major ribosomal genes and telomeric sequences using FISH. Furthermore, we inves- tigated the presence of these DNA fractions in the M. helena genome. Materials and Methods Chromosomal and genomic DNA preparation Six individuals of both sexes (four males and two females) of G. unicolor were captured along the southwestern Sardinian coast and raised in an aquarium. Blood was collected from the dorsal artery of individuals using a heparinized syringe, and blood cells were used for all experiments. Sex was determined by histological analysis of the gonads of each individual. Metaphase chromosomes were obtained by lymphocyte cultures and chromo- some morphology was classified according to arm ratios.28,29 Genomic DNA was obtained using standard phenol-chloroform procedure.30 For comparative purposes, M. helena metaphase chromosomes and genomic DNA were also used in the same experiments. Characterization of genomic DNA: Southern hybridization, cloning, sequencing and sequence analysis A search for repetitive DNAs was conducted using restriction enzyme digestion of the genomic DNA of G. unicolor with different restriction endonucleases (MboI, DdeI, HaeIII and AluI). The endonucleases MboI and DdeI revealed conspicuous bands of about 5 kb and 350 bp, respectively. Briefly, approximately 7 μg of genomic DNA was digested with 14 U of MboI or DdeI at 37°C for 16 h and subse- quently analyzed by gel-electrophoresis using 1% agarose gels containing ethidium bromide (0.5 μg/mL) and TAE buffer (80.04 M Tris- acetate, 0.002 M EDTA) at 25 V for 16 h. The genomic organization of the isolated repeti- tive fragment was determined using Southern blot hybridization. Genomic DNA was digest- ed with different restriction enzymes (MboI and DdeI), separated by electrophoresis in 1.5% agarose gel, and the DNA fragments were transferred onto a Hybond N+ nylon membrane (Amersham Biosciences, Uppsala, Sweden) by capillarity. The MboI purified band (called GuMboI family) was used as a probe and hybridized under conditions of high stringency, using the non-radioactive ECL method (Enhanced Chemioluminescent Kit, Amersham Bioscences) while the DdeI 350-bp isolated band (called GuDdeI family) was cloned in the One Shot Top 10 competent E. coli (Invitrogen Life Technologies, Carlsbad, CA, USA) using the Ready To Go pUC18 BAP+ligase kit (Amersham Biotech, Inc., Uppsala, Sweden) and following the manufacturer’s recommendations. Five posi- tive clones of the GuDdeI family were sequenced using the automated Alpha express DNA sequencer (Pharmacia, Uppsala, Sweden) and the sequences aligned in CLUSTAL W using the default parameters.31 MEGA version 4 was used to determine nucleotide composition and the genetic dis- tance between sequences calculated using the Kimura 2-parameter (K2P) method.32 Moreover, a GenBank search was performed in order to compare DdeI satDNA with other sat DNAs in the database. One of the clones (called GuDdeI 313) was employed as a probe in filter experiments, following the same pro- cedure described for GuMboI DNA hybridiza- tion. Fluorescence in situ hybridization procedure and karyotype analysis FISH procedure was effected using the same probes employed in the filter hybridiza- tion (GuMboI family and GuDdeI 313 clone), the GuDdeI family and Xenopus laevis 45S rDNA. The probes were labeled with biotin- 16-dUTP (Roche Applied Science, India- napolis, IN, USA) by nick-translation, using the Nick Translation kit (Roche Applied Science) and following the manufacturer’s instructions. Hybridization was performed overnight at 37°C in 50% formamide/2xSSC. Post-hybridization washes were carried out at 37°C in 50% formamide/ 2xSSC for 15 min fol- lowed by 15 min washes in 2xSSC at 37°C. Detection was carried out using fluorescein isothiocyanate-conjugated avidin (Vector Laboratories, Inc., Burlingame, CA, USA), with two amplification steps using anti-avidin biotin coniugated (Vector Laboratories, Inc). Chromosomes were counterstained using propidium iodide (Sigma Aldrich, St. Louis, MO, USA) (1 μg/mL) and DAPI (4’,6-diamidi- no-2-phenylindole) (Sigma Aldrich) (0.5 μg/mL). The same procedure was effected for all probes. Telomeric sequences (TTAGGG) were mapped by two-hour-FISH using a telom- ere PNA (peptide nucleic acid) probe conju- gated with FITC (Telomere PNA FISH kit/FITC, DakoCytomation, Glostrup, Denmark), following the manufacturer’s instructions. More than twenty metaphase plates were analyzed for each probe using a Zeiss Imager M1 fluorescence microscope; images were captured with a Hamamatsu digital camera C8484 and processed using a karyotyping- and FISH-dedicated image analysis system (Cromowin Plus, TESI Imaging, Pianiga (VE), Italy). Original paper Results Characterization ofMbo I- and Dde I- restricted DNA fractions, Southern hybridization and sequencing Genomic DNA of G. unicolor digested with either MboI or DdeI showed a high mw band of about 21 Kb (Figure 1). Moreover, MboI enzyme produced a band of about 5 Kb (GuMboI) and a smear composed of DNA frag- ments of about 4 kb to a few hundred base pairs (Figure 1). Cleavage of genomic DNA of Figure 1. Agarose gel electrophoresis of Gymnothorax unicolor genomic DNA digested with restriction endonucleases MboI (lane 1) and DdeI (lane 2). The arrow indicates the 5kb band detected by MboI, and the arrowhead indicates the 350-bp band detected by DdeI. M, phage lambda DNA cleaved with HindIII, used as a molecular size marker. [European Journal of Histochemistry 2011; 55:e12] [page 69]