The Life and Death of Elysia Chlorotica

Since the end of university I’ve had some trouble letting go of science as a constant occupation. Fortunately I was given the chance to post here about an old journal club presentation rather than go cold turkey, so here is a short piece on one of the most interesting organisms I’ve read about.

The picture on the left is of a sea slug of the genus Elysia which has evolved to blend in with its surroundings by looking almost exactly like a small leaf. The green colour that makes this camouflage so convincing is produced when the slug feeds on algae and steals their chloroplasts, subsequently storing them throughout its body. The use of chloroplast pigments to produce a leaf colour is common among sea slugs but the subject of this post is exceptional among the rest of its family. While in the majority of sea slugs the chloroplasts stop working within days or weeks of incorporation, those taken up by Elysia chlorotica can remain stable and active within the slug for at least ten months. As a result the slug is a rare example of a photosynthetic animal and it can survive without food for a long time, as long as it has light and carbon dioxide.

E. chlorotica has coevolved with a specific species of algae named Vaucheria litorea, which is a source of food and chloroplasts for the slug. The metamorphosis of E. chlorotica larvae into their slug form takes place only if the larvae are attached to filaments of V. litorea and in the absence of this algal species the larvae will die. Immediately after metamorphosis the slugs feed on the algae to which they are attached and take up their chloroplasts, incorporating them into their branched digestive tract.

As mentioned previously, most species of sea slug lose their chloroplast functions soon after obtaining them from algae. This is expected to be because the majority of genes required for chloroplast survival and function are encoded on the algal nucleus. Since animals do not have genes involved in chloroplast maintenance the question of how E. chlorotica manages to sustain its stolen plastids was investigated by several labs. In a recent study, the chloroplast genome of V. litorea was sequenced to determine whether it had greater genetic autonomy than previously sequenced chloroplasts, however, the chloroplast genome was found to be largely normal and was missing many genes essential for photosynthesis.

One of these missing genes, psbO, encodes a component of photosystem II which is vulnerable to damage during photosynthesis and needs to be regularly resynthesised for chloroplast function. Rumpho et al. were able to amplify the whole of this gene from sea slug DNA using primers based on seqeuence databases. The amplified sequence was shown to correspond exactly to the version of psbO from V. litorea, indicating that lateral gene transfer had taken place between the algae and the slug. The same sequence could be amplified from sea slug eggs which had not yet encountered algae, confirming that the sequence had entered the E. chlorotica germline. The authors behind this study speculate that all of the other genes needed for chloroplast survival and function have also been transferred to the slug germline, explaining the unique ability of E. chlorotica to maintain its chloroplasts for its entire life. So it seems that the sea slugs have stolen both DNA and organelles from algae, which is quite an achievement.

Another interesting property of E. chlorotica is that its generations are separated from each other. Whether slugs are collected in the lab or monitored in their natural environment, the entire adult population undergoes a synchronous death every year after they have laid their eggs. Pierce et al. observed that viral particles could be seen in the cytoplasm and nuclei of slugs just before their mass death and suggested that they may be responsible for the mortalities. The morphology of the viruses and the presence of reverse transcriptase activity suggest that they are retroviruses and, since the particles were found even under controlled laboratory conditions, it is likely that the virus is encoded on the genome of the sea slug. The group who made this observation suggested that the viruses may respond to annual environmental changes and kill the sea slugs in the spring, though this hypothesis has not been tested so far.

Unfortunately, the last paper I found on research into the viruses within the sea slugs was in 1999 and so research into this interesting aspect of the organism may not be ongoing. However I'm hoping to find some more about it in the future.

Rumpho, M. (2000). Solar-Powered Sea Slugs. Mollusc/Algal Chloroplast Symbiosis PLANT PHYSIOLOGY, 123 (1), 29-38 DOI: 10.1104/pp.123.1.29

Pierce, S., Maugel, T., Rumpho, M., Hanten, J., & Mondy, W. (1999). Annual Viral Expression in a Sea Slug Population: Life Cycle Control and Symbiotic Chloroplast Maintenance Biological Bulletin, 197 (1) DOI: 10.2307/1542990

Pierce, S., Massey, S., Hanten, J., & Curtis, N. (2003). Horizontal Transfer of Functional Nuclear Genes between Multicellular Organisms Biological Bulletin, 204 (3) DOI: 10.2307/1543594

Rumpho, M., Worful, J., Lee, J., Kannan, K., Tyler, M., Bhattacharya, D., Moustafa, A., & Manhart, J. (2008). From the Cover: Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica Proceedings of the National Academy of Sciences, 105 (46), 17867-17871 DOI: 10.1073/pnas.0804968105

Flu flu go away, come again another day (when I don't have exams)

Right I should be revising for those finals but with my arm hurting I am using it as an excuse not to write practice essays today and typing doesn't put as much strain on it (I think).

