We cloned a cDNA and the gene for Japanese flounder TNF. The TNF cDNA consisted of 1217 bp, which encoded 225 amino acid residues. The identities between Japanese flounder TNF and members of the mammalian TNF family were ∼20–30%. The positions of cysteine residues that are important for disulfide bonds were conserved with respect to those in mammalian TNF-α. The Japanese flounder TNF gene has a length of ∼2 kbp and consists of four exons and three introns. The positions of the exon-intron junction positions of Japanese flounder TNF gene are similar to those of human TNF-α. However, the length of the first intron of Japanese flounder is much shorter than that of the human TNF-α gene. There are simple CA or AT dinucleotide repeats in the 5′-upstream and 3′-downstream regions of the Japanese flounder TNF gene. Southern blot hybridization indicted that Japanese flounder TNF exists as a single copy. Expression of Japanese flounder TNF mRNA is greatly induced after stimulation of PBLs with LPS, Con A, or PMA. These results indicated that Japanese flounder TNF is more like mammalian TNF-α than mammalian lymphotoxin-α, with respect to its gene structure, length of amino acid sequence, number and position of cysteine residues, and regulation of gene expression.

Tumor necrosis factor-α is a 17-kDa protein that is synthesized by different cell types upon stimulation with endotoxin, inflammatory mediators, or cytokines such as IL-1 and, in an autocrine manner, upon stimulation with TNF itself (1, 2, 3, 4). The biological effect of TNF-α may vary depending on the relative concentration, the duration of cell exposure, and the presence of other mediators, and plays roles in immune and inflammatory responses and in the pathogenesis of many diseases (1, 2, 3, 4).

There are also several TNF-like proteins, which together are referred to as the TNF ligand superfamily. The members of the TNF ligand superfamily (TNF-α, lymphotoxin-α (TNF-β), lymphotoxin-β, CD27 ligand, CD30 ligand, CD40 ligand, Fas ligand, OX40 ligand, and TRAIL) share common biological activities, but some properties are shared by only some ligands, whereas others are unique. The biosynthesis of TNF family proteins is largely regulated at the posttranscriptional level (5). Bacterial endotoxin, which strongly induces TNF-α production, thus seems to elicit at least some of its effects by altering macrophages. It has previously been shown that a 3′-untranslated TTATTTAT element that is present in numerous cytokine genes and protooncogenes is capable of repressing the translation of mRNA molecules in which it occurs (6).

The nucleotide sequences of the cDNA and genes encoding TNF-α have been reported for several mammalian species (7, 8, 9, 10, 11, 12, 13, 14). Human secreted-type mature TNF-α is a nonglycosylated protein of 17 kDa with a length of 157 amino acid residues and forms dimers and trimers (4). The gene of human TNF-α has a length of ∼3 kbp and is interrupted by three introns (15). The structure of human TNF-β is different from that of TNF-α (15). The protein coding regions of the human TNF-β gene are separated into three parts (15).

Thus, mammalian TNF family proteins have been well characterized. However, no information is available on TNF proteins in fish. To understand the role and mechanism of TNF in the immune system in fish, it is essential to identify and characterize the TNF. In this study, we isolated a cDNA and gene for the TNF from Japanese flounder Paralychthys olivaceus.

Peripheral blood samples were taken from a single homocloned Japanese flounder P. olivaceus (16). Leukocytes were isolated by centrifugation, at 400 × g for 20 min, with Percoll solution (1.072 g/ml). Leukocytes were cultured in RPMI 1640 containing Con A (70 μg/ml) and PMA (0.35 μg/ml) and were sampled after 1, 2, and 3 h. mRNA was isolated using a micro mRNA purification kit (Amersham Pharmacia Biotech, Piscataway, NJ). The purified mRNAs of three different time periods were pooled and used to construct a cDNA library. cDNA was synthesized using a cDNA synthesis kit (Amersham Pharmacia Biotech) with an oligo(dT) primer. The cDNA library was constructed in λZAPII vectors (Stratagene, La Jolla, CA) according to the instructions of the manufacturer.

We conducted an expressed sequence tag analysis of the cDNA library prepared from Con A/PMA-treated leukocytes. Conversion of the recombinant λZAPIIs into the pBluescript plasmid was conducted by in vivo excision according to the protocol of the manufacturer (Stratagene). After conversion of phage clones into plasmids, we randomly selected clones from the library and sequenced them. cDNA clones were sequenced using ThermoSequenase (Amersham Pharmacia Biotech) with M13 forward and/or M13 reverse primers and an automated DNA sequencer LC4200 (Li-Cor, Lincoln, NE). Each determined sequence was compared with all sequences available in DDBJ/EMBL/GenBank using the BLAST version 2.0 (17, 18) (http://www.ncbi.nlm.nih.gov).

