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Wednesday, June 27, 2007

Nobel Laureate: Albert von Szent-Györgyi Nagyrapolt

The Nobel Prize in Physiology or Medicine 1937.
"for his discoveries in connection with the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid"

Albert von Szent-Györgyi Nagyrapolt (1893 - 1986 ) received the Nobel Prize in Physiology or Medicine for his work on the biochemistry of combustion, or respiration as we now know it. In most cases when we read the presentation speeches for past Nobel Prizes we recognize the seminal insights that have led to a better understanding of science. This recognition is usually even more obvious when we review the Nobel Lecture of the prize winner [Szent-Gyorgyi Lecture].

In this case the presentation speech seems hopelessly confusing and it's difficult to see any connection between our current understanding of metabolism and the view presented in 1937. Szent-Gyorgi worked on the metabolism of vitamin C and on the role of fumarate in basic metabolism. (Fumarate was Monday's Molecule #32.) Szent-Gyorgi thought that vitamin C was an essential cofoactor in metabolism but we now know that it plays only a minor role [Scurvy and Vitamin C]. He also though that fumarate was a cofactor bound to the surface of an enzyme—it is not. Furthermore he thought that fumarate was part of a reductive pathway where hydrogen atoms were taken up from the food and passed from oxaloacetate to malate to fumarate to succinate and then to a cytochrome complex where they were eventually combined with water. We now know that this pathway part of a large pathway (the Krebs cycle [Nobel Laureates: Hans Krebs]) that operates in the opposite direction. We also know that it is electrons, not protons, that carry the energy of metabolism.

Read the presentation speech below to see the state of knowledge in 1937. Keep in mind that at the time there were other scientists, notably Hans Krebs, who were on the right track.

Your Majesty, Your Royal Highnesses, Ladies and Gentlemen.

The Staff of Professors of the Caroline Institute, pursuant to the task devolving upon them by the terms of the will of Alfred Nobel, have awarded the Prize for Physiology or Medicine for the year 1937 to Professor Albert von Szent-Györgyi, in recognition of his discoveries concerning the biological combustion processes with especial reference to vitamin C and to the fumaric acid catalyst. The wording of the above sentence indicates that the mechanism of biological oxidation has been investigated beyond the great discoveries in this field made by Otto Warburg, Heinrich Wieland, and their successors. Their systems of catalysts for oxidation have been shown to be dependent on Szent-Györgyi's new catalysts.

It was generally known before that combustion liberates energy in living cells which can be employed there without loss - directly for the building up of new substances - for storage or for the building of functioning cell structures. The building up of living organisms then is dependent in essential respects on combustion, which is guided by catalyst systems. Thus catalyst systems are conditional for the building up of living organisms. Consequently in the unknown period during which organic life originated, the formation of these and other catalyst systems must have preceded the completion of the living animal organisms.

Preferably I should wish to confine my remarks to the new conquistador from Szeged. The survey is however of higher importance on this occasion, and moreover the course of the events is dramatically concentrated. Each one of the three has conquered new ground by intuitive daring and skill. Szent-Györgyi's greatest achievement has intimately linked up the accomplishments of the two others and of their successors, giving us for the first time a picture of a coherent oxidation process - of the interplay of three catalyst systems and the oxidation thereby in metabolism.

Warburg, who always stood alone with some few faithful co-workers, is the foremost pioneer, and he had to overcome the greatest difficulties. At this day there is none who can any longer throw doubt on his discoveries, but that was not so, when in 1931 underestimated by the majority, he was awarded the Nobel Prize by the Caroline Institute. He has shown that the inert oxygen, with which the red blood corpuscles are fully loaded, is taken up from them by a catalyst system to which many red pigments belong (for brevity's sake called «the red system»). These are related to the red blood pigment. They contain as active groups (for the most part) iron and specific proteins. In this system the oxygen combines with the iron during varying periods of time. In the case of the most rapid catalysts, it combines with the iron, is converted into a lively, reactively disposed form and is delivered - all at a speed that gives a flowing stream of active oxygen from the catalyst system. One thought that this active oxygen oxidized directly. That is not the way however. On the contrary, the active oxygen meets hydrogen - but that is another story, belonging to Szent-Györgyi's great discovery. The manner in which the life-giving active oxygen's dramatic encounter in the darkness of the cells ensues, had been unknown ever since the morning of time until, in 1933, Szent-Györgyi carried out some experiments which proved to be the prelude to the revelation of the secret.

