Friday, March 25, 2011

Of Mice and Mountains

If you have ever ascended Mount Everest, or even visited a city of high elevation, then you are personally aware of the physical discomforts, such as chest constriction, labored breathing and dizziness, which can result upon entering a high altitude, hypoxic (low levels of oxygen) environment.  We all know that oxygen is required for life (with the exception of those extremeophiles living deep underwater or in hot geysers somewhere).  Going only a few minutes without it can lead to permanent brain damage in newborn babies and adult humans alike.  But what about in environments with low levels of oxygen? What happens on a biochemical level to allow organisms to persist in such alpine environments?  Well, Storz et al. (2008) have recently identified specific amino acid changes in hemoglobin genes that account for differences in high and low altitude adaptation in deer mice populations.  Before delving further into this research, however, some background information is warranted: first, considering hemoglobin function in most vertebrates, and second, summarizing what is known to date about high altitude adaptations.

In ideal conditions, that is, at sea level, oxygen concentration is approximately 21%, which, in healthy individuals, saturates hemoglobin (the oxygen-binding protein found in red blood cells). Hemoglobin transports oxygen and other gases from the lungs to various tissues throughout the body, where the oxygen is then released for cell processes.  Hemoglobin in turn carries carbon dioxide, a waste product, back to the lungs for expulsion.  At sea level, our saturated hemoglobin molecules can transport the maximum amount of oxygen throughout the body and cellular mechanisms can function ideally.  This does not remain true when we ascend to higher elevations, where less oxygen is available. Eventually, we may acclimatize to these high altitude conditions, but what about organisms that permanently inhabit such an environment? Various species of plants, animals and microbes inhabit and thrive in environments thousands of feet above sea level. Adaptations must exist to allow them to persist in these conditions. Clearly, something must be different between sea level-dwelling creatures and alpine ones.

Hemoglobin represents a likely source of variation underlying altitude adaptations to low and high elevations as it is directly linked to oxygen transportation and utilization.  This idea was extensively investigated in the 1970s and 1980s by L.R.G. Snyder and M.A. Chappell, who studied hemoglobin biochemistry and genetics. From these efforts, we now know that the variations in blood chemistry at different elevations are due to a complex hemoglobin polymorphism (more than one form of the same thing). Specifically, Chappell and colleagues linked differences in blood oxygen affinity (the ability of oxygen to bind to hemoglobin) with variation at two gene duplicates that code for a hemoglobin subunit.  Both gene duplicates are polymorphic for two main classes of protein alleles (different forms of a gene), which we can call “A” and “B.”  The three combinations of these two allele types correspond to different blood oxygen affinities: mice with two “A” alleles exhibit a high oxygen affinity, “BB” mice exhibit a low affinity, and mice with both “A” and “B” (known as a double heterozygote) have an intermediate affinity. Additionally, mice with the high affinity genotype (combination of alleles for a certain characteristic) performed best at high elevations under hypoxic conditions, but poorly at sea level; and vice versa for mice with the low affinity genotype.  Mice with the heterozygote genotype were intermediate in oxygen affinity and performance. So we know that specific hemoglobin genes differ between high and low altitude populations, and that these differences confer fitness advantages: in this case, variation in the physiological performance of the mice. With this extensive empirical background and modern molecular genetic techniques, researchers are now address the specific molecular changes responsible for these physiological adaptations to high altitude environments.

In a 2008 paper published in PLoS Genetics, Jay Storz et al. investigate just that: the genetic mechanisms of alpine adaptation in the wide-ranging deer mouse, Peromyscus maniculatus.  The deer mouse is an excellent species in which to study high altitude adaptation, since it has the widest vertical range of any mammal in North America, spanning from below sea level (Death Valley, CA) to alpine environments higher than 4,300 meters.  At these extreme heights, the partial pressure of oxygen (pressure exerted by oxygen alone) is only 55% of that at sea level; thus the deer mouse experiences vastly different oxygen environments. In this paper, the authors aim 1) to identify the specific genetic mutation responsible for hemoglobin divergence across varying altitudes and 2) to test for evidence of diversifying selection (different traits favored in different environments) between high and low altitude populations.

To do this, the authors collected 41 mice from three areas along an altitudinal transect spanning nearly 4,000 vertical meters and across a linear distance of 547 km, from the lowlands of Kansas to the mountains of Colorado. From the mouse blood, they cloned copies of adult α-globin (one of the protein parts of hemoglobin), and by examining amino acid (organic building blocks of proteins) variation, were able to identify six amino acid substitutions (a swap of one amino acid for another) that distinguish the two gene duplicates discovered in the earlier research mentioned above. Hemoglobin, as a protein, is composed of a linear sequence of amino acids, which, if altered, may affect the properties of the molecule, including oxygen-binding affinity. Of the six substitutions, the authors identified one of significant functional consequence, which is referred to as 58(E7)HisàGln, but we will just call it E7 (His and Gln represent two distinct amino acids). In all mammals studied to date, E7 serves a critical role in iron-oxygen binding in hemoglobin. In humans, this substitution results in increased oxygen affinity in low oxygen environments.

