Isolation, characterization and chromosomal localization of repetitive DNA sequences in Physalaemus ephippifer (Anura Leiuperidae) / Isolamento, caracterização e localização cromossômica de sequências de DNA repetitivo de Physalaemus ephippifer (Anura Leiuperidae)

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

17/03/2010

RESUMO

Previous cytogenetic studies of Physalaemus ephippifer, a species currently allocated to the group P. cuvieri, showed an interesting female-specific chromosome heteromorphism. In 14 females of P. ephippifer, chromosome pair 8 was heteromorphic with regard to the occurrence of an NOR anda terminal C-band. Such heteromorphism was not found in the seven analyzed male specimens. These findings suggest that the chromosomes of pair 8 may be sex chromosomes in P. ephippifer, characterizing a ZZ ♂/ ZW ♀ sex-determination system in this species. Despite these important data, conventional cytogenetic techniques performed in this and other species of the Physalaemus group were not sufficient to clarify the processes involved in the karyological divergence in this anuran group. Aiming to look for new cytogenetic markers that may help in the study of P. ephippifer sex chromosomes and in the cytogenetic analysis of this group of Physalaemus, we studied repetitive DNA sequences isolated from P. ephippifer. Genomic DNA extracted from two females of P. ephippifer from Belém (Pará) was digested with the restriction enzyme BamHI. The restriction fragments were separated by electrophoresis in agarose gel. DNA fragments, which were ultimately named Pep194, Pep165, and Pep320, were isolated from the gel, cloned, sequenced, and localized in situ in the karyotype of P. ephippifer. The sequence Pep320 was very similar to the fragment EU343727.1 isolated from P. cuvieri. Although Pep320 and Pep165 were totally different in nucleotide sequence, they were mapped on the same chromosome site, which corresponded to the pericentromeric C-band in the short arm of chromosome 3. This raises doubts about the molecular organization of these sequences, which can be arranged in independent clusters, but can also represent partial regions of the same repeat unit. The sequence Pep194 was mapped in regions that coincided with the NORs, located in the long arm of Z and W chromosomes and in the short arm of the W chromosome. However, the Pep194 sequence had no similarity with the coding regions of the nucleolar rDNA or with intergenic spacers associated to them. The restriction fragment Pep194 had an interesting internal arrangement, being composed of two terminal direct repeats, each with 63 bp, flanking an internal region of 68 bp. To further investigate the organization of this sequence in the genome of P. ephippifer, we amplified some Pep194 segments from genomic DNA by PCR using specific primers designed to anneal in inner.

ASSUNTO(S)

cromossomos sexuais physalaemus heterocromatina dna satélite sexual chromosomes physalaemus heterochromatin satellite dna

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