So Swine flu/Mexican flu/H1N1 flu has brought Mexico to a halt and scared the rest of the world into a panic. Big mouth Vice-president Biden of the USA has done it again saying he wouldn't trust travelling etc. fair point but you are trying to calm the masses as a leader. From what is humble virologist wannabe can see is this new hybrid strain made from genes of avian, swine and human flu is doing is spreading very quickly among people. For some reason it has killed lots in Mexico (allegedly) but only causing mild cases elsewhere. Today however the researcher at Mill Hill have suggested this strain is not very deadly. This paints a rather paradoxical picture. We see many deaths in Mexico from a fast spreading flu. Yes the world should be worried and has done the right thing by preparing for the worse. But we have only seen mild cases in most people outside Mexico and its genome suggests it is only mild. It doesnt have the make-up that causes cytokine storm that kills young people (like Spanish flu did). That is one thing not to worry about (right now). Plus it only infects cells near the top of the respiratory track so doesn't cause infections deep in the lungs, making it easy to spread but not as deadly as some.

So two things need to be thought about 1) why has it killed so many people in Mexico? and 2) what shall we call it? I am not going to tackle the first but I have some thought. The second I think should be Mexican flu. Swine flu is hurting the pig farming industry (some countries have banned pork imports!) while H1N1 is just rubbish. Loads of flu strains are called H1N1. Some seasonal flu is H1N1 and Spanish flu of 1919 was H1N1!!! I personally think the H and N naming system is out dated and inaccurate. It is not the H and N proteins that make a virus what is is per se but the many other changes with them.

Closing thought though, Flu has a high mutation rate with its RNA genome (no where near HIV but still more than what we would like) so changes may occur soon that could be game changers.

http://news.bbc.co.uk/1/hi/health/8028371.stm

When a bit more fat is not that bad

Nothing is as simple as it looks in Biology. We can’t even rely on gene or protein sequences to tell us what is going to happen. At the chromosomal level, the new field of epigenetics is showing how the nucleotide sequence does not define everything. At protein level we have to consider post-translational modifications. My final year project, now at the final write up stages, concentrated on an aspect of Trypanosoma brucei molecular biology which I very pompously described as a potential drug target and the cure for African sleeping disease. This bit was probably ‘paper talk’ though. In any case, the project focused on protein palmitoylation.

Palmitoylation is part of a group of several lipid modifications that can occur in proteins. Examples include N-myristoylation, prenylation and GPI-anchors. N-myristoylation is the reason why I thought it was not pushing it to much to say that we can kill parasites by targeting these modifications. N-myristoylation corresponds to the co-translational addition of myristate to an N-terminus glycine, and is catalyzed by N-myristoyltransferases (NMT). It so happens that NMTs were shown to be essential for parasite viability, and several antifungal NMT inhibitors apparently work on reducing NMT activity in T.brucei as well(1).

But back to palmitoylation, the post-translational addition of palmitic acid to a cysteine residue (reviewed in 2 and 3). It can occur in any place in the protein, and so far no consensus palmitoylation motif has been identified. Often it occurs in an N-terminus cysteine, just next to a myristoylation site. These proteins are said to be dually acylated. Palmitoylation can also happen in close proximity to prenylation sites. Palmitoylation can perform many functions in the cell, the most obvious being protein tethering to membranes and cellular localization. It has also been shown to interfere with protein-protein interactions and even with protein degradation. For example, the yeast SNARE Tgl1, when is not palmitoylated, can interact with the ubiquitin ligase Tul1(4).

Those paying attention (or still reading) will think that it is a bit strange that palmitoylation can regulate protein degradation like that. In fact, most of these lipid modifications, N-myristoylation for example, last for the life time of the protein. Well, palmitoylation is characterized by a unique feature: it is reversible. This means that it can determine processes that other lipidations cannot. Dynamic trafficking of proteins of proteins, for example. This is the case of Ras protein, which through an deacylation/reacylation cycle is able to exchange between the plasma membrane and the Golgi apparatus (5). Reversibility can also be important for signalling. Regulator of G-protein signalling 2 (RGS2) was shown to increase or decrease its GTPase-activating ability depending on which of its cysteine residues are palmitoylated(6).

The process of palmitoylation in itself is catalyzed by the enzymes palmitoyl-acyltransferases (PATs). These were pretty hard to identify, apparently. Not only they were difficult to purify, but the reaction itself can occur spontaneously with biological significance(7), which made many sceptic on whether these enzymes existed at all. However, they were identified, first in yeast, now in mammals and in my dear T.brucei.

