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In most mammals pituitary GH is encoded by a single gene with no close relatives. However, in man the GH gene has been shown to be one of a cluster of five closely related genes, four of which are expressed in the placenta. Rhesus monkey also expresses at least five closely related GH-like genes, although the genomic organisation of these has not been fully reported. Here we describe the cloning and characterisation of GH-like genes in a new-world monkey, the marmoset (Callithrix jacchus). This species possesses a cluster of eight GH-like 'genes'. The gene at the 5' end of this cluster encodes pituitary GH and is similar to that encoding human GH. Five of the eight marmoset 'genes' are probably pseudogenes, since they include mutations which would prevent normal expression, including stop codons and small insertions/deletions that would change the reading frame. In one case a large part of a gene is deleted, and in another a large insertion is introduced into an exon. The remaining two marmoset genes are potentially expressible, as proteins with sequences substantially different (at 25-30% of all residues) from that of marmoset GH itself; whether and in which tissue(s) such expression actually occurs is not yet known. None of the marmoset genes is clearly equivalent to any of the human GH-like genes expressed in the placenta, and this and phylogenetic analysis suggest that the duplications that gave rise to the marmoset GH gene cluster occurred independently of those that gave rise to the corresponding cluster in man. Although it includes more 'genes', the marmoset cluster extends over a shorter region of chromosomal DNA (about 35 kb) than does the human GH gene cluster (about 50 kb).

A number of analogues of ovine growth hormone (GH), in which regions of the hormone had been deleted, were produced by site-directed mutagenesis, and characterised by radioimmunoassays and radioreceptor assays. These analogues were based on a previously described variant (oGH1) in which an 8-residue extension replaces the N-terminal alanine of pituitary-derived ovine GH. Three analogues with deletions near the N-terminus were studied, with shorter extensions of 7 or 1-2 residues (oGH14, oGH5) or with the N-terminal sequence Ala-Phe-Pro- of pituitary-derived ovine GH replaced by Thr-Met-Ile-Thr- (oGH11). These modifications had little effect on potency in radioimmunoassays based on a polyclonal antibody and five different monoclonal antibodies (MABs), or in a radioreceptor assay, indicating that the N-terminal sequence was not included in the epitope binding to any of the monoclonal antibodies, or a major epitope binding to the polyclonal antibody, or in receptor binding site 1. A variant in which residues 133-139 were deleted retained full binding to 4 of the 5 MABs, suggesting correct folding, but markedly reduced binding to MAB OA16, suggesting that the epitope for this MAB includes some or all of these residues. This variant also failed to displace about 35% of labelled hormone from the polyclonal antibody studied, suggesting that residues 133-139 may be involved in a major epitope for this antibody. This variant showed slightly lower receptor binding activity than ovine GH. Two other deletion variants - oGH1Delta33-46 (equivalent to the naturally occurring 20K variant of human GH) and oGH1Delta180-191 (lacking the C-terminal 12 residues) showed poor folding efficiency and solubility, and low binding to all MABs except OA15, which has a linear epitope. The results suggest that these variants were incorrectly folded, but interestingly they did retain some activity in the receptor-binding assay (respectively about 5% and 0.5% of the activity of ovine GH itself).

Pituitary growth hormone (GH), like several other protein hormones, shows an unusual episodic pattern of molecular evolution in which sustained bursts of rapid change are imposed on long periods of very slow evolution (near-stasis). A marked period of rapid change occurred in the evolution of GH in primates or a primate ancestor, and gave rise to the species specificity that is characteristic of human GH. We have defined more precisely the position of this burst by cloning and sequencing the GH genes for a prosimian, the slow loris (Nycticebus pygmaeus) and a New World monkey, marmoset (Callithrix jacchus). Slow loris GH is very similar in sequence to pig GH, demonstrating that the period of rapid change occurred during primate evolution, after the separation of lines leading to prosimians and higher primates. The putative marmoset GH is similar in sequence to human GH, demonstrating that the accelerated evolution occurred before divergence of New World monkeys and Old World monkeys/apes. The burst of change was confined largely to coding sequence for mature GH, and is not marked in other components of the gene sequence including signal peptide, 5' upstream region and introns. A number of factors support the idea that this episode of rapid change was due to positive adaptive selection. Thus (1) there is no apparent loss of function of GH in man compared with non-primates, (2) after the episode of rapid change the rate of evolution fell towards the slow basal level that is seen for most mammalian GHs, (3) the accelerated rate of substitution for the exons of the GH gene significantly exceeds that for introns, and (4) the amino acids contributing to the hydrophobic core of GH are strongly conserved when higher primate and other GH sequences are compared, and for coding sequences other than that coding for hydrophobic core residues the rate of substitution for non-synonymous sites (K(A)) is significantly greater than that for synonymous sites (K(S)). In slow loris, as in most non-primate mammals, there is no evidence for duplication of the GH gene, but in marmoset, as in rhesus monkey and man, the putative GH gene is one of a cluster of closely related genes.

In mammals the structure of pituitary GH is generally strongly conserved, reflecting a slow basal rate of molecular evolution. However, on a few occasions the rate has increased - markedly during the evolution of primates and artiodactyls, and to a small extent during the evolution of rodents and rabbit - giving rise to marked differences between GH sequences of these species. In order to extend knowledge of rodent GHs we have cloned and characterised part of the GH gene of the Eurasian mole rat (Spalax ehrenbergi) using genomic DNA and a PCR technique. The sequence of all of the coding region and 5' untranslated region (UTR), most of the 3' UTR and part of the promoter region is described. The overall organisation of the mole rat GH gene is similar to that of GH genes from other mammals. The proximal Pit-1 sequence in the gene promoter differs somewhat from that of rat or mouse. The deduced sequence for the mature GH from mole rat differs from that of pig GH (thought to be identical to the ancestral placental mammal GH sequence) at 7 residues and from rat, mouse and hamster GHs at 9 to 12 residues. Only one or two of these substitutions involve residues close to the receptor-binding sites of the hormone.