For the moment I will leave oxygen, and direct the attention to the first, apparently unimportant, experiments carried out by Wieland. These led him to the conception of an idea, which was destined to carry him on to the disclosure of an extensive part of the mechanism of oxidation. A large number of investigators were soon attracted by Wieland's opinion. This seemed to be at variance with the oxygen activation - at any rate that was the view of a majority. This apparent inconsistency was never considered by Szent-Györgyi, nor by Warburg.

Wieland had observed that palladium is capable of absorbing hydrogen from certain organic compounds, which means their partial combustion or oxidation. Through the cooperation of many investigators the presence was revealed of extensive metal-free catalyst systems, the effect of which was shown to consist in the removal of hydrogen from metabolic substances, in agreement with Wieland's concept. These catalysts were given a name in common: dehydrogenases (hydrogen-removers, hydrogen-absorbers, or hydrogen-transporters) and the idea was held pretty generally that the hydrogen activated by this system would be capable of reacting directly with the inert oxygen molecules. Hydrogen superoxide was supposed to form an intermediate product. That is not the highway of oxidation however. On the contrary, the hydrogen first meets Szent-Györgyi's catalyst system from a different side to the one where the activated oxygen flows into it from the «red system». That again is another story, which also belongs to Szent-Györgyi's great discovery. From 1925 onwards he had been investigating a number of hydrogen-absorbers. Previously to anyone else he formed the view of these as members of a catalyst system in the service of oxidation (in other quarters loosely conceived of as being auxiliary catalysts of some kind for fermentation). He was also occupied with experiments on a yellow substance, termed flave by him, while his investigation regarding vitamin C was being completed, and conducted on to the isolation of that substance, enabling him later to insert it in the catalyst system of certain hydrogen-removers. Vitamin C and another substance, containing sulphur as a hydrogen-removing group and defined by Sir Frederick Gowland Hopkins and others, were however until 1934 the only substances belonging to the hydrogen-transporters in the oxidation-chain that had been isolated. The rank that they possess as catalysts is dependent on the velocity of the hydrogen-transportation and the degree of the activation of the hydrogen - problems that still await a satisfactory solution. On the other hand, Hugo Theorell succeeded in 1934 in isolating, in Warburg's laboratory, the first really rapid hydrogen-transporter, called «the yellow enzyme». He could also show that it was a phosphoric-acid ester of vitamin B2, linked to a specific protein. Warburg and Christian, in 1935, defined the nature of the active group in two other dehydrogenases, colourless and metal-free (co-ferment and co-zymase), which had long frustrated the attempts of other investigators. One of them was the catalyst that Szent-Györgyi had placed in this section of the oxidation-chain.

The magnificent series of Szent-Györgyi's discoveries commenced in 1933. They were carried out and pursued at Szeged with extraordinary rapidity and precision. His clear vision for essentials induced him, in spite of his isolation of ascorbic acid and of his identification of it with the so-termed vitamin C - a feat that was justly hailed with enthusiasm - to hand over to others for the time being the tempting pursuit of the further development of that discovery, and to devote the whole of his energy to the problem of combustion, notwithstanding the difficulties it presented. Many investigators had been working at the so-called plant acids in the muscular system, and had observed their capacity for intensifying oxidation in that tissue. The readiest explanation however of how that came about, viz. that they are easily combusted themselves, simply did not fit in with Szent-Györgyi's intuitive perception. By elaborating reliable methods of analysis for the substances in question, and by means of consistent experiments, he and his co-workers proved that the plant acids were not consumed by combustion, were not ordinary nutrient substances, but were on the contrary themselves active groups of catalysts which served to maintain the combustion without themselves suffering any diminution thereby. The process involves a peregrination of hydrogen more intricate than the adventurous journeys of Odysseus, though more rapid. Hydrogen is released out of the metabolic substances, probably through cooperation between Szent-Györgyi's and Warburg's co-ferment and Theorell's yellow enzyme, and encounters the plant acids, entering in that way Szent-Györgyi's's system. These acids transfer the system into the order: oxalacetic acid, malic acid, fumaric acid, and succinic acid, then, in the form of active hydrogen, to encounter the active oxygen from «the red system» and form water and free energy - a series of providentially subdued explosions which I alluded to before as a dramatic encounter. The plant acids act as catalysts by cooperation with specific proteins, and the effect of the yellow enzyme probably extends some way into this Szent-Györgyi's intermediate system.

