Sticky proteins, complexity drama and selection's blind eye

*For your entertainment, rejected titles:
[Sticky proteins and complex relationships]
[(protein) Relationship drama: promiscuous proteins in small populations]
[Not all is good that sticks: non-adaptive complexity gain through compensatory protein adhesion]
[Man, I suck at titles]

NB: This post can be considered as part 2.5 of my In defense of constructive neutral evolution series; also recommended for some background are part 1, discussing selection, drift and Neutral Theory, and part 2, discussing Constructive Neutral Evolution; to answer a popular question, part 3 *will* materialise eventually once I get off my ass and write it.

Constructive neutral evolution is one mechanism of complexity increase without any associated increase in fitness – or, in other words, non-adaptive complexity gain. Basically, a random interaction between two proteins can lead to a fixed dependency if this interaction compensates for a mutation that was otherwise lethal – termed 'pressuppression'. In this way, previously unnecessary dependencies accumulate to make a very bulky, bureaucratic system that essentially does the same thing. We've all seen it in our institutions, and evolution is about as efficient.

Now, one bottleneck in this model is waiting for proteins to actually interact. Proteins are quite sticky and non-specific by nature, but usually not too much as that can be quite deleterious. Piling up a bunch of proteins on each other has a non-negligible chance of interfering with their function, and one would expect for chance interactions to not be excessively promiscuous, although those who have done regulatory genetics and protein work are probably aware just how annoyingly non-specific some of the protein binding can get. Luckily, there is now a possibly mechanism boosting these chance interactions, and thus alleviating that particular bottleneck in the Constructive Neutral Evolution process, rapidly accelerating complexification and protein network obfuscation to the extent where the interaction map looks like a web; not a finely organised web of an orb-weaver but rather one of those clumpy webs that are a clusterfuck of stickiness and silk. Enter this week's Fernández and Lynch 2011 Nature paper, from here onwards referred to as "the paper".

Protein 'stickiness' can be enhanced by biochemical means. Proteins vary in stability, and themselves come in populations – generally, most are in the optimal conformation that is presumably functional, but some individuals are messed up. This happens well past the sequence and folding errors, and some perfectly 'normal' proteins can be in a suboptimal state at any given time. Clearly, this affects the overall efficiency of the protein – even if it's enzymatically awesome, the overall 'protein' as we biologists understand it (sans population aspect) would decline in efficiency if a large chunk of its population is in a misfolded state.

One aspect that pushes around the proportion of the protein in the 'right' conformation is how well it plays with water. It shouldn't be too surprising that hydrophobic regions induce instability. What was new to me, but perhaps old news to those who actually understood chemistry, is that the exposure of the polar(hydrophilic) protein backbone to water also has a destabilising effect – and not only that, but often more significant than that of exposed hydrophobic regions! This may seem counterintuitive – doesn't water like hydrophilic regions? And there lies our problem.

Water molecules are attracted to polar groups, and the amino acid backbone is quite polar. This means little water molecules wander in towards the backbone and form hydrogen bonds with it. The problem is twofold: first of all, the protein, like all molecules, likes to 'jiggle'. The more it can jiggle in its given conformation, the more favourable that conformation is thermodynamically since its satisfied by more states. Entropy, etc. (now we're *really* entering territory I know nothing about, since my phys chem experience is locked away by PTSD...). Hooking up this backbone with water molecules reduces its 'jiggle' room, and makes it less thermodynamically stable – making change to other conformations more probable, therefore possibly leading to more errors in the protein population.

Secondly, as detailed further in the paper, water likes to hang out with more of itself. Water molecules are happiest in foursomes, sharing four hydrogen bonds with their neighbours. When a creepy protein backbone emerges and lures an unsuspecting water molecule away into the protein's murky depths, the water molecule cannot form as many bonds with its fellows (or as many hydrogen bonds, period), and is really sad and lonely. Or, in proper terms, the system becomes less stable, since thermodynamics will favour an arrangement where these water molecules are all happily coordinated with each other, and not being molested in a corner by an amino acid polar group. In other words, exposing the polar backbone (Solvent-Accessible Backbone Hydrogen Bonds, SABHBs in the paper) to water induces what is called Protein-Water Interfacial Tension (PWIT).

One way this tension can be released and the backbone exposure ('coded for' by genes, by the way) can be compensated for is if a random other protein (or more of its own kind) are recruited to cover that exposed backbone. This would help stabilise the protein conformation, and allow this potentially deleterious drawback to be tolerated (and get fixed in the population). Ultimately, the second (and third, etc) protein can become exapted for something useful, although just an eventual dependency is good enough to make sure these proteins stick together permanently. The crazy web of interactions gets crazier.

Fernández & Lynch's fig1a suffices perfectly but I like making diagrams, so I made one anyway. See text.

(Disclaimer: I'm horrible at chemistry, this may all have been thoroughly wrong...read the paper.)

Now I'm about the last person to willingly blog about biochemistry, and this seems to have little only a distant relevance to evolution, particularly the non-adaptive kind that fascinates yours truly. It will make sense in a bit. Recall from a few seconds ago (hey, already difficult for some of us) that protein instability leads to reduced protein efficiency. This reduction is generally tolerated, however, until it's bad enough to have a higher chance of being removed. Recall from [what should be] introductory population genetics that selection acts probabilistically, with true slightly deleterious mutations have a lesser, but still significant, chance of fixation than strongly deleterious mutations, which selection has a higher chance of taking care of before drift quietly fixes it. (more detail in older post here) Since proteins are, quite unsurprisingly, also governed by fundamental principles of population genetics, drift becomes involved there too.

