Chromosome Evolution in the Free-Living Flatworms:First Evidence of Intrachromosomal Rearrangements in Karyotype Evolution of Macrostomum lignano (Platyhelminthes, Macrostomida)

The free-living flatworm Macrostomum lignano is a hidden tetraploid. Its genome was formed by a recent whole genome duplication followed by chromosome fusions. Its karyotype (2n = 8) consists of a pair of large chromosomes (MLI1), which contain regions of all other chromosomes, and three pairs of small metacentric chromosomes. Comparison of MLI1 with metacentrics was performed by painting with microdissected DNA probes and fluorescent in situ hybridization of unique DNA fragments. Regions of MLI1 homologous to small metacentrics appeared to be contiguous. Besides the loss of DNA repeat clusters (pericentromeric and telomeric repeats and the 5S rDNA cluster) from MLI1, the difference between small metacentrics MLI2 and MLI4 and regions homologous to them in MLI1 were revealed. Abnormal karyotypes found in the inbred DV1/10 subline were analyzed, and structurally rearranged chromosomes were described with the painting technique, suggesting the mechanism of their origin. The revealed chromosomal rearrangements generate additional diversity, opening the way toward massive loss of duplicated genes from a duplicated genome. Our findings suggest that the karyotype of M. lignano is in the early stage of genome diploidization after whole genome duplication, and further studies on M. lignano and closely related species can address many questions about karyotype evolution in animals

The free-living flatworm Macrostomum lignano

Wudarski J, Egger B, Ramm SA, et al. The free-living flatworm Macrostomum lignano. EvoDevo. 2020;11(1): 5

Cysteine residues 244 and 458-459 within the catalytic subunit of Na,K-ATPase control the enzyme's hydrolytic and signaling function under hypoxic conditions

Our previous findings suggested that reversible thiol modifications of cysteine residues within the actuator (AD) and nucleotide binding domain (NBD) of the Na,K-ATPase may represent a powerful regulatory mechanism conveying redox- and oxygen-sensitivity of this multifunctional enzyme. S-glutathionylation of Cys244 in the AD and Cys 454-458-459 in the NBD inhibited the enzyme and protected cysteines' thiol groups from irreversible oxidation under hypoxic conditions. In this study mutagenesis approach was used to assess the role these cysteines play in regulation of the Na,K-ATPase hydrolytic and signaling functions. Several constructs of mouse α1 subunit of the Na,K-ATPase were produced in which Cys244, Cys 454-458-459 or Cys 244-454-458-459 were replaced by alanine. These constructs were expressed in human HEK293 cells. Non-transfected cells and those expressing murine α1 subunit were exposed to hypoxia or treated with oxidized glutathione (GSSG). Both conditions induced inhibition of the wild type Na,K-ATPase. Enzymes containing mutated mouse α1 lacking Cys244 or all four cysteines (Cys 244-454-458-459) were insensitive to hypoxia. Inhibitory effect of GSSG was observed for wild type murine Na,K-ATPase, but was less pronounced in Cys454-458-459Ala mutant and completely absent in the Cys244Ala and Cys 244-454-458-459Ala mutants. In cells, expressing wild type enzyme, ouabain induced activation of Src and Erk kinases under normoxic conditions, whereas under hypoxic conditions this effect was inversed. Cys454-458-459Ala substitution abolished Src kinase activation in response to ouabain treatment, uncoupled Src from Erk signaling, and interfered with O2-sensitivity of Na,K-ATPase signaling function. Moreover, modeling predicted that S-glutathionylation of Cys 458 and 459 should prevent inhibitory binding of Src to NBD. Our data indicate for the first time that cysteine residues within the AD and NBD influence hydrolytic as well as receptor function of the Na,K-ATPase and alter responses of the enzyme to hypoxia or upon treatment with cardiotonic steroids

The origin of B chromosomes in yellow-necked mice (Apodemus flavicollis)—Break rules but keep playing the game

