Hidden evolution: How the mammoth kept its cool

Last weekend I went to see an exhibition on mammoths in the Natural History Museum in London (unfortunately now closed). The descriptions and posters did a very good job of explaining our current knowledge of mammoths and how that knowledge had been gained. In particular, the curators were very keen to highlight the evolutionary relationships between different mammoth species and modern elephants.

A lot of the differences between the woolly mammoths and modern elephants are quickly apparent. The woolly mammoths are larger, hairier, and of course there are the tusks.

The tusks can be a big giveaway for a mammoth

The tusks can be a big giveaway for a mammoth

But one little sign caught my eye. It said that from the genetic material gathered from mammoth remains (we’ve recorded about 70% of their genome, so maybe not too far off until we can clone mammoths back into existence!), we can tell that woolly mammoths had a mutated form of haemoglobin that worked better in cold temperatures.

This haemoglobin discovery was published in 2010, so it isn’t anything new. But for me, this is evolution at its most interesting – not apes walking upright or fish breathing on land, but the tiny internal changes that adapt the body’s complex machinery.

Haemoglobin has a difficult balancing act to achieve – it needs to grab hold of oxygen from the lungs and it needs to release it around the body. If it holds the oxygen too tightly, the muscles can’t receive enough. If the haemoglobin holds on too weakly, it won’t be able to carry enough oxygen to where it is needed.

Elephant haemoglobin, like the human form, more readily releases oxygen at higher temperatures. That comes down to the energetics of breaking its hold, but it also means that harder-working (and therefore warm) muscles can take a greater share. This can be a serious issue if body-parts get too cold – they can suffocate to death. This is a danger for trekking around Siberia, even if you are large and hairy.

Researchers from the University of Manitoba, Canada, compared the haemoglobin genes of woolly mammoths to those of an Asian elephant from a nearby zoo. They noticed that there were four mutations that led to changes in the amino acid chains of the haemoglobin proteins.

Without any mammoth blood to hand, it can be very difficult to work out the effect of these changes. Fortunately, although we can’t resurrect a full mammoth now, we can insert the haemoglobin DNA into bacteria and resurrect one small part of them.

With the bacterium-produced mammoth haemoglobin, the researchers could run the tests needed to confirm that it was indeed adapted to the cold. Rather than the strong link to temperature seen in elephants, the mammoths’ haemoglobin can release oxygen at a similar level over a wide range of temperatures.

The structure of haemoglobin, with the blue regions showing where the mammoth mutations are

The structure of haemoglobin, with the blue regions showing where the mammoth mutations are (Credit: K Campbell et al, 2010)

The blue regions in the picture above show where the changes are in the structure of haemoglobin. These changes don’t appear to make any direct contact with the heme group (the part that carries the oxygen, shown in orange sticks), but they alter the shape of the protein subtly. This change in shape is enough to give mammoths a better chance of surviving freezing cold environments.

Mutations in haemoglobin are also seen in other animals adapted to cold environments, such as the arctic fox. The mutations seen in these animals are all different but lead to similar effects – an example of convergent evolution.

Just last year, the same researchers who made this discovery started work on an exciting new find: well-preserved mammoth blood. This could give them the chance to test the same idea on haemoglobin straight from a mammoth. There is even a (very small) chance that intact cells could be found in the sample, which would make cloning a full mammoth a lot easier.

So before long we might find out a lot more about these legendary creatures.

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2012 Nobel prize in chemistry: G protein-coupled receptors

I couldn’t let today pass without writing something about the Nobel prize. Before long I’ll write a full piece about G protein-coupled receptors (GPCRs) but that’s quite a big story. For now, I just want to stress their importance and their place in current research.

A cell requires isolation from the outside world; maintaining different concentrations of ions inside and outside the cell is a vital part of how it functions. GPCRs play a hugely important role in detecting signals sent from outside the cell and relaying them to the inside. They are known as trans-membrane proteins because they extend from outside the cell, through the membrane and into the inner region. This means that when they bind a ligand on the outside of the cell (e.g. a hormone), the resulting structural rearrangement can trigger a chain of signals through to the inner machinery of the cell.

