A centromeric highly repeated DNA and its function in spatial arrangement of mitotic and meiotic chromosomes in birds

Elena Gaginskaya, Alsu Saifitdinova, Svetlana Derjusheva, Andrei Malykh*, Vladimir Zhurov
Biological Institute of Saint-Petersburg University, RUSSIA (chromas@paloma.spbu.ru)
*Fidelity Systems Inc., USA (www.fidelitysystems.comfsi@fidelitysystems.com)
The problem of spatial chromosome arrangement in the nucleus has been attracting attention since the famous works by Rabl (1885) and Boveri (1888). It is now clear that centromeric associations between non-homologous chromosomes in mitosis must exist, and these may serve to maintain non-random spatial chromosome arrangement within the cell. Nevertheless, the structural and mechanistic bases of interchromosomal associations remain so far uncertain. The data presented provide a new evidence for involvement of satellite DNAs in interchromosomal interconnections.

FCP (Fringilla coelebs PstI element)

A highly repetitive centromeric FCP sequence was cloned and sequenced. The FCP tandem repeats with a unit length 505/506 bp accounted for about 0,9% of the chaffinch genome. Direct genomic sequencing with FCP-specific primers and ThermoFidelaseTM 2A enzyme complex revealed the consensus sequence and the five most common single nucleotide polymorphisms (SNPs) for the FCP unit.

SNPs
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A, B, E, F — direct sequencing of genomic DNA; arrows show positions of SNPs.In A — SNP1 (T/G); in B — SNP2 (C/G); in E — SNP3 (C/G) and SNP4 (G/C); in F — SNP5 (A/C). C, D, G, H — sequencing from a plasmid containing one copy of FCP.
The direct genomic sequencing also revealed the head-to-tail arrangement of FCP repeated units.

Scheme I. RE and SNP map of FCP
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Scheme J. Domains of FCP
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Scheme I illustrates FCP tandem repeat. The main G+C component in the FCP repeated unit is 57%. According to the primary structure FCP unit may be divided into two domains (Scheme J), one of them being CpG enriched, the other contains the A-T bendable blocks.

Scheme K. Regulatory elements in FCP unit
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FCP satellite DNA transcribes during the lampbrush phase. Scheme K illustrates the positions of potential transcription regulatory elements in the FCP repeat.

FCP centromeric repeat is involved in spatial arrangement of mitotic chromosomes in chaffinch

DNA was revealed in interchromosome connectives after immunostaining, nick-translation and random-primed labeling in situ. The FCP repeated DNA was localized by FISH and PRINS in centromeric regions of all mitotic chromosomes and, most importantly, in threads connecting centromeres of non-homologous chromosomes. We demonstrated that the threads do contain DNA, and concluded that the FCP centromere repeat is responsible for interchromosome interactions during mitosis in chaffinch.

FISH with FCP probe on metaphase
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Metaphase chromosomes after FISH with biotinilated FCP probe. FITC/PI

Anti-dsDNA immunostaining
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Nick-translation in situ
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Random-PRINS
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DNA visualization in mitotic chromosomes and interchromosome threads (arrowheads) by immunostaining with anti-dsDNA AB (A), by nick-translation in situ (B), and random-primed labeling in situ (C). FITC (A, B), Cy3 (C).

PRINS with FCP probe
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FCP localization on chaffinch mitotic chromosomes by PRINS with FCP-specific primers. FITC/PI

FCP in intrephase nuclei. A - FISH, B - PRINS
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Patterns of FCP localization in the interphase nuclei revealed by FISH with FCP probe (A) and by PRINS with FCP specific primers (B). Cy3/DAPI (A), FITC/PI (B)

Loop extensions from scaffold

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The scheme illustrates one of possible ways of the intercentromeric DNA threads formation by an expansion of linked FCP loop domains from the chromosome scaffolds.

FCP centromeric repeat seems to be responsible for meiotic chromosome association at late stages of chaffinch oogenesis

FISH on LBC
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Lampbrush chromosomes after FISH with FCP probe. FISH signals are visible at the sites of protein bodies (arrowheads) localization. A — one of macrobivalents, B — a few lampbrush chromosomes joined by the fusion of protein bodies. Cy3 detection.

Karyosphere
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The karyosphere from late oocyte of chaffinch. Compact chromosomes (arrows) are visible on the sufaces of fusing protein bodies. CMA/PI.

Scheme of karyosphere formation
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The scheme illustrates chromosome association by the fusion of centromeric protein bodies at the stage following the lampbrush phase in chaffinch oocyte nuclei.

© Laboratory of Chromosome Structure and Function, 2001