Characterisation of the GH gene cluster in a new-world monkey, the marmoset (Callithrix jacchus).

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).

Molecular evolution of growth hormone (GH) in Cetartiodactyla: cloning and characterization of the gene encoding GH from a primitive ruminant, the chevrotain (Tragulus javanicus).

In mammals the sequence of pituitary growth hormone (GH) is generally strongly conserved, indicating a slow basal rate of molecular evolution. However, on two occasions, during the evolution of primates and that of cetartiodactyls, 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. To define further the burst of GH evolution that occurred in cetartiodactyls, the GH gene of the chevrotain (Tragulus javanicus) has been cloned and characterized by use of genomic DNA and a polymerase chain reaction technique. Two very similar gene sequences, which probably reflect allelic variation, were isolated. The deduced sequence for the mature chevrotain GH differs from that of the bovine or red deer GH at only two to three residues, and phylogenetic analysis shows that the burst of rapid evolution of GH that occurred in the Cetartiodactyla must have been completed before the divergence of the Tragulidae and the advanced ruminants (Pecora). The rate of evolution during this burst must therefore have been greater than previously estimated. In other aspects (including signal sequence, 5' upstream sequence, and synonymous substitutions in the coding sequence), the chevrotain GH gene differs considerably from the GH genes of other ruminants and here there is no evidence for the period of accelerated evolution that is seen for GH itself.

Molecular evolution of GH in primates: characterisation of the GH genes from slow loris and marmoset defines an episode of rapid evolutionary change.

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.

Production and characterisation of deletion mutants of ovine growth hormone.

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).

Cloning and characterisation of the gene encoding red deer (Cervus elaphus) growth hormone: implications for the molecular evolution of growth hormone in artiodactyls.

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.

Cloning and characterisation of the rabbit growth hormone-encoding gene.

The gene encoding growth hormone (GH) has been cloned from a rabbit genomic library, and its sequence has been determined. The rabbit GH gene is similar to other mammalian GH, being comprised of five exons and four introns. As in rodents and artiodactyls, the rabbit GH occurs as a single gene, with no evidence for a cluster of GH-like genes, as is found in primates. The amino acid sequence of rabbit GH is similar to that of pig GH and other conserved mammalian GH, and, like these, differs markedly from the available sequences of ruminant and primate GH. This provides further support for the idea that, in mammals, GH show a slow underlying rate of evolution which has increased markedly on at least two occasions.

The effect of changes in nucleotide sequence coding for the N-terminus on expression levels of ovine growth hormone variants in Escherichia coli.

The expression levels of coding sequences for pituitary growth hormone, introduced into Escherichia coli by genetic manipulation techniques, vary markedly according to the precise sequence introduced. In order to understand the basis of this variation more fully, we have studied the relationship between the level of expression in E. coli of a series of ovine growth hormone variants and the nucleotide sequences coding for their N-terminal regions. Sequence variation resulted from the introduction of deletions, or site-directed mutations, into a plasmid containing the coding sequence for ovine growth hormone preceded by the initiation codon and 25 bases derived from beta-galactosidase or linker regions of plasmid pUC8. The expression levels of the variants varied from less than 0.01% to over 34% of the total cell protein, indicating that changes in the nucleotide sequence close to the initiation codon had a marked effect on expression level. The results of a comparison of closely related sequences in pairs of plasmids giving poor or good expression are consistent with the hypothesis that poor translation of growth hormone mRNAs is caused by the presence of secondary structures close to the initiation codon. Secondary structures are identified that appear to explain the variation in expression levels.

Preparation and characterization of a recombinant DNA-derived ovine growth hormone variant internally labelled with sulphur-35.

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 microCi/micrograms, 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and properties of a recombinant DNA-derived ovine growth hormone analogue (oGH1) expressed in Escherichia coli.

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 6M 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 gel-filtration chromatography. Purified oGH1 had an Mr 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.

Cloning, sequence and expression in Escherichia coli of cDNA for ovine pregrowth hormone.

cDNA prepared from mRNA from ovine anterior pituitary glands was cloned in Escherichia coli and the sequence of a clone encoding the full coding sequence of ovine pregrowth hormone (preGH) determined. The predicted sequence for ovine GH agrees with that determined previously on the protein, except that residue 99 is asparagine rather than aspartic acid. The cDNA sequence also accords with one of the two genomic sequences for the ovine GH gene that have been reported. Expression plasmids using trp and lac promoters were constructed which allowed expression at low levels of ovine preGH in E. coli, as detected by immunoblotting and immunoassay.

Production of plasmids giving high expression of recombinant DNA-derived ovine growth hormone variants in Escherichia coli.