The authors subsequently propose that, being found in both high and low altitude environments, deer mice have BOTH E7 forms (one with His and one with Gln) present in their blood, each with a unique oxygen binding affinity, and some mechanism to regulate the ratio of one E7 form to the other in response to metabolic demand. Why is this so interesting? Well, the possession of multiple hemoglobin forms has been documented in only one other mammal, the Yak, which lives high in the Tibetan Plateau.  Also, these different forms of the same protein represent functional divergence (genes duplicate and then diverge in function), rather than the concerted evolution (pair of genes evolves in concert) that was demonstrated in other mammal systems. This alone is pretty interesting, but the authors also wanted to test for evidence of the predicted mode of selection, diversifying selection, responsible for this difference. 
To do this, the allele frequencies at the two genes were compared between high and low altitude individuals; significant polymorphisms were observed that fit with a model of diversifying selection.  Continuing down the road of diversifying selection, the authors also found a significant excess of linkage disequilibrium (when alleles group in a nonrandom manner), and higher levels of altitudinal differentiation in the two genes of interest than in a group of unlinked nuclear loci (locus=gene site) that were used as a neutral comparison.  Both findings are consistent with a model of diversifying selection.  The authors then identified five replacement polymorphisms based on two things: 1) a high value of site-specific differentiation (between low and high elevations) and 2) a high frequency of the derived allele at high elevations. Of these five candidates, the amino acid substitution 64(E13) AspàGly (E13 for our sake) was singled out and predicted to increase oxygen-binding affinity; in fact, this is the case in human hemoglobin (The E13 in deer mice is shown in the image to the right). In one last hoorah for diversifying selection, the authors found a high level of amino acid divergence between the two classes of alleles, thus supporting the maintenance of two functionally distinct protein alleles as a long-term balanced polymorphism (AKA two distinct groups continue to exist).

So what is the take-home message here?  Overall, the authors were able to pinpoint the specific amino acid changes responsible for high altitude hemoglobin adaptation.  The authors confirmed the E7 substitution as responsible for differences in the two gene duplicates, but also revealed a series of mutations, one of particular interest (E13), that are responsible for allelic differences in oxygen affinity between high and low altitude populations of deer mice. Lastly, the authors determined that the distinct alleles are maintained via diversifying selection between high and low elevation populations. Who knows, in the distant future perhaps energy drinks derived from mouse-hemoglobin will allow mountaineers to climb just a bit higher…

The main paper:
Storz, J. F. et al. The molecular basis of high-altitude adaptation in deer mice. PLoS Genet. 3, e45 (2007).

The others:
Snyder LRG (1978) Genetics of hemoglobin in the deer mouse, Peromyscus maniculatus. I. Multiple α- and β-globin structural loci. Genetics 89: 511–530.
Snyder LRG (1978) Genetics of hemoglobin in the deer mouse, Peromyscus maniculatus. II. Multiple alleles at regulatory loci. Genetics 89: 531–550.
Snyder LRG (1980) Closely linked alpha-chain hemoglobin loci in Peromyscus maniculatus and other animals: Speculations on the evolution of duplicate loci. Evolution 34: 1077–1098.
Snyder LRG (1981) Deer mouse hemoglobins: Is there genetic adaptation to high altitude? Bioscience 31: 299–304.
Snyder LRG (1985) Low P50 in deer mice native to high altitude. J Appl Physiol 58: 193–199.
Snyder LRG, Born S, Lechner AJ (1982) Blood oxygen affinity in high- and low-altitude populations of the deer mouse. Respir Physiol 48: 89–105.
Chappell MA, Snyder LRG (1984) Biochemical and physiological correlates of deer mouse α chain hemoglobin polymorphisms. Proc Natl Acad Sci U S A 81: 5484–5488.
Chappell MA, Hayes JP, Snyder LRG (1988) Hemoglobin polymorphisms in deer mice (Peromyscus maniculatus): Physiology of beta-globin variants and alpha-globin recombinants. Evolution 42: 681–688.
Higgs DR, Vickers MA, Wilkie AOM, Pretorius I-M, Jarman AP, et al. (1989) A review of the molecular genetics of the human α-globin gene cluster. Blood 73: 1081–1104.

Second Mouse:
Mighty mouse:

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