Green Effin' Protein!!

I mentioned last time that green fluorescent protein is one of these sort of serendipitous scientific discoveries that happens every so often, like penicillin.  What, you might ask, is green fluorescent protein?

There have been lots of news stories over the past decade about scientists making various animals glow in the dark.

Glow in the dark bunny!!

Glow in the dark kitties!!!!!!

Glow in the dark adorable baby monkey licking a blanket!!

What these glow in the dark dudes have in common is green fluorescent protein, also affectionately known as GFP.  Each of these organisms has had a bit of DNA encoding the gene for GFP embedded in their cells  so that, in addition to all the other proteins that they normally make, their cells also produce GFP.

But what is GFP and where did it come from?

Green fluorescent protein is, as the name implies, a type of protein that happens to give off fluorescent light when another blue or ultraviolet light source hits it.  Protein may seem like an unlikely candidate to be the agent of biological fluorescence-- after all, who would think that stuff that's in muscle milk and egg whites would be able to glow?  One of the cool things about GFP is that it exemplifies the amazing diversity of tasks that proteins carry out.

As to the where question- the answer is not your local Michael's craft supply shop, though certainly there is no shortage of fluorescent things to be found there.  GFP, despite the rather unearthly glow, is, in fact, a natural product.  It was discovered by a scientist named Osamu Shimomura and it comes from a species of jellyfish that is naturally fluorescent.  That species is called Aequorea victoria.

Aequorea victoria, doing its thing

Osamu Shimomura has been studying Aequorea victoria since the 1960s and shared in a Nobel Prize awarded for the discovery and development of GFP.  Shimomura grew up in 1930s Japan and was a teenager when the atomic bomb fell at Nagasaki; he was 15 miles from ground zero.  The flash blinded him for 30 seconds.

Shimomura went on to study organisms that glow, including Vargula hilgendorfii, whose Japanese name, umi-hotaru, means "sea firefly."  The agent that causes the sea firefly to glow, he found, was a protein he named luciferin.  Luciferin is not fluorescent- it is bioluminescent, which means that it makes light.  Fluorescent proteins don't create their own light- they only appear to "fluoresce" when light of a certain color hits the protein. This work with the sea-fireflies led Shimomura to Princeton to work for a scientist named Frank Johnson.

It was at Princeton that Shimomura began working with jellyfish.  It took 10,000 jellyfish to purify GFP and aequorin, another bioluminescent protein that glows blue.  In jellyfish, the aequorin is the source of bioluminescent light that the GFP uses to give off its own fluorescence, resulting in that cool greenish-bluish glowing color you can see above.  Cool!

And the crazy thing is, Shimomura doesn't really care about all of the applications that GFP has (and it has been adapted for use in thousands of experiments, most of which do not involve cute and cuddly animals).  He just thinks glowing jellyfish are cool and wants to understand how they do their thing.

Here is a fun site devoted to GFP that was a very useful source for this post:  http://www.conncoll.edu/ccacad/zimmer/GFP-ww/

This one's for you, Carl

So, Nature just published a news story with the headline: The search for alien intelligence: SETI is dead - long live SETI.  For the uninitiated (read: undweeby), SETI stands for the Search for Extaterrestrial Intelligence, and they have been combing the sky for signs of intelligent life in the heavens for the last 60 years with their preferred methodology: radio astronomy.

Well, the headline was a little misleading.  SETI, it turns out, is not really dead.  Thanks for the little scare, though, Nature.  You got me to read the whole article.  Are you happy?  They just can't afford to keep their largest array of telescopes going anymore, but there are still plenty of active SETI projects that are collecting data from other sources.

The Allen Telescope Array: Where SETI used to eavesdrop on the cosmic dialog

SETI started in the 60s with a big push from big name astronomers- chiefly Frank Drake, who invented the eponymous equation laying out the probability of the existence of extraterrestrial life.

The Drake equation according to xkcd.com- note that I don't actually share this viewpoint!

