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.


Monarch butterflies use magnetic compass to migrate

New research has shown that monarch butterflies can follow the Earth’s magnetic field to make their annual migration.

Each autumn, millions of monarch butterflies make an epic journey from Canada to the Michoacan mountains in Mexico. Despite making this 2500 mile journey for the first and only time, the butterflies find their way to the same place as earlier generations. Researchers have long known that the butterflies follow the sun to head south, but this does not explain how the butterflies can still find their way on overcast days. This has led to suspicion that the butterflies could be using an inbuilt magnetic compass, like other migratory species.

The small but mighty monarch butterflies fly 2500 miles across North America

The small but mighty monarch butterfly flies 2500 miles across North America

A new experiment, published in Nature Communications, is the first to show convincing evidence of the butterflies relying on a magnetic field to choose direction. During the 2012 and 2013 autumn migration seasons, researchers placed butterflies in a flight simulator surrounded by magnets. As expected, the butterflies headed ‘south’, following the inclination of the magnetic field. When the researchers flipped the inclination of the magnetic field, the butterflies started to fly in the opposite direction.

Monarch butterflies use a range of techniques for finding their way across the continent

Crucially, this internal compass is dependent on violet light, which was not considered in past experiments. Previous attempts to study the butterflies have used light sources or filters that may not let through enough violet light. In those studies, the researchers could not see any response from the butterflies when they altered the magnetic field.

This light dependence strongly suggests that the butterflies use the same mechanism as fruit flies and European robins for detecting magnetic fields. This relies on proteins known as cryptochromes, which are present in the antennae or eyes. When these proteins are hit by light, they trigger a chain reaction that is influenced by weak magnetic fields.

Last month, a different group of scientists showed that even extremely weak radio waves can disrupt the magnetic compass of European robins. The strengths and frequencies of the electromagnetic noise studied were the equivalent of being within five kilometres of an AM radio station. If this can affect the birds’ ability to migrate, it could also have implications for the monarch butterfly and other migratory species.

‘Humanised’ mice used to study Hepatitis C infection

Over 120 million people worldwide are chronically infected with Hepatitis C and most of them don’t know it. The only signs will be decades after infection, when liver cancer or cirrhosis set in. By then it is too late. There is no cure and there is no vaccine.

Development of treatments for Hepatitis C has been hampered in no small part because of a lack of animal models for research. Only humans and chimpanzees can be infected with the virus. Scientists have learnt a lot about the Hepatitis C virus (HCV) from research on chimpanzees over the past few decades, but now the US (the last developed country to conduct chimpanzee research) will dramatically reduce the use of federally-funded chimpanzees.

‘I think that’s a good thing. Chimpanzees have taught us a lot about the hepatitis C virus, without them it would have taken significantly longer to even identify it as a virus. So there were a lot of benefits from using chimpanzees in biomedical research in general,’ says Marcus Dorner, researcher at Imperial College London. ‘Even though they were allowed previously, chimpanzee studies have always been ethically questionable. They weren’t all in vain; we’ve learned a lot, but ultimately it was a good thing that we got rid of chimpanzee research. I’m totally supportive of that.’

Dorner and his colleagues at Rockefeller University, USA, have succeeded in developing a genetically-engineered mouse where the virus can infect cells and replicate as it would in humans. This could have a big impact on the development of a vaccine, where scientists need to see how the immune system responds, which is not possible with cell cultures. ‘You can do a lot of things with in vitro systems; they have developed quite a bit. There are now a lot of tools that allow extensive studies of a lot of aspects of the hepatitis C virus. But the problem with that is you can only answer a certain subset of questions – you cannot do immunology if you only have liver cells. For a lot of things you a need a functional model system that allows you to answer these questions.’

The new mouse has been genetically modified so that its liver cells have the same receptors on their surface as human liver cells, which allow HCV to recognise it and infect the cells. However, infection is only the first stage of the virus’s lifecycle – it still needs to replicate.

Once the virus infects the ‘humanised’ mouse, it is attacked by the innate immune system. This simply recognises that a virus is present and produces interferon proteins that prevent the virus from replicating. HCV has evolved to evade the human innate immune system, but it is vulnerable in the mouse. By blocking the mouse’s genes for producing interferon, Dorner could see HCV surviving past the initial infection and start to replicate in the mouse’s liver cells.