Previously, we constructed and arrayed a genomic bacterial artificial chromosome (BAC)3 library (19). The average insert size of clones in this library is ∼165 kbp. The arrayed genomic BAC clones were screened for a TNF gene by using a TNF cDNA as a DNA probe. Hybridization was done as previously reported (19). BAC DNAs were isolated by the alkaline lysis method and then digested with EcoRI and subcloned into the pUC119 plasmid vector. The subclones were screened by the colony-hybridization method (20). The genomic clone was sequenced as described above.

Genomic DNA of two different lines of homocloned Japanese flounder, cloned lines 1 and 8, and two noncloned Japanese flounder were isolated as previously reported (19). Total RNA was prepared from either untreated PBLs or PBLs that had been stimulated with LPS (500 μg/ml), Con A (50 μg/ml), or PMA (0.05 μg/ml) by Trizol (Life Technologies, Rockville, MD). Five micrograms of total RNA per lane was denatured at 65°C for 5 min in 50% formamide, electrophoresed through a 1.5% agarose gel containing 6.6% formaldehyde, and transferred to a nylon membrane (NEB, Beverly, MA). Perfect RNA markers 0.2–10 kb (Novagen, Madison, WI) were used for size marker of agarose gel electrophoresis. The probe was the full length of a Japanese flounder TNF cDNA fragment and was labeled with [α-32P]dCTP using a random primer labeling kit (Takara Shuzo, Kyoto, Japan). Southern and Northern blot hybridizations were done as described previously (21).

Total RNA was extracted from healthy Japanese flounder brain, head kidney, trunk kidney, liver, spleen, erythrocytes, leukocytes, skin, muscle, gill, heart, intestine, gonad, and LPS-treated leukocytes using Trizol (Life Technologies). The purified total RNA (10 μg) was reverse transcribed into cDNA using the AMV Reverse Transcriptase First-strand cDNA Synthesis kit (Life Science, Arlington Heights, IL). The final volume of the cDNA synthesis reaction was 25 μl. The reverse-transcribed sample (1 μl) was used in 50 μl of PCR mixture. The PCR primers used in this study were 5′-ggtttaaagtctcaaagtgc-3′ and 5′-agttgactgtgagcatggtg-3′. The β-actin primer set was used for a positive control of RT-PCR (22). PCR was performed with an initial denaturation step of 2 min at 95°C, and then 20 cycles were run as follows: 30 s of denaturation at 95°C, 30 s of annealing at 55°C, and 1 min of extension at 72°C. The reacted products were electrophoresed on a 2.0% agarose gel.

From the expressed sequence tag analysis, we found a clone whose amino acid sequence had significant identity to that of human TNF-α (data not shown). This clone was used as a probe for screening the cDNA library and BAC genome library. The sequences of Japanese flounder TNF cDNA and gene have been deposited in the DDBJ/GenBank/EMBL database (accession numbers AB040448 and AB040449). The TNF cDNA consisted of 1217 bp, which coded 225 amino acid residues (Fig. 1). There is an N-glycosylation site on the 26th amino acid residue, but the region is not included in the mature TNF. Fig. 2 shows an alignment of the amino acid sequence of Japanese flounder TNF with the human TNF-α, lymphotoxin-α, and -β. The identities between Japanese flounder TNF and previously reported mammalian TNF-α, lymphotoxin-α, and -β ranged from 20 to 35%. The Japanese flounder TNF shows 29 and 31% amino acid identity to human TNF-α and lymphotoxin-α, respectively, although the length of the amino acid sequence of the Japanese flounder sequence is more similar to that of human TNF-α than to that of lymphotoxin-α, whereas these two human proteins are themselves 30% identical. The phylogenetic analysis suggests that the TNF-α and lymphotoxin-α diverged after the divergence of mammals from teleosts (Fig. 3).

FIGURE 1.

Nucleotide and deduced amino acid sequence of Japanese flounder TNF cDNA. The TA-rich motifs (TTATTTAT) are underlined.

FIGURE 1.

Nucleotide and deduced amino acid sequence of Japanese flounder TNF cDNA. The TA-rich motifs (TTATTTAT) are underlined.

Close modal
FIGURE 2.

Alignment of Japanese flounder TNF with human TNF-α, lymphotoxin-α (TNF-β), and lymphotoxin-β. Sequences were obtained from DDBJ/EMBL/GenBank database. Amino acids identical with Japanese flounder are shown by dots. The position of residues identical in all sequences are shown with asterisks. Gaps (dashes) have been placed to maximize the identity.

FIGURE 2.

Alignment of Japanese flounder TNF with human TNF-α, lymphotoxin-α (TNF-β), and lymphotoxin-β. Sequences were obtained from DDBJ/EMBL/GenBank database. Amino acids identical with Japanese flounder are shown by dots. The position of residues identical in all sequences are shown with asterisks. Gaps (dashes) have been placed to maximize the identity.

