Not-Monsters Adding Not-Poison to Sugar

Andrew Kimbrell is a goddamn bio-Luddite, one of many.

It embarrasses me that certain liberals can be so staunchly and irrationally opposed to technology, based upon paranoia over corporate interest, a weirdly conservative adherence to the simple purity of "Nature," and their own naked ignorance. One of the major victims of bio-Luddite oppression is genetically modified (GM) foods, sometimes referred to as "Frankenfoods" (but not by me).

In a column today in the Huffington Post, Kimbrell sows paranoia over a specific GM crop, the Roundup Ready sugar beet developed by Monsanto. These sugar beets are genetically modified to be resistant to glyphosate, the active ingredient in the weed killer Roundup.

GM opponents often have a hard time explaining just what makes GM food so dangerous. Sometimes it's argued that the introduced genes themselves are somehow pollutive, despite the fact that it's all the same adenine guanine cytosine thymine, baby. Kimbrell makes a particularly poor argument here, based on glyphosate:

At the request of Monsanto, the U.S. Environmental Protection Agency increased the allowable amount of glyphosate residues on sugar beetroots by a whopping 5,000% -- glyphosate is the active ingredient in Roundup. Sugar is extracted from the beet's root and the inevitable result is more glyphosate in our sugar. This is not good news for those who want to enjoy their chocolate morsels without the threat of ingesting toxic weed killer.

He then goes on about how seed farmers could start making seeds from Roundup Ready sugar beets so the GM crop spreads, and how sugar from GM beets gets mixed in with regular beets, and how GM beet pollen could contaminate other crops' genetics, and how there could be a huge consumer backlash, and how Big Science is putting poison in your dear mother's chocolates OMG!!!

Notice a problem here? How about the fact that the glyphosate isn't coming from the beets, moron!

I repeat, these GM beets do not produce glyphosate. What they do is allow farmers to use glyphosate on their crops with greater confidence in killing off weeds and maintaining good crop yield. The GM beets may increase the incentive to use glyphosate, but if that's a problem, then can be kept in check by regulation. Kimbrell's glyphosate beef isn't with the beets, it's with the EPA's change in tolerable glyphosate limits.

But is that even a legitimate concern? He makes it sound as if Monsanto asked, "Could we please put deadly poison on our beets?", and the EPA said, "Sure, since you asked so nicely!" This is just a guess, but I'd bet the EPA actually looked at some of the science behind glyphosate and its associated risks before raising the tolerable limit.

Glyphosate actually appears to be a very safe chemical. (Please forgive me for referencing Wikipedia here, but seriously, it just goes to show that you don't need to dig too deep to uncover the stupid.) It acts by inhibiting an enzyme in plants called 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Normally, EPSPS kicks off a pathway to synthesize aromatic amino acids like phenylalanine, tryptophan, and tyrosine. In the presence of glyphosate, this pathway is inhibited, so the plants can't make these amino acids, and therefore die. The GM beets contain a copy of the EPSPS gene found in a strain of Agrobacterium. EPSPS made from this gene is resistant to glyphosate, so the aromatic amino acid synthesis pathway is uninhibited. The gene is already widely used in GM soybeans.

Glyphosate does not have this kind of effect in animals because we don't have that synthesis pathway; we get aromatic amino acids from our diet. And at a glance, the evidence seems to suggest minimal other side effects from glyphosate. The EPA would know better than I.

So Kimbrell is getting his knickers in a twist over genetically modified sugar beets that aren't producing a dangerous chemical that actually isn't that dangerous. Yeah... Next time he wants to play bioethics, maybe he should get the "bio-" part straight first.

Creationist Resistance to Antibiotic Resistance, Part II

When last we left our story of evolution and antibiotic resistance, we looked at the importance of random mutation to the development of resistance. Now that we’ve addressed (at least in a small way) the origin of these resistance genes, we can take a look at their greater role in evolution.

Commenter Dan Gaston got us off to a great start on the last post, noting that lateral gene transfer is in fact a primary mechanism of bacterial evolution, even though it doesn’t explain the origin of the genes being transferred.

At the end of the day, evolution is about change, plain and simple. Creationists don’t seem to realize this, as evidenced by their second objection to resistance as evidence of evolution: “If resistance DOES result from random mutation, it doesn’t count as evolution, because there’s always a price to be paid for gaining resistance.”

This is the sort of approach taken by Michael F. Behe, who likens the development of resistance genes to “trench warfare” and genome degradation, rather than an “arms race” of increasing buildup. Like many things that Behe believes, this is baloney.

First of all, it misses the point of evolution, which (as I said before) is change. Whether or not these changes meet Behe’s mystical criteria for “increasing complexity” doesn’t matter. It’s still evolution.

But more importantly, it’s short-sighted. As we’ll see, evolution of resistance means more than just having a resistance gene.