Beta-2 adrenergic receptor

Beta-2 adrenoceptor, the first structure of a human GPCR. It was solved by Brian Kobilka’s lab in 2007. The red portion indicates the membrane-bound section and the green indicates the intra-cellular section [the extra-cellular region was not observed].

Trans-membrane proteins are poorly studied, largely because it is so difficult to keep them stable when studying them in the lab. The parts of the protein that are outside the membrane will have positive and negative charges pointed outwards, interacting with water molecules. However, the membrane is made from fatty chains that repel water and so the region of the protein that stays in the membrane will be structured in a way that avoids water by having its hydrophobic amino acids pointing outwards. Keeping both of these regions stable in the same solution is tricky, but manageable. Detergents can mimic the membrane and are widely used.

Detergents can make it difficult to study the structure of GPCRs. Most protein structures are solved using X-ray crystallography or nuclear magnetic resonance (NMR). However, crystallography requires all the protein molecules to be very uniform which is difficult with the added complexity of a detergent that may form different shapes and sizes. NMR begins to lose its sensitivity as the size of the protein increases. In this case, the detergent counts towards the protein’s size which makes it difficult to get data.

This has meant that despite the huge importance of GPCRs in biochemistry and medicine (over a third of all drugs act via GPCRs), our understanding of them is still relatively poor. However we would be a lot further behind if it wasn’t for Lefkowitz and Kobilka – they are very deserving Nobel laureates.

Pearls of Wisdom: Oysters genome reveals hardy defences

The oyster genome has been sequenced, making it the first mollusc to have its whole DNA mapped out. Molluscs (more specifically the phylum Mollusca) are largest group of marine species, making up nearly a quarter of all known species, and naturally play an important part in maintaining the ecosystem. Analysis of the genome has revealed a highly developed response to environmental stresses.

Oysters tend to live in estuaries or by the seashore – both very changeable environments. Estuaries continuously vary in salinity, requiring a highly adaptable system for life there. The seashore, or intertidal region, also has varying salinity but also has the problem of exposure to air when the tide goes out. Although exposure to air might be the least of your concerns if you find yourself stuck in the full glare of the midday sun at low tide.

The Pacific Oyster (Crassostrea gigas) lives a difficult life, but has the tools to help it survive.
Credit: David.Monniaux

Protecting against heat is common concern and so we have specialised proteins to defend against damage, aptly known as heat shock proteins. These proteins are found in nearly all forms of life, that’s how important they are. Researchers found 88 HSP70 (heat shock protein 70) genes in the oyster genome, significantly more than the 17 genes found in humans. Not only are there many of these genes, but they are highly responsive; 14 times more Hsp70 was produced under heat stress overall, with some genes activating over 2000-fold.

Hsp70 is usually used in the cell to protect newly synthesized proteins. Proteins are often highly dependent on being folded properly to prevent them from aggregating. Aggregation not only means the protein is useless, but it may be damaging to the cell. As the protein leaves the ribosome it is in a vulnerable position until it has time to fold into shape. Hsp70 acts by cradling the protein strand and protecting it until it can be passed on for further processing.

Hsp70 is found in nearly all forms of life, that’s just how important it is

The protective ability of Hsp70 is also why it is known as a heat shock protein. When the temperature gets high, proteins begin to unfold and are again threatened by aggregation. Hsp70 recognises loose strands of protein and binds to them, protecting them in the same fashion. This may help to explain the oyster’s ability to survive at temperatures up to 49°C (120°F).

Heat is only one factor that oysters have to deal with and their genome shows just how hardy they are. They appear to have a high rate of mutations and a large number of transposons, both resulting in high genetic diversity. This has allowed them to accumulate many mechanisms to prevent apoptosis (programmed cell death) in times of stress. In fact, over 4000 genes (16% of the whole genome) responded to air exposure.