As populations get smaller, drift becomes a more dominant force relative to selection, and the window of 'effectively neutral' mutations – slightly beneficial and slightly deleterious, but unlikely to be dealt with by selection – increases. More mess is tolerated. This means more protein inefficiencies are allowed to fix in the population, those induced by backbone exposure among them. Since there are now more proteins that are no longer happy with themselves (or, rather, have an increased Protein-Water Interfacial Tension), they are more likely to stick together for biochemical stability. And here Constructive Neutral Evolution can come in too, allowing further deleterious mutations that are now presuppressed by the recruited proteins. In a way, this greases the presuppression process, rather than competing with it as this BBC news piece made Ford Doolittle appear to suggest.

Now, this is all great in theory, but is there any real data in support of this? For one thing, there is a clear increase of interactome (set of all interactions in an organism) complexity correlating with decrease in effective population size, suggesting a link between lax selection and accumulating complexity. Furthermore, the proteins in organisms of these smaller populations have more blistering backbone exposures to water. Supporting the relationship with population size further yet with the advantage of more phylogenetically independent events (but less interactome data), bacterial intracellular endosymbionts consistently exhibit higher protein backbone exposure (hydration) than their free-living counterparts. Selection appears to disfavour not only polar backbone exposure (also described as 'poorly wrapped proteins' in the paper), but once again, the rise of interaction complexity as a whole. (Fernández and Lynch 2011 Nature, in case you somehow managed to miss that)

Obviously I like this paper because it adds another mechanism to the arsenal of evolutionary processes happening independently of adaptation. But moreover, I don't think one can find too many examples of biochemistry mixed with population genetics. You hardly find cell and developmental biologists thinking about population genetics, and perhaps many biochemists have never even been exposed to such a subject. When fields that should never come that close together do, some really nice explosions of insight can occur (my sad attempt at chemical metaphors). We really need to talk to other more, and maybe even wander over to other departments from time to time. It's sometimes (often) frustrating to communicate with those strange ones from afar, but just like ethnic xenophobia, its interdisciplinary counterpart must also be overcome.

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Figure 2a annoyed me a little as it ignored phylogenetic relationships, which is a big no-no when comparing properties of taxa. The figure is technically fine, especially since there aren't any correlation analyses there, but it's hard to discount phylogenetic history as being the cause behind the correlation of the traits without actually the characters on a tree. Anyway, since I like playing with data and running statistical analyses on things, especially when I didn't actually have to go through the pain of obtaining the data myself, I mapped some characters (interactome complexity from fig2a) on a phylogeny:

Unfortunately, even the most basic statistical operations become an epic headache when trees are involved, and very quickly things become painfully complicated, for the human as well as the computer. Especially when you're handed a dataset of mixed categorical and continuous characters, as I learned the hard way last night. After fighting Mesquite for a good many hours, I finally had to resort to extracting the Ne*µ (effective pop size * mutation rate; roughly put, both lead to increased selection efficiency) estimates from Lynch & Conery 2003 – relying on an intersection of two datasets meant that our taxon sampling was quite sad by the end of this enterprise. Anyway, I ran a pairwise comparison test (Maddison 1999 J Theor Biol) on the data, which probably isn't the best thing ever, but I got something resembling significance: p = 0.019. Depending on how statistically noisy your field is, you may even deem this acceptable. In any case, not too bad given my crude (and somewhat clueless) analysis and limited taxon sampling:

Moral of the story: the inverse correlation between interactome complexity and effective population size is unlikely to be a mere artefact of shared phylogenetic history. In other words, Fernández & Lynch's hypothesis stands strong.

I mostly did this because I thought it'd take a couple hours max. If hours meant days, that wasn't too far off... but hey, I learned something!

Acknowledgments: thanks to Lucas Brouwers for helping me wade through the heavy biochemical stuff, and to Mike Lynch for explaining the key idea of the paper a while earlier. Otherwise I would've probably been too daunted to even read it, let alone blog about it...
Oh, and my Twitter people for random phylogenetics advice ;-)

Reference
Fernández, A., & Lynch, M. (2011). Non-adaptive origins of interactome complexity Nature DOI: 10.1038/nature09992

[will add some supplementary refs once I return to internet on Monday...]

Ratcheting up some splice leaders: a note on directionality

In the sea of eukaryotic genetic diversity also lurk different manners of doing day-to-day genome work itself. Ciliates run two nuclear genomes, trypanosome kinetoplasts contain a chainmail suit of RNA editing circles and dinoflagellates are just weird in every genome compartment they have. Their plastids contain tiny minicircles often containing but a single gene, capable of "rolling" transcription where the minicircle is much like a Mesopotamian cylindrical seal, leaving a concatenated repeated string of genes on the transcript. The mitochondria have linear genomes with short fragmented repeated chunks of important genes all over them. But the nuclear genome is the most fucked up: for one thing, dinoflagellates lack a few histones, and have enormous genomes stored in absolutely bizarre chromosomes. More importantly for our story: every single gene must be trans-spliced with a 'splice leader', a short sequence that attaches at the beginning of the mRNA transcript and brings to it the 3' cap necessary for transcription to work. Oddly enough, Euglenozoans like the trypanosomes and euglenids seem to have a very similar system, evolved entirely by chance* convergence (Lukes et al. 2009 PNAS goes over this remarkable convergence in more detail).

