did. The male hybrids showed a significantly higher frequency of asynapsis and recombination failure between the heterochromatic arms of the X and Y chromosomes than the males of the parental species. Female hybrids as well as the females of the parental species demonstrated a high incidence of centromere misalignment at the XX bivalent and partial asynapsis of the ends of its heterochromatic arms. In all three karyotypes, recombination was completely suppressed in the heterochromatic arm of the X chromosome, where the pseudoautosomal region is located. We propose that this recombination pattern speeds up divergence of the X- and Y-linked pseudoautosomal regions between the parental species and results in their incompatibility in the male hybrids.

Next Generation Sequencing of Chromosome-Specific Libraries Sheds Light on Genome Evolution in Paleotetraploid Sterlet (Acipenser ruthenus).

Several whole genome duplication (WGD) events followed by rediploidization took place in the evolutionary history of vertebrates. Acipenserids represent a convenient model group for investigation of the consequences of WGD as their representatives underwent additional WGD events in different lineages resulting in ploidy level variation between species, and these processes are still ongoing. Earlier, we obtained a set of sterlet (Acipenser ruthenus) chromosome-specific libraries by microdissection and revealed that they painted two or four pairs of whole sterlet chromosomes, as well as additional chromosomal regions, depending on rediploidization status and chromosomal rearrangements after genome duplication. In this study, we employed next generation sequencing to estimate the content of libraries derived from different paralogous chromosomes of sterlet. For this purpose, we aligned the obtained reads to the spotted gar (Lepisosteus oculatus) reference genome to reveal syntenic regions between these two species having diverged 360 Mya. We also showed that the approach is effective for synteny prediction at various evolutionary distances and allows one to clearly distinguish paralogous chromosomes in polyploid genomes. We postulated that after the acipenserid-specific WGD sterlet karyotype underwent multiple interchromosomal rearrangements, but different chromosomes were involved in this process unequally.

Intrachromosomal Rearrangements in Rodents from the Perspective of Comparative Region-Specific Painting.

It has long been hypothesized that chromosomal rearrangements play a central role in different evolutionary processes, particularly in speciation and adaptation. Interchromosomal rearrangements have been extensively mapped using chromosome painting. However, intrachromosomal rearrangements have only been described using molecular cytogenetics in a limited number of mammals, including a few rodent species. This situation is unfortunate because intrachromosomal rearrangements are more abundant than interchromosomal rearrangements and probably contain essential phylogenomic information. Significant progress in the detection of intrachromosomal rearrangement is now possible, due to recent advances in molecular biology and bioinformatics. We investigated the level of intrachromosomal rearrangement in the Arvicolinae subfamily, a species-rich taxon characterized by very high rate of karyotype evolution. We made a set of region specific probes by microdissection for a single syntenic region represented by the p-arm of chromosome 1 of Alexandromys oeconomus, and hybridized the probes onto the chromosomes of four arvicolines (Microtus agrestis, Microtus arvalis, Myodes rutilus, and Dicrostonyx torquatus). These experiments allowed us to show the intrachromosomal rearrangements in the subfamily at a significantly higher level of resolution than previously described. We found a number of paracentric inversions in the karyotypes of M. agrestis and M. rutilus, as well as multiple inversions and a centromere shift in the karyotype of M. arvalis. We propose that during karyotype evolution, arvicolines underwent a significant number of complex intrachromosomal rearrangements that were not previously detected.

De novo assembling and primary analysis of genome and transcriptome of gray whale Eschrichtius robustus.

BACKGROUND: Gray whale, Eschrichtius robustus (E. robustus), is a single member of the family Eschrichtiidae, which is considered to be the most primitive in the class Cetacea. Gray whale is often described as a "living fossil". It is adapted to extreme marine conditions and has a high life expectancy (77 years). The assembly of a gray whale genome and transcriptome will allow to carry out further studies of whale evolution, longevity, and resistance to extreme environment. RESULTS: In this work, we report the first de novo assembly and primary analysis of the E. robustus genome and transcriptome based on kidney and liver samples. The presented draft genome assembly is complete by 55% in terms of a total genome length, but only by 24% in terms of the BUSCO complete gene groups, although 10,895 genes were identified. Transcriptome annotation and comparison with other whale species revealed robust expression of DNA repair and hypoxia-response genes, which is expected for whales. CONCLUSIONS: This preliminary study of the gray whale genome and transcriptome provides new data to better understand the whale evolution and the mechanisms of their adaptation to the hypoxic conditions.

Rapid Karyotype Evolution in Lasiopodomys Involved at Least Two Autosome - Sex Chromosome Translocations.

