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. 2023 Apr 12;80(5):121. doi: 10.1007/s00018-023-04770-3

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2023

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Fig. 1 — a Schematic representation of the acrocentric human chromosomes. Scheme of an acrocentric chromosome with the rDNA portion and distal (DJ) and proximal (PJ) junction sequences. rDNA repeats are located on q Arm of the chromosome. The number of rDNA repeats varies on the chromosomes. Each ~ 13.3 kb rDNA repeat is composed of the transcribed region encoding 45S rRNA (5′-ETS, 18S, ITS1, 5.8S, ITS2, 28S and 3′-ETS) and a ~ 30.7 kb IGS (intergenic spacer sequence). The 13.3 kb transcribed human 45S rDNA region is shown in blue. Telo-telomere regions. CEN-centromere region. b Southern blot analysis of six human/mouse hybrid cell lines containing human chromosomes 13, 14, 15, 21 and 22 [A9 (#13) 89-2 (chr13), A9 hygro14 10F (chr14), A9 (Neo15)-3 (chr15), A9 #21-16 (chr21), A9#22 (γ2) (chr22γ2) and A9HyTK-22 (chr22TK)]. Genomic DNA possessing one of the acrocentric chromosomes was isolated from each hybrid cell line, digested by EcoRV and separated by CHEF gel electrophoresis (range 50 kb–1.1 Mb). The rDNA repeats were detected with a radioactively labeled probe specific to the rDNA intergenic spacer (IGS). Red arrowheads indicate the fragments that contain rDNA repeats. M1 a marker [CHEF DNA Size Lambda Ladder (BIO-RAD)]. c FISH analysis of human/mouse hybrid cell line A9#22 (γ2) containing chromosome 22 with the human IGS FISH probe specific to the intergenic spacer of the rDNA unit (see “Materials and methods”). Chromosome 22 (green) is indicated by a white arrowhead. d A copy number of the rDNA repeats in a human acrocentric chromosome in each hybrid cell line was estimated by quantitative real-time PCR (qPCR). e 3D immuno-FISH was performed on human/mouse hybrid cells. Anti-RRP1 antibodies (Nop52) (red) used to visualize nucleoli were combined with a BAC FISH probe containing the IGS sequence (green). Nuclei were stained with DAPI (blue). Nucleolar- and non-nucleolar-associated NORs are indicated by white arrowheads. f Quantification of 3D immuno-FISH showing the percentage of cells in which the NORs from human acrocentric chromosomes and human non-acrocentric chromosomes 4 and 18 associated with mouse nucleoli. For each cell line, the experiment was replicated three times with the total number of the nucleolar localized and non-localized chromosomes ranged from 141 to 317 (Table S3)