*Or perhaps something happened to both that made them prone to evolve this bizarre system.

Genomic quirks are not just interesting in their own right as some arcane oddities, but can reveal a great deal about the dynamics of genomes in general. The dinoflagellate splice leader system turns out to yield a very crisp illustration of the power of ratchets and the toll of reverse transcription on genomes.

To reiterate, every single nuclear gene transcript in a dinoflagellate must be spliced with the 3'cap-bearing 'splice leader', or else it simply won't work. This means that the dino is full of mature transcripts with splice leaders attached to the transcribed genes. Enter reverse transcriptases, which are prevalent in probably most, if not all, eukaryotic genomes, thanks to viruses and their partners in genomic parasitism crimes, transposons. When they're not busy moving transposons around and helping viruses move in, they reverse transcribe random gene transcripts for fun, that may then, on occasion, be successfully recombined back into the genome. This process probably doesn't happen [successfully] every day, but over thousands or millions of years (and countless individuals) is rampant enough to leave a noticeable trace in the genome.

So we have a load of transcripts floating around with an extra sequence stitched onto them from the splice leader. Do the reverse transcriptases care in the slightest? Of course not: to them, a ribonucleotide is a ribonucleotide, give or take some trace biophysical stuff that might make a couple people cringe at what I just said. (meaning, I wouldn't be surprised if there could be some slight but ultimately detectable biases there too) This means that splice leader, on occasion, actually makes its way back into the nuclear genome attached to the beginning of the gene.

However, this splice leader does not substitute for the usual splice leader trans-splicing, since the 3' cap must be added again, or else the transcript will not be translated. That now-nuclear gene-attached splice leader ends up being completely useless, and is able to gradually degrade into benign junk, provided it doesn't mess with the translation of the gene. What is really cool is that one can actually see this gradual degradation, as shown in Slamovits and Keeling 2008 Current Biol:

Mmmm, actual data! Note how the oldest SL piece closest to the gene (on the right) is the most degraded. (Slamovits & Keeling 2008 Curr Biol)

Once the unnecessary splice leader chunk becomes part of the gene, the gene gets transcribed and trans-spliced like any other – meaning it is once again susceptible to replaying that same process of reverse transcription, except this time it already has a relict sequence. It can acquire a second one on top of that. This explains how there can be several concatenated splice leader relics tagging along, like in the above figure.

Splice leader trans-splicing not necessarily promoting reverse transcription – only makes it easier to detect. In other words, it inadvertently makes for a wonderfully convenient system where you can actually track what happens to a gene after it gets reverse transcribed. Once the gene makes its new home, the old gene copy is still present and they generally would be functionally redundant, so the dual-copy state is extremely unstable as ultimately the loss of one of the copies will be tolerated. If the newly transcribed copy is lost, we never see it and thus don't talk about it in the first place. However, once the clean original is lost, only the gene with the crap from the splice leader remains, and reversal to the original state is so improbable it's practically impossible. In other words, this process is a wonderful example of an evolutionary ratchet.

Ratchets are interesting because they confer intrinsic directionality to a system, even in the absense of external pressures (like selection). The accumulation of splice leader junk in the dinoflagellate's genes isn't particularly healthy, nor is it particularly deleterious – it's effectively neutral. However, one can argue that we do have an example of bloated complexity here. Since you can't go back and lose chunks of splice leaders, this ratchet essentially ensures that left to its own devices, this aspect of genome complexity will increase on its own. At a certain point, there will probably be ever-increasing selection against accumulating further splice leaders, and those lineages that go too far will simply die off – the central tendency doesn't care, and the ratchet will keep on going regardless of what selection 'wants'.

This ratchet example is therefore an elegant case of evolutionary direction that's not particularly well explained by the central dogmas of Modern Synthesis or (neo)Darwinism, where selection is the force that crafts order and directionality, with mutation a mere passive provider of material to be molded. I will go into a deeper discussion of this in another post (there's a cool paper coming out soon), but I think it's worth briefly mentioning here too while we're at it. The "mutation" step (to which, I guess, this trans-splicing and reverse-transcription process can be awkwardly attached) here is what provides a drive, a push in a certain direction, and towards increasing complexity, no less (although that last detail is irrelevant). While selection is present and provides constraints (if both genes are lost, for example, the organism dies), it does not do the 'driving' or 'forcing' in this system. Very crudely put, selection here is the passive phenomenon, and mutation is at the wheel.

Another case of intrinsic directionality, but where reversal is allowed, is your garden variety directional bias – where proceeding in one direction is more probable than going backwards. A very basic example of that is if the replication machinery favours a certain type of nucleic acid – left to its own devices, the genome base composition would be skewed in that direction. Boundaries can also induce an apparent directionality, but in this case it's no longer intrinsic... that's, again, a topic for another day.

This idea was a part of the Mutationism theories in the early 20th century, which were a little extreme and perhaps premature, since mutation was far from being even marginally understood at the time. In the usual melodramatic manner characteristic of academia and the scientific community, the pendulum swung far to the opposite extreme, and Modern Synthesis was born. It became heresy to think that mutation itself can actively contribute to direction and order. The field became engulfed in a false dichotomy, where either selection or mutation can actively provide direction, with the modern folk siding with the former. That is a serious mistake and an unnecessary waste of great explanatory potential – you can go so much farther with selection, drift, mutation and recombination all at the wheel, each pulling with different magnitudes in various directions. Well, technically, you wouldn't if you were the thing being pulled – which resonates so well with the absense of 'ascension' or general active directionality in the evolutionary system as a whole. Evolution is a slow, painful, inefficient and rather stochastic process, partly because the cart is being pulled in so many ways.

