Thursday 2 December 2010

Arsenic Life - ZOMG! The straight dope on extremophiles and arsenic.

By now you may have heard today's big announcement in Science1 of the discovery of extremophile bacteria that use arsenic in their DNA rather than phosphorous. This discovery has been played up by the media (as usual) as being a major discovery that will cause scientists to "rewrite the story of life" or other such hyperbole-based headlines. And while definitely interesting, this discovery, unfortunately, is just not a major finding.

What the discovery is:

The team of researchers were concerned with a lake in California called Mono Lake. This lake itself is particularly interesting: it is highly alkaline, with a pH at around 10, so it is very basic (remember that water has a neutral pH of 7, and that the pH scale is logarithmic, so the lake is 1000 times more alkaline than a regular freshwater lake). The lake also contains quite high levels of arsenic, an element that is highly poisonous to life. The scientists, aware that extremophile bacteria just love to live in what we would normally consider absolutely hostile conditions, were curious about what kind of microbial life lived in the lake.

What they found were microbes that not only lived happily in the highly alkaline, arsenic-laden conditions, but actually utilized arsenic in place of phosphorous in their biochemistry: their DNA contained arsenic where it normally would have a phosphate backbone and even some of their amino acids contained arsenic instead of phosphorous. They did this by growing the bacteria in media containing radiolabelled arsenic, and after allowing the bacteria time to utilize the arsenic as they pleased, found that the radiolabelled atoms ended up in the DNA, amino acids and other normally phosphorus-containing molecules.

What this DOESN'T mean:

This does not mean we have discovered "alien" life, like some media outlets have been saying. Such life easily fits in with what we know about extremophile bacteria - they are resilient enough to find a way to use toxic environments to their advantage.

It also doesn't mean that we need to re-examine the way we think early life evolved. It is unlikely this discovery says anything about evolutionary history (though it might say volumes about how evolution can be incredibly innovative!).

What it DOES mean:

It does mean that evolution can work in wondrous ways that we hadn't even imagined. Make no mistake, however - this finding surely fits within current evolutionary theory.

It also means, on a more hypothetical level, that astrobiologists may need to broaden their definitions of "life" when searching for the signs of life outside of our planetary system.

Why this discovery isn't as big of a deal as it first seems:

Although at first the announcement of life that uses arsenic as a building block is exciting, a little critical thinking shows that there is reason for skepticism. The researchers studied the microbes by slowly increasing the level of arsenic in the media they were grown in. After multiple rounds of this, they were left with microbes that were found to have arsenic in their DNA and amino acids. However, this method leaves open the possibility that they were simply selecting for microbes that could use arsenic under high arsenic/low phosphorous levels. In other words, this experiment does not show that using arsenic is the natural way these microbes live. It is entirely possible that they use phosphorous for their important biochemical molecules like ever other microbe, and under stressful conditions, switch to different mode where they use arsenic instead. It's even possible that after successive rounds of increasing arsenic content, the team artificially selected for microbes that had this ability: in effect, they may have forced the microbes to evolve a new pathway that does not exist in nature.

This finding doesn't say alot about evolutionary history either: Lake Mono is a relativley recent geological feature, so it's likely the microbes adapted to such an environment relatively recently also.

Furthermore, the team did not attempt to determine HOW the bacteria incorporate arsenic into their DNA and amino acids: what biochemical pathways are involved? Do they use NTAs instead of NTPs? How stable would such molecules be - phosphate plays an important role in stabilizing the structure of DNA, so would the use of arsenic affect this? Are such molecules recognised by the microbes' DNA polymerase? Or by any important enzymes, for that matter? Arsenic is a larger atom than phosphorous - will this alter the shape and size of the major and minor grooves and if so, how does this affect the binding of proteins? Is arsenic ALWAYS used by these microbes, even in low arsenic/high phosphorous levels? These questions are just begging to be answered, and until they are, I think the vast majority of molecular biologists and biochemists will remain skeptical.

An interesting find, for sure - the very fact that organisms can use arsenic to live is exciting - but a "new form of life" or even "arsenic-based life"? Well, there's really no evidence to support such claims at the moment.

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PZ over at Pharyngula has a great rundown of why calling this "arsenic based life" is silly, echoing many of the points I made above.

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1. Wolfe-Simon F, Blum JS, Kulp TR, Gordon GW, Hoeft SE, Pett-Ridge J, Stolz JF, Webb SM, Weber PK, Davies PCW, Anbar AD, Oremland RS (2010) A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus. Science DOI: 10.1126/science.1197258.