One thing is true about the creationists’ claim: in the majority of cases, resistance comes with a cost. Antibiotics work by disrupting some normal process within the cell. Resistance genes can operate by two different mechanisms: they can either disrupt the normal cellular process so the antibiotic can’t target it anymore, or they can create new proteins that actively do something to inhibit the antibiotic (like export it or degrade it). The latter is typically a plasmid-bound resistance gene, the former more typical of chromosomal mutations. But in the absence of antibiotic, these resistance mechanisms tend to lead to decreased growth. Mutations that disrupt antibiotic activity also decrease the efficiency of the targeted cellular process; proteins synthesized from plasmid might have a side effect on normal cell function; even just copying an unused plasmid is a waste of energy. From am medical perspective, this suggests that when antibiotic resistance crops up, we can just take away the antibiotic for a little while and the antibiotic-sensitive bacteria will eventually outbreed the resistant bacteria, and we can start over.

Behe and other creationists quit there and call it a day. But the problem is that all these studies of the cost of resistance were performed in naïve bacteria. That is, one minute the bacteria didn’t have the resistance gene, the next minute they did, and we looked at the difference.

What would happen if we let the bacteria and their new resistance genes get accustomed to each other for a while? Evolution would predict that, in the absence of antibiotics, there would be pressure to ameliorate the cost of resistance through mutation. Either you get rid of the resistance gene causing the problem, or you keep the resistance gene but acquire new cost-compensatory mutations that reduce its side effects.

Several studies were performed to test that hypothesis. Richard Lenski published a great review article in 1998 covering several of them.[1] You can read it for yourself here [PDF]; I’ll do my best to summarize some of the major findings.

Cost-compensation of plasmid-bound resistance:

A strain of E. coli was transformed with a plasmid carrying resistance to the antibiotics tetracycline and chloramphenicol. For this generation of bacteria, the cells with the plasmid were slightly less fit than those without (in the absence of antibiotics).

The researchers then grew the plasmid-carrying bacteria for 500 generations (75 days) in a culture containing chloramphenicol, to make sure the cells didn’t just ditch the plasmid. They then took those bacteria out of the chloramphenicol and isolated a colony of cells without the plasmid. For this generation of bacteria, the cells with the plasmid were slightly more fit than those without!

Further study showed that it was the bacterial chromosome that had changed, not the plasmid. Over just five hundred generations, enough cost-compensatory mutations had accumulated on the bacterial genome to make the resistance plasmid a boon rather than a bane, even in the absence of antibiotic.

Cost-compensation of chromosomal resistance mutations:

Here, researchers started with mutations of rpsL, a gene that encodes part of the bacterial ribosome (a little blob that synthesizes protein), that result in streptomycin resistance in E. coli. Streptomycin is a type of antibiotic called an aminoglycoside; it binds to the ribosome, preventing protein synthesis and killing the cell. Certain rpsL mutations prevent streptomycin from binding to the ribosome, thus making the cell streptomycin-resistant. However, this change to the ribosome also slows the rate of peptide (protein) elongation.

The researchers grew streptomycin-resistant bacteria in the absence of streptomycin (since it’s on the chromosome, not a plasmid, they don’t have to worry about the gene just being lost), and after a mere 180 generations they found that the rate of peptide elongation was back up to what it had been in wild-type cells. What’s more, they found that the bacteria still had the mutation conferring streptomycin resistance. Rather than mutating back to wild-type, the cells had acquired cost-compensatory mutations elsewhere in the chromosome.


These two studies indicate that fighting antibiotic resistance would be a LOT harder than was previously thought. It isn’t as easy as just taking away the antibiotic and letting the resistant bacteria fade into obscurity. Rather than ditch their costly genes for resistance, the bacteria are evolving cost-compensatory mutations so they can have their cake and eat it, too.

Hm… multiple naturally-selected mutations leading to a benefit with little or no noticeable cost? Sounds like evolution to me.


PS - One final note on the matter of antibiotic resistance, via Greg Laden: apparently there is some hesitation on the part of biomedical journals to refer to the “evolution” of antibiotic resistance, preferring instead to use terms like “emergence.” Head over to Greg’s place to check it out.

Resource:
[1] Lenski, R.E. (1998). Bacterial evolution and the cost of antibiotic resistance. International Microbiology, 1(4), 265-270.

Reality Is Not Subject to Vote

Today, we have more evidence that creationist attacks on evolution are scientifically bankrupt, and are nothing more than a desperate appeal to the opinion of a misinformed public.

Last week, PZ Myers reported on comments by Florida Board of Education member Linda Taylor:

[Quoting Taylor:] I would support teaching evolution, but with all its warts. I think that some of the facts have been questioned by evolutionists themselves. I would want them taught as theories. That's important. They could be challenged by others and the kids could then be taught critical thinking and they can make their own choices.

Thank you, Linda Taylor. Warts: name two. Theory: define the term. Answer the following multiple choice question:
Who is best qualified to make informed choices about complex scientific theories?

A: Scientists with years of training in the subject, and qualified science teachers who understand the fundamentals of the theory.
B: Creationists who won't even commit to an estimate of the age of the earth.
C: Members of the board of education who have absolutely no training in the sciences.
D: Children who are just being introduced to the topic for the first time, haven't read any of the primary literature, and who are entirely dependent on the competence of the instructors who have given them an outline of the general story.