The paper in Nature detailing these results had 85 authors. This gives a good indication of how much work goes into whole genome sequencing and analysis. There is a lot to learn from studying the genomes of other organisms, but it is still difficult work.

Listening in on Lysozyme

Researchers from the University of California have used a ‘molecular microphone’ to listen to a single protein molecule at work.

One of the world’s smallest transistors, constructed from a single carbon nanotube, was attached to a molecule of lysozyme, a bacterium-fighting protein.

Lysozyme has been the subject of thousands of publications ever since Alexander Fleming, who was suffering from a cold at the time, first tried cultivating a sample of nasal mucus and discovered its antibacterial properties.

Proteins, which are very long, string-like molecules, need to be folded in a specific way in order to function properly. Lysozyme was the first enzyme to have its structure solved, back in 1965. However, knowing the structure alone is not enough to explain how lysozyme works – the way it moves is important too.

Lysozyme folds to form two ‘jaws’, which allow it to hold sugar chains in place in order to cut them apart by breaking a chemical bond. The development of the nano-transistor allowed scientists to study how this protein behaves in unprecedented detail. The team were able to detect lysozyme cutting through chemical bonds at a rate of 15 per second, before it got stuck and started opening and closing its jaws 300 times per second trying to find more bonds to cut (think Pac-Man!).

 

As the transistor is attached to a single molecule, the researchers can see how each protein behaves, rather than just looking at the average of millions of molecules. “Our circuits are molecule-sized microphones,” says Philip Collins, associate professor of physics and astronomy, who led the project with Professor Gregory Weiss from the department of biochemistry. “It’s just like a stethoscope listening to your heart, except we’re listening to a single molecule of protein.”

This research is the first to use carbon nanotubes for studying the activity of a single molecule. This would previously have been achieved by attaching a fluorescent dye to the protein but these often wear out within seconds. By using the nano-transistor, the team were able to ‘listen’ to the protein for ten minutes, which was crucial for them to observe the protein switching between its different forms.

This ‘microphone’ can be applied to countless other proteins, giving scientists a chance to listen to the machinery of our body at work.

Bird’s Eye View: How to see magnetic fields

The ancient Greeks, like many people since, were confounded and fascinated by the migration of birds. Homer recognised that cranes “flee the winter and the terrible rains and fly off to the world’s end”. Meanwhile, Aristotle wrongly asserted that each year summer redstarts would transform into robins come winter, as the two species were never seen in Greece together. In modern times, we’ve come to appreciate the vast distances covered by migratory animals and the remarkable precision with which they make the journey. How is this feat achieved?

It is known that animals use sounds, landmarks or even smells to guide and navigate their way across continents. But the most intriguing and least understood navigation ability is magnetoreception: the detection of the Earth’s magnetic field through an internal, biological compass. This ability has been seen in a variety of animals, from ants to crocodiles. In fact, wildlife rangers in Florida resorted to taping magnets to the heads of crocodiles to prevent them from finding their way back after being relocated. Magnetoreception has even been suggested in the humble cow, after researchers using Google Earth accidentally discovered that cows tend to line up with the Earth’s magnetic field.

Magnetoreception was first observed in captive robins in 1957. In autumn, when it was time for them to migrate from Frankfurt to Spain, they kept flying southwest in their cage. This happened even though the room was isolated from any outside visual stimuli with which the robins could orientate themselves. This led to the idea that robins might use an internal magnetic compass to migrate. Many studies have been conducted since, but controversy still rages over the exact underlying mechanism of magnetoreception.

