Original paper the same species with DdeI produced an elec- trophoretic smear, where a band of about 350 bp (GuDdeI) was present (Figure 1). When GuMboI DNA was used as a probe in G. unicol- or MboI- digested DNA, a band of about 5 Kb and other fainter bands of lower mw, ranging from 5 Kb to about 200 bp, were observed (Figure 2a). The Southern hybridization of GuMboI probe on M. helena genomic DNA digested with the same enzyme produced a band of about 10 Kb and other bands of lower mw (Figure 2a). The cloning and sequencing of the GuDdeI band revealed DNA fragments of 248-340 bp. Analysis of the sequenced segment of the clones showed that the sequences were very different, and the GuDdeI 313 clone was select- ed for studies. Southern hybridization using GuDdeI 313 probe on G. unicolor genomic DNA cleaved with DdeI showed a ladder of bands, whose mw ranged from about 6.5 Kb to about 400 bp; furthermore, a large band of 313 bp (i.e. the GuDdeI 313 band) was observed (Figure 2b). These results indicate a tandem arrangement of repeating units typical of satel- lite DNA. When this probe was used on M. hele- na genomic DNA cleaved with DdeI, a series of bands ranging from about 6.5 Kb to about 600 bp and a band of about 310 bp was observed, the latter being thinner than that observed in G. unicolor (Figure 2b). The complete nucleotide sequence of the GuDdeI 313 clone is shown in Figure 2c; it was 313 bp long and composed of 58,1% AT bases. No similarity between the GuDdeI 313 and any sequence deposited in the nucleotide sequence databas- es was detected. Cytogenetic mapping of repetitive DNA sequences FISH using the GuMboI probe produced pos- itive signals in the centromeric region of most chromosomes of G. unicolor, including a large acrocentric pair (Figure 3a), the latter region being DAPI negative (Figure 3c). 45S rDNA sites were located in the pericentromeric region of a single acrocentric pair (Figure 3b) and this region was also dull after DAPI stain- ing (Figure 3d). The hybridization of GuDdeI 313 probe produced bright positive signals in the centromeric region of some G. unicolor chromosomes, mostly acrocentric ones (Figure 3e), while GuDdeI probe showed positive sig- nals in all centromeres (Figure 3f). None of the probes (GuMboI, GuDdeI and GuDdeI 313) produced any positive signal on M. helena chromosomes (data not shown). FISH with the telomeric probe (TTAGGG)n revealed hybridization signals on each telomere of all chromosomes, and interstitial telomeric sites (ITS) were not detected (Figure 3g). [page 70] [European Journal of Histochemistry 2011; 55:e12] Figure 2. Southern blot of the genomic DNA samples of Gymnothorax unicolor (1) and Muraena helena (2) after digestion with the restriction enzyme MboI and hybridization to the GuMboI repetitive probe (a) and after digestion with the restriction enzyme DdeI and hybridization to the GuDdeI 313 repetitive probe (b). In (a), note the 5 Kb band in G. unicolor, and the 10 Kb faint band in M. helena. Base sequence of the GuDdeI 313 clone (c), its length cor- respond to 313 bp. Figure 3. Fluorescence in situ hibridization of Gymnothorax unicolor metaphase chromo- somes hybridized with different repetitive DNA sequences (yellow signals) and counter- stained with propidium iodide. They include GuMboI (a), 45S rDNA sites (b), the sequen- tial DAPI counterstaining of the same metaphases (c) and (d), GuDdeI (e), GuDdeI 313 (f ) and (TTAGGG)n sequences (g). The arrows indicate the DAPI-region in the chromosome no.9 (c,d), which corresponds to the NOR site (a,b). Scale bars: 10 μm.