Today, creationist fuckwit Michael Egnor posts a response:

Because this is a democracy and Myers doesn’t actually get to dictate the choices, the question is really ‘fill in the blank,’ not multiple choice.

Here’s my suggestion for the answer to the question "Who is best qualified to make informed choices about complex scientific theories in public schools in Florida?":

The people of Florida, through their elected school boards.

Darwinists like Myers find democracy so frustrating.

Yes, because clearly the average Florida voter spends every other day simply immersed in the primary biology literature! They know so much about biology, it hurts!

Democracy doesn't get to determine science; the best it can do is decide how we (at the level of government and society) respond to science. No matter how much the creationists want everyone to get together and say evolution is wrong, you can't vote away reality.

We need government officials who recognize that distinction. Good leadership doesn't mean knowing all the answers. It means knowing where to find the answers and how to employ them. A responsible school board must defer to the scientific community, not public opinion nor their own meager understanding, for advice concerning the teaching of evolution. That's what PZ's questions are meant to demonstrate: The school board members most qualified to decide policy are those who recognize that scientists are most qualified to decide science.

The creationists, of course, don't want responsible leadership. Their only hope is that ignorance breeds ignorance, which is why they are constantly trying to sabotage our children's education.

Creationist Resistance to Antibiotic Resistance, Part I

Earlier last week, GilDodgen of Uncommon Descent wrote about his plans to revolutionize medicine and save us all from antibiotic-resistant bacteria. He exhibits some spectacularly BAD logic, even for a cdesign proponentsist. For a few excellent takes on WHY he is so stupid, check out these fine bloggers:

Humble Monkey
Sandwalk
Respectful Insolence
Panda’s Thumb
ERV

Now, I don’t want to go into Dodgen’s points and claims specifically; those have already been torn to ribbons in the links above. Instead, I want to address the more general trend of creationist denial regarding antibiotic-resistant bacteria.

Resistance to antibiotics (henceforth just “resistance”) is one of the starkest examples we have of the power of evolution by natural selection of random mutation. So it only stands to reason that creationists will fall over each other to deny that resistance has anything to do with evolution. To do so, they employ two main talking points lies (embodied in this *shudder* Answers in Genesis article):

1) “The genes for resistance are not the result of random mutation; they’ve been there all along, we just didn’t notice them!”

2) “Even if resistance DOES occasionally result from random mutation, it doesn’t count as evolution, because there’s always a price to be paid for gaining resistance.”

We’ll deal with that first lie today.

Even if the creationist grudgingly admits the importance of natural selection to the growth of a resistant population, they vehemently deny that what’s being selected is the result of random mutation. Instead, they say that either there were a handful of resistant bacteria around to begin with, or they inherited the genes for resistance from a different kind of bacteria via lateral gene transfer. Dodgen’s post falls into this camp, sort of, since he’s downplaying the power of random mutation. Another, perhaps clearer, example would be a recent UD post by idnet.com.au discussing divergence of E. coli in the human gut versus that of the baboon. Although resistance is not specifically addressed, it is claimed that any new genes found in either population of bacteria must be the result of lateral gene transfer.

Real science, of course, is chock FULL of examples of the power of random mutation. Let’s look at just one example: Pseudomonas aeruginosa infections in patients with cystic fibrosis, from a paper published by Antonio Oliver et. al. in the journal Science in 2000.[1] (See that little BPR3 icon? That’s the sign that things are about to get good.)

Cystic fibrosis (CF) is a genetic disorder resulting from a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). Normally, CFTR adjusts ion concentrations in order to make water flow out of the cell via osmosis. In the lung, this source of water is what keeps your mucus nice and wet and fluid. But if CFTR is broken, the mucus in the lung (and elsewhere, such as the GI tract) becomes dry and super-thick, causing all kinds of hell for the CF patient’s body.

P. aeruginosa is an opportunistic bacteria; it can't and won't infect a healthy adult. It can, however, cause serious infection in certain scenarios if exposure is high and the host's defenses are already down. Two major targets for serious acute (short-term) infection are mechanically ventilated patients and victims of serious burn wounds. But for patients with CF, P. aeruginosa instead causes chronic (long-term) infection. For most such patients, it’s not the CF that kills you, it’s the Pseudomonas.

As it turns out, P. aeruginosa infections from CF lungs show a lot more colony diversity than colonies grown from patients with acute infections. Oliver’s group hypothesized that this diversity was due to the conditions within the CF lung, which as a result of hyperosmolar viscous mucus is highly compartmentalized and continually changing. Such an environment would favor the growth of bacteria that could keep on their toes, so to speak, quickly and readily adapting to changing environmental niches within the lung. And evolutionary biology predicts that, in order to adapt to that kind of environment, you have to be really good at mutating.