Close modal
FIGURE 3.

Phylogenetic tree of TNF-α, lymphotoxin-α (TNF-β), and lymphotoxin-β amino acid sequences. Sequences were aligned using CLUSTAL W, with gap opening penalty 10. Gap-extension penalty 0.05, delay divergent sequences 40%, and protein weight matrix (BLOSUM series). The tree was generated from this alignment. Bootstrap probabilities (%) on interior branches are for the UPGMA tree. Human Fas ligand, which is a member of TNF superfamily, was used as the outgroup.

FIGURE 3.

Phylogenetic tree of TNF-α, lymphotoxin-α (TNF-β), and lymphotoxin-β amino acid sequences. Sequences were aligned using CLUSTAL W, with gap opening penalty 10. Gap-extension penalty 0.05, delay divergent sequences 40%, and protein weight matrix (BLOSUM series). The tree was generated from this alignment. Bootstrap probabilities (%) on interior branches are for the UPGMA tree. Human Fas ligand, which is a member of TNF superfamily, was used as the outgroup.

Close modal

Southern blot hybridization indicated that the homocloned Japanese flounder TNF gene exists as a single copy and not as a member of a multigene family (Fig. 4). Interestingly, digestion of EcoRI showed the presence of polymorphic variant for Japanese flounder TNF. We cloned the ∼9-kb EcoRI DNA fragment from the isolated BAC clone and sequenced entirely. The Japanese flounder TNF gene has a length of ∼2 kb and consists of four exons and three introns (Fig. 5). The positions of the exon-intron junctions of Japanese flounder TNF gene are similar to those of the human TNF-α gene. The lengths of the introns of the Japanese flounder TNF gene are shorter than those of the human TNF-α gene. This is especially true of the first intron of the Japanese flounder TNF gene, which is one-sixth the length of the first intron in the human TNF-α gene. The Japanese flounder TNF gene has six simple dinucleotide repeats, three in the 5′-upstream region and three in the 3′-downstream region (Fig. 5). Approximately 1 kb upstream from the ATG start codon, there are four repeats of TGGGGG (Fig. 5).

FIGURE 4.

Southern blot hybridization analysis of genomic DNA from four individual fish. Genomic DNA was digested with EcoRI (E), PstI (P), and SacI (S).

FIGURE 4.

Southern blot hybridization analysis of genomic DNA from four individual fish. Genomic DNA was digested with EcoRI (E), PstI (P), and SacI (S).

Close modal
FIGURE 5.

Nucleotide sequence of the Japanese flounder TNF gene. The simple dinucleotide sequences and TGGGG repeats are underlined. Sequences identical with the cDNA sequence are indicated in capital letters.

FIGURE 5.

Nucleotide sequence of the Japanese flounder TNF gene. The simple dinucleotide sequences and TGGGG repeats are underlined. Sequences identical with the cDNA sequence are indicated in capital letters.

Close modal

As shown in Fig. 6, mRNA from fresh PBLs and unstimulated PBLs in the medium did not express TNF-α mRNA. When the PBLs were stimulated with LPS, PMA, and a calcium ionophore, or Con A, for 1 and 3 h, TNF mRNA expression was greatly induced. However, when PBLs were stimulated with LPS, PMA, and the calcium ionophore, or Con A, for 6 h, the expression level of TNF mRNA was less than it was after 1- and 3-h stimulation. All RNA samples were intact because rehybridization of the same membrane with a Japanese flounder β-actin cDNA probe (23) revealed a 1.4-kb band in all lanes.

FIGURE 6.

Northern blot hybridization of the Japanese flounder TNF. Lane 1, freshly isolated Japanese flounder PBLs; lanes 2–5, Japanese flounder PBLs were incubated for 1 h with medium only (lane 2), LPS (lane 3), PMA (lane 4), or Con A (lane 5); lanes 6–9, Japanese flounder PBLs were incubated for 3 h with medium only (lane 6), LPS (lane 7), PMA (lane 8), or Con A (lane 9); and lanes 10–13; Japanese flounder PBLs were incubated for 6 h with medium only (lane 10), LPS (lane 11), PMA (lane 12), or Con A (lane 13).

FIGURE 6.

Northern blot hybridization of the Japanese flounder TNF. Lane 1, freshly isolated Japanese flounder PBLs; lanes 2–5, Japanese flounder PBLs were incubated for 1 h with medium only (lane 2), LPS (lane 3), PMA (lane 4), or Con A (lane 5); lanes 6–9, Japanese flounder PBLs were incubated for 3 h with medium only (lane 6), LPS (lane 7), PMA (lane 8), or Con A (lane 9); and lanes 10–13; Japanese flounder PBLs were incubated for 6 h with medium only (lane 10), LPS (lane 11), PMA (lane 12), or Con A (lane 13).

