How to afford a big sloppy genome

i-e88a953e59c2ce6c5e2ac4568c7f0c36-rb.png

My direct experience with prokaryotes is sadly limited — while our entire lives and environment are profoundly shaped by the activity of bacteria, we rarely actually see the little guys. The closest I’ve come was some years ago, when I was doing work on grasshopper embryos, and sterile technique was a pressing concern. The work was done under a hood that we regularly hosed down with 95% alcohol, we’d extract embryos from their eggs, and we’d keep them alive for hours to days in tissue culture medium — a rich soup of nutrients that was also a ripe environment for bacterial growth. I was looking at the development of neurons, so I’d put the embryo under a high-powered lens of a microscope equipped with differential interference contrast optics, and the sheet of grasshopper neurons would look like a giant’s causeway, a field of tightly packed rounded boulders. I was watching processes emerging and growing from the cells, so I needed good crisp optics and a specimen that would thrive healthily for a good long period of time.

It was a bad sign when bacteria would begin to grow in the embryo. They were visible like grains of rice among the ripe watermelons of the cells I was interested in, and when I spotted them I knew my viewing time was limited: they didn’t obscure much directly, but soon enough the medium would be getting cloudy and worse, grasshopper hemocytes (their immune cells) would emerge and do their amoeboid oozing all over the field, engulfing the nasty bacteria but also obscuring my view.

What was striking, though, was the disparity in size. Prokaryotic bacteria are tiny, so small they nestled in the little nooks between the hopper cells; it was like the opening to Star Wars, with the tiny little rebel corvette dwarfed by the massive eukaryotic embryonic cells that loomed vastly in the microscope, like the imperial star destroyer that just kept coming and totally overbearing the smaller targets. And the totality of the embryo itself — that’s no moon. It’s a multicellular organism.

I had to wonder: why have eukaryotes grown so large relative to their prokaryotic cousins, and why haven’t any prokaryotes followed the strategy of multicellularity to build even bigger assemblages? There is a pat answer, of course: it’s because prokaryotes already have the most successful evolutionary strategy of them all and are busily being the best microorganisms they can be. Evolving into a worm would be a step down for them.

That answer doesn’t work, though. Prokaryotes are the most numerous, most diverse, most widely successful organisms on the planet: in all those teeming swarms and multitudinous opportunities, none have exploited this path? I can understand that they’d be rare, but nonexistent? The only big multicellular organisms are all eukaryotic? Why?

Another issue is that it’s not as if eukaryotes carry around fundamentally different processes: every key innovation that allowed multicellularity to occur was first pioneered in prokaryotes. Cell signaling? Prokaryotes have it. Gene regulation? Prokaryotes have that covered. Functional partitioning of specialized regions of the cell? Common in prokaryotes. Introns, exons, endocytosis, cytoskeletons…yep, prokaryotes had it first, eukaryotes have simply elaborated upon them. It’s like finding a remote tribe that has mastered all the individual skills of carpentry — nails and hammers, screws and screwdrivers, saws and lumber — as well as plumbing and electricity, but no one has ever managed to bring all the skills together to build a house.

Nick Lane and William Martin have a hypothesis, and it’s an interesting one that I hadn’t considered before: it’s the horsepower. Eukaryotes have a key innovation that permits the expansion of genome complexity, and it’s the mitochondrion. Without that big powerplant, and most importantly, a dedicated control mechanism, prokaryotes can’t afford to become more complex, so they’ve instead evolved to dominate the small, fast, efficient niche, leaving the eukaryotes to occupy the grandly inefficient, elaborate sloppy niche.

Lane and Martin make their case with numbers. What is the energy budget for cells? Somewhat surprisingly, even during periods of rapid growth, bacteria sink only about 20% of their metabolic activity into DNA replication; the costly process is protein synthesis, which eats up about 75% of the energy budget. It’s not so much having a lot of genes in the genome that is expensive, it’s translating those genes into useful protein products that costs. And if a bacterium with 4400 genes is spending that much making them work, it doesn’t have a lot of latitude to expand the number of genes — double them and the cell goes bankrupt. Yet eukaryotic cells can have ten times that number of genes.

Another way to look at it is to calculate the metabolic output of the typical cell. A culture of bacteria may have a mean metabolic rate of 0.2 watts/gram; each cell is tiny, with a mass of 2.6 x 10-12g, which means each cell is producing about 0.5 picowatts. A eukaryotic protist has about the same power output per unit weight, 0.06 watts/gram, but each cell is so much larger, about 40,000 x 10-12g, that a single cell has about 2300 picowatts available to it. So, more energy!