And what was supposed to be a short post if already long enough. I finish by adding that my project was about finding proteins that were palmitoylated and that would change their localization in the absence of this modification, in an attempt to identify substrates for RNAi PAT studies in T.brucei. I must say that the project was not particularly successful, but I developed the important skill of writing long discussions based on few results, which I bet will come handy in the future.

I now challenge Mel to continue in this post-translational modification topic and tell us about his project on glycosylation.


References:

1. Price, H. (2002). Myristoyl-CoA:Protein N-Myristoyltransferase, an Essential Enzyme and Potential Drug Target in Kinetoplastid Parasites Journal of Biological Chemistry, 278 (9), 7206-7214 DOI: 10.1074/jbc.M211391200

2. Nadolski MJ, Linder ME. Protein lipidation. FEBS J 2007;274:5202-10.

3. Greaves J, Chamberlain LH. Palmitoylation-dependent protein sorting. J Cell Biol 2007;176:249-54.

4. Valdez-Taubas J, Pelham H. Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation. EMBO J 2005;24:2524-32.

5. Rocks O, Peyker A, Kahms M, et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 2005;307:1746-52.

6. Ni J, Qu L, Yang H, et al. Palmitoylation and its effect on the GTPase-activating activity and conformation of RGS2. Int J Biochem Cell Biol 2006;38:2209-18.

7. Kummel D, Heinemann U, Veit M. Unique self-palmitoylation activity of the transport protein particle component Bet3: a mechanism required for protein stability. Proc Natl Acad Sci U S A 2006;103:12701-6.

Faith of a different sort

So having finals upon us should be no excuse to stop the advancement of scientific and philosophical thought on here. So when I wandered into town today to do a favour for my dad (30 min walk for me, 3 hour drive for him) I saw a stall with a sign talking about truth. I still have no idea what a truth is so I thought I would have a look at what truths they were talking about. It was a stall full of Jehovah’s witnesses and I was very nice and polite before you say anything! I always am to there face. Being nasty and telling them, ‘you are probably was wrong as you can be’ doesn’t help win them over to your cause! Richard Dawkins QED. But over our chat I mentioned I supported evolution and was an atheist. But they suggested trusting some of the gaps in evolution had an explanation needed faith. This is an interesting one. It doesn’t but it requires something. I know very little physics and geology. If someone was to question the age of the earth in front of me I would have to turn around I say ‘I don’t know how they worked it out and don’t understand the sciences behind it’ so is it faith in these people I have in other scientists? Probably. But is it bad or wrong for me. I think it is a different sort of faith. First of all it is not blind absolute trust. I know I can easily be wrong about many things (it doesn’t happen often enough though) and I know other can be.

We all need to have this kind of faith to get through life. We cannot know everything so we need to trust others or ‘the system’. Imagine a friend offers you a life in there car but you have never seen them drive before in your life. You don’t know if they are good or bad or will get you killed or not. But you get in anyway. Perhaps because you are so lazy you don’t care if you could die as long as it gets you there faster like me. Or because you have faith that having passed a driving test and having belief in there abilities means you can trust them. It is like knowing peer reviewing produces mostly good knowledge and the author is confident in the results even if you don’t understand the experiments or the maths used to analyse it. This evidence based faith is not a bad thing but needs use to take the results with a large pinch of results. It is not perfect. Sometimes someone who you think would be a good driver turns out not to be while taking a ‘fact’ at face value because you trust the scientist you get it from could mean you are wrong but sometimes we need to take these ‘leaps of faith’. Just know that is what you are doing when you do.

Agree or not?

Drosophila: Virgins, balancers and jump-outs

A wise man once said that the sooner you find your model organism, the better. Ignoring the fact that the wise man I am quoting is actually me, I think I generally agree with him. This year I am working on an exciting project which extensively uses the model organism Drosophila melanogaster, geneticists’ favourite little invertebrate. In the 15 short weeks I have been working with flies, I have learned a great deal from these wonderful creatures and experienced emotions from adoration to amusement to frustrated rage in my dealings with them.

Briefly, this is your average everyday “fruit fly” (even though a more appropriate name would be “vinegar fly”) with the complete package of a head, a thorax with 2 wings and 6 legs, an abdomen, and everything else that makes flies flies. I will spare you the tediously detailed anatomy and physiology lecture and will instead jump straight to the cool stuff that interests us geneticists and you readers of genetics blogs.