Thursday 25 November 2010

Next-Generation DNA sequencing: The Future is Now! Part 1: Pyrosequencing

Are you tired of sequencing your gene of interest 800bp at a time? Sick of straining your eyes staring at a fuzzy chromatogram? Fed up with waiting hours for your PCR to finish only to realize you forgot to add ddNTPs to your reaction mix? Well, nex-gen DNA sequencing is for YOU!

Cheesy sales-schtick aside, next-generation DNA sequencing technologies are on the rise (and are poised to soon become current-gen technologies - some already are!). The days of loading your PCR'd sequencing mix onto an automated capillary sequencer may soon be numbered. So, to make the change of power of our mighty sequencing overlords a little easier, this series of posts will be dedicated to how the upcoming technologies work, their advantages over conventional sequencing technologies, and their problems.


Today's post: Pyrosequencing

Pyrosequencing is perhaps the one Next-Gen sequencing technology that is the most like current generation automated sequencing (if you need a reminder on how that works, I've written about it in the past). It still requires primer design, and rounds of PCR, but the method of detection of incorporated nucleotides differs.

Pyrosequencing starts by adding your PCR'd sequence to a reaction mix that contains DNA polymerase, and three important enzymes: DNA sulfurylase, apyrase and luciferase. DNA sylfurylase is an enzyme that converts a pyrophosphate molecule into ATP. Luciferase is an enzyme which uses ATP to convert luciferin into oxyluciferin, resulting in the emission of light. Apyrase's job is to degrade unincorporated nucleotides. Given that the addition of a nucleotide into a growing DNA strand results in the release of a pyrophosphate molecule, keen readers might already see how pyrosequencing works.

With conventional automated chain-termination sequencing techniques, we add all of our dNTPs to the reaction mix at once; they can easily be distinguished because each base has a different fluorescently labelled dye bound to it. With pyrosequencing, however, one cannot toss in all the nucleotides at the same time. Rather, we have to add each one sequentially for each nucleotide in the sequence. That is, we first do a reaction with A, then with T, then with C and lastly with G. We then repeat this cycle over and over until the sequencing reaction is complete (if this sounds confusing, hopefully the diagram and video below will clarify it). Why we do this will be apparent in a moment.

When a nucleotide is incorporated into the DNA strand, a pyrophosphate molecule is liberated1. This pyrophosphate molecule can then be converted by the DNA sulfurylase into a molecule of ATP. Luciferase picks up this ATP2 molecule and uses it to catalyze the conversion of luciferin to oxyluciferin. This chemical conversion results in the emission of a photon. This chain of events, then, means when a nucleotide is added by DNA polymerase to the growing DNA chain, we get the emission of light. A computer with a suitable detector could detect this light, and we would have an indication of when a nucleotide was added in our sequencing reaction.

But if we add all of our dNTPs at once, then how do we know which ones are being ? This is why we only add one nucleotide to the mix at a time. First the reaction is run using dATP. We then add apyrase to remove any remaining nucleotides and repeat with dCTP, and so on. If we add the nucleotides one at a time, then they will be incorporated (or not incorporated) into the sequence one at a time, and consequently we get one light signal at a time.

The light signals are recorded on a chart called a pyrogram. This chart records not only which nucleotides resulted in the emission of light but also the intensity of that signal. If three dTTP molecules were incorporated at once, then there would be three photons emitted and three times as much light; this would result in a triple peak on the pyrogram. From the pyrogram, then, one could easily read off the exact DNA sequence. The figure to the left shows one such pyrogram. The sequence in this example would be GCAGGCCT.




The following video puts the whole process together nicely.



So why would one choose pyrosequencing over automated chain-termination methods? Well, for one, it's cheaper (though, not as cheap as some of the other upcoming next-generation sequencing methods). Practically, it's easier to do, since it doesn't require running through gels or capillaries. Analyzing the resulting pyrogram is also easier than analyzing a chromatogram. Chromatograms can often be spotted with "N"s when the computer cannot tell if the wavelength of light from a dye is one color or the other; however, with pyrosequencing, detection is binary - either a photon is emitted or not - so results are more accurate and clearer.

Though, pyrosequencing does have it's drawbacks. It requires a greater number of PCR cycles than traditional sequencing does, so it may take longer to complete, especially for longer sequences. Currently, a typical read of sequence data from pyrosequencing is about 300 to 500bp - shorter than the typical 800 to 1200bp you get from chain-termination methods. This, however, is likely to improve as the technology advances and becomes more refined. The shorter reads, though, make it tough to sequence genomic regions containing high amounts of repetitive DNA.

