Some units of the 5S rDNA of Solea senegalensis were amplified by PCR and sequenced. Three main PCR products (227, 441, and 2166 bp) were identified. The 227- and 441-bp fragments were characterized by highly divergent nontranscribed spacer sequences (referred to as NTS-I and NTS-II) that were 109 and 324 bp long, respectively, yet their coding sequences were nearly identical. The 2166-bp 5S rDNA unit was composed of two 5S rRNA genes separated by NTS-I and followed by a 1721-bp spacer containing the U2, U5, and U1 small nuclear RNA genes (snRNAs). They were inverted and arranged in the transcriptional direction opposite that of the 5S rRNA gene. This simultaneous linkage of 3 different snRNAs had never been observed before. The PCR products were used as probes in fluorescence in situ hybridization experiments to locate the corresponding loci on the chromosomes of S. senegalensis. A major 5S rDNA chromosomal site was located along most of the short arm of a submetacentric pair, while a minor site was detected near the centromeric region of an acrocentric pair.

The 5S ribosomal DNA (rDNA) consists of one transcriptional unit of about 120 base pairs, which is separated from the next unit by a non-transcribed spacer (NTS). The coding sequence and the NTS together form a repeat unit which can be found in hundreds to thousands of copies tandemly repeated in the genomes. The NTS regions seem to be subject to rapid evolution. The first general model of evolution of these multigene families was referred to as divergent evolution, based on studies using hemoglobin and myoglobin as model systems. Later studies showed that nucleotide sequences of different multigene family members are more closely related within species than between species. This observation led to a new model of multigene family evolution, termed concerted evolution. Another model of evolution, named the birth-and-death model, has been found to be more suitable to explain the long-term evolution of these multigene families. According to this model, new genes originate by successive duplications, and these new genes are either maintained for a long time or are lost, or else degenerate into pseudogenes. In this review we describe different sources of variability in the 5S rDNA genes observed in several distinct fish species. This variability is mainly referred to NTSs and includes the presence of other multigene families (mainly LINEs, SINEs, non-LTR retrotransposons, and U snRNA families). Different types of microsatellites have also been found to contribute to the increase of variability in this region. Our recent results suggest that horizontal transfer contributes to the increase of diversity in the NTSs of some species. Variability in the 5S rDNA coding region affecting the stability of the structure, but without effects on the function of the 5S rRNA, is also described. Retrotransposons seem to be responsible for the high dynamism of 5S rDNA, while microsatellites acting as recombination hot spots could stabilize a wide variety of unusual DNA structures, affecting DNA replication and enhancing or decreasing promoter activity in gene expression. The relationship between the high variability found at molecular level and the low variability found at chromosomal level is also discussed.

A cytogenetic analysis of the sole Solea senegalensis was carried out using silver staining for the nucleolus organizer region (Ag-NOR) identification, one-color FISH for chromosomal mapping of 45S and 5S ribosomal DNAs (rDNAs), (GATA)n, and (TTAGGG)n, and two-color FISH for co-localization of both rDNAs. The Ag-NORs and the 45S rDNA were mapped to a medium-sized submetacentric chromosomal pair. Hybridization with the 5S rDNA showed a major signal on the short arm of a medium-sized submetacentric chromosome pair and a minor signal on a centromeric site of a small acrocentric chromosome pair. Differences in the Ag-NOR and 45S and 5S rDNAs FISH signal sizes were observed between homologous chromosomes and among individuals. A two-color FISH co-localized 45S and 5S rDNAs to a medium-sized submetacentric chromosomal pair. The hybridization with the telomeric (TTAGGG)n repeat displayed small signals at all chromosomal telomeres. Finally, the (GATA)n probe produced dispersed and small hybridization signals on all chromosome spreads, showing its ubiquitous existence in the genome. These results were compared with those from other Pleuronectiformes and discussed in terms of karyotype evolution.

The karyotype of Halobatrachus didactylus presents 46 chromosomes, composed of eight metacentric, 18 submetacentric, four subtelocentric, and 16 acrocentric chromosomes. The results of FISH showed that the major ribosomal genes were located in the terminal position of the short arm of a large submetacentric chromosome. They also showed a high variation in the hybridization signals. The products of amplification of 5S rDNA produced bands of about 420 pb. The PCR labeled products showed hybridization signals in the subcentromeric position of the long arm of a submetacentric chromosome of medium size. Double-color FISH indicated that the two ribosomal families are not co-located since they hybridized in different chromosomal pairs. Telomeres of all the chromosomes hybridized with the (TTAGGG)n probe. The GATA probe displayed a strong signal in the long arm of a submetacentric chromosome of medium size, in the subcentromeric position. The double-color FISH showed that the microsatellite GATA and the 5S rDNA gene are located in different chromosomal pairs. The majority presence of GATA probes in one pair of chromosomes is unusual and considering its distribution through different taxa it could be due to evolutionary mechanisms of heterochromatine accumulation, leading to the formation of differentiated sex chromosomes.

The physical location of 18S-5.8S-28S rDNA, telomeric sequences with (TTAGGG)n DNA probe and (GATA)n microsatellites were performed by fluorescence in situ hybridization in chromosomes of red abalone Haliotis rufescens. The karyotype of red abalone showed a diploid number of 36 (8M+9SM+1ST). FISH performed with rDNA probe, showed the location of major ribosomal clusters in the terminal region of the large arms of two submetacentric pairs (chromosome 4 and 5). Localization of heteromorphisms of FISH-rDNA was found between chromosome homologues and sister chromatids in all metaphases analyzed. This indicates that rDNA clusters are variable within the red abalone genome. The variability in the NOR-bearing reported using silver staining in other gastropods and our result are discussed. In addition, the presence of microsatellite (TTAGGG)n and (GATA)n was demonstrated after FISH treatment by DNA probes. The telomeric sequence occurred at the ends of all mitotic chromosomes, while the (GATA)n repetitive was found on chromosomal interstitial zones as well as at the telomeres in abalone chromosomes.