(The latter part, concerning directional biases and Mutationism, is based on various publications and conversations with Arlin Stoltzfus and Dan McShea, whom I gratefully acknowledge. =D)

References:
McShea, D. (2001). The minor transitions in hierarchical evolution and the question of a directional bias Journal of Evolutionary Biology, 14 (3), 502-518 DOI: 10.1046/j.1420-9101.2001.00283.x

Slamovits, C., & Keeling, P. (2008). Widespread recycling of processed cDNAs in dinoflagellates Current Biology, 18 (13) DOI: 10.1016/j.cub.2008.04.054

Stoltzfus A (2006). Mutationism and the dual causation of evolutionary change. Evolution & development, 8 (3), 304-17 PMID: 16686641

MolBiol Carnival #10: Assays, cyanobacteria and metabolism regulation

Welcome to the 10th edition of the MolBiol Carnival!

Apologies for the delay – am behind on pretty much everything and frantically trying to tie up loose ends of my degree, fun times. Also, it's kinda awkward to write up a carnival post with only THREE submissions – you guys really need to submit more and/or write more MolBiol posts!

It seems molecular biology doesn't get blogged about specifically as much as evolution and diversity – perhaps because molecular biologists are usually busy troubleshooting their PCRs and RNA work for weeks on end, and have little time left over to write. In fact, judging from recent woes experienced by some of my lab buddies, I'm beginning to doubt the existence of RNA and believe it may all be a giant elaborate hoax invented to enslave more grad students. Have any of you ever *seen* RNA? That's what I thought...

This month we have a very biochemical (post-translational, if you will) MolBiol Carnival featuring enzyme spec, cyanobacterial biofuel precursors and some sweet diastereomer metabolism regulation.

Enzyme Assay
Christopher Dieni at BitesizeBio has a nice write-up on measuring enzyme kinetics using UV spectrophotometry, complete with procedure, tips and troubleshooting – the kind of thing you wish accompanied every assay you've been assaulted by. Not being anything close to a biochemist, I had no idea you could actually observe enzyme action using something as simple as a spec, so this is quite cool!

Cyanobacteria and biofuel production
With growing concerns with using land plants for biofuels (for one thing, kind of odd to use food to power cars when not everyone has enough of it...), increasing attention has been turned towards algae eukaryotic and not. For one thing, algae are already quite good at photosynthesising and are vastly more abundant than plants, and arguably have the largest contribution to global photosynthesis – not surprising given the earth's surface is 70% ocean. Michael Scott Long at a NASW.org blog explains recent developments in genetic engineering and domestication of cyanobacteria for fatty acid production.

Diastereomers and regulation of metabolism
Stereoisomers are the beginning chemistry student's worst nightmare – they're so similar and easy to mix up, particularly if you're like me and can't tell left from right to begin with. However, a bacterium (rather, its enzymes) would have little trouble with the stereochemistry portion of a intro biochem class – to them, stereoisomers are day and night (and other things). Glucose and galactose are 'close enough' to each other for a biochem student, but a flipped arrangement at just a single stereocentre is enough to require a whole new set of enzymes and drastic changes in the pathway. E.coli prefers glucose, but can also process galactose (compromising its growth rate) by embellishing its metabolic pathways a little – the products of galactose digestion are sent to the tricarboxylic acid cycle via the glycoxylate shunt. Becky Ward at It Takes 30 discusses how sugar type availability affects the transcriptional regulation of this glycoxylate shunt, among other things, featuring a galactose-loving mutant.

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This was fun. Wish there were more submissions – having to write up random blog posts forces me to revisit forgotten subjects and explore new ones: I'd never brave a post on metabolic regulation on my own! By not submitting, y'all are having a deleterious effect on my education... ;-)

The next edition will be hosted by our resident microbiologist @labratting at Lab Rat, and she better get more than three submissions... come on, we do so much molecular biology in almost every field of biology! Write 'er up, dammit!

MolBiol Carnival #04

Welcome to the fourth edition the MolBiol Carnival!

With a GRE to write this Thursday, don't have much time or energy for creativity, so I'll just run the submitted posts on a gel this time. The submitted posts were measured for word count* and run on a gel in Excel (didn't know you could do that, did you?) – aside from a contaminant (and mysterious primer dimers despite not using any primers), the gel seems to have worked and is displayed below:
(Sorry, was too lazy to make a fake ladder too...)
*Ideally, one would use character count instead, but that requires extra clicking and I'm lazy energetically-challenged.

Now I'll do some gel extractions and run the resulting sketchy solution through a sequencer (ie dumped them off at the sequencing unit, to be glared at by the personnel as usual). Ooooh, it came back like this: nnnnnnynnnnnrmnnnnhrnnnnnnrannnnnn. That's right, I have a single base pair recognised! And that was my longest read! nnnn's are so easy to assemble into contigs. Am I doing it right? Nothing wrong with sequencing single posts without amplification, you see?

And this is why wise ones keep hardcore molecular biology preferrably in an entirely separate building from cell biologists like me. Preferably with security. For the greater good of Science.

