RESUMO
A highly repetitive centromeric Fringilla coelebs PstI (FCP) element was cloned and sequenced. The FCP tandem repeats with unit 505 or 506 nt accounted for about 0.9% of the entire genome and had 57% GC. Direct genomic sequencing with FCP-specific primers and ThermoFidelase 2A revealed the consensus sequence and the five most common single-nucleotide polymorphisms (SNPs) for the FCP unit. FCP may be transcribed and may play a role in spatial arrangement of the genome.
Assuntos
Aves/genética , Centrômero , Sequências Repetitivas de Ácido Nucleico , Animais , Sequência de Bases , Primers do DNA , Polimorfismo de Nucleotídeo ÚnicoRESUMO
A new family of avian centromeric satellites is described. The highly repeated sequence, designated FCP (Fringilla coelebs PstI element), was cloned from the 500-bp PstI digest fraction of the chaffinch (Fringilla coelebs L.) genomic DNA, sequenced, and characterized. The FCP repeat was found to have 505-506 bp length of monomer, 57% content of GC, to compose about 0.9% of the chaffinch genome, and to be highly methylated. Results of Southern-blot hybridization of cloned FCP element onto genomic DNA digested with different restriction enzymes, and sequencing directly from total genomic DNA using FCP-specific primers and ThermoFidelase enzyme (Fidelity Systems Inc.) were in agreement with a tandem arrangement of this repeat in the chaffinch genome. Five positions of single-nucleotide polymorphism (SNP) were found in the FCP monomers using direct genomic sequencing. Fluorescence in situ hybridization (FISH) with FCP probe and primed in situ labelling (PRINS) with FCP specific primers showed that the FCP elements occupy pericentric regions of all chaffinch chromosomes. On chromosome spreads, the fluorescent signals were also observed in the intercentromeric connectives between nonhomologous chromosomes. The results suggest that the centromeric FCP repeat is responsible for chromosome ordering during mitosis in chaffinch.
Assuntos
Centrômero , Aves Canoras/genética , Sequências de Repetição em Tandem , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA , Feminino , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
A highly repeated FCP (Fringilla coelebs PstI element) sequence was localized by FISH in centromeric regions of all chromosomes of the chaffinch. Besides, FISH signal was found also in interchromosomal connectives linking centromeres of non-homologous chromosomes in mitotic cells. The presence of DNA in the connectives was confirmed by immunostaining with anti-dsDNA antibodies as well as in experiments on nick-translation and random primed labeling in situ. Non-denaturing FISH with FCP probe and random primed labeling of non-denatured chromosomes resulted in fluorescence signal on both centromeres and intercentromeric connectives, thus providing evidence for the availability of single-strand DNA tracts in FCP sequence. It is suggested that the highly repeated FCP centromeric sequence may be respondible for interconnection of mitotic chromosomes and may by involved in nuclear architecture maintenance in the chaffinch.