One of the most interesting things about Drosophila is in fact the people that use them in research. These people are commonly known as “fly pushers”, since they do in fact spend a lot of their time pushing flies around under a microscope using an old half-mangled paintbrush. One thing that you will inevitably hear about once you start hanging out with fly pushers is how interested they are in virgin females. Flies, that is. You see, since experiments in fly genetics involve crossing male flies from one line to female flies of another in order to obtain progeny of the required genotype, one must ensure that the females have not already been fertilised by males before picked out and crossed to a different line. Collecting virgins involves looking for flies that have a dark spot in their abdomen called the meconium, which is visible for 2-3 hours from eclosion (i.e. emergence of the adult from the pupa). Females will not be receptive to males for up to 8 hours from eclosion so any fly that has a meconium can be safely considered a virgin. The most efficient way to collect virgins is to empty a vial of eclosing flies in the morning, selecting females with visible meconium, and returning in the evening. Any females in that vial will be virgins even if the meconium isn’t visible, since less than 8 hours will have passed since your last collection. Much like the world we live in these days, virgins can be rather hard to find, so if you ever hear a scientist worrying about the fact that they need as many virgins as possible, you now have an idea of what they are talking about.




Now let’s delve into some actual genetics. One of the multitude of reasons Drosophila are an excellent test tube for genetic experimentation is something called a “balancer chromosome”. A line of flies carrying a recessive lethal mutation can be maintained by tracking the mutation using dominant markers, or by “balancing” it over a different lethal mutation on its homologous chromosome. This way each chromosome “rescues” the mutation the other is carrying. However, if recombination takes place between these chromosomes, it is likely that you will then have one chromosome carrying both mutations over a wild type chromosome, which will quickly take over the stock and the mutations will be lost. You could always outcross males every generation (there is no recombination in male Drosophila) but that would be very laborious. In order to get around this problem, fly geneticists initially discovered and subsequently intentionally generated balancer chromosomes. These are full chromosomes which contain multiple inversions (essentially all the genes are still there and intact, but their order is jumbled up) in order to suppress recombination with its homologous chromosome and make any gametes with recombined chromosomes non-viable due to aneuploidy. They also carry some dominant visible markers such as Cy (curly wings), Tb (tubby), Sb (stubble) and others, and some recessive lethal markers, meaning that they can never go homozygous.

So, to illustrate this, if you have a recessive lethal mutation on chromosome 2 (let’s now call that chromosome “Yorf”), in order to establish a line that will self-perpetuate and not need constant attention, all you need to do is cross it to a line that carries a balancer second chromosome, for example the chromosome CyO. You will now have a line whose flies will always have the same genotype, i.e. Yorf/CyO. As the Yorf/CyO flies interbreed, any Yorf/Yorf progeny will not survive because the mutation will be lethal, and any CyO/CyO flies will also not survive because of the recessive lethal marker that CyO carries. There will also never be any recombination between the Yorf and CyO chromosomes because of the multiple inversions in CyO that prevent homologous pairing. Wonderful, isn’t it?

Another great thing about Drosophila is insertional mutagenesis. P-elements are transposons, short stretches of DNA which, in the presence of transposase and absence of inhibitor, are excised from the genome and reinserted randomly into another site. Some P-elements are rendered inactive by having the transposase gene deleted. This temporarily immobilises the transposon, until that fly is crossed to a fly carrying an active transposase gene. In the progeny, the P-element will jump out, and if it inserts into the coding sequence of a gene, it can disrupt its function. A mutagenesis screen can then be carried out and the insertion locus can be mapped using standard complementation or cytological mapping. There is however an application of P-element mutagenesis that has accelerated Drosophila genetics in recent years.

The targeted gene knock-out system developed in mice is an excellent way to study the role of single genes, but the long generation time and ethical concerns make the mouse a rather cumbersome model organism. An analogous targeted gene disruption tool has been developed in Drosophila, taking advantage of a property of P-element transposition. When a P-element is transposed, its excision from the genome can sometimes be imprecise, taking some of its flanking DNA along with it, thus causing a deletion in that gene. Since an extensive library of mapped P-element inserts already exists (in over half the fly genome), a line carrying a P-element insert in a particular gene can be obtained and the P-element “jumped out” by crossing to a transposase line. In the cases where the jump-out has been imprecise, the result will effectively be a targeted gene knock-out.

This is, of course, only scratching the surface of the range of genetic tools that Drosophila offers. The best way to understand fly genetics is to try some crosses on paper yourself. Some quite amazing things can be achieved within a few weeks’ time simply by performing a series of crosses and studying the progeny. The waiting time is still a little too long for impatient people like myself, but careful time planning can ensure that you always have some flies to play with and are never stuck staring at a pupa, waiting for the damn fly to emerge.