Thus, the oxygen-activation in the red iron system and hydrogen-transfer from nutrients by the yellow metal-free system along with co-agents have been united by Szent-Györgyi through the discovery of this intermediate system. The interplay of «the red system»'s cytochrome-group and the yellow enzyme might probably also, according to Theorell, proceed directly. The flaws are numerous, but not of a character to constitute any essential breach in the highway of the oxidation-chain. Numerous ramifications of the latter however already begin to be discernible.

It is of especially great importance that at least two vitamins - C and B2, and possibly B1, and P - are in cooperation in the oxidation chain and are catalysts, illustrating the way in which these vitamins act in the organism. It may be that development in the near future will reveal the importance for our organism of copper concerning oxidation and of vitamin C with certain followers in plants, viz. oxidating enzyme, and oxidizable and reducible substances (Szent-Györgyi's flavonoles, termed vitamin P), which are capable of forming a sensitively attuned system with the vitamin, hydrogen-superoxide and proteins, or parts of them, with active and activating sulphur in the molecule. The sulphur of the alchemists of old, out of which everything was to radiate, is destined to experience a renaissance.

Professor Albert von Szent-Györgyi. As a representative for the Caroline Institute on this occasion, I am commissioned to give expression to our high estimation of your researches.

You never swerved from your unyielding purpose to study the primary and fundamental processes of biological oxidation. Entering upon this difficult field of biochemical research you soon became a pioneer by interpreting the position and real function of co-ferment as an important link in the chain of dehydrogenating catalysts. Not even your important discoveries regarding vitamin C could deter you from following a certain strain of thought. I am deducing now from a close observation of your work that you were drawing distinctions in your mind at this occasion between your interesting discovery of ascorbic acid and the bare possibility of some other audacious plans of yours coming true. At this early stage they must have involved the investigation of the fundamental mechanism of connecting hydrogen activation with that of oxygen activation. Your intuitive mind decided in favour of the possibility of success, and you won through. In the year 1933 the first signs became visible for outsiders, and from then on the pace set by you and your co-workers at Szeged was astonishing, and your results were fundamentally new and highly important. In the midst of fervent research work with most promising aspects you are the discoverer and idealist to the mind of Alfred Nobel.

I ask you, Professor Szent-Györgyi, to receive the prize from the hands of His Majesty, our gracious King.

Tuesday, June 26, 2007

Darwin Still Rules, but Some Biologists Dream of a Paradigm Shift

That's the title of an article in today's New York times. It's written by Douglas H. Erwin, "a senior scientist at the National Museum of Natural History at the Smithsonian Institution and a research professor at the Santa Fe Institute." You can read the entire article here.

Erwin says some rather silly things about "paradigm shifts" but I'll leave that to people like John Wilkins who know what Thomas Kuhn really meant. Suffice to say, the term is very much abused these days.

Putting that aside, Erwin makes some good points about the controversies in modern evolutionary theory. He points out that most of the old "paradigm" about evolution by natural selection is being challenged in one way or another. For example,
In the past few years every element of this paradigm has been attacked. Concerns about the sources of evolutionary innovation and discoveries about how DNA evolves have led some to propose that mutations, not selection, drive much of evolution, or at least the main episodes of innovation, like the origin of major animal groups, including vertebrates.
This is correct. There's a resurgence of "mutationism" that puts more emphasis on the role of mutation in evolution. This re-emphasis is not popular among evolutionary biologists but it's gaining ground.
Comparative studies of development have illuminated how genes operate, and evolve, and this places less emphasis on the gradual accumulation of small genetic changes emphasized by the modern synthesis. Work in ecology has emphasized the role organisms play in building their own environments, and studies of the fossil record raise questions about the role of competition. The last major challenge to the modern synthesis came in the 1970s and 1980s as my paleontological colleagues, including the late Stephen Jay Gould, argued for a hierarchical view of evolution, with selection occurring at many levels, including between species.
Again, these new ideas are all in play in modern evolutionary theory. The first refers to "evo-devo" and there's no question that some evolutionary biologists are rethinking evolution in light of the discoveries in developmental biology. (I think they're wrong, but that's not the point here.)

