Molecular mechanisms and topological consequences of drastic chromosomal rearrangements of muntjac deer

Muntjac deer have experienced drastic karyotype changes during their speciation, making it an ideal model for studying mechanisms and functional consequences of mammalian chromosome evolution. Here we generated chromosome-level genomes for Hydropotes inermis (2n = 70), Muntiacus reevesi (2n = 46), female and male M. crinifrons (2n = 8/9) and a contig-level genome for M. gongshanensis (2n = 8/9). These high-quality genomes combined with Hi-C data allowed us to reveal the evolution of 3D chromatin architectures during mammalian chromosome evolution. We find that the chromosome fusion events of muntjac species did not alter the A/B compartment structure and topologically associated domains near the fusion sites, but new chromatin interactions were gradually established across the fusion sites. The recently borne neo-Y chromosome of M. crinifrons, which underwent male-specific inversions, has dramatically restructured chromatin compartments, recapitulating the early evolution of canonical mammalian Y chromosomes. We also reveal that a complex structure containing unique centromeric satellite, truncated telomeric and palindrome repeats might have mediated muntjacs’ recurrent chromosome fusions. These results provide insights into the recurrent chromosome tandem fusion in muntjacs, early evolution of mammalian sex chromosomes, and reveal how chromosome rearrangements can reshape the 3D chromatin regulatory conformations during species evolution.

Intraspecies multiple chromosomal variations including rare tandem fusion in the Russian Far Eastern endemic evoron vole Alexandromys evoronensis (Rodentia, Arvicolinae)

The vole Alexandromys evoronensis (Kovalskaya et Sokolov, 1980) with its two chromosomal races, “Evoron” (2n = 38–41, NF = 54–59) and “Argi” (2n = 34, 36, 37, NF = 51–56) is the endemic vole found in the Russian Far East. For the “Argi” chromosomal race, individuals from two isolated populations in mountain regions were investigated here for the first time using GTG-, GTC-, NOR methods. In the area under study, 8 new karyotype variants have been registered. The karyotype with 2n = 34 has a rare tandem fusion of three autosomes: two biarmed (Mev6 and Mev7) and one acrocentric (Mev14) to form a large biarmed chromosome (Mev6/7/14), all of which reveal a heterozygous state. For A. evoronensis, the variation in the number of chromosomes exceeded the known estimate of 2n = 34, 36 and amounted to 2n = 34, 36, 38–41. The combination of all the variations of chromosomes for the species made it possible to describe 20 variants of the A. evoronensis karyotype, with 11 chromosomes being involved in multiple structural rearrangements. In the “Evoron” chromosomal race 4 chromosomes (Mev1, Mev4, Mev17, and Mev18) and in the “Argi” chromosomal race 9 chromosomes (Mev6, Mev7, Mev14, Mev13, Mev11, Mev15, Mev17, Mev18, and Mev19) were observed. Tandem and Robertsonian rearrangements (Mev17/18 and Mev17.18) were revealed in both chromosomal races “Evoron” and “Argi”.

Genome collinearity analysis illuminates the evolution of donkey chromosome 1 and horse chromosome 5 in perissodactyls: A comparative study

Background It is important to resolve the evolutionary history of species genomes as it has affected both genome organization and chromosomal architecture. The rapid innovation in sequencing technologies and the improvement in assembly algorithms have enabled the creation of highly contiguous genomes. DNA Zoo, a global organization dedicated to animal conservation, offers more than 150 chromosome-length genome assemblies. This database has great potential in the comparative genomics field. Results Using the donkey (Equus asinus asinus, EAS) genome provided by DNA Zoo as an example, the scaffold N50 length and Benchmarking Universal Single-Copy Ortholog score reached 95.5 Mb and 91.6%, respectively. We identified the cytogenetic nomenclature, corrected the direction of the chromosome-length sequence of the donkey genome, analyzed the genome-wide chromosomal rearrangements between the donkey and horse, and illustrated the evolution of the donkey chromosome 1 and horse chromosome 5 in perissodactyls. Conclusions The donkey genome provided by DNA Zoo has relatively good continuity and integrity. Sequence-based comparative genomic analyses are useful for chromosome evolution research. Several previously published chromosome painting results can be used to identify the cytogenetic nomenclature and correct the direction of the chromosome-length sequence of new assemblies. Compared with the horse genome, the donkey chromosomes 1, 4, 20, and X have several obvious inversions, consistent with the results of previous studies. A 4.8 Mb inverted structure was first discovered in the donkey chromosome 25 and plains zebra chromosome 11. We speculate that the inverted structure and the tandem fusion of horse chromosome 31 and 4 are common features of non-caballine equids, which supports the correctness of the existing Equus phylogeny to an extent.