So the experimenters collected a whole bunch of P. aeruginosa samples from 30 CF patients, and a whole bunch of P. aeruginosa samples from 75 patients with acute infections, and compared the mutation frequencies of those isolates. Both the CF and non-CF patients had a whole bunch of P. aeruginosa isolates with low mutation frequencies. But in addition, the CF patients had a whole bunch of isolates with mutation frequencies 100 times as great! Genetic analysis of these mutator isolates over several years indicated that they were all different and persistent; these were bacteria that evolved into mutators within the host and stuck around, and not mutators transmitted between patients. Further investigation demonstrated that, for several of the mutator isolates, one or more error-avoidance genes were mutated or deleted altogether, explaining their high mutation rate.

This is the point in the presentation when a creationist might say, “Aha! Nothing new was created; mutation was only capable of breaking genes that were already there!” But this is only the first half of the story; as we’ll see, the mutator phenotype opens the doors to further beneficial mutations.

You see, cells normally keep those error-avoidance genes around for a reason. If the mutator phenotype is so prevalent in CF patients, it must be conferring some advantage in that environment. That is, given the conditions of the CF lung, it’s apparently more important to be able to get beneficial mutations than to prevent detrimental ones.

Patients with P. aeruginosa infections, especially those with CF, are subject to extensive treatment with a broad range of antibiotics. So the experimenters took a look at whether the mutator phenotype had any effect on the evolution of antibiotic resistance:



This is the key figure for our discussion. In case you can’t tell, black bars are mutator strains from CF patients, grey bars are non-mutator strains from CF patients, and white bars are strains from non-CF patients (all non-mutator). For a broad range of antibiotics, mutator strains showed a much higher frequency of antibiotic resistance compared to non-mutator strains.

The only way this makes sense is if antibiotic resistance is the result of naturally-selected random mutation. Mutator strains have higher mutation rates, and are therefore more likely to acquire the mutations necessary for resistance. The non-CF non-mutators serve as a control. The important comparison is between CF mutators and CF non-mutators, because they had everything in common except their mutation rate. They were derived from the same ancestral strains that first infected the patient. They were subject to the same antibiotic therapies. They grew in the same environment, shared space with the same other species of bacteria, and therefore had the same potential for lateral gene transfer. If resistance were the result of anything other than mutation, then we should see no statistical difference between the black and grey bars. But we do see a difference.

That’s evolution. The CF lung is a dynamic environment with different selective pressures than sites of other, acute infections. CF lungs favor selection of bacteria that can mutate rapidly. This increased rate of mutation results in other selectable beneficent mutations, such as resistance to antibiotics.

It’s important to note that Oliver and company weren’t trying to convince anyone that bacteria evolve; real scientists understood that already. They were trying to use that understanding to save people’s lives. Their insight into how a Pseudomonas infection behaves within the lung is the first step to fighting that infection. Armchair physicians who don’t understand antibiotics and deny the power of random mutation are of no help to the dying.

Next time we’ll look at that second creationist claim, and what random mutation can do to bacteria that are already resistant to antibiotics. That’s when things get really intense.

Resource:
[1] Oliver, A. (2000). High Frequency of Hypermutable Pseudomonas aeruginosa in Cystic Fibrosis Lung Infection. Science, 288(5469), 1251-1253. DOI: 10.1126/science.288.5469.1251

SCIENCE AGAIN!

I SAID SCIENCE AGAIN!

I've just accepted a position as research assistant with the Broad Institute of Harvard/MIT. I'm going to be a professional scientist!

The lab in which I'll be working is dedicated to creating deletion libraries for a few different bacteria, including Mycobacterium (the pathogen responsible for tuberculosis) and Pseudomonas (an opportunistic pathogen--meaning it tends only to cause problems if you're already sick--that's especially fond of causing respiratory infections in people with cystic fibrosis). Creating a deletion library means deleting a single gene from the bacterial chromosome, and repeating the process for every gene in the genome. So in the end you have thousands of bacterial colonies, each with a different single gene deleted.

What's really great about this particular project is that we'll be making our deletion library freely available to other researchers. So if a researcher somewhere is interested in a specific gene and wants to know what happens when it's deleted, they don't need to go through creating the deletion themselves, they can just look up the gene in our library and request a sample.

How excited am I to be making that kind of contribution to the scientific community?

Pretty damn excited.

But how, you may be wondering, does one delete a gene from a chromosome? The trick is homologous recombination. If two strands of DNA have a region that matches the other in sequence, that region is said to be homologous between the two. In a cell, two homologous DNA regions can line up, break at the same place, switch pieces, and fuse back together; this is called homologous recombination, or "crossing-over."

Let's say the section of chromosome in which we're interested looks like this:

. . . ====[ Upstream DNA ][ GENE ][ Downstream DNA ]==== . . .

We can build a plasmid (little circle of DNA, kind of like a mini-chromosome for bacteria) that has a section looking like this:

. . . ====[ Upstream DNA ][ Downstream DNA ]==== . . .

