T t

Figure 2. Genomic organizations of the five GH and GH-related genes on human chromosome 17q22-24. The tissue in which each gene is expressed and the mRNA level are shown. (Adapted from Figure 1 of Barrera-Saldana, 1998.)

Genes

Tissues

Also, the divergence of the duplicate genes into the pituitary-expressed GH gene and the placenta-expressed GH-like genes supports the view that gene duplication allows the opportunity of tissue specialization (Force et al., 1999; Lynch et al., 2001).

We note that an episodic mode of molecular evolution has occurred following in the duplicated GH loci. Was it due to adaptive evolution? Ohta (1993) analyzed the ratio of nonsynonymous to synonymous substitutions among species and among duplicated copies of GH and GHR. She found that the ratio was significantly greater in the comparisons of GH-related genes within species than that between species, implying that there were more non-synonymous substitutions between the duplicated GH-related genes than between the same genes in different species. However, it is not clear whether the accelerated amino acid substitution was due to relaxation of functional constraints or positive Darwinian selection. In the next section, we describe the pattern of molecular evolution of the GH-N locus, which is expressed in the pituitary and has retained the same function of mammalian GH.

Episodic Molecular Evolution of GH in Mammals

It was noticed early on that there is a considerable rate variation in the evolution of GH among mammalian lineages (Gillespie, 1991; Wallis, 1981). Wallis (1994) extended the investigation to include 16 mammalian species belonging to 7 eutherian orders. He inferred the "ancestral" mammalian sequence (which is identical to the pig GH) and showed that human and rhesus monkey GH differ dramatically from the ancestral sequence, by 62 and 64 residues, respectively. This is in a stark contrast to the observation that the GH sequences from other mammalian species differ from the ancestral sequence by only a few amino acids (Wallis, 1994). The accelerated amino acid substitution observed in the evolution of GH in the hominoid lineage is among the fastest rate of molecular evolution of known mammalian proteins so far (see the compilation in Li, 1997). Wallis (1994) also noted that following a brief period of rapid "burst" of substitutions, the rate of evolution soon returned to the slow "basal" rate.

Subsequently, the DNA sequences of GHs from other primate species were obtained to pinpoint the time where the rapid evolution of GH began (Adkins et al., 2001; Liu et al., 2001; Wallis et al., 2001). The GHs from two prosimian species, the bushbaby (Adkins et al., 2001) and the slow loris (Wallis et al., 2001), show a slow rate of evolution comparable to that in other mammalian species, whereas three New World monkey species studied (Liu et al., 2001; Wallis et al., 2001) have evolved at a rate close to that in human GH. These observations concluded that the rapid "burst" of evolution of primate GHs occurred before the divergence of the New and Old World monkey lineages.

What is the basis of this rapid evolution is a puzzling question. A selectionist view is that it is due to positive Darwinian selection (Ohta, 1993; Wallis, 1994, 2001). If so, what is the functional basis of this phenomenon? The other extreme is a neutralist interpretation: for some reason, possibly related to the functional redundancy conferred by the gene duplication(s), the selective constraints upon the coding sequences of GH were lifted briefly before the split of Old World and New World monkey lineages, allowing rapid amino acid substitutions to occur.

Test of Positive Selection in DNA Sequences of Primate GHs and GHRs

Liu et al. (2001) investigated the above two hypotheses using DNA sequence data from the bushbaby, tarsier, squirrel monkey, rhesus monkey, and human. They conducted a maximum-likelihood analysis of the nonsynonymous rate/synonymous rate ratio (the dN/ ratio; see an earlier section). We also performed a similar analysis, adding the sequence data from the slow loris and marmoset, and obtained virtually the same result (Figure 3A). The free-ratio model, which assumes an independent dN/dS ratio for each branch, fitted the data significantly better than the one-ratio model, which assumed the same dN/dS ratio for all the species considered. Rapid evolution started after the divergence between the tarsier and simian lineages. The highest rate occurred in branch s, which connects the common ancestor of the simian and tarsier lineages and the common ancestor of simians. However, the estimated dN/dS ratio along this branch was still less than 1; so this analysis did not decisively detect positive selection.

Liu et al. (2001) also examined a subset of amino acid sites: the "functionally important" sites, which include sites that form salt bridges and hydrogen bonds with GHRs and sites that are involved in the interaction with the prolactin receptor (Cunningham and Wells, 1989, 1991; Somers et al., 1994). They showed that significantly more amino acid changes have accumulated at functionally important sites than the other sites in simian GHs. This suggests the presence of positive selection in the evolution of these sites. The GHR sequences from primate species were also analyzed using two mammalian out-groups: the pig and the rabbit (Liu et al., 2001). Again, the dN/dS ratios for the primate lineage and outgroups were significantly different by the likelihood ratio test, best described as episodic molecular evolution (see Figure3B). The ancestral branch of higher primates showed a significantly accelerated nonsynonymous substitution rate. The dN/dS ratio of GHR along the human lineage was even greater than 1.