B chromosomes (Bs) are known for more than hundred years but their origin, structure and pattern of evolution are not well understood. In the past few years new methodological approaches, involving isolation of Bs followed by whole DNA amplification, DNA probe generation, and fluorescent in situ hybridization (FISH) or the B chromosome DNA sequencing, has allowed detailed analysis of their origin and molecular structure in different species. In this study we explored the origin of Bs in the yellow-necked wood mouse, Apodemus flavicollis, using generation of microdissected DNA probes followed by FISH on metaphase chromosomes. Bs of A. flavicollis were successfully isolated and DNA was used as the template for B-specific probes for the first time. We revealed homology of DNA derived from the analyzed B chromosomes to the pericentromeric region (PR) of sex chromosomes and subtelomeric region of two pairs of small autosomes, but lower homology to the rest of the Y chromosome. Moreover, all analysed Bs had the same structure regardless of their number per individual or the great geographic distance between examined populations from the Balkan Peninsula (Serbia) and Eastern Europe (south region of Russia and central Belarus). Therefore, it was suggested that B chromosomes in A. flavicollis have a unique common origin from the PR of sex chromosomes, and/or similar evolutionary pattern.PloS one (2017), 12(3): e017270

Low-pass single-chromosome sequencing of human small supernumerary marker chromosomes (sSMCs) and Apodemus B chromosomes.

Supernumerary chromosomes sporadically arise in many eukaryotic species as a result of genomic rearrangements. If present in a substantial part of species population, those are called B chromosomes, or Bs. This is the case for 70 mammalian species, most of which are rodents. In humans, the most common types of extra chromosomes, sSMCs (small supernumerary marker chromosomes), are diagnosed in approximately 1 of 2000 postnatal cases. Due to low frequency in population, human sSMCs are not considered B chromosomes. Genetic content of both B-chromosomes and sSMCs in most cases remains understudied. Here, we apply microdissection of single chromosomes with subsequent low-pass sequencing on Ion Torrent PGM and Illumina MiSeq to identify unique and repetitive DNA sequences present in a single human sSMC and several B chromosomes in mice Apodemus flavicollis and Apodemus peninsulae. The pipeline for sequencing data analysis was made available in Galaxy interface as an addition to previously published command-line version. Human sSMC was attributed to the proximal part of chromosome 15 long arm, and breakpoints leading to its formation were located into satellite DNA arrays. Genetic content of Apodemus B chromosomes was species-specific, and minor alterations were observed in both species. Common features of Bs in these Apodemus species were satellite DNA and ERV enrichment, as well as the presence of the vaccinia-related kinase gene Vrk1. Understanding of the non-essential genome elements content provides important insights into genome evolution in general.This is a post-peer-review, pre-copyedit version of an article published in Chromosoma. The final authenticated version is available online at: [http://dx.doi.org/10.1007/s00412-018-0662-0

Sequence Composition and Evolution of Mammalian B Chromosomes

B chromosomes (Bs) revealed more than a hundred years ago remain to be some of the most mysterious elements of the eukaryotic genome. Their origin and evolution, DNA composition, transcriptional activity, impact on adaptiveness, behavior in meiosis, and transfer to the next generation require intensive investigations using modern methods. Over the past years, new experimental techniques have been applied and helped us gain a deeper insight into the nature of Bs. Here, we consider mammalian Bs, taking into account data on their DNA sequencing, transcriptional activity, positions in nuclei of somatic and meiotic cells, and impact on genome functioning. Comparative cytogenetics of Bs suggests the existence of different mechanisms of their formation and evolution. Due to the long and complicated evolvement of Bs, the similarity of their morphology could be explained by the similar mechanisms involved in their development while the difference between Bs even of the same origin could appear due to their positioning at different stages of their evolution. A complex analysis of their DNA composition and other features is required to clarify the origin and evolutionary history of Bs in the species studied. The intraspecific diversity of Bs makes this analysis a very important element of B chromosome studies

