Communication in Genomics and Proteomics
Cloning and characterisation of the GH gene from the common dolphin (Delphinus delphis)

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Abstract

The sequence of growth hormone (GH) is generally strongly conserved in mammals, but episodes of rapid change occurred during the evolution of primates and artiodactyls, when the rate of GH evolution apparently increased at least 50-fold. As a result, the sequences of human and ruminant GHs differ substantially from those of other non-primate GHs. Recent molecular studies have suggested that cetaceans are closely related to artiodactyls and may be deeply nested within the artiodactyl phylogenetic tree. To extend the knowledge of GH in Cetartiodactyla (Artiodactyla plus Cetacea), we have cloned and characterised a single GH gene from the common dolphin (Delphinus delphis), using genomic DNA and a polymerase chain reaction technique. As in other mammals, the dolphin GH gene comprises five exons and four introns. The deduced sequence for the mature dolphin GH differs from that of pig at two residues only, showing that the apparent burst of rapid evolution of GH occurred largely after the separation of cetaceans and ruminants.

Introduction

Growth hormone (GH), prolactin, and several related placental proteins share various structural and biological features, belong to the family of somatogenic and lactogenic hormones, and are thought to have arisen from a common ancestral gene by successive duplications (Goffin et al., 1996; Rand-Weaver et al., 1993). They are included in the cytokine/growth factor structural superfamily, members of which have a similar 3D structure comprising a four-helix bundle connected in an unusual `up–up–down–down' fashion (Abdel-Meguid et al., 1987; De Vos et al., 1992; Kossiakoff and de Vos, 1999). GH is secreted by the anterior pituitary gland in vertebrates. Its main action is promotion of somatic growth, but it also plays a role in various aspects of metabolism. These actions involve binding to a cell surface receptor, a member of the cytokine receptor superfamily. Receptor dimerization initiates signal transduction, which leads to activation of various intracellular pathways, particularly that involving the non-receptor tyrosine kinase, Jak2 (Thomas, 1998). In most non-primate mammals, GH appears to be encoded by a single gene, although some caprine ruminants have two GH genes (Wallis et al., 1998). In all mammals, the GH gene extends over 2–3 kb and comprises five exons split by four introns. In higher primates, the GH gene has expanded by a series of gene duplications to give a gene cluster (Chen et al., 1989).

In vertebrates, the molecular evolution of GH appears to have involved long periods of slow basal change, which were interrupted by bursts of rapid evolution. Two such bursts of accelerated evolution occurred during GH evolution in eutherian mammals, one during the evolution of artiodactyls and the other during the evolution of primates (Liu et al., 2001b; Wallis, 1981, Wallis, 1994, Wallis, 1996; Wallis et al., 2001). These periods of rapid change appear to have been due to adaptive natural selection, possibly involving changes in receptor-binding and biological properties (Wallis, 1996, Wallis, 1997; Wallis et al., 2001).

To define further the burst of GH evolution that occurred during artiodactyl evolution, GH genes from several species of this order have been cloned and characterised. The mature pituitary GH proteins of sheep and goat are identical and differ from that of ox at one residue. The mature GH protein from the red deer (Cervus elaphus) is identical to that of ox (Lioupis et al., 1997) and the sequence of GH from a primitive ruminant, the chevrotain (Tragulus javanicus), is very similar to that of ox (Wallis and Wallis, 2001). The sequences of these ruminant GHs differ from those of pig and alpaca at 18–19 residues. Thus, the episode of accelerated change in artiodactyl GH evolution must have occurred after divergence of camelids and pigs from ruminants, but before divergence of extant ruminant groups.

Recent molecular phylogenetic studies (e.g., Arnason et al., 2000; Gatesy et al., 1996; Graur and Higgins, 1994; Liu et al., 2001a; Madsen et al., 2001; Murphy et al., 2001; Nikaido et al., 1999; Shimamura et al., 1997; Smith et al., 1996) have suggested that cetaceans are nested within the artiodactyl phylogenetic tree; such an arrangement is not in agreement with paleontological evidence, though recent fossils support the close relationship between artiodactyls and cetaceans (Thewissen et al., 2001). The term Cetartiodactyla has been suggested for the clade combining Artiodactyla and Cetacea (Montgelard et al., 1997). To assess the significance of the burst of rapid evolution of GH in Cetartiodactyla, it is clearly important to consider GH from Cetacea. GH (protein) sequences have been reported from two cetacean species, fin whale (Tsubokawa and Kawauchi, 1985) and sei whale (Pankov et al., 1982), and prove to differ markedly, but a cetacean GH gene has not been studied previously. Here, we describe the cloning and characterisation of the GH gene of the common dolphin, Delphinus delphis.

Section snippets

Preparation of genomic DNA

Genomic DNA was isolated from liver of a female common dolphin, D. delphis (tissue provided by Dr. Paul Jepson, Institute of Zoology, London), using the SDS–proteinase K method of Towner (1991). The DNA obtained ran as a single band of high molecular weight on 1% agarose gel.

Polymerase chain reaction (PCR)

Two sets of oligonucleotide primers were used for PCR. The sense and the antisense primers were based on the conserved sequences of bovine, ovine, and goat GH genes at positions indicated in Fig. 1. The sense primer common

The dolphin GH gene

Two pairs of PCR primers designed on the basis of known sequences for artiodactyl GH genes allowed amplification of fragments of dolphin genomic DNA corresponding to sizes expected for the GH gene (1749 and 1900 bp, Fig. 1). The amplified DNA was cloned and two clones containing the larger fragment (prepared in two separate PCRs) were subjected to DNA sequencing. Sequencing was hindered by the presence of secondary structure, a problem which was overcome by analysing the products of SacII

Acknowledgments

We thank Paul Jepson for kindly providing us with a sample of dolphin liver, Mrs E. Korneeva for carrying out the automatic DNA sequencing, and the BBSRC for a studentship to Z.M.

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