O. Caryl Wallis

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.

Molecular Evolution of Prolactin in Primates

Pituitary prolactin, like growth hormone (GH) and several other protein hormones, shows an episodic pattern of molecular evolution in which sustained bursts of rapid change contrast with long periods of slow evolution. A period of rapid change occurred in the evolution of prolactin in primates, leading to marked sequence differences between human prolactin and that of nonprimate mammals. We have defined this burst more precisely by sequencing the coding regions of prolactin genes for a prosimian, the slow loris (Nycticebus pygmaeus), and a New World monkey, the marmoset (Callithrix jacchus). Slow loris prolactin is very similar in sequence to pig prolactin, so the episode of rapid change occurred during primate evolution, after the separation of lines leading to prosimians and higher primates. Marmoset prolactin is similar in sequence to human prolactin, so 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 (nonsynonymous sites) for mature prolactin and is not marked in other components of the gene sequence. This and the observations that (1) there was no apparent loss of function during the episode of rapid evolution, (2) the rate of evolution slowed toward the basal rate after this burst, and (3) the distribution of substitutions in the prolactin molecule is very uneven support the idea that this episode of rapid change was due to positive adaptive selection. In the slow loris and marmoset there is no evidence for duplication of the prolactin gene, and evidence from another New World monkey (Cebus albifrons) and from the chimpanzee and human genome sequences, suggests that this is the general position in primates, contrasting with the situation for GH genes. The chimpanzee prolactin sequence differs from that of human at two residues and comparison of human and chimpanzee prolactin gene sequences suggests that noncoding regions associated with regulating expression may be evolving differently from other noncoding regions.

Episodic molecular evolution of pituitary growth hormone in Cetartiodactyla.

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 substantially. As a result the sequences of higher primate and ruminant GHs differ markedly from sequences of other mammalian GHs. In order to increase knowledge of GH evolution in Cetartiodactyla (Artiodactyla plus Cetacea) we have cloned and characterized GH genes from camel (Camelus dromedarius), hippopotamus (Hippopotamus amphibius), and giraffe (Giraffa camelopardalis), using genomic DNA and a polymerase chain reaction technique. As in other mammals, these GH genes comprise five exons and four introns. Two very similar GH gene sequences (encoding identical proteins) were found in each of hippopotamus and giraffe. The deduced sequence for the mature hippopotamus GH is identical to that of dolphin, in accord with current ideas of a close relationship between Cetacea and Hippopotamidae. The sequence of camel GH is identical to that reported previously for alpaca GH. The sequence of giraffe GH is very similar to that of other ruminants but differs from that of nonruminant cetartiodactyls at about 18 residues. The results demonstrate that the apparent burst of rapid evolution of GH occurred largely after the separation of the line leading to ruminants from other cetartiodactyls.

Evolution of Growth Hormone in Primates: The GH Gene Clusters of the New World Monkeys Marmoset (Callithrix jacchus) and White-Fronted Capuchin (Cebus albifrons)

The GH gene cluster in marmoset, Callithrix jacchus, comprises eight GH-like genes and pseudogenes and appears to have arisen as a consequence of gene duplications occurring independently of those leading to the human GH gene cluster. We report here the complete sequence of the marmoset GH gene locus, including the intergenic regions and 5′ and 3′ flanking sequence, and a study of the multiple GH-like genes of an additional New World monkey (NWM), the white-fronted capuchin, Cebus albifrons. The marmoset sequence includes 945 nucleotides (nt) of 5′ flanking sequence and 1596 nt of 3′ flanking sequence that are “unique”; between these are eight repeat units, including the eight GH genes/pseudogenes. The breakpoints between these repeats are very similar, indicating a regular pattern of gene duplication. These breakpoints do not correspond to those found in the much less regular human GH gene cluster. This and phylogenetic analysis of the repeat units within the marmoset gene cluster strongly support the independent origin of these gene clusters, and the idea that the episode of rapid evolution that occurred during GH evolution in primates preceded the gene duplications. The marmoset GH gene cluster also differs from that of human in having fewer and more evenly distributed Alu sequences (a single pair in each repeat unit) and a “P-element” upstream of every gene/pseudogene. In human there is no P-element upstream of the gene encoding pituitary GH, and these elements have been implicated in placental expression of the other genes of the cluster. The GH gene clusters in marmoset and capuchin appear to have arisen as the consequence of a single-gene duplication event, but in capuchin there was then a remarkable expansion of the GH locus, giving at least 40 GH-like genes and pseudogenes. Thus even among NWMs the GH gene cluster is very variable.

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.

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.

Studies on the subunit structure of the adenosylcobalamin-dependent enzyme ethanolamine ammonia-lyase

The clostridlal adenosylcobalammdependent enzyme ethanolamme ammonia-lyase (EC 4.3.1.7) is reported to have mol. wt 520 000 [l] and to contam 2 active &es/molecule [2] On the basis of results obtained by ultracentrlfugatlon m the presence of guanidinium hydrochloride, it was proposed [l] that the native enzyme molecule 1s composed of 8-10 subunits of mol. wt 5 1 000. There was however some evidence of heterogeneity of subunits in these results. The present mvestlgatlon was mitlated to reevaluate the subunit structure of the enzyme usmg sodium dodecyl sulphate (SDS)-acrylamide gel electrophoresls [3] . This techmque is a very powerful tool for such studies and is capable of resolving species which cannot be dlstmguished by the procedure m [l] Two different subunits, mol. wt 5 1 000 and 36 000, were found m equimolar proportions mdlcatmg that one molecule of native enzyme 1s made up of 12 subunits, 6 of each type.

Cloning and characterisation of the GH gene from the common dolphin (Delphinus delphis).

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.

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.

Stereochemistry of the coenzyme B12-mediated rearrangement of 2-aminoethan-1-ol by ethanolamine ammonia-lyase

Reaction kinetics and 2H n.m.r. spectra of reaction products show independently that the coenzyme B12-mediated rearrangement of 2-aminoethan-1-ol by the enzyme ethanolamine ammonia-lyase proceeds with migration of the (1S)-hydrogen.