Heterochromatic regions in Japanese quail chromosomes: comprehensive molecular-cytogenetic characterization and 3D mapping in interphase nucleus
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Chromosomes of Japanese quail (Coturnix coturnix japonica, 2n=78), a galliform domestic species closely related to chicken, possess multiple heterochromatic segments. Due to the difficulties in careful analysis of such heterochromatic regions, there is a lack of data on their DNA composition, epigenetic status, as well as spatial distribution in interphase nucleus. In the present study, we applied giant lampbrush chromosome (LBC) microdissection for high-resolution analysis of quail centromeric regions of macrochromosomes and polymorphic short arms of submetacentric microchromosomes. FISH with the dissected material on mitotic and meiotic chromosomes indicated that in contrast to centromeres of chicken macrochromosomes, which are known to harbor chromosome-specific and, in some cases, tandem repeat-free sequences, centromeres of quail macroautosomes (CCO1–CCO11) have canonical organization. CCO1–CCO11 centromeres possess massive blocks of common DNA repeats demonstrating transcriptional activity at LBC stage. These repeats seem to have been subjected to chromosome size-correlated homogenization previously described primarily for avian microchromosomes. In addition, comparative FISH on chicken chromosomes supported the previous data on centromere repositioning events during galliform karyotype evolution. In interphase nucleus of different cell types, repetitive elements specific for microchromosome short arms constitute the material of prominent centrally located chromocenters enriched with markers of constitutive heterochromatin and rimmed with clusters of microchromosomal centromeric BglII-repeat. Thus, clustering of such repeats is responsible for the peculiar architecture of quail interphase nucleus. In contrast, centromere repeats of the largest macrochromosomes (CCO1 and CCO2) are predominantly localized in perinuclear heterochromatin. The possible involvement of the isolated repeats in radial genome organization is discussed.
KeywordsCentromere repeats Chromocenters Chromosome microdissection Heterochromatic regions Japanese quail and chicken chromosomes Lampbrush chromosomes
Bacterial artificial chromosome
Japanese quail (Coturnix coturnix japonica) chromosomes
Chicken nuclear-membrane-associated repeat
Degenerate oligonucleotide-primed PCR
Fluorescence in situ hybridization
Chicken (Gallus gallus) chromosome
Long terminal repeats
short arm of submetacentric microchromosomes
Quail embryonic fibroblasts
Saline-sodium citrate buffer
The authors sincerely thank Felix A. Habermann (Ludwig-Maximilians-University of Munich, Germany) for providing the chicken chromosome-specific paint F12, Richard Crooijmans and Martin Groenen (Wageningen University, The Netherlands) for providing the chicken BAC clone WAG29F23, and Svetlana Galkina for providing the oligonucleotide probe to BglII-repeat.
The research of AZ, AM, and AK was supported by a grant of the President of the Russian Federation (MK-1630.2017.4). The research of NK, ABHA-R, and TL was supported by the DAAD University Partnership Programme between Friedrich Schiller University (Jena, Germany) and Saint Petersburg State University (Saint Petersburg, Russia). The work was partially performed using experimental equipment of the Research Resource Center “Molecular and Cell Technologies” of St. Petersburg State University.
AZ designed the study, carried out the principal molecular-cytogenetic experiments, and drafted the manuscript. AM carried out 3D molecular-cytogenetic experiments on somatic cells and tissues and drafted the manuscript. NK, ABHA-R, AZ, and TL performed microdissection procedure. AK designed the study, coordinated the project, and revised the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
None of the authors have any competing interests in the manuscript.
All international, national, and institutional guidelines for the care and use of laboratory and farm animals were followed (institutional Ethical Committee approval no. 131-03-2, 14 March 2016).
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