The luminescence allows researchers to track the infection in one mouse over several days

The luminescence allows researchers to track the infection in one mouse over several days

The mice and the virus were genetically engineered so that the liver cells would glow when the virus replicated. This meant that the progress of the infection in a mouse could be followed from day to day by placing the anaesthetised mouse in an imager (see picture above). Without this system, biopsies would need to be taken at set time intervals from many more mice.

The previous ‘gold standard’ for HCV mouse models is mice with human liver cells. This requires genetically modifying the mice to suffer liver damage so that implanted human liver cells can grow to make up 90% of the liver. This comes with the heavy cost of kidney damage, weakened immune system and a 60% attrition rate for the mice. As well as the ethical cost, the treatment for the mice is very expensive and it is difficult to raise enough mice to produce reliable results. It is hoped that the new mouse model will replace this and reduce the number of animals used.

Dorner has recently moved to Imperial College and is now turning his hand to other chronic diseases like hepatitis B, Dengue fever and HIV. “I’m bringing this mouse model with me to Imperial, and here it will most likely be a collaborative tool for people to use. These mice are a tool to answer questions that they have in a more relevant model system.”

One aim is to apply the same technique to developing a similar mouse model for hepatitis B. Even though there is already a vaccine for hepatitis B, over 300 million people are chronically infected worldwide. As Dorner points out: “If you look at the total numbers it is a huge problem. For everybody that is chronically infected it is a lifelong burden, even if it doesn’t develop any disease they need to take drug treatments for the rest of their lives and those drugs don’t come cheap.” Unlike hepatitis C, there are no cell cultures for studying hepatitis B so a new animal model could make a big difference towards developing new treatments for these millions of people.

Last Sunday was World Hepatitis Day, a day to highlight the ‘forgotten’ diseases of hepatitis B and C which kill over 1 million people per year, as many as HIV/AIDS. New advances are sorely needed and these new mouse models could have a big impact.

Another Brick in the Wall: Turning cancer’s strengths against it

A mutation that allows cells to grow out of control could also provide a new way to target and destroy cancer cells. This potential Achilles’ heel comes from a mutation in a gene called PTEN, which is found in a wide range of cancers.

PTEN is one of many tumour suppressor genes that we have to prevent our cells from growing out of control. If the PTEN gene stops working because of a mutation, it can cause tumours to develop – indeed many tumours have a mutated form of PTEN. However when a door closes, a window opens: the PTEN mutation helps the tumour to grow, but it could also mark it out as a target.

Researchers from the Institute of Cancer Research, London, found that switching off another gene known as NLK killed tumour cells that had the PTEN mutation. This makes NLK a good target for drug developers to create a new cancer treatment.

The difficult thing about cancer is that it is made of us – it is our own cells that have mutated and grow wildly out of control. That means it is unlikely there will ever be a quick fix. Antibiotics work efficiently because bacteria are so different to us that we can develop drugs that target their weaknesses yet barely affect our own cells. But how do you kill something that is the same as you? Current treatments for cancer cause a lot of side-effects in patients because as they try to kill the cancer they also do damage everything else in the body. This is why finding ways to target cancer specifically is so important.

There are several proteins which we cannot live without, and our cells die if the genes responsible for producing those proteins are mutated or switched off. Targeting these proteins and genes are rarely going to be useful for treatments, as they will kill the patient about as quickly as they kill the cancer. So Alan Ashworth and colleagues set out to find proteins that are not essential in healthy cells, but cells with the PTEN mutation cannot live without. This would pave the way for designing drugs that target the tumour and leave healthy cells alone.

The researchers took samples of tumour cells with and without the mutation, and switched off genes for important proteins that are used for regulating lots of processes in the cell. To do this they used small molecules of RNA (DNA’s less famous cousin) which interfere with the processes of specific genes. This is why these molecules are known as small interfering RNA (or siRNA). They block the chain of events that allow a gene to produce a protein, effectively switching it off. By switching off 779 genes individually, they could look for ones where cells with the PTEN mutation died and cells without the mutation survived.

This is how the researchers discovered the powerful effect of switching off the NLK gene. They are not certain how this works but it appears to protect a protein called FOXO1 that can act as a backup tumour suppressor and cause the cancer cell to die. When PTEN is mutated, the FOXO1 protein becomes vulnerable to a process called phosphorylation, which means it is ejected from the cell nucleus and destroyed. NLK is one of the proteins that phosphorylates FOXO1 and so by switching off the NLK gene, FOXO1 is able to do its job.

