Molecular cytogenetic analysis of the highly repetitive DNA in the genome of Apodemus argenteus, with comments on the phylogenetic relationships in the genus Apodemus

Abstract

The DNA of Apodemus argenteus was digested with DraI, and the resultant DraI fragment of highly repetitive DNA was isolated and analyzed by DNA filter hybridization, cloning, sequencing, and fluorescence in situ hybridization (FISH). Southern blot hybridization and nucleotide sequencing revealed that most of the DraI fragment consisted of a 230-bp repeating unit and contained no sex-chromosome-specific nucleotide sequences. The DraI fragment included the CENP-B box-like sequence, with a strong homology to the human CENP-B box sequence. FISH revealed that the DraI fragment was specific to all pericentromeric C-band-positive regions, as well as to the C-block of the X chromosome. No hybridization signals were obtained from A. speciosus, A. peninsulae peninsulae, A.p. giliacus, A. agrarius, A. sylvaticus, A. semotus, or Musmusculus when the DraI fragment was used as probe. Peptide nucleic acid (PNA)-FISH using the CENP-B box-like sequence in the DraI fragments as probe suggested that this nucleotide sequence was also specific to all pericentromeric C-heterochromatic regions of A. argenteus chromosomes. Zoo-blot hybridization using DraI-digested genomic DNA from three species of Apodemus (namely, A. argenteus, A. speciosus, and A. peninsulae) and from Mus musculus strongly suggested that the consensus DraI fragment contained nucleotide sequences that were species-specific for A. argenteus. These results also suggest that A. argenteus is phylogenetically distant from other Apodemus species examined, as well as the possibility that the DraI fragment might be related directly to the delayed quinacrine mustard fluorescence of many pericentromeric C-heterochromatic regions of the chromosomes in A. argenteus.