So that is pyrosequencing in a nutshell. Next time: Helicos sequencing!
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1. NTPs are triphosphates (that's what the TP stands for!), meaning each nucleoside (base+sugar) is attached to a phosphate molecule. To add a nucleotide to a growing DNA strand, the reaction requires an input of energy. This energy comes from the breaking of the triphosphate chain in each nucleotide; two phosphates are broken off and released as a pyrophosphate (PPi) molecule, and the remaining portion of the nucleotide is attached to the hydroxyl group on the 3' carbon of the previous nucleotide in the sequence.

2. Observant readers might be confused here. Since luciferase requires ATP to convert luciferin to oxyluciferin, won't this cause a problem when we add dATP to the sequencing reaction? Won't there be competition between DNA polymerase and luciferase? Well, if you thought that, then you'd be right! For this reason, we use dATPαS instead of dATP for pyrosequencing. This molecule has a sulfur atom attached to the α phosphate of the nucleotide, and cannot be used as a substrate by luciferase. Problem solved!

Sunday 14 November 2010

Dawkins' Answers Some Questions

His answers are interesting enough, but the best part starts at 11:42, when he reads out some of his hate mail.

Sunday 7 November 2010

"Science knows it doesn't know everything. Otherwise, it'd stop."

Dara O'Briain is great. Gotta love his takedown of homeopaths, nutritions and other snake oil salesmen.

Wednesday 20 October 2010

Have an hour to spend and nothing to do?

Then I suggest watching this video of Richard Dawkins having a chat with Neil deGrasse Tyson.
Two brilliant minds and amazing popularizers of science. DeGrasse Tyson is as charismatic as ever...I'd love to get a chance to meet him. If anyone can get people excited about sicence, it's him.

The Stupid, It Burns....

The ever laughable (and pitiable) Ken Ham has a little segment on the Answers In Genesis website called Kids Answers, where he takes time out of his busy day of ignoring evidence and thinking up new ways to sidestep logic and reason to answer questions sent to him by children. A little rascal named Brendon asked him a very good question that points out just one aspect of the absurdity that Young Earth creationists believe, and Ham has responded with this little gem (emphasis mine):

Q-If God created the world 6,000 years ago or so, why are stars millions of light years away?

A-Brendon, what a question! Yes, we know from the dates God gives us in the Bible that He did create the whole universe about 6,000 years ago. When we hear the term light-year, we need to realize it is not a measure of time but a measure of distance, telling us how far away something is. Distant stars and galaxies might be millions of light-years away, but that doesn’t mean that it took millions of years for the light to get here, it just means it is really far away!

Really, Ken? Way to show that you really are absolutely ignorant. I'm sure I don't need to explain it to anyone reading this, but a lightyear is defined as the distance that light travels in one year. That's why it's called a light year. It takes one year for light to travel one lightyear, it takes 10 years for it to travel ten lightyears, and so on. So yes, if we observe an object a million lightyears away, it means precisely that it took the light a million years to reach us.

Do creationists like Ham really want to be taken seriously? Because time and time again they display such a complete dearth of understanding even the most simple and fundamental concepts of science. There's hardly a better example than that above.


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Super nerdy Star Wars-geek aside: Ham isn't the only one who confuses measurements of space and time - George Lucas is guilty of this as well! In Star Wars: A New Hope, Luke and Obi-wan meet Han Solo in the Mos Eisley cantina for the first time. When Luke admits he's never heard of the Millennium Falcon, Han tells him that the Falcon completed the Kessel Run in "less than twelve parsecs", obviously meaning to brag that he was able to complete the course quicker than any other pilot. However, a parsec is a measurement of distance, equal to 3.26 lightyears, and not a measurement of time.

Hardcore Star Wars nerds such as myself, though, will argue that what Han meant was that he was able to navigate the course by taking a shorter route that took him dangerously close to a black hole instead of flying the entire 18 parsec course. He was thus bragging about his piloting skills rather than his speed.

Monday 4 October 2010

2010 Nobel in Medicine/Physiology goes to...

Robert Edwards for his work on the development of in vitro fertilization.

This came as a bit of a surprise, since his work on IVF was completed some 3 decades ago, and the prize is generally given to work done in the last 10 years.

Anyway, it is work that is worthy of a Nobel prize, if a bit unexpected. Now to guess who wins the prize in Physics tomorrow...