Over the years there had been some federal support for SETI efforts through the National Radio Astronomy Observatory and NASA (though NASA funding was quickly cut by a senate bill for being "an utter waste of taxpayers' money").  Then dot-com backers filled the SETI coffers in the late 90s, providing the initial capital to build the Allen Telescope Array (named for Microsoft bigwig Paul Allen) in collaboration with UC Berkeley... until that bubble burst.  The Array remained unfinished at 46 telescopes out of a planned 350, and as a result Berkeley lost the NSF grant money that would have covered operational costs.  The Array hobbled along for a while, but this year it was powered down for good.

Now, the search for extraterrestrial intelligence has got to be one of the most out-there branches of science that still has some semblance of mainstream credibility.  It's sort of at the logical limit of science, taking an established methodology (radio astronomy) and applying it to test the hypothesis that we are not alone in the universe.  The nature of the payoff is, admittedly, completely beyond our anticipation or comprehension.  We just don't know anything about when we could hope to find intelligent life, what the signal of intelligent life might look like, or how finding little green men might impact affairs on earth.

At the other end of the sprectrum, there is science that is eminently fundable.  Projects where there are few dots to connect between the research and a drug or a weapon are perennial favorites among the large federal funding agencies (though DARPA is surely known for funding some really "out-there" stuff- psychic spies, anyone?).

However, science is not always predictable.  Hugely important discoveries-penicillin! green fluorescent protein!- have come from unexpected and relatively impoverished corners.  And there are well-funded projects that promised great things for society that have not delivered as anticipated.  Take the human genome project, for instance, which has scarcely begun to fulfill its goal of revolutionizing medicine even now, over a decade after the first draft of the genome was published.

Ultimately, no matter how safe a bet a project might seem, science is, at it's heart, is all about characterizing the unknown.  You never know which pursuits will pay off big time as opposed to those that yield bubkes.  It's kind of a crap shoot.

So where should the taxpayer dollars be funneled?  Are the serendipitous discoveries just happening at a low level all the time, fueled by enthusiastic amateurs who wouldn't know what to do with a heap of grant money if it was laid in their laps?  Daniel Werthimer, a part-time SETI scientist, thinks that scaling back operations isn't necessarily a bad thing.  "It's naive to think that we know what ET, a billion years ahead of us, is going to be doing. So we want to be a small-scale science, trying lots of things."

The funding question is really difficult.  I've been trying to address it but everything I write comes out too philosophical, and being a bit of a romantic about science my inclination is to say FUND EVERYTHING NEAT!!!  So I'll leave you with this.  I wonder if Carl Sagan liked this song.

A Tale of Two Strawberries

 As promised: a brief history of the cultivated strawberry.

Meet Amédée-François Frézier, 18th century spy, mathematician, cartographer, strawberry smuggler, and general international man of mystery.

Amédée-François Frézier.  Handsome, no?

Frézier's most lasting work would be a royally-commissioned reconnaissance mission for Louis XIV in South America between 1712 and 1714.  After carefully characterizing coastlines, flora, fauna, and, most importantly, Spanish assets, Frézier hopped back onto his merchant ship to Marseilles with all kinds of loot, including five specimens of a new species of strawberry whose fruits he described for posterity as being "as large as a whole Walnut, and sometimes as a Hen's Egg."  This was good news for Old World strawberry cultivation since the few species currently available to Europeans bore delicious but rather lilliputian fruits.

Frézier's illustration of Chilean strawberry, enormous fruit and all.

It could very well have been familial pride that drew Frézier to the familiar trifoliate-leaved plant with the unusually large berries that he encountered during his time in Peru.  Frézier is a bastardization of fraise, French for strawberry, and was purportedly derived from a knighthood bestowed upon Frézier's tenth century ancestor Julius de Berry (I'm not making this up, I promise) by King Charles the Simple in return for serving up an excellent crop of them for a feast at a time of year (early May) that seemed somewhat miraculous.