(For ease of visualization later, DNA from the chromosome is red and DNA from the plasmid is green.) We can stick that plasmid in the bacteria, and hope for homologous recombination between the chromosome and the plasmid. Recombination only occurs in a few of the cells, though, so we need a means of selecting for those cells that do cross-over.

We need two different recombination events to occur, one on either side of the gene. So we'll stick two marker genes on our plasmid: a strength (like resistance to ampicillin, an antibiotic) and a weakness (like a gene that makes sucrose toxic). First we grow the bacteria on plates containing ampicillin; only those cells that have the plasmid (meaning they've had one cross-over event) will survive. Then we take those survivors and grow them on plates containing sucrose; now only those cells that got rid of the plasmid (meaning they had a second cross-over event) will survive.

So after two rounds of selection, the bacterial chromosomes hopefully look something like this:

. . . ====[ Upstream DNA ][ Downstream DNA ]==== . . .

However, that assumes that one recombination event occurred in the upstream region and one occurred in the downstream region. If both events occurred in the same region, then the chromosome could look like:

. . . ====[ Upstream DNA ][ GENE ][ Downstream DNA ]==== . . .

or

. . . ====[ Upstream DNA ][ GENE ][ Downstream DNA ]==== . . .

So after all that selection, about 50% of our bacteria colonies still have the gene! We therefore need to add one more step: we pick a bunch of bacteria colonies and sequence that part of the chromosome to see if the gene was deleted successfully. The colonies that pass sequencing get added to our deletion library!

So that's how you delete a single gene from a bacterial chromosome. Part of my job will be figuring out how to optimize that process to handle tens or hundreds of genes at once. That means I get to use robots!

This is going to be awesome.

(Oh, and in case you were curious, the opening to this post comes from this Homestar Runner cartoon.)

Tangled Bank #87

The 87th edition of the Tangled Bank is up and running over at Balancing Life. Be sure to head on over and check out a samplin' of fine science blogging from the last fortnight. (Yours truly even has a submission this time around!)

Evolution of the X and Y Chromosomes, Part 3

Last time, we looked at how Lahn & Page determined the evolutionary ages of various regions of the X chromosome. Since then, more refined techniques have distinguished the following evolutionary strata in the X chromosome:



PAR stands for pseudo-autosomal region. This is a tiny region of DNA that still allows recombination between the X and Y chromosomes; that means the PAR retains high homology between the X and Y. The strata are numbered in order of evolutionary age; S1 is the oldest (in terms of divergence from Y), S5 is the youngest.

A 2005 paper by Carrel et. al. looked at a whole bunch of genes on the X chromosome, where they were, and whether they escaped X-inactivation. This resulted in the following figure, which shows the amount of escape from inactivation for each stratum:



The red end of the spectrum represents inactivated genes; the purple end represents genes that escape inactivation. Basically, the further away you get from the PAR, the fewer genes you'll find that have escaped X-inactivation.

It's important to note that the genes that escape X-inactivation don't necessarily have high Y-homology themselves; sometimes they just hang out with other genes that have high homology and ride their coattails.

So finally, what does this all tell us about how dosage compensation evolved in mammals? Recall that the active X is hyperactive compared to autosomes. When genes started decaying on the inverted pseudo-Y, the male cells started ramping up expression from their other X chromosome to compensate. But this heightened X expression carried over to females, too. That meant the female cells were getting too much X expression; to compensate, they evolved a mechanism for silencing one of their chromosomes.

Conclusion:
We noted that, though dosage compensation in mammals occurs via inactivation of one of the two X chromosomes in XX cells, some genes escape X-inactivation. Furthermore, expression from the active X is twice as great as expression from the autosomes. We wanted to know how this ties into evolution of the X and Y chromosomes.

X and Y homology experiments have demonstrated that the X chromosome can be broken into a pseudo-autosomal region plus five evolutionary strata based on time since divergence from the Y chromosome.

It turns out that genes found in regions with higher Y-homology are more likely to escape X-inactivation. This tells us that gene silencing likely evolved as a response to up-regulation of X expression, which in turn evolved in response to degradation of homologous genes on the pseudo-Y.

This is, of course, a fairly general model. There are exceptions, genes with homology that are silenced and genes without homology that escape silencing. But we still see a profound evolutionary trend. It goes to show that evolution isn't just about inventing genes for new proteins; it's also about changing regulation of the genes you already have.

Evolution of the X and Y Chromosomes, Part 2

Let's turn to research on X and Y chromosome evolution to explain some of what we saw in Part 1. Once upon a time, the Y chromosome used to be just another X chromosome. But at some point, the X-that-would-be-Y (I'll call it pseudo-Y for now) suffered a major inversion; that is, a big chunk of the chromosome was broken off, flipped around, and stuck back on. This causes a problem with recombination. During meiosis, homologous chromosomes line up and swap pieces of DNA. This can only occur if the pieces being swapped are pretty much the same. Recombination is an important means of swapping around alleles (different versions of genes) and therefore a big player in evolution.

