Genetics of Adaptation Graduate Seminar
Author: Elizabeth Brown
Woolly mammoths are iconic animals in North American and Eurasian lore. Paintings of these amazing animals line caves dating back over 10,000 years, and their ivory has been used to create figurines as long as 35,000 years ago. Woolly mammoths, now extinct, lived during the last ice age and were well adapted to cold environments. Some of these adaptive features include small ears and tail, and thick fur covering their body. Although woolly mammoths displayed these features, because they are extinct one would think investigating the genetic basis of cold adaptation should be impossible. However, the use of ‘ancient DNA’ has enabled DNA analyses to be performed on mummified tissues and skeletal material dating back up to 1 million years.
The woolly mammoth belongs to the Elephantid lineage, which includes the African and Asian elephants. This lineage originated in Africa approximately seven million years ago and is well described. Woolly mammoths, unlike the African and Asian elephants, moved from a tropical to an Arctic environment, which facilitated possible physiological specializations to cold climates.
One such adaptation to a cold environment is the differential binding and unloading of oxygen, a requirement for many metabolic processes, to locations throughout the body. Hemoglobin is the protein responsible for such transport. This protein is a four-part molecule located in the blood. In most mammals the process of oxygen unloading is temperature-dependent, such that small increases in temperature can cause large decreases in the affinity of hemoglobin for oxygen. This allows for increased oxygen unloading to warmer exercising muscles that have increased metabolic requirements.
In Substitutions in woolly mammoth hemoglobin confer biochemical properties adaptive for cold tolerance, Campbell et al. explain the methodology used to conclude that the hemoglobin of woolly mammoths possess unique attributes that have enabled them to thrive in cold environments (Nature Genetics 2010). They first extracted DNA from a 43,000 year old Siberian mammoth specimen. Despite the obvious advantage of sequencing ancient DNA in the analysis of genes of extinct organisms, this is a very difficult and sensitive process. It is not uncommon for ancient DNA samples to be degraded and/or contaminated with modern DNA. So, after careful handling, the DNA sequence of the area encompassing the α-globin and β/δ globin genes, genes coding for any one of four parts of the hemoglobin molecule, was located and then compared to blood samples obtained from both African and Asian elephants.
The authors found three amino acid substitutions located within the β/δ fusion gene of the woolly mammoth. A substitution is a type of mutation in which single amino acid is altered. Two of these mutations occur on the surface of the protein, whereas the last is located at the interface between two of its subunits. Since each subunit slides and rotates in conjunction with the other parts of the protein, the location of the latter mutation causes extensive conformational modifications to the complex as a whole.
This same mutation is also found in Rush hemoglobin, a mutant human hemoglobin protein. Since the woolly mammoth is extinct, the true function of this mutation can only be inferred from extant species with similar mutations. In humans, this mutated protein has been found to cause hemolytic anemia, a disease in which red blood cells are abnormally broken down. Within the Rush protein, two additional chlorine (Cl-) binding sites are found. Cl- negatively regulates the affinity of hemoglobin for oxygen, so when more Cl- atoms bind to hemoglobin, the attraction between hemoglobin and oxygen decrease and when fewer Cl- atoms bind, attraction increases. When higher numbers of Cl- atoms can bind, changes in temperature have a smaller effect on hemoglobin-oxygen affinity. This would be energetically advantageous in cold-tolerant mammals, ensuring a consistent delivery of oxygen to the extremities, where the temperature of these regions may significantly differ from the core body temperature.
The authors wanted to determine whether the functional characteristics of the human Rush protein are in fact similar to that of the woolly mammoth hemoglobin protein. To do this, they inserted woolly mammoth-specific substitutions into an Asian elephant hemoglobin protein and then compared it to a protein without the mutations. Next, they compared the functions of these proteins at multiple temperatures, and striking functional differences were observed. Interestingly, the woolly mammoth hemoglobin possessed higher oxygen affinity than the Asian elephant hemoglobin at all temperatures tested. This is shown in the upper figure at 37°C. In the absence of Cl- atoms, woolly mammoth hemoglobin become saturated with oxygen much sooner than those of the Asian elephant. This would drastically impair the ability of hemoglobin to distribute oxygen throughout the body. However, upon the addition of Cl- atoms, there was a significantly reduced effect of temperature compared to that of Asian elephant hemoglobin. This is shown in the figure below, in which there are substantial differences between proteins without any molecules that alter protein function compared to those that have Cl- or other effector molecules. These results, in conjunction with those of the Rush protein, confer the adaptive role of this specific substitution for increased cold tolerance.
Campbell et al. also determined that the location of the substitution resulting in increased Cl- binding is very specific. Changing the mutated amino acid to a structurally different and chemically distinct amino acid all produce radically increased oxygen affinities, however there was no decrease in the effect of temperature, a feature of woolly mammoth hemoglobin. This demonstrates that the functional properties of the hemoglobin molecule at this location are mediated by the size and specific chemistry of the substituted amino acid.
Since woolly mammoths have been extinct for approximately 10-12,000 years, the true adaptive significance of this single amino acid substitution cannot causally be tested. However, the physiological innovations that have evolved in the past are of fundamental importance in the study of evolutionary biology and can provide insight on functional traits in both past and present species.