But alas!  Frézier was in for an unwelcome surprise upon his return home.  The strawberry plants that had once borne such incredibly large fruits in Peru bore absolutely none on French soil.  The plants propagated themselves perfectly well by sending out vine-like runners with small satellite plants that were sent all over Europe for horticulturalists to coddle and fuss over.  But the fact remained that for 50 years no one could consistently entice Chilean strawberries to, well, make strawberries.  When they did produce fruit, no one seemed to know why.

The problem?  In choosing plants with the largest fruits from Peru, Frézier had inadvertently selected all females.  The Chilean strawberry, it turns out, is one of the rare species in the plant kingdom to have separate sexes, a state known to botanists as dioecy (Greek: di- two, oikia- house).  Most plants are hermaphroditic- the flowers on an individual plant possess both male and female sexual apparatus, and most of these are capable of self-pollinating.  Perhaps these odds were the reasoning behind Frézier's oversight- he just assumed that the plants he took back with them were able to self-pollinate in order to produce fruit.  (See this wiki for a brief primer on the dizzying world of plant sexuality: "It's complicated" doesn't quite begin to cover it.)

The plants that Frézier had overlooked in Peru- those that infrequently bore small, irregular fruits, or worse yet, none at all- were not evolutionary duds, they were simply the males of the species whose chief reproductive duty is to produce pollen, not to bear fruit.  The solution was suggested by leading strawberry expert Antoine Nicholas Duchesne in 1766: to find a suitable pollen donor among strawberry species available in Europe so that the female flowers could be pollinated and thus bear fruit.  This solution was accidentally implemented by gardeners who planted other species of strawberry amongst the Chilean strawberry, allowing bees to cross-pollinate all the species.  The winning candidate for consistently pollinating the Chilean strawberry was the Virginian strawberry, brought to Europe from the meadows of what are now the eastern United States some 100 years before the Chilean strawberry's landing.

Eventually, these mid-18th century crosses of North and South American strawberries in European gardens yielded the grand prize: modern cultivated strawberry, or Fragaria x ananassa (the "x" denotes the hybrid nature of the species), available in markets across the globe.  Voilà!  French spies, mistaken identities, and an ultimate, highly profitable and delicious redemption.

Fragaria x ananassa: modern cultivated strawberry

*Fun fact*: the success in the pairing of the Chilean and Virginian strawberries may in part be attributed to both species being octoploid.  That means that instead of having two copies of each of their chromosomes like we do (a state called diploidy), these strawberry species have eight.  This also makes them good for home DNA extraction since they have a lot of DNA.

I should also note that G.M. Darrow's book The Strawberry: History, Breeding and Physiology, was an indispensable resource for this post- it is available in full through the USDA website, here.

Nettie and the Sex Chromosomes

In my last year as an undergraduate I had the good fortune to work on a very interesting project where I helped to map a novel sex chromosome: that of Fragaria chiloensis, a species of strawberry native to the west coast of the Americas.  F. chiloensis is one of the forebears of cultivated strawberry, along with another American species called Fragaria virginiana.  The history of cultivated strawberry is pretty fascinating (mistaken identities!  French spies!) and probably deserves a space of its own here.

Fragaria chiloensis on the beach: Photo by Claus Holzapfel

While working on this project I became interested in the history of our understanding of sex chromosomes.  I was excited to learn from a visiting lecturer of a scientist working during the turn of the century whose contributions were very important to that understanding.  In fact, some scholars cite her as the unsung discoverer of sex chromosomes whose untimely death likely robbed her of the recognition that she deserves.  Her name was Nettie Stevens.

Nettie Stevens, 1904.  Photo from the Carnegie Institution of Washington.