A method for the production of plasmids giving different levels of expression of ovine growth hormone (oGH) variants in E. coli is described. The cDNA sequence coding for mature oGH was inserted into the multiple cloning site of plasmid pUC8 and random deletions were then introduced 3' to the initiation codon. Clones producing GH (with varying N-terminal extensions) were identified by immunological screening. Levels of expression of GH-related protein, measured by immunoassay or on SDS-polyacrylamide gels, varied from over 20% to less than 0.05% of total cell protein. The coding sequence of plasmid pOGHe101, giving very high expression of variant oGH1, was determined.

Stereochemistry of the rearrangement of 2-aminoethanol by ethanolamine ammonia-lyase.

(1R)-2-Amino[1-2H1]ethanol and (1S,2RS)-2-amino[1,2-2H2]ethanols have been synthesised by decarboxylation of (2S,3R)-[3-2H1]serine and (2S,3S)-[2,3-2H2]serine respectively. The stereochemical integrity of these labelled 2-aminoethanols has been ascertained from the 1H-NMR spectra of their N,O-dicamphanoyl derivatives. This assay has also been used to confirm that samples of (2R)- and (2S)-2-amino [2-2H1]ethanols prepared from (2R)- and (2S)-[2-2H1]glycines are stereochemically pure. Ethanolamine ammonia-lyase rearranges (1R)-2-amino[1-2H1]ethanol to acetaldehyde at approximately the same rate as it rearranges unlabelled 2-aminoethanol, whilst (1S,2RS)-2-amino[1,2-2H2]ethanol is rearranged at the same rate as the (1,1-2H2)-labelled substrate. The isotope effect is approximately kH/k2H = 8. The 2H-NMR spectra of the 3,5-dinitrobenzoates of the ethanol produced by reduction in situ of the acetaldehyde formed in the rearrangements show that the 1-2H1 label migrates in (1S,2RS)-2-amino-[1,2-2H2]ethanol and 2-amino[1,1-2H2]ethanol but not in (1R)-2-amino[1-2H1]ethanol. The above results indicate that the adenosylcobalamin-dependent ethanolamine ammonia-lyase catalyses the rearrangement of 2-aminoethanol with migration of the 1-pro-S-hydrogen atom.

The extents of formation of cobalt(II)-radical intermediates in the reactions with different substrates catalysed by the adenosylcobalamin-dependent enzyme ethanolamine ammonia-lyase.

1. The reactions of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, with the 'good' and 'relatively poor' substrates 2-aminoethanol and (S)-2-aminopropanol respectively, under conditions of saturation with substrate were investigated by rapid freezing in conjunction with electron paramagnetic resonance (e.p.r.) spectroscopy and by stopped-flow spectrophotometry. 2. In disagreement with earlier reports [Babior et al. (1972) J. Biol. Chem. 247, 4389-4392], it was found that the reaction of 2-aminoethanol gave an e.p.r. signal observed in rapid freezing experiments characteristic of a coupled Co(II)-free radical system. This signal was similar to, though not identical with, that obtained with (S)-2-aminopropanol. The steady-state level of the signal with 2-aminoethanol as substrate was 0.56 of that attained with (S)-2-aminopropanol. 3. The results of these e.p.r. experiments were shown to be consistent with stopped-flow data obtained under closely similar reaction conditions, the latter indicating a corresponding ratio of 0.64. The results also are consistent with those of a rapid wavelength scanning, stopped-flow spectrophotometric study [Hollaway et al. (1978) Eur. J. Biochem. 82, 143-154].

The number of functional active sites per molecule of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, as determined by a kinetic method.

1. A kinetic approach to the determination of the number of functional active sites per molecule of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, is described. 2. Time courses for formation and breakdown of a cob(II)alamin intermediate during reaction of the enzyme, fully saturated with adenosylcobalamin, with L-2-aminopropanol as substrate, were followed using a stopped-flow spectrophotometer under two conditions: (a) enzyme concentration much greater than that of substrate, (b) substrate concentration much greater than that of enzyme. 3. Results were analysed in terms of a three-step mechanism involving binding of substrate (k+1 step), cob(II)alamin formation (k+2 step) and cob(II)alamin breakdown (k+3 step). the kinetic scheme was shown to be sufficient to account for the observed time courses and rate constants of 80 s-1 (k+2) and 1.5 s-1 (k+3) were determined. 4. The number of active sites per enzyme molecule (n) was calculated from the kinetic data in three ways: (a) calculation from amplitude of absorbance measurement, (b) calculation from measurements of the values of rate constants and (c) analysis by computation of the kinetic data using the computer program FACSIMILE. A value for n close to 6 was calculated by each of these methods. This value is in disagreement with the literature value of about two sites per molecule but is consistent with the I6II6 subunit structure of the enzyme. 5. Kinetic analysis of data from experiments in which the adenosylcobalamin concentration was varied while substrate and enzyme concentrations remained constant showed that all the active sites function with identical rate constants. 6. The principle and mathematical basis of the kinetic method for determining the value of n is given as an Appendix.