The inversion event threw a wrench into X chromosome recombination. The un-inverted sections could still recombine, but the inverted section couldn't, and therefore these formerly homologous stretches of DNA diverged, each gradually mutating in different ways. The pseudo-Y experienced a number of other inversion events, each time taking another chunk of the homologous region and rendering it incapable of recombination with X.

How do we know this? Because the inversion events left behind fingerprints:



This image is from a 1999 paper by Lahn & Page. We would expect that inverted sections of the pseudo-Y would diverge from the corresponding regions on the X. The more time has passed since the inversion, the more divergence we should expect.

Lahn & Page looked at 19 X-linked genes that were known to have homologous sections of DNA on the Y chromosome. For each of these DNA regions, they measured the differences between the X and Y versions. The x-axis in the figure shows location on the chromosome. The y-axis shows the estimated mean number of substitutions per synonymous site. What we see are four main age blocks; genes in Group 1 are more diverged from their Y counterparts than those in Group 2, which suggests the Group 1 region of DNA inverted earlier.

Next time, we'll look at how these evolutionary regions of the X chromosome relate to which genes escape X-inactivation.

Evolution of the X and Y Chromosomes, Part 1

So far we know that dosage compensation in humans and mice involves silencing one of the two X chromosomes in XX (normally female) animals (see Part 3 of the Development Primer). But that isn't the whole story.

Dosage compensation requires either up-regulation of X expression in cells with one X chromosome, or down-regulation in cells with two X chromosomes. At first, we just compared the two Xs to each other, and saw that one was silenced while the other was expressed. But when we compare expression from the active X chromosome to a normal autosome, we see that the active X is twice as active as any autosome.

Next, consider Turner syndrome (XO women) and Klinefelter's syndrome (XXY men). These syndromes are caused by a rare event called nondisjunction. Normal human cells are diploid; that is, they have two copies of each chromosome (one from Mom, one from Dad). Gametes (sperm and egg) need to be haploid, having one copy of each chromosome. So as the cell prepares to divide during meiosis, the chromosomes are lined up in the center of the cell and are pulled in opposite directions. Normally, one chromosome of each pair goes to each side. But sometimes nondisjunction occurs; a pair of chromosomes get stuck together, so one gamete gets no copy and the other gets an extra copy (like those old Twix commercials: "Two for me, none for you!"). If those gametes then get to become part of a zygote, then the zygote will have an abnormal number of chromosomes.

If the inactive X chromosome were completely inactive, then we should expect to see no difference between XO and XX females, nor between XY and XXY males. But since Turner and Klinefelter's are syndromes, the lack of X in the former and extra X in the latter must be doing something.

As it turns out, not all the genes on the inactive X are silenced; some escape inactivation. Recall from the Development Primer that Xist-RNA forms a transcription-free zone in the nucleus, and the inactive X crawls inside. But it seems that, like a loose bundle of yarn stuck hastily in your pocket, some loops of DNA dangle out of this zone, and the genes on those loops get expressed. But which genes, and why?

At this point, we turn to research on X and Y chromosome evolution.

Stay tuned.

Development Primer: 3 Models of Sex Determination and Dosage Compensation, Part 3

Mus (mouse)

Of our three models, the mouse (being a mammal) is most closely analogous to humans. Here, as with the flies, XX animals are female and XY animals are male. This time, however, sex determination depends upon the Y-linked gene Sry instead of the X:A ratio. If you have Sry, you become a male. Note the implications of this for individuals with an abnormal number of sex chromosomes. An XO fly would become a (pseudo)male due to the X:A ratio, whereas an XO human would become a female (with Turner syndrome) due to lack of Sry. An XXY fly would be female, whereas an XXY human would be male (with Klinefelter's syndrome).

Overall sex determination in mammals relies primarily on differentiation of the gonad. The embryonic gonad is bipotential (neither male nor female but capable of differentiating into either) and contains both somatic cells and germ cells (the cells that will give rise to the germ line, i.e. sperm or eggs). The somatic cells of the gonad will differentiate into either Sertoli cells (for testes) or Follicle cells (for ovaries); the hormone production of the gonad will then affect development of the rest of the body.

So we're looking for a cellular mechanism affecting gonad differentiation. As it turns out, there are two primary exogenous growth factors (proteins excreted from a cell that affect growth and development) expressed by the somatic cells: FGF9 and WNT4. FGF9 causes the somatic cells to differentiate into Sertoli cells, and inhibits Wnt4 expression. WNT4 causes the somatic cells to differentiate into Follicle cells, and inhibits Fgf9 expression. Initially, the somatic cells express both factors, and they balance each other out. But as development progresses, the balance gets tipped one way or the other.

In mammals, SRY promotes expression of FGF9, thus tipping the balance toward male development. However, a similar mechanism in other vertebrates might not require a single gene like Sry. For instance, for many animals (such as crocodiles) sex determination is affected by environmental factors like temperature. It's entirely possible that these environmental factors could be affecting a balance such as that between FGF9 and WNT4.