Nettie Stevens was not your typical academic scientist.  She was born in 1861 to a carpenter in Cavendish, Vermont and was educated at the Westford Academy in Massachusetts, which still exists today as a public prep school outside of Boston.  The Westford Academy at that time was open to people of any nationality, age or sex.  That disclaimer has since grown considerably and is displayed at the bottom of the school's website: "Westford Public Schools does not discriminate on the basis of race, color, sex, religion, national origin, sexual orientation, disability or homelessness."  After graduating from Westford, Nettie spent the next fifteen years alternating between work as a teacher and librarian and continuing her studies, all the while saving up to pursue bachelor's and master's degrees across the country at Stanford.

After four years in California and at the age of 39, Nettie returned to the east coast in 1900 to complete her doctoral work at Bryn Mawr College outside of Philadelphia.  Around that time, Bryn Mawr, which had been established some 15 years prior, supported an enviable biology faculty despite being an institution for the education of women.  Successively chairing the biology department were Edmund B. Wilson and Thomas Hunt Morgan, the so-called father of modern genetics, whose work with fruit flies would later cement the chromosomal theory of inheritance and earn him the name of a chromosomal unit of measure- the centiMorgan.  Morgan mentored Nettie during her doctoral studies and eventually would write her a glowing recommendation when she was applying for a fellowship through the Carnegie Institute in order to continue her research at Bryn Mawr after graduating.  He wrote:

"Of the graduate students that I have had during the last twelve years I have had no one that was as capable and independent in research work as Miss Stevens and now that she has her degree she is devoting all of her time to research...  Miss Stevens has not only the training but she has also the natural talent that is I believe much harder to find. She has an independent and original mind and does thoroughly whatever she undertakes. I fear to say more lest it may appear that I am overstating her case."

Morgan had good reason to talk up his protegé; in his recommendation he goes on to describe the collaborative work they would undertake in characterizing sex determination upon her successful receipt of the fellowship- they would study the "accessory chromosome" that C. E. McClung had in 1901 proposed to be linked to sex determination.  He concludes his plea: "It is also of the greatest importance to me to have someone working with me on this problem and I know of no one who is so well suited to carry out work of this sort as Miss Stevens."

Stevens and Morgan did not end up publishing their work together; Morgan was still clinging to the idea that traits were passed from one generation to the next by the mixing of parental cytoplasm, the fluid inside of cells, upon the union of sperm and egg at fertilization.  To Edmund B. Wilson, a colleague of Stevens and Morgan, the purpose of the "accessory chromosome" was, at best, to carry hereditary information important for, but not determinant of, sexual traits.

Stevens' work suggested otherwise.  While studying the lowly mealworm, she found that cells from males of the species had 19 large and one small chromosome, while cells from females had 20 large chromosomes.  Moreover, during the creation of sperm cells, half of the sperm were endowed with 10 large chromosomes, while the other half received 9 large and one small chromosome.  In this observation lay a tantalizing explanation for sex determination that fit Mendelian expectations- the small chromosome was in fact a male determinant.  These findings were published in 1905 in her book, Studies in Spermatogenesis, alongside hundreds of painstakingly hand-drawn images of her observations under the microscope.

Mealworm sperm progenitor cell with 19 large
and one small chromosome (highlighted)

Mealworm egg progenitor cell
with 20 large chromosomes

Four mealworm sperm cells:
two with 9 large and one small chromosome (above)
and two with 10 large chromosomes (below)

Nettie clearly recognized the significance of this finding.  She summarizes it in the text of her book:

"Since the somatic cells of the female contain 20 large chromosomes, while those of the male contain 19 large ones and 1 small one, this seems to be a clear case of sex-determination... the spermatozoa which contain the small chromosome determining the male sex, while those that contain 10 chromosomes of equal size determine the female sex."

This is an important point because in the same year, Edmund B. Wilson published a paper making a similar inference about a type of leaf-footed bug in which he had found males to have one less chromosome than females.  Wilson, however, was less willing to call a spade a spade than was Stevens, relegating his comment in support of a chromosomal hypothesis of sex determination to a footnote.  Morgan at this time was still convinced of the primacy of cytoplasm in inheritance and in his private correspondence expressed that his colleague Wilson had gone "wild over chromosomes."

