Novel selectively amplified DNA sequences in the germline genome of the Japanese hagfish, Eptatretus burgeri

Abstract

In the Japanese hagfish Eptatretus burgeri, 16 chromosomes (eliminated [E]-chromosomes) have been lost in somatic cells (2n = 36), which is equivalent to approx. 21% of the genomic DNA in germ cells (2n = 52). At least seven of the 12 eliminated repetitive DNA families isolated in eight hagfish species were selectively amplified in the germline genome of this species. One of them, EEEb1 (eliminated element of E. burgeri 1) is exclusively localized on all E-chromosomes. Herein, we identified four novel eliminated repetitive DNA families (named EEEb3–6) from this species through PCR amplification and suppressive subtractive hybridization (SSH) combined with Southern-blot hybridization. EEEb3 was mosaic for 5S rDNA and SINE element. EEEb4 was GC-rich repeats and had one pair of direct and inverted repeats, whereas EEEb5 and EEEb6 were AT-rich repeats and composed of several sub-repeats. Interestingly, all but EEEb3 were transcribed in the testes, although no functional open reading frame (ORF) was found. We conducted fluorescence in situ hybridization (FISH) to examine the chromosomal localizations of EEEb3–6 and EEEb2, which was initially isolated from the germline DNA of this species. All of them were completely colocalized on all EEEb1-positive E-chromosomes. The estimation of the copy number by slot-blot hybridization revealed that (i) EEEb1–6 family members occupied 39.9% of the total eliminated DNA, and (ii) a small number of repeats were retained in somatic cells, suggesting the incomplete elimination. These results provide new insights about the mechanisms involved in the chromosome elimination and the evolution of E-chromosomes.