Tuesday 28 September 2010

2010 Nobels - time to place your bets.

Next week the Nobel Prizes for 2010 will be announced. Who are your picks for this year?

A lot of people seem to be putting their money on Craig Venter for the prize in Medicine/Physiology this year. Probably because of all the media attention he's received in the last little while. And though he has done some important things for the field, I'm a bit unsure. Perhaps it's a bit soon for a Nobel for him? Though I think he might be a good choice, I'm kinda hoping it goes to Maire-Claire King for her work on detecting the variants of the BRCA1 and BRCA2 genes that are associated with breast cancer. This fits all the criteria for the prize - revolutionary work which has had a practical and important real-world application. Also, it would be the second year in a row that the prize in Medicine has gone to a female (women laureates are few and far between, unfortunately).

I dont really know much about Physics and Chemistry to make a guess at who the prizes in those categories will go to. Im unlikely to even understand the work of whoever wins the Physics prize but Im looking forward to seeing who wins in Chemistry.

We molecular biologist types are lucky: our work often qualifies us for both the Medicine/Physiology and the Chemistry prizes!

Wednesday 28 July 2010

Long absence

Woah, it's been a long time since I've posted anything on this lovely ol' blog of mine.
I guess it's been a combination of factors - being pretty busy with work, not having much to write about and general laziness. Hopefully, I'll get back into making regular posts soon enough.


Hopefully.

Saturday 23 January 2010

Viral Catapults

It's been quite some time since I've written any posts here (not exactly true, I have about a dozen unfinished drafts of posts that I may get around to finishing eventually), so I thought I'd break the silence with the details of some interesting research on viruses recently published in Science1.

How viruses spread and infect new cells within the body is just as important as learning how viruses are spread from person to person (or from other animals to humans, as the case may be). There are lots of ways viruses can spread to new cells once a host has become infected, and some of them are pretty interesting. Some viruses churn out massive amounts of viral particles and cause the host cell to rupture, and the swarms of progeny will infect nearby healthy cells. A team of researchers in the UK, however, have discovered a new method of viral cell to cell transmission that's a bit...different: viral catapults.

The team from Imperial College London, lead by Geoffery Smith, fluorescently tagged vaccinia poxvirus and using live-imaging techniques, were able to visualize how vaccinia is able to move from cell to cell. What they found is pretty interesting.

Once a cell has been infected by vaccinia, the virus hijacks the cell's replication machinery and uses it to rapidly produce massive amounts of two particular viral proteins called A33 and A36. These two proteins are then transported to the cell's membrane and are expressed on the outer surface, forming a sort of mesh around the cell. This mesh acts kind of like a tag that tags the cell as having been infected. When another vaccinia virus comes along and contacts the cell, it isn't able to get inside because of the A33/A36 complex. Instead, the virus gets lodged in the mesh.

Now for the really cool part. Once the virus has become lodged in the protein complex, this triggers a cascade within the host cell that rearranges its actin microfillaments (microfillaments, composed of action, are a part of the cell's cytoskeleton, the network of fibers that allows cells to maintain their shape and structure, as well as playing a role in cellular locomotion, division and intracellular transportation). The fillaments are rearranged into one long fillament that protrudes out at the site of viral attachment and sends the virus flying off into the intercellular medium, hopefully to find an uninfected host cell far away from the site of infection. Think of it kinda like hitting a billiard ball with a cue and knocking it to the other end of the pool table.

Totally cool.

This explains some observations made by Smith and colleagues about the rate of infection of the vaccinia virus. Vaccinia is seemingly able to move through populations of cells at a faster rate than would be predicted by viral reproduction rates alone (they were able to film a short video of the virus spreading quickly through a culture of cells, too! It certainly does move fast.) This ability of vaccinia to catapult itself to new potential hosts provides an adequate explanation.

Smith and co. went even further with their research. They knocked out the genes for A33 and A36 from the virus and found that these mutant viruses had significantly increased infection times. Furthermore, they inserted the A33 and A36 genes into healthy human cells, and found that it was sufficient to induce the virus-flinging reaction. This suggests that the whole mechanism - reception of the viral docking into the complex, signaling to trigger the rearrangement of the actin filaments and recruitment of the filaments to the proper location - is mediated by only two proteins! The team is not sure how the two proteins are able to pull off this task, but I'll be keeping my eye out for more research from their lab to find out.

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1. Doceul, V., Hollinshead, M., van der Linden, L. & Smith, G. L. Science advance online publication doi:10.1126/science.1183173 (2010).