This sex determination mechanism takes place primarily in the gonad; a different mechanism entirely is needed for dosage compensation in all the cells of the body. Unlike the worm mechanism of reducing each X by half in hermaphrodites or the fly mechanism of doubling expression of the male X, dosage compensation in mice and humans works by (almost) completely silencing one of the two female X chromosomes.

In both males and female cells, the autosomes produce enough of a certain blocking factor (BF) to bind to one X chromosome and block expression of the gene Xist. In a cell with only one X chromosome (i.e. a normal male), that' the end of the story. In a cell with two X chromosomes (i.e. a normal female), Xist is expressed on the chromosome that did not receive BF. Xist is a gene that does not code for protein; its end product is untranslated RNA. Xist RNA aggregates to form a region in the nucleus that excludes RNA ploymerase II and transcription factors. The X chromosome migrates into this region, thus silencing it.

The paternal X chromosome carried by the sperm is imprinted so it will always be chosen for inactivation in the zygote. Once the embryo reaches the blastocyst stage of development, X-inactivation is temporarily turned off. As the cells then differentiate, X-inactivation is reinitiated. This time, the decision of which X chromosome to deactivate is random. Thus, the adult animal will be mosaic for X-inactivtion; some of the animal's cells will have the paternal X deactivated, and some cells will have the maternal X deactivated.

The location where BF binds to the X chromosome, since the region runs opposite to Xist, is called Tsix. If Tsix is deleted from one X chromosome, then that chromosome will always be chosen for X-inactivation, since BF cannot bind it. A Tsix deletion in an XY cell will result in ectopic (out-of-place) X-inactivation, which is lethal.

Mouse summary:
Two sex chromosomes -- XY male, XX female. FGF9 and WNT4, mutually inhibitory growth factors, are expressed in the gonad somatic cells. Y chromosome carries Sry, which promotes FGF9 and tips the balance to testis development; otherwise, WNT4 tips balance to ovary development. X-inactivation occurs by transcription of Xist RNA, which forms a nuclear domain excluding transcription factors. Autosomes produce enough blocking factor (BF) to rescue one X chromosome from inactivation, by binding Tsix and thereby blocking Xist. The paternal X is imprinted to be always chosen for inactivation in the zygote; at the blastocyst stage, X-inactivation is reset and randomized.

Development Primer: 3 Models of Sex Determination and Dosage Compensation, Part 2

Drosophila

Drosophila's sex chromosomes are perhaps more familiar-looking, in that it has an X and a Y chromosome, with XX animals being phenotypically female and XY animals being phenotypically male. As we'll see, however, the mechanisms are vastly different from what we see in humans and other mammals.

Sex determination and dosage compensation begin as they did in C. elegans with reading the X:A ratio. This time, however, the X-linked numerator factors (like daughterless and sisterless) drive Sxl (sex lethal) expression from a promoter that results in the code for a fully-active SXL protein (nomenclature note: italics are typically used for the name of the gene, capital letters are used for the corresponding protein). The A-linked denominator factors (like deadpan) bind to the numerator factors to inhibit their activity. Thus, if the fly has only one X chromosome, the denominator factors will titrate out the numerator factors, so Sxl only gets up and running in XX animals.

SXL a protein that alters RNA splicing. Most mRNA when it is first transcribed from DNA must first be processed within the nucleus before it is shipped out for protein translation. This processing includes removing introns, non-coding regions of DNA within a gene. Normal Sxl mRNA has a premature stop codon in its third intron; if the cell tries to make protein from this mRNA, it will stop translation short, resulting in a non-functioning protein. SXL protein is responsible for altering splicing to remove this intron so the cell can make more fully functional SXL.

Thus, SXL drives its own positive feedback loop. Both XX and XY animals express Sxl mRNA from the normal promoter. In XY animals, there isn't ever any SXL around to splice this mRNA, so no SXL is made. In XX animals, the numerator factors drive SXL expression at the beginning of development from a special promoter that doesn't require splicing, so there's enough SXL to kick-start splicing of the normal mRNA and keep generating more SXL long after the numerator factors stop working.

SXL is then responsible for proper splicing of tra to its active form, which in turn (in conjunction with tra-2) alters splicing of the transcription factor dsx, which causes differentiation to the female phenotype.

SXL also inhibits msls, a gene which otherwise would initiate dosage compensation by increasing expression from the male X. Note that in C. elegans, dosage compensation meant scaling down expression in the female, but in Drosophila, dosage compensation means scaling up expression in the male.

Drosophila summary:
Two sex chromosomes -- XX female, XY male. Numerator factors drive expression of fully-functional SXL in XX animal, kicking off SXL positive feedback loop. SXL activates pathway leading to female development. SXL inhibits msls pathway to dosage compensation. In the absence of SXL (i.e. in XY animals), msls drives increased expression from the single X chromosome.

Development Primer: 3 Models of Sex Determination and Dosage Compensation, Part 1

You're probably aware that a person's sex is typically determined by their combination of sex chromosomes. In humans, females have two X chromosomes, whereas males have an X and a Y. But how do you go from X and Y to boy and girl? And what does the cell have to do to compensate for the chromosome differences between the sexes?