Close modal

We tested several organs of healthy Japanese flounder for the presence of TNF mRNA by RT-PCR. None of the organs, tissues, or cells examined expressed TNF mRNA. Only LPS-treated leukocytes expressed TNF mRNA (data not shown).

This is the first report of cloning and characterization of TNF from a teleost. The amino acid sequence alignment showed that the Japanese flounder TNF is equally homologous (∼30%) to human TNF-α and lymphotoxin-α, although the identity between human TNF-α and lymphotoxin-α is ∼30%. The human TNF-α has two cysteine residues in the mature protein region, which form a single disulfide bond, whereas the lymphotoxin-α does not have a disulfide bond (Fig. 2). These two cysteine residues are also conserved in the Japanese flounder TNF. The phylogenetic analysis suggests that the teleostei have one ancestral TNF gene and the mammalian TNF-α and lymphotoxin-α gene were duplicated and evolved after mammals diverged from teleosts (Fig. 3). The number of exons and introns and the positions of the exon-intron junctions of the Japanese flounder TNF gene are similar to those of the human TNF-α gene. The protein coding regions of the human lymphotoxin-α gene consist of three exons (15). We speculate that the Japanese flounder TNF has a role similar to that of the mammalian TNF-α based on similarities in the structures of the genes, the lengths of the amino acid sequences, and the existence of cysteine residues that form a disulfide bond.

There is no complete polyadenylation signal AATAAA in the 3′-untranslated region (UTR). However, TA-rich motifs (TTATTTAT) within the mammalian TNF-α 3′-UTR that were shown to influence TNF-α mRNA half-life (6) and translational efficiency (5) are also present in the Japanese flounder TNF cDNA (Fig. 1). Caput et al. (6) reported that the consensus sequence TTATTTAT is present in the 3′-UTR of both human and mouse TNF mRNAs, as well as the mRNAs encoding human lymphotoxin, human CSF, human and mouse IL-1, human and rat fibronectin, and most of the sequenced human and mouse IFNs. All of these mRNAs, except the lymphotoxin mRNA, lack homology to the TNF mRNAs in the coding region (6). It is particularly prevalent among mRNAs encoding proteins related to the inflammatory response (6). Using constructs in which the chloramphenicol acetyltransferase (CAT) coding sequence is followed by varying segments of the TNF 3′-UTR, it was possible to demonstrate that a downstream sequence present in the TNF-α mRNA is sufficient to induce a more than 200-fold increase in CAT synthesis in response to activation by endotoxin. The induction of CAT activity is due to a marked enhancement of translational efficiency, rather than to change in cytoplasmic mRNA concentration (5). The Japanese flounder TNF gene has two complete TTATTTAT sequences and some incomplete sequences of this type in the 3′-UTR. This suggests that the expression of Japanese flounder TNF is regulated at the translation level.

The transcription of the mammalian TNF-α gene is also regulated by various inducers, such as viruses, LPS, and PMA (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). In contrast, the TNF-β gene is not produced in response to LPS (2). The human TNF-α gene contains NF-κB-binding motifs. The NF-κB is thought to have a roll in constitutive high level baseline expression of the human TNF-α gene (24). Recently, a new regulation mechanism for TNF-α gene expression involving LPS-induced transcription factor has been reported (35). There are some potential NF-κB-binding motifs in the 5′ upstream region of the Japanese flounder TNF gene (Fig. 4). Interestingly, these regions are close to the simple dinucleotide repeat sequences (Fig. 4). These sequences might have a role in controlling the expression of the Japanese flounder TNF gene. Expression of the Japanese flounder TNF gene was induced by LPS, Con A, and PMA, but the gene was not expressed in the cell culture medium (Fig. 6). Interestingly, the expression level of the TNF gene in Japanese flounder PBLs after a 6-h incubation with LPS, Con A, or PMA was lower than it was after 1 h and 3 h of incubation. The highest induction were observed after a 1- and 3-h incubation with PMA (Fig. 6). The expression pathway of the Japanese flounder TNF gene might respond quickly to some inducers of TNF gene transcription. These results indicate that the Japanese flounder TNF gene, like the human TNF-α gene, is regulated by various inducers, such as LPS, PMA, and Con A. The present results also indicate that the Japanese flounder TNF is more like mammalian TNF-α than mammalian lymphotoxin-α.

In conclusion, all of the characteristics of the cloned gene in this study, i.e., its gene structure, amino acid sequence, and expression pattern, are similar to those of human TNF-α, and thus indicate that it is TNF-α. This is the first report of the TNF-α cDNA and gene from a nonmammalian vertebrate.

1

This work was supported in part by a grant from the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS-RFTF97L00902).

3

Abbreviations used in this paper: BAC, bacterial artificial chromosome; UTR, untranslated region.

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