RNA preparation

The rainbow trout (130 ± 20 g in body weight) purchased from a local dealer were maintained under LD cycles of 14 hr:10 hr (LD 14: 10; light on 06:00 – 20:00) at 14°C for at least a week. Fish were anesthetized with 0.06% 2-phenoxyethanol and sacrificed by decapitation at mid-day (12:30 – 13:30) or mid-night (00:30 – 01:30). After dissection, the retina was used for total RNA extraction immediately, while the liver was frozen in liquid nitrogen and stored at −80°C until RNA preparation. Total RNA was extracted with an RNA extraction solution, Isogen (Nippongene, Toyama, Japan) according to the manufacture's protocol. Poly(A)⁺ RNA was prepared from total RNA using Oligotex dT30<Super> (Takara, Tokyo, Japan) according to instructions of the manufacture.

Primers

The following oligonucleotide primers were chemically synthesized: primer 1, 5′-TG(C/T)CCIGA(A/G)(C/T)TI(T/A)(C/G)I(C/T) TIGGITGG-3′; primer 2, 5′-GGIACIA(A/G)IGC(A/G)T(C/T)(C/T)TC(A/ G)CACAT-3′; primer 3, 5′-CTCACACATGAGCACTGCAC-3′; primer 4, 5′-CATCAGGATAGGGCCCTTG-3′; primer 5, 5′-TTGAGGAGGGGAGACTAGTAGC-3′; primer 6, 5′-CCAAGAGAGAAGACTTGTGC-3′; primer 7, 5′-GGTGTGCACTTTAGACTACC-3′; anchor primer, 5′-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGG IIGGGIIG-3′; AUAP primer, 5′-GGCCACGCGTCGACTAGTAC-3′; Not I d(T) primer, 5′-AACTGGAAGAATTCGCGGCCGCAGGAAT₁₈-3′; RTG primer, 5′-AACTGGAAGAATTCGCGG-3′.

Polymerase chain reaction (PCR)

Each PCR amplification was performed by using GeneAmp PCR System 2400 (Perkin Elmer, Norwalk, CT, USA) in a total volume of 20 μl with Taq DNA polymerase (Takara) and primers (250 nM each).

Amplification of a trout AANAT cDNA fragment by PCR

PCR was first performed with degenerated primers (the primers 1 and 2) deduced from the conserved amino acid sequences of AANAT of rat (GenBank Accession number U40803), ovine (U29663) and human (U40347). The primers 1 and 2 correspond to the amino acid positions 75–82 (CPELSLGW) and 157–164 (MCENALVP) of the rat AANAT, respectively. First strand cDNA was synthesized from the retinal total RNA of the rainbow trout collected at mid-night with Ready-To-Go^TM T primed first strand cDNA synthesis kit (Pharmacia Biotech, Tokyo, Japan) and subjected to 30 cycles of PCR amplification with the primers 1 and 2. Each reaction cycle consisted of incubations at 94°C for 1 min, 50°C for 1 min and 72°C for 1 min. The final elongation step was carried out at 72°C for 7 min.

5′-Rapid amplifications of cDNA ends (RACE)

First strand cDNA was synthesized from the retinal total RNA of the rainbow trout collected at mid-night using 5′-RACE PCR kit (GIBCO/BRL, Gaitherburg, MD, USA) according to the manufacture's instructions. The primer 3 was used to prime cDNA synthesis. Subsequently, a dC tail was added to the 3′-end of the synthesized single-stranded cDNA and PCR amplification for 35 cycles was performed with the primer 4 and 5′-anchor primer (GIBCO/BRL). Each reaction cycle consisted of incubations at 94°C for 30 sec, 55°C for 30 sec and 72°C for 1 min. The final elongation step was carried out at 72°C for 7 min.

3′-RACE

First strand cDNA was synthesized with Ready-To-Go^TM T primed first strand cDNA synthesis kit as described above and used as a template for 30 cycles of PCR amplification with the primer 5 and RTG primer containing the complementary sequence to the Not I d(T) primer for 3′-RACE. Each reaction cycle consisted of incubations at 94°C for 30 sec, 55°C for 30 sec and 72°C for 2 min. The final elongation step was carried out at 72°C for 7 min.

Confirmation of the obtained sequence by PCR

The obtained nucleotide sequence by 5′- and 3′-RACE over the coding region (nucleotide position 29-915 of the trout AANAT sequence) was reconfirmed by PCR amplification using the primers 6 and 7.