So I have up on 'sequencing' the posts from scratch and went directly to 'GenBank' (reads, The Internet) where these posts were strangely already deposited. As they say, "Two months in the lab can save two hours in the library." – source unknown.

Saving the best for last, or going straight for the best first, we start with our friendly and not-so-friendly prokaryotes. First off we have an explanation of the effect of ATP on bacterial biofilms in the medical context by Michael Scott Long at Phased. Next we have another biofilm-forming bacterium, beautifully-named Golden Staph, with a really nice SEM, seen at the right. It has a close relative which may actually be quite helpful to us medially, explained at James Byrne's Disease of the Week!. Another post from his blog features Pseudomonas aeruginosa and amazing antiseptic honey action. Apparently, biofilms don't fell so well after being smothered with honey. Last item from Disease of the Week this issue comes just in time for flu season – an explanation of how vaccines work, in two parts.

Next up we hear about bacteria being 'floxed'. To find out what the Cre-lox system and Streptomyces have to do with each other, head over and read LabRat's post for your daily flox. She also has a nice post on bacterial division.

Taking a break from small things, we have a post on thoroughbred horses: turns out, while the pedigree of the stallions was well-maintained, it did not dawn upon the ancients that the mares contribute half of the phenotype. Thus, while the males were imported from various exotic locations, any local female was considered to suffice...find out more at GrrlScientist's Punctuated Equilibrium.

Next we have Lucas Brouwers on tinkering and the evolution of novelty at Thoughtomics, tracing the story of the metazoan nuclear receptor. Of course, this receptor could be misfolded upon formation, like any other protein. How are defect proteins removed before they wreck havoc upon the cell? Enter E3 ubiquitin ligases and their role in removing proteins originating from mRNAs devoid of stop codons, in a post by David Weinberg at You'd Prefer an Argonaute (a title that makes me feel oddly...silenced *groan*).

And last but not least, all the proteins must fit somewhere. Well, the genes that code for them anyway. Often, these genes have a very spacious home in a massive genome(amid piles of junk), as discussed in Iddo Friedberg's Byte Sized Biology. Here I must shamelessly add a plug for 'my' kingdom: the coolest genomic gymnastics happen among the protists, aka "the other 99% of eukaryotic diversity, that you don't hear about". We have the smallest eukaryotic genomes, called nucleomorphs, as relict algal nuclei remaining after secondary endosymbiosis (in cryptomonads and chlorarachniophytes). We also have [arguably] the largest eukaryotic genomes: Amoeba proteus and Amoeba dubia, the latter around 670GBp, as well as dinoflagellates with their unusual low-histone nuclei. And size is not all that matters – some nuclei, eg. of euglenozoans, have polycistronic messages consisting of many eukaryotic genes riding off a single promoter. They rely upon splice leader trans-splicing to work, and that is only the beginning of awesome... feel free to stick around here for more! ;)

That's it for this month's edition of The MolBio Carnival. You can check future hosts and past editions on the Carnival's home page. Be sure to subscribe to the RSS feed to receive notifications and summaries when new editions of the Carnival are posted. Also, you are welcomed to submit your best molbio blog articles to the next edition of The MolBio Carnival which will be hosted by LabRat. More info here. The previous edition was hosted by Alexander Knoll at Alles was lebt.

My favourite thing about carnivals is the exposure you get to various topics and writers as a reader, and the forced exposure to various topics as a host. Thus, submit, submit, submit – feel responsible for enlightening the next host as well as the readership about the existence of topics they never come across! Molecular biology is everywhere – let's see more of that!

PS: Carnival of Evolution #19 came out yesterday at Byte Sized Biology.
Also, if hungry and poor, Sci at Neurotic Physiology has an awesome compilation of recipes at her The Grad Student Eating in Style Carnival.

PPS: Apologies for slight delay. Reason – my life looks roughly like this comic. INTERNET! FOREVER...

Some cited references:
[Will add as soon as I wake up + get to a computer tomorrow. Don't want to be walked in on still being in the lab at 6am...]

Obligatory synthetic genome post: clearing up some confusion

I wasn't gonna bother writing anything about this, considering that pretty much the entire blogging community has sucked the topic dry and written about it much better than I could've. But one little detail still bugs me enough to fail at keeping my trap shut: the phrases "synthetic cell" and "synthetic bacterium". And the brutal media misrepresentation of the whole thing. Also note there will be bias as I tend to be rather skeptical towards synthetic biology in general, partly because the sheer magnitude of their difficulties are underplayed in the media, and replaced with some naive fantasies about "custom life" or irrational fears of Frankenstein-like creatures taking over the world or something. I think we are faaaaar too behind in our understanding of biology attempts at understanding biology for any of these fantasies and fears to be worth considering.

With the recent hype about the synthesis of a new bacterial chromosome (based on an existing one with a few minor modifications), it seems like media and bloggers alike are confusing 'genome', 'cell' and 'organism', using all three interchangeably. In fact, one does get the feeling that lately the existence of the cell has been largely eclipsed by the genome. As it largely has been outside the field of cell biology, sadly. I think a nice sketch of this majority viewpoint can be represented in this quote from Pharyngula:

"So, if after a period of time, you've got a cell whose DNA was produced by a machine, and whose membranes, enzymes, structural proteins, and metabolic by-products were all produced by that machine-generated DNA or the protein products of that DNA, what makes it a non-synthetic cell?" PZ Myers 22 May 2010

Granted, this was said in defense before some utterly ridiculous claims by crazy people, eg. that this somehow proves creationism. Still, as a biologist, PZ Myers should know better - only the proteins and nucleic acids (incl. ribozymes) have been synthesised by the genome. The membranes and non-protein metabolic products, while influenced by genomic activity, also have a life of their own, having been inherited and modified since the origin of life itself. Furthermore, systems like cellular organisation are also not entirely 'programmed' by the genome, and are also inherited extragenomically.

