Introduction

Highly repetitive sequences are interspersed throughout virtually all eukaryotic genomes. They can be classified into two categories based on their size: long interspersed repetitive elements (LINEs) and short interspersed repetitive elements (SINEs). They have been isolated from the genomes of many multicellular organisms from invertebrates to vertebrates (Ohshima et al., 1993; Okada & Ohshima, 1995). SINEs are typically less than 500 base pairs in length and can be unique to a taxonomic rank, for example a family, a genus or a few species (Smit, 1996). Almost all SINEs reported to date are derived from tRNA (Okada, 1991a,b; Okada & Ohshima, 1995), with the exception of the primate Alu and the rodent B1 families which are derived from 7SL RNA (Ullu & Tschudi, 1984). SINEs are believed to be amplified by a process of retrotransposition (Singer, 1982). In contrast to DNA transposable elements, SINEs appear to be inserted irreversibly and thus they should serve as ideal evolutionary and phylogenetic markers (Okada, 1991b).

In the genome of salmonid fishes both classes of sequences have already been identified (Moir & Dixon, 1988). Kido et al. (1991) have characterized three families of SINE sequences derived from tRNA: SmaI, FokI and HpaI families. The salmon SmaI family is restricted to the genomes of chum salmon (Oncorhynchus keta) and pink salmon (O. gorbuscha). The FokI family is only present in species from the genus Salvelinus, and the HpaI family is present in all the species of the salmonid family. The presence of specific elements of the different families has been used to infer phylogenetic relationships among salmonids (Murata et al., 1996) and more recently to obtain DNA fingerprint patterns (Spruell & Thorgaard, 1996). In this work, we present the results of our fluorescent in situ hybridization (FISH) experiments on the chromosomes of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) using highly repetitive sequences belonging to the HpaI family.

Materials and methods

PCR amplifications

A set of primers based on the Hpa-345 locus of Kokanee salmon (Oncorhynchus nerka adonis) (Murata et al., 1993) was designed for specific amplification of the HpaI family in the genome of Atlantic salmon and rainbow trout. PCR was performed with a Perkin Elmer 2400; the amplification mixture for PCR comprised 5–20 ng of genomic template DNA, 150 pmol of each primer, 200 μM of dNTPs and 1 unit of Taq polymerase (Boehringer). Amplification reactions were performed in a thermal cycler (Perkin Elmer 2400) with 30 cycles consisting of 95°C for 1 min, 58°C or 60°C for 1 min, and 72°C for 1 min.

Cloning and nucleotide sequence

The PCR amplified products were treated with kinase, electrophoresed, excised from the gel using a silica matrix (Bio 101) and ligated in the SmaI site of the pUC18 cloning vector, then sequenced in both strands by using the Sequenase Kit (USB) with universal primers. The reaction products were fractionated by electrophoresis on a 6% polyacrylamide gel. DNA sequences were searched for homologies using the nonredundant amino acid sequence database at the NCBI, with the BLAST program.

Cosmid library

A total genomic library of Atlantic salmon was constructed in a cosmid vector (sCos-1, Stratagene). Cosmids were isolated by plating out the library at low density and selecting after hybridization with the HpaI unit sequence obtained from the rainbow trout PCR amplification. The probe was labelled by a random priming technique using (α32P)dATP (Amersham). Filters were hybridized at 65°C in 3× SSPE, 0.5% dried milk, 1% SDS and 6% polyethylene glycol overnight, and washed in 1× SSC, 0.1% SDS at 65°C. Positive bands isolated from the genomic cosmid were excised, cloned and sequenced in both strands.

Chromosome preparation and FISH

Blood was collected from the dorsal artery of 10 large fish species, anaesthetized with MS-222, using a heparinized syringe. Metaphases were obtained from blood cultures as described by Sanchez et al. (1990).

Probes were labelled with biotin-11-dUTP using a nick translation kit (Boehringer). Details of probe preparation, in situ hybridization methods and the analysis of metaphases are described in Pendas et al. (1993a,b).