Robin in the winter

Robins can find their way with only the Earth’s magnetic field to guide them, but how do they achieve this?
Photo credit: Christine Matthews

Over fifty animal species have been found to use an internal magnetic compass so far, and several different mechanisms have been proposed and observed. The most established mechanism relies on the presence of small crystals of magnetite, a naturally magnetic mineral, in either the nose or the beak, surrounded by receptor nerves. Magnetite has been found in many animals, including humans, where it could be used to sense the magnetic field of the Earth and create a magnetic field map for migration. However, in experiments on birds where this magnetite receptor was deliberately disrupted by anaesthetic or a strong magnetic pulse, the birds could still orientate themselves along the magnetic field. This suggests that there is an alternative mechanism at work. Even more intriguingly, this alternative mechanism only works when there is light present, and didn’t appear to be influenced by reversing the direction of the field.

In 1978, Klaus Schulten suggested a mechanism for this type of magnetoreception, known as the radical pair mechanism. This proposes that there is a light-activated reaction in the bird’s eye that is affected by magnetism. By detecting the rate of the reaction, birds can sense the strength and alignment of Earth’s magnetic field. The problem with this idea is that the Earth’s magnetic field is incredibly weak, and so its influence on a normal reaction is a million times less than the energies involved in a normal chemical reaction. How could it possibly have a detectable effect?

The secret to detecting the magnetic field lies in generating a pair of radicals, which are molecules with unpaired electrons that interact strongly with magnetic fields. Creating these radicals requires a burst of energy, as provided when the molecules are exposed to light. Within a suitable molecule or protein, two radicals can form what is known as a ‘spin-correlated pair’ that exists in two different states. Conversion between these two states is affected by a magnetic field, and the rate of conversion can be monitored through the concentration of the radicals. In this way, a weak magnetic field can become detectable by cells in an organism.

The radical pair mechanism fits with the observations that cannot be reconciled with magnetite receptors—it is both dependent on the presence of light and unresponsive to the polarity of the field. Experimental evidence was lacking in 1978 when Schulten proposed the mechanism, so the idea received little attention for twenty years.

In 2000, a research group from Illinois suggested that proteins known as cryptochromes may be behind this source of magnetoreception. Cryptochrome proteins are found in the eye of robins, and absorb blue light to start a radical reaction—the perfect candidate to generate biologically detectable spin-correlated radical pairs. This led to renewed interest in the area, including the development of a proof-of-principle artificial magnetoreceptor system by a team of researchers at Oxford University. This was the first man-made chemical compass; the first artificial chemical system sufficiently sensitive to detect the Earth’s weak magnetic field on the planet’s surface.

Cryptochrome protein with a flavin radical initiator

Cryptochrome proteins are found in many creatures and absorb blue light through a co-factor known as FAD (shown in yellow)

The contribution of cryptochrome and the radical pair mechanism to magnetoreception in animals is still being investigated. Despite initial scepticism, evidence from model systems and computational work has shown that this mechanism is feasible for detecting magnetism. Cryptochromes are primarily responsible for maintaining circadian rhythms in many animals, including humans. Like many proteins throughout evolution, cryptochromes have found a new role in a different part of the body. From their presence in the eye, it has even been suggested that robins could sense the results of the radical reaction along the optic nerve and actually ‘see’ the magnetic field.

With growing evidence of weak magnetic fields affecting biological processes, there is increasing interest in how they might affect us. Numerous studies have shown a significant correlation between proximity to high-voltage power lines—which carry a low-frequency magnetic field­­—and increased rates of childhood leukaemia. In 2001 the International Agency for Research on Cancer classified extremely low-frequency magnetic fields as a possible carcinogen. Yet several attempts to demonstrate magnetic field induced carcinogenesis or tumour promotion in cells have failed, so this issue is still surrounded by uncertainty.

Perhaps in years to come our suspicions of magnetic fields transforming healthy cells into cancerous ones might be viewed just as fanciful as Aristotle’s redstarts to robins hypothesis. While we cannot be sure yet that power lines cause cancer, further analysis of Google Earth has shown that they can certainly disrupt the ability of cows to line up with the Earth’s magnetic field—tricking them into aligning with the magnetic field of the power line instead.