Morgan eventually came around a few years after this work was published.  Not only did he concede that chromosomes are the vehicles of hereditary information, he also went on to conduct truly brilliant work in fruit flies that showed that genes for specific traits were strung like beads along the lengths of chromosomes- physically attached to one another in a phenomenon called linkage.  The closer two genes are to one another on a chromosome, the more likely they are to be inherited together.  This fact can be used to map the distance (now measured in centiMorgans) between genes on a chromosome, and this is exactly what Morgan did, generating the first genetic maps in his lab.  This work established the fruit fly species Drosophila melanogaster as the most important model organism of the 20th century- thousands of researchers study Drosophila to this day.

Nettie Stevens did not fare so well.  Her career was off to a solid start at Bryn Mawr, where she continued her research on sex chromosomes throughout the first decade of the 20th century.  In 1912, Bryn Mawr offered Nettie a professorship, but she was unable to accept the position.  She had fallen ill with breast cancer and moved from Philadelphia to Baltimore to receive state-of-the-art treatment at Johns Hopkins Hospital.  William S. Halsted, the father of modern surgery and pioneer of many surgical practices including radical mastectomy, likely treated her there.  In a summary of Bryn Mawr president M. Carey Thomas' personal papers in the College archive is the following clue: "Writing of Nettie Stevens's illness in May 1912, [Halsted] asserted that he would never accept a fee from a college professor."  Nettie died on May 4, 1912.

Nettie was remembered warmly at Bryn Mawr, eulogized by one of her students as a caring mentor, "taking great personal interest in her students... always eager to help them, especially in making a start with any research work."  In the same eulogy is an anecdote in which Nettie reassures an inquisitive student, "How could you think your questions would bother me?  They never will, so long as I keep my enthusiasm for biology; and that, I hope, will be as long as I live."

Morgan's assessment of Stevens' work was not as favorable.  He certainly acknowledged the importance of her work in characterizing the chromosomes of 59 species of flies and beetles, stating in a summary of her career published in Science:

"Such an extensive study will not seem superfluous when the reception of this important discovery in regard to sex is remembered, for the profound significance of the results were by no means generally appreciated, and it is not going too far to say that many cytologists assumed a sceptical or even antagonistic attitude for several years towards the new discovery."

However, Morgan goes on to criticize her careful thoroughness, writing that her "work is characterized by its precision, and by a caution that seldom ventures far from the immediate observation.  Her contributions are models of brevity- a brevity amounting at times almost to meagerness."  Stevens was perhaps not outwardly romantic about her love of science since Morgan felt that "She was a trained expert in the modern sense- in the sense which biology has ceased to be a playground for the amateur and a plaything for the mystic."  But perhaps the most scathing remark is the combination of these two perceived faults: "...she was careful to a degree that makes her work appear at times wanting in that sort of inspiration that utilizes the plain fact of discovery for wider vision."

This harsh judgment seems unfair.  I cannot doubt that Stevens sat daily before her microscope in awe of what lay before her, for she was so singularly devoted to her work- a career for which she worked so hard even just to begin.  She paid her way through school with money she had spent 15 years saving and fought for funding to continue her research once she had finished her training.  I am inclined to believe that her caution was necessary- Stevens did not have the reputation to help her weather a false step.  If a wild extrapolation from her data proved incorrect, her hard-won career would be over.

There is some scholarly work out there about Nettie Stevens (in addition to her own body of work) on which I relied heavily in researching this post. There are two biographical sketches- one by Stephen G. Brush and another by Ogilvie and Choquette- and I went to their sources as well as additional ones for some of the information on Morgan and Wilson.  However I feel there is more to this story.  Ogilvie and Choquette concede that personal biography is difficult because so few sources exist regarding Stevens' life, but I am intrigued by that little nugget in the M. Carey Thomas papers at Bryn Mawr about Halsted treating Nettie.  Perhaps a research trip to Philadelphia and Baltimore is in order?