As seen from these analyses, statistical analyses done without functional and structural information often do not produce clear-cut results. This is because positive selection may not continue for a very long time. For example, it is easy to imagine a period of rapid evolution driven by adaptive natural selection for some time in a specific lineage. If this period was short compared to the total length of the branches used in the analysis, then the conventional analyses will not have enough statistical power to detect such a period, unless there is an a priori assignment of branches to be considered. If the background rate of evolution was very low, due to strong purifying selection, then even in the presence of positive selection for the majority of sites, some sites will still be governed by the negative selective force and, therefore, reduce the overall dN/dS ratio. Even though several statistical models implementing more realistic likelihood assumptions have been devised, the power of such

2JÎ49 Galago

Slow loris

Human

Rhesus monkey

Squirrel monkey

Marmoset

Tarsier

Human

Baboon

Rhesus

46.8/106.7

Rabbit

Figure 3. (A) The maximum-likelihood estimates of the numbers of nonsynony-mous and synonymous substitutions in the GH genes under the "free-ratio" model. S indicates the branch of the simian common ancestor. Branches are proportional to the total numbers of substitutions. (B) The maximum-likelihood estimates of the numbers of nonsynonymous and synonymous substitutions in the GHR genes under the "free-ratio" model. (Adapted from Figure 2B of Liu et al., 2001.)

tests is usually low (see above). Therefore, an informative and complementary approach will be to directly investigate the functional consequences of specific substitutions that have occurred.

A Case Study of Functional Evolution—The Emergence of Species Specificity

In this section, we describe a series of studies in an effort to elucidate the underlying mechanism of specific amino acid substitutions that are responsible for the evolution of a functional trait. One of the major differences between primate GH and GHRs, from those of other mammalian species, is as follows: GHs from humans and rhesus monkey can bind and activate nonprimate GHRs, as well as primate GHRs. In contrast, nonprimate GHs have extremely low affinities for human GHR and, therefore, cannot stimulate growth in rhesus monkey (Carr and Freisen, 1976). This phenomenon is referred to as the "species specificity" of primate GHR (Carr and Freisen, 1976; Peterson and Brooks, 2000).

A simple hypothesis of the evolution of species specificity of primate GHR entails that an amino acid substitution specific for the primate lineage is responsible for the emergence of species specificity. With this idea, Souza et al. (1995) examined the GHR sequences from various species to find residues at the GH-GHR interfaces that differentiate primate GHRs from nonprimate GHRs. The interaction between Asp171 of GH and Arg43 of GHR caught their attention. Arg43 of GHR forms two hydrogen bonds with Asp171 and Thr175 of hGH (De Vos et al., 1992). While the Thr175 residue is conserved between primates and nonprimates, Asp 171 is not; nonprimates have His instead of Asp at the site equivalent to 171. As for GHR, nonprimates have Leu instead of Arg at position 43. These two are the only pair of complementary residues at the site1 interface that differ between primates and nonprimates. Based on this observation, Souza et al. (1995) proposed that the incompatibility between GH171 and GHR43 is a major determinant of species specificity between primate GHRs and nonprimate GHs.

Biochemists have tested the validity of this proposal directly by engineering changes in GH and GHR and performing binding experiments (Behncken et al., 1997; Gobius et al., 1992; Laird et al., 1991; Souza et al., 1995). For example, Souza et al. (1995) showed that a change from Leu43 to Arg43 in bovine or rat GHR greatly reduced their site1 affinity for bovine GH and rat GH but had little effect on their affinity to hGH, whereas, a change from Arg to Leu in hGHR enabled it to bind bGH (also see Laird et al., 1991). The results from these experiments showed that residues GH171His and GHR43Arg are mainly responsible for the incompatibility of these two proteins in distantly related species.

Some interesting questions arise. First, "When did the critical substitution that caused the species specificity occur during primate evolution?" Liu et al. (2001) and Wallis et al. (2001) showed that the change from His to Asp at site 171 of GH occurred in New and Old World monkeys. Liu et al. (2001) also found that the Arg substitution at site 43 of GHR occurred only in the Old World monkeys; the New World monkey species still have Leu—the "nonprimate" residue. This implies that the His171Asp change in GH preceded the Leu43Arg change in GHR, supporting the hypothesis based on stereochemical inferences that the His ^ Asp mutation in the hormone must have preceded the Leu ^ Arg mutation in the receptor (Behncken et al., 1997).

Second, since the GH of the New World monkeys has the "primate" form but the GHR still has the "nonprimate" form, can the GHR of New World monkeys bind to both primate and nonprimate GH? Recently, in vitro assays showed that the GHBP of squirrel monkey can indeed bind to both hGH and rat GH (Yi et al., 2002). Thus, the GHR of New World monkeys represent a transitional phase in the emergence of the species specificity of GHR in Old World primates.

Third, was the emergence of species specificity driven by positive selection? If the GHR43Leu ^ Arg substitution had a positive effect on the binding affinity between GH and GHR, then this should be measurable from the binding interactions of GH and a mutant encoding this new mutation. Yi et al. (2002) therefore engineered the GHR of the squirrel monkey to encode the Leu43Arg mutation and determined the binding affinity of this GHR toward the GH of the squirrel monkey, in comparison with that of the wildtype squirrel monkey GHR. The mutant GHR performed no better than the wild type GHR; in fact, the binding affinity was about twofold lower than that of the wild-type receptor. Therefore, the new mutation may have no selective advantage over the "nonprimate" residue Leu, at least for the squirrel monkey GHR. This suggests that the emergence of species specificity was due to random drift. A second line of evidence supporting this view is the distribution of the intermediary stage (GH171Asp - GHR43Leu). Liu et al. (2001) reported that the intermediary phase persists in both the squirrel monkey and the spider monkey—the two most diverged New World monkey species. These two species are estimated to have diverged about 25 MYA (Goodman et al., 1998). If the emergence of species specificity had a selective advantage, the intermediary state should have been short lived. From these two lines of evidence, they proposed that the emergence of species specificity, which is an example of dramatic functional difference between two variants of the same protein, may not be due to an adaptive evolution (Yi et al., 2002).

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