Chromosomal relationships among the native rodents (Cricetidae: Oryzomyini) of the Galápagos Islands, Ecuador

Although the Galápagos Islands are recognized for their contribution to our understanding of evolutionary theory and have received the attention of scientists for over 185 years, our understanding of the native rodents there has been minimal relative to many other groups of organisms. Much of what we knew through most of the 20th century was based solely on species descriptions. Chromosome data has been limited to only Nesoryzomys narboroughi (2n = 32, FN (number of autosomal arms) = 50) and Aegialomys galapagoensis (2n = 56; FN = 58). We present the karyotypes of the only remaining extant species in the genus, N. swarthi (2n = 56; FN = 54) and N. fernandinae (2n = 44; FN = 54). Chromosomal banding reveals that extensive rearrangement has occurred within Nesoryzomys, including Robertsonian fusion and tandem fusion events but these alone cannot account for the diverse diploid numbers found within the genus. We propose that 1) N. swarthi represents the ancestral karyotype for the genus, similar to A. galapagoensis, 2) N. swarthi and N. fernandinae share the same fundamental number, suggesting divergence by Robertsonian fusions, and 3) N. narboroughi has the most derived karyotype, based on banding morphology and low diploid number.

Comparative Chromosome Painting in Genets (Carnivora, Viverridae, Genetta), the Only Known Feliforms with a Highly Rearranged Karyotype

Mammalian carnivores have been extensively studied by cross-species chromosome painting, which indicated a high degree of karyotypic conservatism in the cat-like suborder Feliformia relative to the ancestral carnivore karyotype (ACK). The first exception to this high degree of karyotypic conservation in feliforms was recently confirmed in genets, mesocarnivores belonging to the basal family Viverridae. Here, we present a comparative analysis of the chromosome rearrangements among 2 subspecies of the small-spotted genet Genetta genetta (the Iberian nominate and the Arabian grantii) and the panther genet G. maculata, the 2 most common and widespread genets, using whole-chromosome paints from the domestic cat (Felis catus). The chromosome homology maps and the presence of numerous interstitial telomeric sites in both genet species strengthen the hypothesis that a highly rearranged karyotype compared to the ACK may occur throughout Genetta. The karyotype of G. maculata appears to have undergone more rearrangements than that of G. genetta, which is an older lineage. Notably, we identified a tandem fusion distinguishing G. g. genetta and G. g.grantii. As G. g. grantii is morphologically and genetically distinctive, and tandem fusions have been associated with substantial postzygotic isolation in mammals, this cytogenetic finding flags the subspecies for future taxonomic investigations.

Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

Genetically encoded Förster Resonance Energy Transfer (FRET) based biosensors report on changes in biochemical states in single living cells. The performance of biosensors depends on their brightness and dynamic range, which are dependent on the characteristics of the fluorescent proteins that are employed. Cyan fluorescent protein (CFP) is frequently combined with yellow fluorescent protein (YFP) as FRET pair in biosensors. However, current YFPs are prone to photobleaching and pH changes. In addition, more efficient acceptors may yield biosensors that have higher contrast. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Förster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mChery and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. The sensor with mScarlet-I as acceptor and mTurquoise2 as donor shows a higher dynamic range in ratiometric FRET imaging experiments and less variability than with mCherry as acceptor, due to the high quantum yield and efficient maturation of mScarlet-I. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor.

A Novel Cellular Spheroid-Based Autophagy Screen Applying Live Fluorescence Microscopy Identifies Nonactin as a Strong Inducer of Autophagosomal Turnover

Dysregulation of the basal autophagic flux has been linked to several pathological conditions, including neurodegenerative diseases and cancer. In addition, autophagy has profound effects on the response of tumor cells to therapy. Hence, the search for pharmacological modulators of autophagy is of great clinical relevance. We established a drug screening assay in which the autophagic flux is measured by recording the fluorescence emission of the tandem fusion protein mRFP-GFP-LC3 by dynamic live-cell imaging. We optimized the assay for the identification of autophagy modulators in three dimensions with U343 glioma cell spheroids, which represent a more realistic cancer model than conventional 2D cell cultures. We validated the assay by screening a library of known autophagy modulators. As the first application, a small library of 94 natural compounds was screened for its impact on autophagy. We discovered the cyclic ionophore nonactin as a new and potent autophagy inducer. This novel autophagy screening assay based on 3D tumor spheroids is robust, reproducible, and scalable. It provides a valuable tool for both basic research and drug screening campaigns.