Molecular Cloning and Sequence Analysis of Growth Hormone Cdna of Neotropical Freshwater Fish Pacu (piaractus Mesopotamicus)

RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH) cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank. Growth hormone (GH) is a protein of approximately 22 kDa secreted from the anterior pituitary gland. It plays important roles in the regulation of growth and development of vertebrates (Moore et al., 1982). In fishes, GH is also involved in different physiological processes, such as reproduction (Trudeau, 1997) and osmoregulation (Saka-moto et al., 1997). Genomic GH sequences and cDNAs from fishes have been characterized and used as a phylo-genetic marker for different taxonomic groups (Koren et al. Piaractus mesopotamicus (pacu) is an important South American freshwater fish, and a native species of the River Plate basin which comprises the Uruguay, Paraguay and Paraná Rivers. In the Paraguay basin, P. mesopotamicus is one of the most important fish for commercial and sports fishery. Moreover, it is the fourth most important species in terms of aquaculture production in Brazil (SEAP, 2006). Pacu belongs to the order Characiformes and, along with the orders Cypriniformes, Gymnotiformes and Siluriformes, is included in the Otophysi series (Fink and Fink 1981). At present, cDNA sequences from GH of nine species of Siluriformes and 27 species of Cypriniformes have been deposited in GenBank. In this report, we present the nucleotide sequence of the GH cDNA from P. mesopotamicus and compare the encoded polypeptide with GHs of other species of the Otophysi group. Our strategy to isolate the GH cDNA was to PCR amplify it from a cDNA library using primers whose sequence was based on the respective cDNA sequence of other members of the Otophysi.

