*Biology Department and Graduate Program in Organismal and Evolutionary Biology, University of Massachusetts;
and
Department of Ecology and Evolution, University of Chicago
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Abstract |
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Introduction |
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Evolutionarily, humans, apes, and Old World monkeys exhibit several unique aspects of their growth hormone (GH), a polypeptide of approximately 190 amino acids in its mature form. Amino acid sequences of growth hormone are available from a number of mammals, including rodents, artiodactyls (i.e., cows, sheep), carnivores, and primates, and from these sequences a consensus, or "ancestral," growth hormone can be inferred (Wallis 1994
). Most mammalian GHs differ from this consensus by 04 substitutions. In contrast, human and rhesus monkey GHs differ from the consensus by 62 and 64 substitutions, respectively. These values are over three times as high as those (1819 substitutions) observed for artiodactyls, the second most divergent set of species. This increased number of amino acid substitutions is correlated with a change in the interaction of the hormone with the growth hormone receptor (GHR). Human GH injected into a nonprimate is biologically active and will stimulate growth. However, mouse or pig GH is completely ineffective in humans. Indeed, in vitro studies have demonstrated that bovine GH is 3,000-fold less potent than is human GH in competitive binding assays for the human GHR (Souza et al. 1995
). Another unique aspect of growth hormone evolution in higher primates is that humans and rhesus monkeys have five tandemly arrayed copies of GH-related genes rather than one, as is found in nonprimate mammals. One of these loci (GH) is expressed by the pituitary gland, while the remaining four are expressed only in the placenta (Chen et al. 1989
; Golos et al. 1993
).
These unique features of higher primate GH evolution raise many questions. (1) When did the duplications which gave rise to multiple GH-like genes occur during primate evolution? (2) Is the increased protein divergence a common feature of all primates, or is it restricted to a subset of primates? (3) Have amino acid substitution rates been high throughout primate evolution, or did most of the changes occur over only a short period of time? (4) What amino acid substitutions are responsible for the species specificity of human GH? Wallis (1996)
demonstrated several cases of rate variation in vertebrate GH evolution and determined that most of the amino acid changes in primates occurred before humans and macaques diverged. In this paper, we demonstrate that Galago senegalensis, a prosimian, has a single GH gene, and we show that the acceleration of the amino acid substitution rate occurred after the separation of the higher primate lineage from the galago lineage. Indeed, the Galago GH sequence is very similar to the "ancestral" mammalian GH and shares none of the amino acid substitutions present in human GH which are expected to affect GHR binding.
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Materials and Methods |
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Southern Hybridization
Fifteen to 20 micrograms of genomic O. crassicaudatus DNA was digested with EcoRI, Pst I, BamH and Sau3A (all obtained from New England Biolabs), electrophoresed through a 0.8% agarose gel (2% for Sau3A digest) and transferred to a Zeta-Probe GT blotting membrane (Bio-Rad). A cloned PCR product obtained with primers 3F and 9R (see above) on genomic DNA of O. crassicaudatus was labelled with [-32P]-dCTP using a Random Primed DNA Labeling kit (Boehringer Mannheim) and used as a probe. Hybridization was performed at 65 degrees overnight in 6 ml of fluid (7% SDS, 0.5 M Na2HPO4, pH 7.2). The membrane was washed twice at 65 degrees in 50 ml of 5% SDS, 0.04 M Na2HPO4, pH 7.2, twice at 65 degrees and once at 65 degrees in 50 ml of 1% SDS, 0.04 M Na2HPO4, pH 7.2. The membrane was exposed to film for 4872 hours.
Sequence Analysis
The new sequence from Galago was aligned with sequences from GenBank (table 1
) using the program CLUSTAL W (Thompson, Higgins, and Gibson 1994
). Numbers of substitutions at nonsynonymous and synonymous sites were calculated by the method of Li (1993)
for the entire prohormone-coding region (218 codons in Galago), and relative-rate tests were performed according to Wu and Li (1985)
. Because the number of substitutions at twofold-degenerate sites was small, rate tests were performed only on nondegenerate and fourfold-degenerate sites. Numbers of nonsynonymous substitutions per site were apportioned among the branches of a user-defined tree by the Fitch-Margoliash method using the program Fitch in the PHYLIP, version 3.572c, package of programs (Felsenstein 1993
). This user-defined tree was also input into the program PAUP, version 3.1.1 (Swofford 1993
), and the ancestral GH nucleotide sequence at each node among primates, rodents, and artiodactyls was inferred by parsimony. Using this inferred sequence, the numbers of synonymous and nonsynonymous substitutions along each branch within these three orders were counted. At those sites where the ancestral state was ambiguous, the pathway which minimized the number of nonsynonymous substitutions was accepted. Fisher's exact test was used to test the equality of the ratio of nonsynonymous to synonymous substitutions in two areas of the tree: the combined human/macaque branches versus the branch preceding their split, and the combined cow/goat/sheep branches versus the branch preceding their split. The nonrandom clustering of amino acid replacements along the length of the GH protein in human GH was tested by a chi-square analysis, with the P value calculated according to a Monte Carlo procedure. For the chi-square test, the growth hormone protein was divided along its length into 10 segments of 19 amino acids. The tests were performed using the programs Fisher6 and Monte Carlo RxC (B. Engels, personal communication).