DNA sequencing

The cDNA fragments amplified by the PCR described above were separated by agarose gel electrophoresis and purified using Prep-A-Gene DNA Purification Kit (Bio Rad, Richmond, CA, USA). The purified cDNA fragments were then subcloned into pBluescript II SK(−) vector (Stratagene, La Jolla, CA, USA). Both strands of the plasmid DNA were sequenced with a Model 373A DNA sequencer using dye terminator cycle sequencing kits (Applied Biosystems, Foster City, CA, USA). The nucleotide sequence was determined from two to four clones obtained by independent reverse transcriptions and amplifications to avoid PCR errors.

Sequence analysis

Sequence analysis was performed with the GeneWorks 2.45 (IntelliGenetics, Mountain View, CA, USA). A phylogenetic tree of vertebrate AANAT was constructed by the neighbor-joining method (Saitou and Nei, 1987) based on the amino acid identities by the PHYLIP program (Felsenstein, 1989) using Drosophila AANAT (Hintermann et al., 1996) as an outgroup.

Isolation of genomic DNA and Southern blot analysis

Genomic DNA (10 μg) isolated from the trout liver was digested with the restriction enzymes (EcoR I, Pst I, or Xba I) and used for Southern blot analysis according to the method as described (Kikuchi et al., 1995). A partial AANAT cDNA (nucleotide position 9-570 of the trout sequence) labeled with [α-³²P]dCTP (Amersham Japan, Tokyo, Japan) by Random primer DNA labeling kit ver. 2 (Takara) was used as a probe (5 × 10⁹ cpm/ml).

Northern blot analysis

Poly(A)⁺ RNA (5 μg) prepared from the pooled retina of 10 individuals and the liver of a individual was separated on a formalde-hyde-agarose gel and blotted onto a nylon membrane (Hybond-N⁺, Amersham Japan). The membrane was UV crosslinked using Spectrolinker^TM XL-1500 (Spectronics Corporation, Westburg, NY, USA) for 30 sec. The membrane was prehybridized in 6 × SSC containing 50% formamide, 1 × Denhardt's solution, 0.5% sodium dodecylsulphate (SDS) and calf thymus DNA (100 ng/ml) at 42°C for 12 hr and then hybridized with the randomly [α-³²P]dCTP-labeled probe (the same cDNA probe as in the Southern blot analysis, 5 × 10⁹ cpm/ ml) in the hybridization buffer (6 × SSC containing 50% formamide, 1 × Denhardt's solution and 0.5% SDS) at 42°C overnight. The membrane was washed in 2 × SSC containing 0.5% SDS at room temperature for 10 min, and then washed at 65°C for 10 min in 1 × SSC containing 0.5% SDS and exposed to Fuji X-ray film (Fuji Film, Tokyo, Japan) at −80°C for 2 weeks. The hybridized membrane was also scanned by FUJIX BAS 1000 Mac Bio-Imaging Analyzer (Fuji Film) to count the hybridization signal. The hybridized probe on the membrane was washed out in the washing buffer (5 mM Tris-HCl containing 2 mM EDTA and 0.1 × Denhardt's solution) at 65°C for 2 hr. Then the membrane was rehybridized with the [α-³²P]dCTP-labeled β-actin probe of masu salmon, O. masou, (4,000 cpm/ml; 444 bp; Gen et al., in preparation) corresponding to the nucleotide position 499-942 of mouse β-actin (GenBank accession number X03672) to confirm the integrity of the mRNA loading and transfer efficiency. The membrane was washed, exposed and scanned as described above.

Molecular cloning of trout AANAT

A trout retinal AANAT cDNA fragment (222 bp) was first obtained by PCR using degenerate primers (the primers 1 and 2) designed from the conserved amino acid sequences of mammalian AANAT. This cDNA fragment is 73 and 72% identical to the corresponding region of the rat sequence at the nucleotide and amino acid levels, respectively. Based on this sequence, we designed trout AANAT specific primers (the primers 3-5) and isolated several clones including 5′- and 3′-franking regions by RACE methods. A pair of primers (the primers 6 and 7) was also synthesized and used in reverse transcription-PCR to amplify a cDNA (888 bp) covering the entire coding region of the trout AANAT. The overlapping cDNAs for the trout AANAT spanned 1,585 bp (Fig. 1). The trout retinal AANAT cDNA contains a 723 bp open reading frame encoding 240 amino acids with a predicted molecular weight of 27,420 and an isoelectric point of 7.78. The 5′- and 3′-untranslated regions were 96 and 766 bp long, respectively.

Fig. 1

The nucleotide and deduced amino acid sequences of the trout retinal AANAT. Nucleotide and amino acid sequences are numbered from the first and indicated in the left and right of each line. Arrows indicate the regions used as the primers in PCR. The stop codon is indicated by the asterisk and a consensus sequence for polyadenylation signal is boxed. The sequence has been deposited in DDBJ, EMBL and GenBank nucleotide databases under accesion number AB007294.