Sequence Composition and Evolution of Mammalian B Chromosomes

B chromosomes (Bs) revealed more than a hundred years ago remain to be some of the most mysterious elements of the eukaryotic genome. Their origin and evolution, DNA composition, transcriptional activity, impact on adaptiveness, behavior in meiosis, and transfer to the next generation require intensive investigations using modern methods. Over the past years, new experimental techniques have been applied and helped us gain a deeper insight into the nature of Bs. Here, we consider mammalian Bs, taking into account data on their DNA sequencing, transcriptional activity, positions in nuclei of somatic and meiotic cells, and impact on genome functioning. Comparative cytogenetics of Bs suggests the existence of different mechanisms of their formation and evolution. Due to the long and complicated evolvement of Bs, the similarity of their morphology could be explained by the similar mechanisms involved in their development while the difference between Bs even of the same origin could appear due to their positioning at different stages of their evolution. A complex analysis of their DNA composition and other features is required to clarify the origin and evolutionary history of Bs in the species studied. The intraspecific diversity of Bs makes this analysis a very important element of B chromosome studies

New insights into the karyotype evolution of the free-living flatworm; Macrostomum lignano; (Platyhelminthes, Turbellaria)

The free-living flatworm; Macrostomum lignano; is a model organism for evolutionary and developmental biology studies. Recently, an unusual karyotypic diversity was revealed in this species. Specifically, worms are either ‘normal’ 2n = 8, or they are aneuploid with one or two additional large chromosome(s) (i.e. 2n = 9 or 2n = 10, respectively). Aneuploid worms did not show visible behavioral or morphological abnormalities and were successful in reproduction. In this study, we generated microdissected DNA probes from chromosome 1 (further called MLI1), chromosome 2 (MLI2), and a pair of similar-sized smaller chromosomes (MLI3, MLI4). FISH using these probes revealed that MLI1 consists of contiguous regions homologous to MLI2-MLI4, suggesting that MLI1 arose due to the whole genome duplication and subsequent fusion of one full chromosome set into one large metacentric chromosome. Therefore, one presumably full haploid genome was packed into MLI1, leading to hidden tetraploidy in the; M. lignano; genome. The study of; Macrostomum; sp. 8 — a sibling species of; M. lignano; — revealed that it usually has one additional pair of large chromosomes (2n = 10) showing a high homology to MLI1, thus suggesting hidden hexaploidy in its genome. Possible evolutionary scenarios for the emergence of the; M. lignano; and; Macrostomum; sp. 8 genomes are discussed

Genome and karyotype reorganization after whole genome duplication in free-living flatworms of the genus; Macrostomum

The genus Macrostomum represents a diverse group of rhabditophoran flatworms with >200 species occurring around the world. Earlier we uncovered karyotype instability linked to hidden polyploidy in both M. lignano (2n = 8) and its sibling species M. janickei (2n = 10), prompting interest in the karyotype organization of close relatives. In this study, we investigated chromosome organization in two recently described and closely related Macrostomum species, M. mirumnovem and M. cliftonensis, and explored karyotype instability in laboratory lines and cultures of M. lignano (DV1/10, 2n = 10) and M. janickei in more detail. We revealed that three of the four studied species are characterized by karyotype instability, while M. cliftonensis showed a stable 2n = 6 karyotype. Next, we performed comparative cytogenetics of these species using fluorescent in situ hybridization (FISH) with a set of DNA probes (including microdissected DNA probes generated from M. lignano chromosomes, rDNA, and telomeric DNA). To explore the chromosome organization of the unusual 2n = 9 karyotype discovered in M. mirumnovem, we then generated chromosome-specific DNA probes for all chromosomes of this species. Similar to M. lignano and M. janickei, our findings suggest that M. mirumnovem arose via whole genome duplication (WGD) followed by considerable chromosome reshuffling. We discuss possible evolutionary scenarios for the emergence and reorganization of the karyotypes of these Macrostomum species and consider their suitability as promising animal models for studying the mechanisms and regularities of karyotype and genome evolution after a recent WGD