Now the question is how that relates to genome size. If the prokaryote has a genome that’s about 6 megabases long, that means it has about 0.08 picowatts/megabase to spare. If the eukaryote genome is 3,000 megabytes long, that translates into about 0.8 picowatts of power per megabase (that’s a tenfold increase, but keep in mind that there is wide variation in size in both prokaryotes and eukaryotes, so the ranges overlap and we can’t actually consider this a significant difference — they’re in the same ballpark).

Now you should be thinking…this is just a consequence of scaling. Eukaryotic power production per gram isn’t any better than what prokaryotes do, all they’ve done is made their cells bigger, and there’s nothing to stop prokaryotes from growing large and doing the same thing. In fact, they do: the largest known bacterium, Thiomargarita, can reach a diameter of a half-millimeter. It gets more metabolic power in a similar way to how eukaryotes do it: we eukaryotes carry a population of mitochondria with convoluted membranes and a dedicated strand of DNA, all to produce energy, and the larger the cell, the more mitochondria are present. Thiomargarita doesn’t have mitochondria, but it instead duplicates its own genome many times over, with 6,000-17,000 nucleoids distributed around the cell, each regulating its own patch of energy-producing membrane. It’s functionally equivalent to the eukaryotic mitochondrial array then, right?

Wrong. There’s a catch. Mitochondria have grossly stripped down genomes, carrying just a small cluster of genes essential for ATP production. One hypothesis for why this mitochondrial genome is maintained is that it acts as a local control module, rapidly responding to changes in the local membrane to regulate the structure. In Thiomargarita, in order to get this fine-tuned local control, the whole genome is replicated, dragging along all the baggage, and metabolic expense, of all of those non-metabolic genes.

Because it is amplifying the entire genomic package instead of just an essential metabolic subset, Thiomargarita‘s energy output per gene plummets in comparison. That difference is highlighted in this illustration which compares an ‘average’ prokaryote, Escherichia, to a giant prokaryote, Thiomargarita, to an ‘average’ eukaryotic protist, Euglena.

i-3ec4f5c02d3305d25182f69119307702-cellpower-thumb-400x336-59593.jpeg
(Click for larger image)
The cellular power struggle. a-c, Schematic representations of a medium sized prokaryote (Escherichia), a very large prokaryote (Thiomargarita), and a medium-sized eukaryote (Euglena). Bioenergetic membranes across which chemiosmotic potential is generated and harnessed are drawn in red and indicated with a black arrow; DNA is indicated in blue. In c, the mitochondrion is enlarged in the inset, mitochondrial DNA and nuclear DNA are indicated with open arrows. d-f, Power production of the cells shown in relation to fresh weight (d), per haploid gene (e) and per haploid genome (power per haploid gene times haploid gene number) (f). Note that the presence or absence of a nuclear membrane in eukaryotes, although arguably a consequence of mitochondrial origin70, has no impact on energetics, but that the energy per gene provided by mitochondria underpins the origin of the genomic complexity required to evolve such eukaryote-specific traits.

Notice that the prokaryotes are at no disadvantage in terms of raw power output; eukaryotes have not evolved bigger, better engines. Where they differ greatly is in the amount of power produced per gene or per genome. Eukaryotes are profligate in pouring energy into their genomes, which is how they can afford to be so disgracefully inefficient, with numerous genes with only subtle differences between them, and with large quantities of junk DNA (which is also not so costly anyway; remember, the bulk of the expense is in translating, not replicating, the genome, and junk DNA is mostly untranscribed).

So, what Lane and Martin argue is that the segregation of energy production into functional modules with an independent and minimal genetic control mechanism, mitochondria with mitochondrial DNA, was the essential precursor to the evolution of multicellular complexity — it’s what gave the cell the energy surplus to expand the genome and explore large-scale innovation.

As they explain it…

Our considerations reveal why the exploration of protein sequence space en route to eukaryotic complexity required mitochondria. Without mitochondria, prokaryotes—even giant polyploids—cannot pay the energetic price of complexity; the lack of true intermediates in the prokaryote-to-eukaryote transition has a bioenergetic cause. The conversion from endosymbiont to mitochondrion provided a freely expandable surface area of internal bioenergetic membranes, serviced by thousands of tiny specialized genomes that permitted their host to evolve, explore and express massive numbers of new proteins in combinations and at levels energetically unattainable for its prokaryotic contemporaries. If evolution works like a tinkerer, evolution with mitochondria works like a corps of engineers.

