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OC Wallis and M Wallis

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).

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M. Wallis

Introduction

A new member of the growth hormone (GH)/prolactin family was characterized in 1984 (Linzer & Nathans, 1984), and named proliferin in recognition of its production by proliferating mouse fibroblasts. Since then there has been a remarkable proliferation of new GH- and prolactin-like proteins in what had seemed to be an old and stable family. Many of these proteins are produced in the placenta, which is now also recognized as a major source of proliferin itself, but a new pituitary hormone has been identified in fish, and possible homology of GH and prolactin with other cytokines is also now apparent. The purpose of this communication is to summarize current knowledge of the new proteins in this family and to assess their relationships to GH and prolactin.

The GH gene family in humans

In humans, the gene for GH (hGH-N) occurs as a member of a cluster of five related genes

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O. C. Wallis and M. Wallis

ABSTRACT

An Escherichia coli JM109 clone containing a plasmid, pOGHe101, based on pUC8 and the ovine GH (oGH) cDNA sequence, showed very high expression (up to 25% of total cell protein) of an oGH analogue (oGH1) after induction. oGH1 was found in the particulate fraction of induced bacteria, where electron-dense granules could be seen by electron microscopy. A simple method for the purification of oGH1 is described. The particulate fraction isolated from sonicated bacteria was dissolved in 6 M guanidinium chloride containing dithiothreitol. After threefold dilution the proteins were reoxidized by gentle stirring overnight in air. Soluble renatured protein, recovered after dialysis, was further purified by ion-exchange and gelfiltration chromatography. Purified oGH1 had an M r of 22 000, an isoelectric point of about 6·7 and an N-terminal sequence corresponding to that of oGH, with an extension of eight amino acids replacing the N-terminal alanine. oGH1 behaved similarly to authentic bovine GH in a radioimmunoassay, a radioreceptor assay and a weight-gain assay in hypophysectomized rats. Thus the renatured hormone appears to be correctly folded and the N-terminal extension has little or no effect on biological activity.

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A Lioupis, OC Wallis and M Wallis

In mammals the structure of pituitary GH is generally strongly conserved, indicating a slow basal rate of molecular evolution. However, on two occasions, during the evolution of primates and of artiodactyls, the rate of evolution has increased dramatically (25- to 50-fold) so that the sequences of human and ruminant GHs differ markedly from those of other mammalian GHs. In order to define further the burst of GH evolution that occurred in artiodactyls we have cloned and characterised the GH gene of red deer (Cervus elaphus) using genomic DNA and a polymerase chain reaction technique. The deduced sequence for the mature GH from red deer is identical to that of bovine GH, indicating that the burst of rapid evolution of GH that occurred in Artiodactyla must have been completed before the divergence of Cervidae and Bovidae and suggesting that the rate of evolution during this burst must have been greater than previously estimated. In other aspects (signal sequence, 5' and 3' sequences, introns and synonymous substitutions in the coding sequence) the red deer GH gene differs considerably from the GH genes of other ruminants. Differences between the signal peptide sequences of red deer and bovid GHs probably explain why N-terminal heterogeneity is seen in bovine, ovine and caprine GHs but not GH from red deer, pig or most other mammals.

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M Wallis, A Lioupis and OC Wallis

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AJ Sami, OC Wallis and M Wallis

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).

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OC Wallis, YP Zhang and M Wallis

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.

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M Wallis, D J Gwilliam and O C Wallis

ABSTRACT

125I-Labelled polypeptide hormones have been extremely valuable for radioimmunoassays, receptor-binding studies and investigation of the processing and metabolism of hormones. However, such externally labelled material has the disadvantage that addition of one or more iodine atoms may alter the properties of the polypeptide. Furthermore, for studies on hormone metabolism and processing, the label may become separated from the hormone or its main breakdown products. Use of internally labelled polypeptides produced by biosynthesis can avoid such problems, but previously such material has usually been of low specific radioactivity, and unsuitable for many purposes. Here we describe the development of a procedure for the production of an internally labelled ovine GH analogue (oGH1) using a plasmid produced by recombinant DNA methods and expression in Escherichia coli.

Bacteria were grown in medium containing a low sulphate concentration, and then incubated in medium containing 35SO4 2− as the sole sulphur source. Under these conditions, the bacteria incorporated 35S into proteins including GH. Purification of such material required considerable modification of previously described methods, because of the need to handle very small amounts of highly radioactive material. The bacteria were lysed using lysozyme, and inclusion bodies were solubilized using 6 m guanidinium chloride. [35S]oGH1 was renatured and then purified by gel filtration on Sephacryl S-100, followed by immunoaffinity chromatography and a second gel filtration step. Material prepared in this way had a specific radioactivity of 6–27 μCi/μg, and showed high 'bind-ability' to polyclonal and monoclonal antibodies and to receptors. 35S-Labelled material bound to receptors more effectively than 125I-labelled GH and showed improved stability. Such material appears to be well suited to receptor-binding studies and studies on the processing and metabolism of GH. The procedure developed should be applicable to other polypeptide hormones.

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A Lioupis, E Nevo and M Wallis

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.

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R. S. Boyd, K. P. Ray and M. Wallis

ABSTRACT

Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulate prolactin and GH release from ovine anterior pituitary cells cultured in vitro. Dopamine and somatostatin inhibit release of prolactin and GH respectively, after stimulation by these agents, but without effects on intracellular cyclic AMP concentrations. In each case the inhibitory effects were reversed by pre-treatment of cells with pertussis toxin, in a dose-related fashion (1–100 ng/ml), again without affecting cyclic AMP levels. The results suggest that the inhibitory effects of dopamine and somatostatin in this system are mediated by one or more pertussis toxin-sensitive G proteins, and that these act by a mechanism which does not involve inhibition of adenylate cyclase.