The phosphate group, shown at the top, consists of one phosphorus atom (in orange) and three oxygen atoms (in red). This can be added to certain amino acids to control the behaviour of the whole protein

Phosphorylation is the process of adding a phosphate group, shown at the top, to certain amino acids to control the behaviour of the whole protein. This group consists of one phosphorus atom (in orange) and three oxygen atoms (in red) and has a strong negative charge.

This is just the start of a long journey from the lab to (potentially) the hospital. The researchers have shown that targeting NLK is more likely to kill mutated cells than normal cells, but that does not mean it is safe. NLK still has a role to play in healthy cells and preventing it from working is likely to have side-effects, but it could be worthwhile if this approach can kill tumours. The next stage is to develop a drug to stop the NLK protein from working, so that it can be tested further in cells and in living organisms.

Promising leads against cancer appear often, yet very few ever make it as treatments. One big hurdle is making it through clinical trials; the new drug has to be better than currently available treatments. Targeting NLK would only work against cancers with the PTEN mutation, but now we can use the mutation as a marker to find out which patients that applies to. We are now in the age of personalised medicine, where we can have 100 different treatments for 100 different people with 100 different cancers. Gradually, we are finding ways to attack cancer in whichever form it appears and build up our range of treatments. The weaknesses that we find are not going to cure all cancers, but each one provides another brick in the wall.

This article was originally written for the Access to Understanding competition run by Europe PubMed Central

Finding a needle in a haystack: How RecA slides in to the rescue

RecA is well-practiced at finding a needle in a haystack – its job is to track down the right sequence of DNA out of 3,000,000,000 letters. But until now, it was not clear how it did this: does it jump from one bit of DNA to the next or does it slide along?

Damage to DNA is one of the biggest threats that we face – it is one the main driving factors in developing cancer. However, evolution has provided us with many tools to prevent and fix any problems. One advantage of DNA being a double helix is that if only one strand of the helix breaks, the DNA is still held in place by the second and so it can be easily fixed. However, if both strands break it becomes far more difficult to piece the DNA back together.

The single strand break (left) can be easily patched up - the DNA helix is still held in one piece. However, the double strand break (right) means that the sequence is split into two and it's not possible to stitch them back together.

The single strand break (left) can be easily patched up – the DNA helix is still held in one piece. However, the double strand break (right) means that the sequence is split into two and it’s not possible to stitch them back together.

In a double-strand break, the broken strands separate from each other because they can’t be put back together. To be repaired, a broken strand needs to line up with an undamaged molecule of DNA and use it as a template. However, finding the right sequence to align with is not easy when there are 3 billion nucleotides to search through.

These broken strands rely on a protein known as RecA to act as matchmaker and find the perfect partner sequence of DNA. Several molecules of RecA attach themselves to the strand, straightening it out and forming what is called the pre-synaptic filament. Taekjip Ha from the University of Illinois wanted to find out if this filament is able to slide along the DNA or if it has to rely on jumping from place to place. To test this they used a technique known as FRET (fluorescence resonance energy transfer), which can tell you the distance between two fluorescent tags that are attached to different molecules. One tag was attached to the filament and the other was attached to a short stretch of DNA that would not match with the filament. The researchers were able to see the distance changing very quickly, showing that the RecA-DNA filament was sliding back and forth along the short length of DNA, unsuccessfully trying to find a match.

Several copies of RecA bind to a stretch of single-stranded DNA

Several copies of RecA bind to a stretch of single-stranded DNA (shown in black) to help it find a match

The ‘bait’ DNA was then changed so that it had two patches that matched the sequence of the filament. By attaching fluorescent tags to each patch, the filament could be seen sticking to one of the two sections, and then sliding along and binding to the other. From the speed of the sliding, the researchers estimated that RecA could help to slide along up to 300 base pairs of DNA before reattaching somewhere else. This could help speed up the search for the matching sequence by over 200-fold, making it a much more efficient technique than randomly hopping from sequence to sequence.

This kind of sliding movement is the first that has been seen in a protein-DNA complex and improves our understanding of this highly important process. Fortunately, one lab has made a (low-budget) video to show how RecA works. Enjoy…