In the paper, the authors use the word 'control' to describe what the genome does, which I think is also not entirely accurate -- would be close enough for most circumstances, but the topic here has become much too philosophical and thus demands careful semantics. Thus, normally I wouldn't've even noticed the slightly misused term. Strictly speaking, as mentioned before, the genome synthesises proteins and ribozymes which act in symbiosis with membranes and cytoplasm to form the cell, also the fundamental level of selection in most cases (eg. see the discussion near the beginning of Cavalier-Smith 2001 J Mol Evol). Thus, the genome cooperates with the rest of the cell, rather than controlling it. Both the extragenomic and intragenomic elements 'seek' to be propagated further, and are mutually co-dependent to achieve said goal, thereby acting as a unit. The gene-centred view popularised by the likes of Dawkins may well be parly responsible for the dismissal of heritable (and thus, evolvable) elements outside the nucleus. While the gene-centred view lays foundations for many important and useful models, one must not get too carried away with it.

There's also a point made that by inserting the synthetic M.mycoides genome into M.capricolum, the latter was essentially transformed into the former - that is, 'changed species', if you will. First of all, the M.capricolum-now-mycoides cell is quite possibly still not identical to M.mycoides, perhaps retaining some cytoplasmic features unique to M.capricolum - this depends on how truly different the two species were to begin with. Which leads us to the second point: the muck that is our attempt to define a species in prokaryotic (that is, asexual) populations. While I personally think that, philosophically, a case can be made for some form of species concept in prokaryotes -- eg. stable 'islands' in the 'fitness landscape' or 'design space' -- the authors have not provided a clear description of the difference between the species (or strains?) in question, and thus it is difficult to evaluate the claim about 'one species taking over another'.

The insertion of nuclei into foreign cytoplasm is not a novel concept. In fact, some red algae have mastered the technique millions of years ago, long before animal cloning and such (remember Dolly?). And genomic fragments overall often tend to be quite promiscuous and not too choosy about their cytoplasmic environment.

My intent is not at all to underplay the achievements. Creating long stretches of custom modified DNA is kind of nice, and could perhaps someday be helpful in, say, generating complex knockouts or modifying multiple gene expression patterns/fusing stuff to them/etc at once. Perhaps someday people will look upon our small-scale molecular genetics work in much the same way we now [try not to] laugh at people who spent years sequencing one gene by hand. I worry whether we are entirely prepared to handle such an onslaught of data, but perhaps 20 years ago they wondered the same thing about us. Perhaps someday organismal biologists will move from molecular genetics to molecular genomics (and thus it is imperative for us to understand both genomics and the tree of life itself!). Again, our current work was beyond fantasy just some two-three decades ago!

But I don't think Venter's paper signals any sort of new era of biological science just yet, let alone humanity or whatever. The world has not ended yet. Nor has utopia begun. Tomorrow is back to lab as usual!


I'm rather overwhelmed by offline stuff right now, especially in the reading and comprehension (and writing!) department, and thus have no time to read over what everyone has to say about the paper, let alone analyse the results in any particular detail, but here's a few recommended musings on the subject, much better written than mine:

Opisthokont - fellow protistologist who kind of scooped me on several points, grrr! =P
(I also wondered about the use of an obligate intracellular parasite in the search for 'minimal life'. Parasites are known to undergo rather extreme reductions both in genome complexity/size and cell structure, and tend to be obscenely derived.)

Lab Rat - bacteriologist who is also cautious about the findings. She also comments that implanting synthetic genomes into bacteria is unlikely to add much to the terrorist's arsenal at this point. She also points out just how much we have yet to know before attempting to create life, as even when an organism emerges from some deep resting stage, it is still equipped with various non-genetic elements necessary for its survival. For the rest, read it yourself! =P

A Russian science news site, elementy.ru, actually got the title somewhat accurate: "The first living organism with a synthetic genome was created" (rather than "OMG SYNTHETIC LIFE!!1!"), and then goes into a detailed history of the project itself, with some insightful comments - apparently at some point Venter's team had some issues with a random deletion in dnaA, kind of important for DNA replication! While I still disagree with their underappreciation of cytoplasmic inheritance, the article overall is well-written, if you speak Russian.

Completely irrelevant to the discussion, but my [poorly informed] impression of Craig Venter is along the lines of this music video from a slightly overfunded (;-)) Harvard lab.


On an unrelated note, Merry and Elio have compiled a summary of the first half-year of microbial blogging for 2010 at Small Things Considered. Anyone interested in microbiology, both nucleated and non, should read their blog if you don't already!

Ok, I've now exhausted my writing juices for the next little while. Hopefully not for long...

Irremediable Complexity: Notes from Ford Doolittle's seminar talk

While I work on polishing up Part II of the Neutral Evolution series, thought I'd write up and post my four pages of notes from Ford Doolittle's seminar talk today yesterday, while I can still remember what my scribbles were supposed to mean. As there's no unpublished data there, and a long-awaited paper on the subject has just been submitted, I believe it should be fair game for blogging. Coming from a vicious field (Arabidopsis, sigh...), I'm generally rather cautious about blogging department talks and such, but Constructive Neutral Evolution is a subject in need to spreading, and not a particularly competitive area at the moment...