That last word is unfortunate, because they really aren’t saying that mitochondria engineer evolutionary change at all. What they are is permissive: they generate the extra energy that allows the nuclear genome the luxury of exploring a wider space of complexity and possible solutions to novel problems. Prokaryotes are all about efficiency and refinement, while eukaryotes are all about flamboyant experimentation by chance, not design.

Lane N, Martin W. (2010) The energetics of genome complexity. Nature 467(7318):929-34.

Hey, isn’t that Kevin Bacon in that remake of The Invisible Man?

This is fun for a little while—Google has made their BodyBrowser available, a handy little tool that lets you explore the anatomy of the human body. It only works with the new Google Chrome web browser, unfortunately, and it doesn’t do much, other than spin and click a rather rigidly fixed anatomy model, and about all you can do with it is click on a bit of something or other and see a label pop up.

i-e2664f55823890275afab24fe2d44caf-bodybrowser.jpeg

What would be more useful is something that demonstrated some physiology, too. Stuff that just sits there is ultimately rather boring. A body with working parts that students could poke at and change around would be far more educational.

Google, get right on that.

My mouse has two daddies

i-e88a953e59c2ce6c5e2ac4568c7f0c36-rb.png

This is awesome news. Biologists have figured out how to enable two male mice to have babies together, with no genetic contribution from a female mouse. I, for one, look forward to our future gay rodent overlords.

It was a clever piece of work. Getting progeny from two male parents has a couple of difficulties. One is that you need an oocyte, which is a large, specialized, complex cell type, and males don’t make them. Not at all. You can tear a boy mouse to pieces looking for one, and you won’t find a single example—they’re a cell found exclusively in female ovaries.

Now you might think that all we’d have to do is grab one from a female mouse, throw out its nuclear contents, and inject a male nucleus into it, but that doesn’t work, either. The second problem is that during the maturation of the oocyte, the DNA has to be imprinted, that is, given a female-specific pattern of activation and inactivation of genes. If that isn’t done, there will be a genetic imbalance at fertilization, and development will be abnormal. What we need to be able to do is grow an oocyte progenitor with male DNA in a female ovary.

So that’s what was done, and here’s how.

i-fc016aa3280ce14e31ca8e0a478350eb-twodaddies.jpeg

Start with Father #1, whose cells all contain an X and a Y chromosome. Connective tissue cells were extracted from the mouse (in this case, an embryo), and then reprogrammed by viral transduction with modified copies of the genes Pou5f1, Sox2, Klf4, and Myc. This step produces induced pluripotent stem cells (iPS cells), or cells that have the ability to develop into all (probably) of the tissues of the body. These cells are then grown in a dish.

The next step is to give Father #1’s cells a sex change operation. This turns out to be trivial: in culture, cells can spontaneously lose a chromosome by non-disjunction, and 1-3% of the cells will lose their Y chromosome, and convert to X0. No Y chromosome means it is now a functionally female cell.

There is a significant difference between humans and mice here. Sometimes (about 1 in 5,000 births) humans are born with only one X chromosome, a condition called Turner syndrome. These individuals appear to be entirely normal females, except for some minor cosmetic differences, an unfortunate predisposition to a few problems like heart disease, and of particular relevance here, are also sterile. Mice are different: Turner syndrome mice are fertile. Apparently, mice have a god-given edge in the gay reproduction race.

Once a population of Father #1’s cells that are X0 are identified, they are then injected into a female mouse blastocyst to produce a chimera, an embryo with a mix of host cells (which are genetically XX) and donor cells (which are X0). That they’re mixed together in the resulting offspring doesn’t matter; it may be a callous way of looking at it, but the only purpose of the host XX cells is to provide a female mouse environment to house Father #1’s X0 cells that end up in the ovaries.

That’s the result of all this tinkering: a female mouse is born with a subset of Father #1’s reprogrammed cells nestled in her ovaries, where they mature in a female body and differentiate into oocytes. The oocytes divide by meiosis, producing egg cells that contain either one X chromosome, or no sex chromosome at all (0).