Growth hormone (GH) is a protein of approximately 22 kDa secreted from the anterior pituitary gland.It plays important roles in the regulation of growth and development of vertebrates (Moore et al., 1982).In fishes, GH is also involved in different physiological processes, such as reproduction (Trudeau, 1997) and osmoregulation (Sakamoto et al., 1997).Genomic GH sequences and cDNAs from fishes have been characterized and used as a phylogenetic marker for different taxonomic groups (Koren et al., 1989;Chang et al., 1992;Lemaire et al., 1994;Marins et al., 2003;Clements et. al., 2004).
Piaractus mesopotamicus (pacu) is an important South American freshwater fish, and a native species of the River Plate basin which comprises the Uruguay, Paraguay and Paraná Rivers.In the Paraguay basin, P. mesopotamicus is one of the most important fish for commercial and sports fishery.Moreover, it is the fourth most important species in terms of aquaculture production in Brazil (SEAP, 2006).Pacu belongs to the order Characiformes and, along with the orders Cypriniformes, Gymnotiformes and Siluriformes, is included in the Otophysi series (Fink and Fink 1981).At present, cDNA sequences from GH of nine species of Siluriformes and 27 species of Cypriniformes have been deposited in GenBank.In this report, we present the nucleotide sequence of the GH cDNA from P. mesopotamicus and compare the encoded polypeptide with GHs of other species of the Otophysi group.
Our strategy to isolate the GH cDNA was to PCR amplify it from a cDNA library using primers whose sequence was based on the respective cDNA sequence of other members of the Otophysi.The designed primers GHP-F (5'-TCAGACAACCAGCGGCTCTTC-3') and GHP-R (5'-CAGGGTGCAGTTGGAATCCAG-3') corresponded to the 5' and 3' regions of mature GH of Cyprinus carpio.(Chiou et al., 1990; GenBank accession number M27000).For the construction of the cDNA library, the hypophysis of a 2 kg fish caught at a fish farm was removed, and total RNA was extracted from homogenized tissue with TRIZOL reagent (Invitrogen) following manufacturer's instructions.M-MULV reverse transcriptase (Invitrogen) was used for synthesis of first-strand cDNA.This reaction was carried out according to the manufacturer's instructions using approximately 2 μg of total RNA, 4 μL of RT buffer (5x), 2.5 μL of dNTPs (0.5 mM), 1 μL of RNase out (40 U/μL), 1 μL of Oligo dT12-18 (500 μg/mL), 1 μL of M-MLVRT.Following the cDNA synthesis, GH cDNA was amplified by PCR with the GHP-F and GHP-R primers.Amplification reactions were performed using 35 μL of cDNA, 0.3 UI of Taq Polymerase, 0.25 mM of each dNTPs, 0.3 μM of each primer, 2 mM MgCl 2. The PCR profile was as follows: 36 cycles of 96 °C for 1 min initial denaturation and 94 °C for 40 s denaturation, 60 °C for 1 min annealing, 72 °C for 2 min extension and a final extension at 72 °C for 5 min.A fragment of approximately 500 bp was cloned into pGEM-T Easy vector (Promega).Sequencing was carried out with universal primers (T7 and SP6) using BigDye Terminator Cycle Sequencing kit (PE Applied Biosystems) according to the protocols provided by the manufacturer.Electrophoresis of the purified samples was performed on an ABI 377 DNA Sequencer (PE Applied Biosystems).
The sequence obtained comprised 534 bp which translated in silico to a polypeptide of 178 amino acids.Alignment of the putative polypeptide of the P. mesopotamicus GH (pmGH) with other GHs from the Otophysi group (Siluriformes and Cypriniformes) shows that the GH amino acid sequence of Pacu and that of other species of Siluriformes lack ten amino acids present in the GH hormone of Cypriniformes (Figure 1).This feature is also found in the flounder Paralichthys olivaceus (Watahiki et al., 1989), a member of the superorder Acanthopterygii.
A neighbor-joining tree of GH nucleotide sequences of fishes from different taxonomic groups was inferred in the program MEGA version 3.1 (Kumar et al., 2004), using different models.In this analysis, the pmGH sequence clustered with GH sequences from other Otophysi fishes, grouping with Siluriformes as expected (Figure 2).
Homology analysis of the mature amino acid GH sequence by BLAST (Altschul et al., 1997) showed the highest identity values between pmGH and GH from species of the order Cypriniformes.Those identities ranged from 87% for Ictiobus bubalus (smallmouth buffalo fish) to 75% for Catla catla (catla).Although the amino acid alignment of pmGH and seven other Siluriformes species showed the lack of the same ten amino acids, the percentage of overall identity between the Characiformes P. mesopotamicus and the Siluriformes was lower than between the Characiformes P. mesopotamicus and the Cypriniformes species.
Appropriate folding and, consequently, the tertiary structure of GH requires the presence of paired Cys residues that participate in the formation of disulfide bonds (Paladini et al., 1983).Four Cys residues are found in GH of several mammalian and in many fish species (Abdel-Meguid et al., 1987;Sato et al., 1988;Yamaguchi et al., 1989).Clements et al. (2004) found two distinct GH cDNAs in the tetraploid small buffalofish (Ictiobus bubalus).One of these has the usual four Cys residues and the other GH has an additional Cys residue.This unpaired Cys residue is also found in other Otophysi species such as the goldfish Carassius auratus (Law et al., 1996)  carpio (Chang et al., 1992), the giant catfish Pangasianodon gigas (Lemaire et al., 1994) and the channel catfish Ictalurus punctatus (Tang et al., 1993).
Five conserved Cys residues were also found in the pacu GH (Cys 49 , Cys 113 , Cys 151 , Cys 168 and Cys 176 ).Based on the proposed structure of GH, four of these are likely to participate in the formation of disulfide bonds.As shown in Figure 1, these are in the same amino acid positions when compared to other Otophysi species.The additional Cys residue is located in position 113 in Siluriformes and Characiformes, and in position 123 in Cypriniformes.One possible role of this fifth Cys residue might be the formation of oligomeric complexes, which might affects the appropriate refolding of GH (Fine et al., 1993).
Glycosylation has been shown to occur in GH of salmon (Wagner et al., 1985).This post-translational modification may also take place in two N-linked glycosylation motifs (N-X-S/T) found in pacu GH (N 123 and N 175 ).These same motifs are also present in other Cypriniformes and Siluriformes species, as shown in Figure 1.Anathy et al. (2001) showed that for the siluriform species Heteropneustes fossilis (an Indian catfish), the first motif for N-linked glycosylation is Ala instead of Thr/Ser at position 147 in the mature GH peptide sequence, which eliminates this N-linked glycosylation motif.
A possible secondary sequence structure of pacu GH was predicted using NNPREDICT Protein Secondary Structure Prediction package.This analysis indicated the formation of four antiparallel α-helixes (data not shown).The first α-helix was positioned on amino acid residues 6 to 34, the second on amino acid residues 74 to 106, the third on amino acid residues 115 to 139, and the fourth α-helix on aminoacid residues 174 to 188 (Figure 1).Cunningham and Wells (1989) verified that the C-terminal helix of GH may act in receptor binding.This fourth α-helix may have important functional activity since there is a high similarity of this domain when pmGH and other fish GH are compared.
Figure 2 -Neighbor-joining tree generated from mature GH nucleotide sequences of several Telostomi species using the Kimura-two-parameter model.Numbers at branch points are bootstrap support percentages based on 5000 pseudoreplicates.Accession numbers for the nucleotides GH sequences are: AY375301 for Ictiobus bubalus, the smallmouth buffalo; M27000 for Cyprinus carpio, the common carp; DQ206404 for Piaractus mesopotamicus, the pacu, L27835 for Pangasianodon gigas, the giant catfish; AF530481 for Silurus meridionalis, the southern catfish; X14305 for Salmo salar, the Atlantic salmon; M24683 for Oncorhynchus mykiss, the rainbow trout; S78253 for Morone saxatilis, the striped sea-bass; M97765 for Oreochromis niloticus, the Nile tilapia; AF236091 for Odontesthes argentinensis, the Argentinean silverside; and S73969 for Amia calva, the bowfin.

Figure 1 -
Figure 1 -Comparison of Piaractus mesopotamicus (pmGH) amino acid sequence to two other siluriform species (pgGH of Pangasianodon gigas, the giant catfish and ipGH of Ictalurus punctatus, the channel catfish) and two cypriniform species (ccGH of Cyprinus carpio, the common carp and hmGH of Hypophthalamichthys molitrix, the silver carp).A ten amino acid deletion in pmGH, pgGH and ipGH is shown.The conserved Cysteine residues are underlined.Vertical boxes indicate potential N-linked glycosylation sites and horizontal boxes are the four predicted alpha helixes for mature GH of pacu.