Rosie has another summary of today's yesterday's talk, with an executive summary therein; she tried to scoop me, so clearly this means I must outdo her in length and verbosity =P

[If anything doesn't make sense, it's very likely an error on my part]
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Irremediable Complexity - Ford Doolittle, 07 Apr 2010
[paraphrased from notes; own comments in grey]
- Is each step in the evolution of a complex machine useful?

Irremediable complexity involves three factors: Tinkering, small populations, ratchets (Constructive Neutral Evolution). [prior to that, must discuss common views on complexity first]

Common views on complexity
- Directionality: 19th century - divine forces guide evolution; 20th century - orthogenesis - evolution exhibits a drive towards perfection (and complexity)
- Progress: Life started off being simple and became increasingly complex
But is there necessarily a trend?
(Gould 1996 Full House) The Drunkard's Walk: if one starts off at a limit that cannot be passed, random steps will eventually lead away from this limit. Presumably, life began more or less at the lower limit of complexity (specialised parasites aside), so it's bound to get more complex as it's the only way to go.
In fact, complexity is increased only in a few lineages, which also happen to be the ones we really like to look at.

So is the evolution of multicellular animals and plants from choanoflagellate-like and chlamydomonas-like organisms, respectively, a drunkard's walk or driven by something (eg. selection)?

Selectionist explanations
- Accumulation of specific adaptations results in complexity (eg. The Eye)
Another example: larger genome size is an adaptation for more gene regulation which is required in more complex organisms. [Note: C-value paradox, etc] Molecular biologists have an obsession with 'mystery DNA' having regulatory roles...
- Arms races, sexual selection
Greater biodiversity/more competition between organisms stimulates greater complexity, eg through niche specialisation and sex selection
- Evolving evolvability
Outdoing one's environmental changes by evolving faster. Eg. Exon shuffling as a function of introns: introns space out exons allowing more novel combinations of these exons to occur, which may be adaptive.
Caveat with evolvability -- it's a clade-selection level trait, not individual level.

Neutral forces - should be our null hypotheses
- Tinkering (François Jacob) -- will still have traces of an apparatus' past functions along with new ones --> life is full of Rube Goldberg machines.
- Small population size -- mildly deleterious traits more likely to be fixed in smaller populations (Michael Lynch; more info in Part I of my Neutral Evolution series) Eukaryotes are a case of smaller population size relative to prokaryotes.
- Ratchets -- 1. Maynard-Smith & Szathmary's Major Transitions; 2. Stoltzfus & Covello's Constructive Neutral Evolution

Major Transitions: There are steps in evolution that are difficult or impossible to reverse -- act as ratchets.

Constructive Neutral Evolution: a previously fortuitous (non-functional) interaction can enable an otherwise-deleterious mutation to occur, resulting in a dependency upon this interaction. [a diagram is in the making]

More interactions evoke more opportunity for Constructive Neutral Evolution to drive (via ratchetting) an increase in complexity. [thus, the result would be an explosion of complexity past a certain threshold]
The dependency can be built up by other mutations, thus further solidifying the requirement for a given interaction.
Note that no positive selection is required at any step [only purifying], would also be more drastic in smaller populations where more deleterious mutations are fixed by drift. That said, positive selection can still play a role in parallel.

Examples:
Lambowitz's maturase-requiring group I self-splicing intron. [will be discussed in further detail in impending post] A derived Neurospora lineage requires a maturase for the splicing of group I introns whereas nearby relatives do not. Lambowitz later (2006) argues that maturase-mediated splicing evolved in response to the splicing problem, as opposed to enabling it to arise. This is putting the cart before the horse.

kDNA editosome (Trypanosomes et al.)
[Can be summed up in one interjection: whyyyyyyyyyyyyy???]
In summary, genes coded by the mitochondrial genome are non-sensical, and right after transcription the pre-mRNAs are edited by the complicated process involving templates and inserting various U's where they are needed.

There are several explanations that have been proposed:
- relic of the RNA world -- for starters, tryps are derived.
- to correct pre-existing mutations -- backwards logic again (cart before the horse)
- regulation
[Digression to discuss the two oft-conflated meanings of function]
1. Selected function -- how a trait got to be there
2. Current function -- what happens if trait is removed

Example from Maynard-Smith: Stiff back of the horse. Removal thereof would prevent humans from riding it, but no one can argue that the horse's back evolved so that humans could ride it in the future!

How editing really arose -- CNE [see Stoltzfus 1999 JME and Lukes et al 2009 PNAS(and subsequent correspondence)]: once the process started, it couldn't be reversed, thus complexity reached an absurd level.
"Absurdly complex spliceosome"
- if you think about it, it's "incredibly stupid" to have such a complex machine for removing introns
- cites "Five Easy Pieces" Sharp 1991 Science laying out a hypothesis for the evolution of group II introns
- see Lambowitz example for how the spliceosome may have arisen through initially-neutral protein interactions

Ribosome
Also by CNE. Roughly put, the RNA does most of the enzymatic work in the ribosome, with the proteins taking on more of a structural function. Presumably, initially the ribosome could've been entirely a ribozyme, picking up various proteins for supporting structural roles, like the Lambowitz intron, with the help of constructive neutral evolution.