Finally, Father #2 comes into the picture. Father #2 is an ordinary male, with testes containing cells that go through meiosis and mature into ordinary sperm containing either one X chromosome or one Y chromosome. These sperm are used to fertilize eggs from the chimeric female, which, by all the shenanigans describe above, are derived from Father #1. Both male (XY) and female (X0) progeny ensue. That this actually occurred was thoroughly confirmed by testing the progeny for genetic markers from both fathers…and it’s true. The only genetic contributions were from the dads, and nothing from the host mother.

Now you may be sitting at home with your dearly beloved gay partner and wondering whether you will be able to have babies together someday. Or perhaps you’re a narcissistic man sitting at home alone, thinking you’d like to have babies with yourself, if only you could convince a few of your cells to make eggs (this is another possibility: there is no barrier to this technique being applied in cases where Father #1 is also Father #2, except that it is incestuous to the max). I expect it will be possible someday, but it isn’t right now. There are a few obstacles to doing this in humans.

  1. We haven’t worked out that genetic reprogramming trick for humans yet, so we don’t have a technique for producing pluripotent stem cells from your somatic cells. Give it time, though, and keep funding adult stem cell research, and it’ll happen.

    Also note the rule of unintended consequences. The fundy fanatics have been anti-embryonic stem cell research for years, and one of their tactics has been to insist that adult stem cell research is far more important. In the long run, it is…and oh, look what we’ll be able to do!

  2. The reprogramming trick involves viral transfection, the insertion of mutant copies of a few specific genes. This is probably not desirable. All kids are mutant anyway, but this is adding a specific, constant kind of mutation to all of the individuals produced by this method.

  3. It still requires a woman, and a woman who has been embryonically modified as a blastocyst at that. Did you know women have rights, including the right to not be a vessel for a scientific experiment? It’s true. They also take years and years to grow to sexual maturity, so even if you got started right now it would be a dozen years before she started making oocytes for you, and by the way, she’d inform you that she only produces eggs for herself, not you.

    There may be ways around this, but the techniques aren’t here yet. To produce eggs, we really don’t need the whole woman, just the ovary: another goal of stem cell research is to regrow organs from cells in a dish, for instance to build a new heart or pancreas for transplantation. Consider ovaries on the list of organs.

  4. That difference between mice and humans, that X0 mice are fertile while X0 women are not, seems like a serious problem. We apparently need the pair of X chromosomes working together to provide the correct gene dosage for normal maturation of the egg. It just means that we need to add an extra step to the procedure for people, though: transfer by injection an extra X chromosome from a donor cell from Father #1 to the X0 cells, producing a composite XX cell derived entirely from a male.

  5. The fundies will go raving apeshit bonkers. So what else is new?

  6. OK, there are also some serious ethical concerns that would need to be worked out, independent of the Bible-thumping theocratic sex police. As you can see from the recipe above, this is a procedure that involves extensive manipulation of embryos, almost all of it experimental, and the end result is…a baby. We should be conscientious in our care in any procedure that can produce human beings, especially if there is risk of producing damaged human beings. This can also only be categorized as a kind of expensive luxury treatment, and it’s difficult to justify such elaborate work for solely egotistical gratification. Especially for you, nerd-boy masturbating alone at home. (But learning more about the mechanisms of reproduction is more than enough to justify this work in mice, at least).

Wait…all this is just for male gay couples. What about nurturing lesbians who want to have children together? That has another tricky problem: you need a Y chromosome to induce normal sperm differentiation, and lesbian couples don’t have any of those. At all. They’re going to have to go to a male donor for a genetic contribution, diluting the purity of the genetic side of the procedure. However, that has a technology in the works to help out already: see obstacle #4 above. We’ll have to isolate iPS cells from Mother #1, inject a donor Y chromosome into them, cultivate chimeric male (or chimeric testis in a dish) to produce sperm, and then fertilize eggs from Mother #2 with the Mother #1-derived sperm. Any sons produced by this procedure would have three parents, Mother #1, Mother #2, and the Male Donor who provided the Y chromosome, and only the Y chromosome. Any daughters, though, would only have two parents: Mother #1 and Mother #2.

Isn’t reproductive biology fun? It’s the combination of exciting science with terrifyingly deep social implications.

Deng JM, Satoh K, Chang H, Zhang Z, Stewart MD, Wang H, Cooney AJ, Behringer RR (2010) Generation of viable male and female mice from two fathers. Biology of Reproduction DOI:10.1095/biolreprod.110.088831.