Analysis of deduced protein

The deduced amino acid sequence of the trout AANAT is aligned with those of the chicken, rat, bovine, ovine and human (Borjigin et al., 1995; Coon et al., 1995, 1996; Bernard et al., 1997) (Fig. 2). The sequence of trout retinal AANAT is very similar in amino acid sequences to those of other vertebrates (65, 54, 54, 53 and 51% identical to chicken, rat, ovine, bovine and human AANAT, respectively) but shows lower identity (14%) to the Drosophila AANAT (Hintermann et al., 1996). The carboxy tail of the trout AANAT is 37 amino acid longer than the avian and mammalian AANAT.

Fig. 2

Alignment of amino acid sequences of AANAT of rainbow trout, chicken, rat, bovine, ovine and human. The sequences in chicken, rat, bovine, ovine, and human were obtained from Gen Bank (accession numbers are U46502, U40803, AD000742, U29663, and U40347, respectively). To maximize homologies, gaps represented by hyphns have been introduced in the sequences. Potential phosphorylation sites are shaded: cnpk, cyclic nucleotide-dependent protein kinase; ck2, casein kinase II; and pkc, protein kinase C. The consensus sequence contains moieties conserved in all six species. The two conserved motifs (A and B) for N-acetyltransferase superfamily (Tercero et al., 1992; Coleman et al., 1996; Lu et al., 1996) are underlined.

A phylogenetic tree of vertebrate AANAT is shown in Fig. 3. The branching patterns are highly reliable and the tree topology agreed well with phylogeny.

Fig. 3

A phylogenetic tree of AANAT in vertebrates based on amino acid identities. The tree was constructed by the neighbor-joining method (Saitou and Nei, 1987) using PHYLIP software (Felsenstein, 1989). The values at the nodes are bootstrap probabilities (%) estimated by 1,000 times replications, and the length of the horizontal lines are evolutionary distances.

The trout AANAT sequence contains putative motifs for acetyl coenzyme A binding, motifs A and B (Tercero et al., 1992; Coleman et al., 1996; Lu et al., 1996), at the amino acid positions of 117–136 and 161–173, respectively. The trout AANAT also contains eight putative phosphorylation sites; two consensus sites for cyclic nucleotide-dependent protein kinase at Thr²⁷ and Ser²⁰¹, four sites for casein kinase II at Ser⁴³, Ser⁹³, Thr¹⁸⁵ and Thr²³³, and two sites for protein kinase C at Thr¹²⁵ and Thr²³⁰.

Southern blot analysis

Southern blot analysis was carried out to analyze the genomic organization of the trout retinal AANAT. Probing the liver genomic DNA with labeled cDNA probe produced one band each of 19, 2.9, and 14 kbp after digestion with EcoR I, Pst I, and Xba I, respectively (Fig. 4). These results suggest the trout retinal AANAT is encoded by a single copy gene.

Fig. 4

Southern blot analysis of the trout retinal AANAT gene. When the trout liver genomic DNA (10 μg) was digested with EcoRI, PstI, or XbaI, hybridization with the labeled cDNA probe produced a single band in each lane (EcoR I, 19 kbp; Pst I, 2.9 kbp; Xba I, 14 kbp), indicating that the trout retinal AANAT is encoded by a single copy gene. Molecular weight markers are EcoT14I digests of lambda phage DNA.

Northern blot analysis

Expression of the AANAT gene in the retina and liver was surveyed by Northern blot analysis (Fig. 5A). A single transcript (1.6 kb) was detected in the retina at both mid-day and mid-night, but not in the liver. The mRNA level of the retinal AANAT exhibited 3.3-fold increase at night under LD cycles (Fig. 5B). A similar result was obtained in another experiment.

Fig. 5

Northern blot analysis for AANAT mRNA in the retina and liver. (A) The rainbow trout reared under LD 14:10 (light on 06:00–20:00) were sacrificed at mid-day (Day, 13:00) or at mid-night (Night, 01:00). Northern blot analysis was performed on Poly(A)⁺ RNA (5 μg) obtained from the retina and liver. Arrowheads on the right mark the positions of 28S (top) and 18S (bottom) rRNA bands. Blots were stripped and reprobed with β-actin of masu salmon to allow normalization of lanes. (B) Day-night changes in the AANAT mRNA in the rainbow trout retina. The abundance of the AANAT transcript has been normalized to β-actin mRNA, to correct for variations in the mRNA loading and transfer efficiency. Data are expressed relative to the mid-day value. The trout retinal AANAT mRNA increased 3.3-fold at mid-night as compared with the mid-day value.