Results and discussion

PCR products showed a band of 395 bp in the case of rainbow trout and a band of 255 bp in Atlantic salmon. Cloning and sequencing of four PCR products of each repeat type indicated that rainbow trout contained one unit of the HpaI family, whereas this family was not present in the PCR band resulting from the Atlantic salmon genome amplification. The complete sequences of the two bands are identical to those obtained by Murata et al. (1993) after the amplification of Kokanee salmon and brown trout, respectively (data not shown).

With the aim of locating the HpaI family in the genome of Atlantic salmon, a total cosmid genomic library from this species was screened with the HpaI unit sequence obtained from the rainbow trout amplification. Positive clones were selected and digested with different restriction enzymes and blotted onto a membrane filter. The filter was then hybridized with the HpaI unit. Two positive bands resulting after the digestion of one of the positive cosmids with AvaII were cloned and sequenced. The larger fragment cloned was 342 bp long (Fig. 1a) and showed 68% homology to the coho salmon (Oncorhynchus kisutch) short interspersed element Hpa-51 that is also present in the genome of other Pacific salmon species (Murata et al., 1993). The smaller band, which was 246 bp long (Fig. 1b), showed 82% identity with the Salmo salar transposon like Tc1-encoded transposase pseudogene isolated by Goodier & Davidson (1994).

Figure 1
figure 1

 Nucleotide sequence of the two bands cloned from the Atlantic salmon cosmid containing a unit of the HpaI family. Similarities with coho salmon short interspersed element Hpa-51 (a) and Salmo salar transposon (b) are underlined.

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In order to determine the chromosomal distribution of the HpaI family, FISH analysis was carried out in the two species using as probes the PCR-amplified 395 bp fragment from rainbow trout and the 255 bp fragment from Atlantic salmon, the positive cosmid isolated for the HpaI family and the cloned band from this cosmid that contained a part of the Hpa-51 locus. As expected, hybridization of the PCR product from the Atlantic salmon lacking HpaI family members did not generate any signal in either species. The hybridization signals obtained with the other three probes revealed that the HpaI sequence was interspersed throughout all the chromosome pairs except in the heterochromatin positive areas, for both Atlantic salmon and rainbow trout (see Fig. 2), providing hybridization patterns complementary to the C-banding. Hybridization patterns obtained using the sequence similar to the Hpa-51 locus were identical to the patterns obtained using the whole genomic cosmid. The absence of the SINE sequence in the heterochromatic chromosome arm where the rDNA is located (Pendas et al., 1993a) indicates that the members of this SINE family are located only in the euchromatic regions of the chromosomes.

Figure 2
figure 2

 Fluorescent in situ hybridization (FISH) showing (a) Atlantic salmon chromosomes hybridized with a PCR amplified HpaI SINE fragment from rainbow trout. (b) Rainbow trout chromosomes hybridized with the same probe. Asterisks indicate the rDNA-bearing chromosome arms where no hybridization sites were detected.

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Nonrandom distribution of SINE elements has been described in human chromosomes. For example, the Alu and L 1 families are preferentially found in R and G bands, respectively (Korenberg & Rykowski, 1988); therefore, the distribution of the SINE element found in our work is not surprising. In addition to this particular distribution, we have observed that the HpaI element contained in the cosmid is located adjacent to a Tc-1 transposon-like element (Goodier & Davidson, 1994). Spruell & Thorgaard (1996) have suggested that these two features of SINEs (nonrandom distribution and integration close to other repetitive elements), together with the occurrence of some SINE elements as tandemly repeated arrays (Nagahashi et al., 1991), could be responsible for the production of fingerprinting patterns instead of a smear when these salmon SINE families are used for Southern blot analyses.

The physical distribution of a member of the HpaI family obtained in this work on Atlantic salmon and rainbow trout chromosomes can probably be extended to the other two SINE families (FokI and SmaI). If assayed on chromosomes from all the other members of the Salmonidae family, FISH of SINE elements could provide new information useful in evolutionary studies (Murata et al., 1993). This method could also contribute to standardizing the Atlantic salmon karyotype, because the fluorescent in situ hybridization of these sequences gives a banding pattern that allows the identification of at least 15 chromosome pairs (Fig. 3).

Figure 3
figure 3

 Homologous pairs identified from the banding pattern of chromosomes shown in Fig. 2(a). This Atlantic salmon karyotype is 2n=58.

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