Cited TW O'Brien 2003 IUBMB Life paper: the mammalian mitochondrial ribosome is smaller than that of its host (eukaryotic) yet larger than the prokaryotic counterpart; furthermore, many of the extra mitochondrial ribosomal proteins do not come from the eukaryote! This is a great example of convergence between the two separate evolutionary paths, both starting off with the small (at least in terms of protein count) prokaryotic ribosome and becoming bloated in complexity. Furthermore, mitochondria also have a greatly reduced effective population size, thereby encouraging the accumulation of complexity, unlike in their free-living bacterial brethren. [would be interesting to compare plastid, mitochondrial and eukaryotic ribosomal evolution, controlling for LGT, replacement, loss, and all the other crap associated with endosymbiosis, of course. A nice system with at least two independent replicates originating from very distantly-related sources]

[Another guy (unsure about citing personal communications without their knowledge) rather eloquently put it along the lines of "The rRNA was fully functional enzymatically on its own until the other shit stuck along for the ride"]


In closing, tinkering leads to klunkiness, smaller populations allow more mildly deleterious mutations, and ratchets make complexity ultimately unavoidable.

For another example of irremediable complexity, think what would happen if an institution was formed with a few small committees with specific functions and left alone for a few decades. Like a university. The bureaucracy and administrative complexity would increase while the overall efficiency of the system wouldn't benefit much. One can think of the complex biochemical pathways as "cellular bureaucracy".

"Complexity is unavoidable and largely irremediable: Resistance is useless"


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My initial impression of Ford Doolittle came from the whole 'missing Tree of Life' debacle, where he did appear rather rash and extremist, perhaps largely due to the media portrayal and associated uproar -- after his seminar talk and a more informal chat at lunch, I fully retract my first impressions, and must say that he's actually quite reasonable and not radical or extremist at all (and very pleasant and interesting to talk to). Naturally, science reporters reporters in general tend to hyperpolarise any slightest argument they chance upon, and turn anyone with a shred of an opinion into a flaming radical; thus it was another lesson to be extremely cautious about impressions you get about someone based on someone else's reporting. But another thing I've noticed is he seems less assertive in person than in his own papers as well. Well-balanced and subtle ideas seldom get the attention they deserve, so they must often be blown out of proportion to be at all noticed.

Perhaps this is another reason neutral theories remain so obscure: the adaptationists are occupied with explaining natural selection to creationists et al., thereby being much noisier and noticeable. The more balanced pluralists don't have much extremist thought to push, so they tend to get ignored (I find the Stoltzfus 1999 paper to be quite subtle and unassertive; perhaps that's why it was largely ignored).

Back to the ToL kerfuffle: If one were to say "LGT is very frequent in bacterial populations and may pose very substantial problems to reconstructing bacterial phylogeny and the deep history of life, perhaps even posing a valid question of whether such a phylogeny may truly ever be found", the statement is too reserved and subtle to make much of a splash beyond a small circle of specialists. However, if one goes all out and shouts "The Tree of Life is dead!", people actually turn around and look. Many misinterpret the message and get their own strange notions on the subject (often believed much more strongly than by the originator of the idea...), but at least some people get it and consider the problem.

Furthermore, the "Darwin was wrong" slant was intended to signal that we should get away from our Darwin fetish. I fully agree. Darwin was a great scientist, but it is outright immoral to ignore the hard work of thousands of people who came after him, and the thousands who are working in the field right now. Darwin may have sparked the field, but he is certainly not its most important element. The term "Darwinism" is vacuous and misleading, and should be abandoned soon. Furthermore, who cares what creationists think? Some of us are sick of having to change our vocabulary just because some religious idiot with an agenda could abuse it. The primary role of evolutionary biologists is to study evolutionary biology; educating the public is secondary (while still important); dealing with creationists is about last. Science educators are responsible for that. We cannot fight religion, especially its fundamentalist forms, with reason (hence why Dawkins fails at it). So what if they misunderstand and misrepresent Darwin? People misunderstand and misrepresent quantum mechanics even more, and yet physicists don't seem to lose sleep over it.

Political agendas will always be there; textbooks will always be influenced by the political (and sadly, religious) climate of the times. It sucks, but such is reality. History and the social sciences are also badly mangled in schools (eg. communism is still a topic not only taught entirely wrong, but also characterised by a blunt refusal to listen to those who may know better). After all, to understand science effectively, one must appreciate the subject, and be willing to not only accept, but commit work to it. The unwilling will never learn unless they want to. What is of a much higher importance than defending Darwin is promoting the overall public image of science and academia in general, and making academic activity 'cool' in the eyes of the public. If scientists were well-respected in 1970's Russia, there's no reason we can't acheive that again and in a different society. But it will take work.

But to get that to work, we must strive to suppress our polarising tendencies and recognise that often people's opinions are more subtle than they appear. We must also pay greater attention to ideas that are not pushed as aggressively as others, such as constructive neutral evolution. Those may well be more refined and ready for acceptance than the ideas cast in apparent simplicity and loudness.

References:
O'Brien, T. (2003). Properties of Human Mitochondrial Ribosomes IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 55 (9), 505-513 DOI: 10.1080/15216540310001626610

Sharp PA (1991). "Five easy pieces". Science (New York, N.Y.), 254 (5032) PMID: 1948046

Stoltzfus, A. (1999). On the Possibility of Constructive Neutral Evolution Journal of Molecular Evolution, 49 (2), 169-181 DOI: 10.1007/PL00006540