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Results and Discussion |
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Single GH locus in bushbabies
Four digestions of O. crassicaudatus genomic DNA each produced a single band in a Southern hybridization (fig. 1
). This indicates that there is only a single GH-like locus in bushbabies. Additionally, we performed Southern blot experiment with O. crassicaudatus DNA digested with Sau3A (fig. 1
). According to the inferred restriction map this frequently cutting enzyme has two recognition sites within the region of the O. crassicaudatus GH gene corresponding to the probe we used and, therefore, we expected the Southern blot experiment to yield three bands one of which should correspond to a 605 bp GH fragment between Sau3A sites. Indeed, as shown in figure 1
, there are three bands one of which is approximately 605 bp in length. Thus, the duplications which gave rise to five GH-like loci in human and macaque occurred after the higher primate line of descent branched off from the bushbaby lineage. On this basis, it is reasonable to assume that the single locus sequenced in this study is the ortholog of the pituitarily expressed locus in human and macaque.
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Divergence of Primate GH at Functionally Significant Sites
Nonprimate GH does not function in humans, indicating that significant amino acid replacements occurred in the GH/GHR complex along the human lineage. It is therefore of interest to examine in humans those amino acid substitutions which occur at sites known to be important for GH/GHR interaction and to determine which of these changes may be shared with Galago, whose divergence precedes the increase in amino acid replacement rate.
Of the 11 residues of GH that form salt bridges or hydrogen bonds with the GHR (Devos, Ultsch, and Kossiakoff 1992
), four (residues 19, 41, 167, and 171) vary in humans and macaques (table 4
). Additionally, residue 64, which is not a ligand for GHR but is known to have a major effect on binding energy (Wells 1996
), varies only in humans and macaques. In each case, Galago exhibits the ancestral amino acid state. Site 167 is Arg in both higher primates and ruminants and thus seems unlikely to be a major determinant of the species specificity of human GH. Residue 171 of GH interacts with site 43 of GHR, and it has been demonstrated that the replacement of His with Asp at site 171, accompanied by the Leu-to-Arg change at site 43, accounts for most, but not all, of the change in binding affinity of nonhuman GH for the human receptor (Souza et al. 1995
; Behncken et al. 1997
). The effect of the remaining three residues in table 4
have not been examined experimentally in terms of species specificity, and this would be an interesting topic for further study. On the basis of its protein sequence conservation and lack of variability at sites important for GHR interaction, Galago GH probably behaves like nonprimate GHs physiologically.
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Paralogy of GH-like Genes and Timing of Duplications
The results reported here are based on the orthology of the single GH gene of Galago with the pituitary-expressed GH-N of humans and rhesus monkeys. There is strong evidence from the distribution of paralogous Alu elements (which are restricted to primates) that all the duplications giving rise to multiple GH-like genes in higher primates occurred after Galago diverged. The published human growth hormone locus contains over 40 Alu elements, most of which are related to each other via the gene duplications that gave rise to the five genes, rather than by unique transposition events (Toda and Tomita 1997
). Based on both positional homology and phylogenetic analysis, it is clear that two AluSg elements inserted before the duplications of GH-N that gave rise to the other four GH-like genes. The approximate age of the AluSg family is 31 Myr (Kapitonov and Jurka 1996
), placing an upper boundary on the age of all the gene duplications somewhere in the Oligocene, at least 20 Myr after Galago diverged from the higher primate line of descent. Correspondingly, all human and rhesus monkey GH-like genes form a single cluster in phylogenetic analysis (results not shown).
At any rate, the results of this study are not altered regardless of which higher primate gene is chosen for comparison with Galago and nonprimates. Galago GH exhibits 61 and 63 amino acid differences from human and rhesus monkey GH-N, respectively, but 6677 differences from the placental GH-like hormones of humans and rhesus monkeys. Additionally, human GH-like genes appear to be undergoing concerted evolution via gene conversion (Giordano et al. 1997
) that artificially increases their similarity, and the same may be true of rhesus monkey genes.
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Acknowledgements |
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Footnotes |
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1 Keywords: growth hormone
primates
rate acceleration
gene duplication
Galago
2 Address for correspondence and reprints: Wen-Hsiung Li, Department of Ecology and Evolution, University of Chicago, 1101 East 57th Street, Chicago, Illinois 60637. E-mail: whli{at}uchicago.edu
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