The generic status of Lasiopodomys and its division into subgenera Lasiopodomys (L. mandarinus, L. brandtii) and Stenocranius (L. gregalis, L. raddei) are not generally accepted because of contradictions between the morphological and molecular data. To obtain cytogenetic evidence for the Lasiopodomys genus and its subgenera and to test the autosome to sex chromosome translocation hypothesis of sex chromosome complex origin in L. mandarinus proposed previously, we hybridized chromosome painting probes from the field vole (Microtus agrestis, MAG) and the Arctic lemming (Dicrostonyx torquatus, DTO) onto the metaphases of a female Mandarin vole (L. mandarinus, 2n = 47) and a male Brandt's vole (L. brandtii, 2n = 34). In addition, we hybridized Arctic lemming painting probes onto chromosomes of a female narrow-headed vole (L. gregalis, 2n = 36). Cross-species painting revealed three cytogenetic signatures (MAG12/18, 17a/19, and 22/24) that could validate the genus Lasiopodomys and indicate the evolutionary affinity of L. gregalis to the genus. Moreover, all three species retained the associations MAG1bc/17b and 2/8a detected previously in karyotypes of all arvicolins studied. The associations MAG2a/8a/19b, 8b/21, 9b/23, 11/13b, 12b/18, 17a/19a, and 5 fissions of ancestral segments appear to be characteristic for the subgenus Lasiopodomys. We also validated the autosome to sex chromosome translocation hypothesis on the origin of complex sex chromosomes in L. mandarinus. Two translocations of autosomes onto the ancestral X chromosome in L. mandarinus led to a complex of neo-X1, neo-X2, and neo-X3 elements. Our results demonstrate that genus Lasiopodomys represents a striking example of rapid chromosome evolution involving both autosomes and sex chromosomes. Multiple reshuffling events including Robertsonian fusions, chromosomal fissions, inversions and heterochromatin expansion have led to the formation of modern species karyotypes in a very short time, about 2.4 MY.

Genome-wide comparative chromosome maps of Arvicola amphibius, Dicrostonyx torquatus, and Myodes rutilus.

The subfamily Arvicolinae consists of a great number of species with highly diversified karyotypes. In spite of the wide use of arvicolines in biological and medicine studies, the data on their karyotype structures are limited. Here, we made a set of painting probes from flow-sorted chromosomes of a male Palearctic collared lemming (Dicrostonyx torquatus, DTO). Together with the sets of painting probes made previously from the field vole (Microtus agrestis, MAG) and golden hamster (Mesocricetus auratus, MAU), we carried out a reciprocal chromosome painting between these three species. The three sets of probes were further hybridized onto the chromosomes of the Eurasian water vole (Arvicola amphibius) and northern red-backed vole (Myodes rutilus). We defined the diploid chromosome number in D. torquatus karyotype as 2n = 45 + Bs and showed that the system of sex chromosomes is X1X2Y1. The probes developed here provide a genomic tool-kit, which will help to investigate the evolutionary biology of the Arvicolinae rodents. Our results show that the syntenic association MAG1/17 is present not only in Arvicolinae but also in some species of Cricetinae; and thus, should not be considered as a cytogenetic signature for Arvicolinae. Although cytogenetic signature markers for the genera have not yet been found, our data provides insight into the likely ancestral karyotype of Arvicolinae. We conclude that the karyotypes of modern voles could have evolved from a common ancestral arvicoline karyotype (AAK) with 2n = 56 mainly by centric fusions and fissions.

Segmental paleotetraploidy revealed in sterlet (Acipenser ruthenus) genome by chromosome painting.

BACKGROUND: Acipenseriformes take a basal position among Actinopteri and demonstrate a striking ploidy variation among species. The sterlet (Acipenser ruthenus, Linnaeus, 1758; ARUT) is a diploid 120-chromosomal sturgeon distributed in Eurasian rivers from Danube to Enisey. Despite a high commercial value and a rapid population decline in the wild, many genomic characteristics of sterlet (as well as many other sturgeon species) have not been studied. RESULTS: Cell lines from different tissues of 12 sterlet specimens from Siberian populations were established following an optimized protocol. Conventional cytogenetic studies supplemented with molecular cytogenetic investigations on obtained fibroblast cell lines allowed a detailed description of sterlet karyotype and a precise localization of 18S/28S and 5S ribosomal clusters. Localization of sturgeon specific HindIII repetitive elements revealed an increased concentration in the pericentromeric region of the acrocentric ARUT14, while the total sterlet repetitive DNA fraction (C0t30) produced bright signals on subtelomeric segments of small chromosomal elements. Chromosome and region specific probes ARUT1p, 5, 6, 7, 8 as well as 14 anonymous small sized chromosomes (probes A-N) generated by microdissection were applied in chromosome painting experiments. According to hybridization patterns all painting probes were classified into two major groups: the first group (ARUT5, 6, 8 as well as microchromosome specific probes C, E, F, G, H, and I) painted only a single region each on sterlet metaphases, while probes of the second group (ARUT1p, 7 as well as microchromosome derived probes A, B, D, J, K, M, and N) marked two genomic segments each on different chromosomes. Similar results were obtained on male and female metaphases. CONCLUSIONS: The sterlet genome represents a complex mosaic structure and consists of diploid and tetraploid chromosome segments. This may be regarded as a transition stage from paleotetraploid (functional diploid) to diploid genome condition. Molecular cytogenetic and genomic studies of other 120- and 240-chromosomal sturgeons are needed to reconstruct genome evolution of this vertebrate group.

Chromosomal localization of six repeated DNA sequences among species of Microtus (Rodentia).

C-banding and fluorescence in situ hybridization (FISH) document the distribution of constitutive heterochromatin and six highly repeated DNA families (MSAT2570, MSAT21, MSAT160, MS2, MS4 and STR47) in the chromosomes of nine species of Microtus (M. chrotorrhinus, M. rossiaemeridionalis, M. arvalis, M. ilaeus, M. transcaspicus, M. cabrerae, M. pennsylvanicus, M. miurus and M. ochrogaster). Autosomal heterochromatin is largely centromeric and contains different repeated families in different species. Similarly, large C-band positive blocks on the sex chromosomes of four species contain different repeated DNAs. This interspecific variation in the chromosomal distribution and copy number of the repeats suggests that a common ancestor to modern species contained most of the repetitive families, and that descendant species selectively amplified or deleted different repeats on different chromosomes.