Discussion The digestion of G. unicolor DNA with the enzymes MboI and DdeI showed the presence of distinct bands in agarose gels, as observed in other fish species after genomic DNA diges- tion with restriction endonucleases.33-36 Analysis of membrane immobilized genomic DNA of G. unicolor hybridized to the GuMboI family and to the GuDdeI 313 clone indicates that they correspond to highly repetitive ele- ments, predominantly clustered in the genome. The comparison of satellite GuDdeI DNA sequences with nucleic acid sequences available on databases revealed no similarities to any known DNA sequence. Comparative Southern hybridization revealed that these repeated DNAs are present in both G. unicolor and M. helena, with species-specific features. Among Muraenidae, repetitive DNA fractions were characterized only in M. helena, and in particular two MboI-restricted DNA fractions, called B2 and B3; Southern blotting compara- tive analysis on the G. unicolor genome point- ed out the presence of these DNA fractions in both species, although showing characteristic features in each species.25,27 The two isolated repetitive elements are clustered in the centromeric heterochromatin of many chromosomes of the complement, pre- viously identified by C- banding.20 The repeti- tive sequences are not randomly distributed in the genome, suggesting a pattern of compart- mentalization on chromosomes. In particular, the GuMboI sequence was localized in two dif- ferent types of C-positive areas: in many cen- tromeres and the pericentromeric region of acrocentric pair no. 9, co-localized with the Ag- NOR localization previously obtained.28 FISH mapping of the major ribosomal gene family confirms the presence of only one NOR-bear- ing pair in the G. unicolor genome; this situa- tion is also found in all the Muraenidae stud- ied and is the most frequent condition among Anguilliformes.18,22,37 The GuMboI fraction might be a heterogeneous repetitive DNA associated with ribosomal sequences; this hypothesis could account for the lack of signals in cross-species GuMboI FISH on M. helena chromosomes. A NOR-related DNA fraction was also obtained in M. helena, and the associ- ation between ribosomal cistrons and repeti- tive DNA is commonly found in eukaryotes.25 The G. unicolor GuDdeI DNA fraction was localized in the centromeric region of all G. unicolor chromosomes, while the GuDdeI DNA 313 probe hybridized in only some cen- tromeres, indicating that this satellite DNA family is composed of several types of cen- tromeric repetitive DNAs, also confirmed by the great differences in nucleotide sequences found among clones. Repetitive DNAs are fre- quently localized in centromeric regions of chromosomes in both animals and plants and represent the main component of functional centromeres.38-42 Although their real function is still being debated, satellite DNA is required for centromere composition and organiza- tion.43 Centromeric location of repetitive DNAs is also the most frequent situation among fish, and the distribution pattern in all centromeres had already been found in M. helena and other fish species, like some species of the genus Sparus and some species of Cichlidae.25,44-48 In the Erythrinid fish, Hoplias malabaricus, 5SHindIII is a satellite DNA family speciﬁc to the H. malabaricus genome, and is located only in the centromeric region of some chromo- some pairs. This satellite family has spread to the centromeric region of several chromo- somes and has been favored during evolution due to a possible role of the repetitive sequences of the centromeric region in cen- tromere structure and function.10,11,34 In order to establish the degree of conservation of the GuMboI and GuDdeI DNA fractions, FISH was performed on M. helena chromosomes; the lack of positive signals suggests that these sequences are species-specific and might have originated after the divergence of G. unicolor from the other Muraenidae species. These results agree with the heterochromatin het- erogeneity detected in the two species by in situ restriction enzyme banding.20 Telomeric (TTAGGG)n sequences are pres- ent in the telomeres of the chromosomes of all vertebrates, and their study allows one to establish the presence of chromosomal rearrangements, such as Robertsonian fusions or inversions, involved in the evolution of the chromosomes.49 FISH with the telomeric probe (TTAGGG)n revealed hybridization signals on each telomere of all chromosomes of G. unicol- or, and interstitial telomeric sites (ITS) were not detected, which could indicate that Robertsonian fusions or chromosomal translo- cations were probably not involved in the kary- otypic differentiation of this species. The absence of ITS had already been reported for the other Muraenidae species M. helena and G. tile.18,26 On the other hand, two Anguilliform species studied (Anguilla anguilla and A. ros- trata) presented telomeric sequences colocal- ized with the NOR region.26 In conclusion, the results obtained provide information useful in comparative genomics at the chromosomal level and add to the cytoge- netic knowledge regarding this fish family, contributing to the molecular and cytogeneti- cal characterization of G. unicolor for studying karyotype evolution in Muraenidae. A more extensive cytogenetic survey is needed to allow evaluation of Muraenidae cytotaxonomy. Original paper References 1. Jurka J, Kapitonov VV, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J. Repbase Update, a database of eukaryotic repetitive elements. Cytogenet Genome Res 2005;110:462-7. 2. Biémont C, Vieira C. Junk DNA as an evo- lutionary force. Nature 2006;443:521-4. 3. Sumner AT. 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