Sex chromosomes pose two interesting questions in the study of development:

1. Sex determination: How does the cell interpret the data from the sex chromosomes to result in phenotypic sex?
2. Dosage compensation: The sex chromosomes carry many genes that aren't sex-specific; that is, both male and female cells need the products of those genes in approximately equal amounts. Without dosage compensation, a cell with two X chromosomes will produce twice as much of a given X-linked gene product as a cell with one X chromosome. How does the cell regulate sex chromosome expression so that cells with unequal sex chromosomes express sex-linked genes equally?

The animal kingdom employs a number of different mechanisms for dealing with these two questions. Let's take a brief look at sex determination and dosage compensation in three model organisms: the nematode worm (Caenorabditis elegans), the fruit fly (Drosophila melanogaster), and the common mouse (Mus musculus).

C. elegans
C. elegans is a tiny invertebrate worm with just one kind of sex chromosome: X. A normal worm with two X chromosomes (XX) is a hermaphrodite, producing both sperm and eggs and capable of self-fertilization. A normal worm with one X chromosome (XO) is a male; they're smaller and capable of mating with hermaphrodites.

The pathway leading to sex determination and dosage compensation is initiated by "reading" the X-to-autosome (X:A) ratio. (Autosomes are any chromosomes that aren't sex chromosomes.) In C. elegans, the first main gene in the signal transduction pathway is xol-1 (XO lethal 1, so named because mutations of the gene are lethal to animals with XO genotype). The autosomes express a number of genes, such as sea-1, that promote xol-1 expression; these are called denominator factors, since they show up on the bottom of the X:A ratio. Each X chromosome carries genes like sex-1 and fox-1 that inhibit xol-1 expression; these are called numerator factors. An XO cell doesn't produce enough numerator factors to "cancel out" the denominator factors, so xol-1 is "turned on." An XX cell has twice as much of each numerator factor, enough to "turn off" xol-1.

Xol-1 is the first in a series of several regulatory genes. Since the genes regulate each other, activity alternates down the chain. If xol-1 is on, then it turns off sdc-2, which means her-1 gets turned on, etc. Alternatively, if xol-1 is off, then sdc-2 gets to turn on, and that turns off her-1, etc. This pathway ultimately leads to expression of transcription factors (gene-regulating proteins) specific for either hermaphrodite or male differentiation.

Loss-of-function mutations of some of the genes in this pathway can cause "transformation" to the wrong sexual phenotype. For example, in XO animals her-1 is normally turned on, and we expect to get a male. But if we mutate her-1 so it can't perform its function, then the rest of the pathway downstream acts as if her-1 is off and we get a hermaphrodite phenotype. (Genes are often named according to the phenotype of the mutation that led to their discovery; thus, her-1 got its name for turning XO animals into hermaphrodites.)

So that's sex determination, but what about dosage compensation? It turns out that dosage compensation is activated by sdc-2. Dosage compensation in C. elegans acts by cutting expression of both X chromosomes in half in XX animals. That way, two X chromosomes at half-expression result in the same amount of product as one X chromosome at full expression. That's also why xol-1 mutations are lethal for XO animals; without xol-1 to regulate it, sdc-2 gets turned on when it shouldn't be. That in turn activates dosage compensation, and with only one X-chromosome at half its normal expression, the cell doesn't have enough X-linked gene product to survive.

C. elegans summary:
One sex chromosome -- XO males, XX hermaphrodites. X:A ratio read by X-linked inhibition of xol-1, affecting downstream chain of regulatory genes. Dosage compensation reduces expression from each X chromosome by half in XX animals.

Branching out with a little cognitive psychology

Taking a cue from the illustrious PZ Myers, I'd like to diversify a little bit, and write about something not directly atheism-related.

As you may or may not know, I occasionally contribute to SUGAA Headquarters, the collective blog of the Scientists United for the Global Advancement of Awesomeness (a pack within Ze Frank's ORG). I've tended to focus on evolution (specifically, debunking creationism) so far on this blog, but that's hardly where my interests begin and end. At Dartmouth, I majored in Biology (concentrating in neurobiology and genetics) with a minor in Physics (primarily electricity & magnetism), and I always keep my ear to the ground regarding developments in all the sciences. Even though grad school is still a year off and I'm not what you might call an expert in any field, I still love sharing what I've learned so far.

For instance, yesterday my fiancee found a cool video on YouTube, and it got me thinking about cognitive psychology.

Problem solving is one of the big questions still facing psychologists. How do we solve problems? And what gets in the way of our problem solving?

One of the big difficulties that people face in problem solving is called functional fixedness. Certain types of problems require that we think of a novel use for an object in order to obtain the solution. Functional fixedness gets in the way by latching on to what we know about an object's normal use and refusing to let us think of anything else. For the ultimate success story in overcoming functional fixedness, think of MacGyver. For him, practically nothing has a fixed mundane use; paper clips become lock picks or radio antennae, and chewing gum can defuse a bomb.

(Read more at SUGAA HQ)