The last item refers to punctuated equilibria and hierarchical theory. There's little doubt that Gould's ideas have shaken up the old "paradigms."

I was disappointed that Erwin omitted the biggest threat to the old view of evolution, namely random genetic drift. While we've known about drift for decades the full scale of it's role in evolution is only now beginning to be appreciated. Most people still don't realize that random genetic drift is by far the most common mechanism of evolution.

[Hat Tip: Greg Laden]

Monday, June 25, 2007

Monday's Molecule #32

Today's molecule is very simple. We don't need a long complicated name this time; the short common name will do. (But you're welcome to supply the IUPAC name if you know it.) The trick here is to recognize why this molecular is important and then connect it to Wednesday's Nobel Laureate(s).

Here's a hint; with hindsight, this is probably one of the most undeserving Nobel Prizes in the biological sciences.

The reward (free lunch) goes to the person who correctly identifies the molecule and the Nobel Laureate(s). Previous free lunch winners are ineligible for one month from the time they first collected the prize. There's only one (Marc) ineligible candidates for this Wednesday's reward since many recent winners haven't collected their prize. The prize is a free lunch at the Faculty Club. I've been eating there a lot recently because we've had many meeting concerning an upcoming big event at the Faculty Club on this Friday evening. (My daughter, Jane, is getting married.)

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Saturday, June 23, 2007

Is antibiotic resistance evidence for Darwinian evolution?

Here's a short video where the IDiots discuss antibiotic resistance in bacteria. This is part of the new attack on evolution. It's the main point of Michael Behe's new book, The Edge of Evolution. The idea is that random mutation and natural selection can only break things but they can't make new structures.

I suspect that very few people can scientifically refute the arguments in this video. It can be done, but it's going to take a bit of effort.

An Interview with Michael Behe

I'm reading The Edge of Evolution, the new book by Michael Behe. I'm not finished but I can tell you it's going to be a challenge to refute Behe's main claims. That's not because he's correct—far from it—but because he has done a clever job of picking out scientific data to support his case. The idea is that random mutation and natural selection are simply not capable of doing the things they have to do in order for large scale changes to accumulate. His probability arguments are more sophisticated than those of the average IDiot and I think we owe it to Behe to address them rather than just dismiss them out-of-hand because we don't like the conclusion. I'm looking forward to the challenge.

Here's a short interview with Behe (#5) where he explains his views. Behe says that antibiotic resistance is an example of breaking things rather than creating new things. As you listen to him make this claim, think about the evolution of β-lactamase activity from a transpeptidase enzyme [Penicillin Resistance in Bacteria: Before 1960].

Click here to get your own player.

Friday, June 22, 2007

Denyse O'Leary Asks a Question about Evolutionary Psychology

It never ceases to amaze me that the IDiots know so very little about the science they criticize. They have this myopic view of "Darwinism" and they refuse to adjust it no matter how many time they're told that it isn't true.

So when one of them shows the beginning of understanding, it's time to sit up and take notice. Denyse O'Leary has recently shown just a tiny glimmer on Uncommon Descent [Okay: So evolutionary biologist Larry Moran does NOT believe in evolutionary psychology].
Responding to something I wrote at the Post-Darwinist about the popularity of evlutionary psychology among atheists, Moran (a textbook co-author you may well have suffered though in school), responds:
Just for the record, Denyse, I’m one of those evil atheists that you like to rant about but I’m totally opposed to evolutionary psychology.

But you already knew that many evolutionary biologist were against evolutionary psychology, didn’t you?
No, I didn’t, Larry, and if that’s true, it’s high time more of them voiced their objections. The only sustained critiques I have seen are Hilary and Steven Rose’s unjustly neglected Alas, Poor Darwin and David Buller’s also unjustly neglected Adapting Minds. Steven Rose is a neurobiologist, but Hilary Rose is a social scientist, and David J. Buller a philosopher.

No doubt, there are many critiques out there that I haven’t seen, but I wonder what proportion comes from evolutionary biologists, as opposed to social scientists who know the difference between research and speculation.
I'm not allowed to post on Uncommon Descent—apparently they don't want to learn about evolution. I imagine that many Sandwalk readers have also been banned so here's your chance to educate Denyse.

The most prominent opponents of evolutionary psychology are Richard Lewinton, Steven Rose, and the late Stephen J. Gould. I find it hard to believe that the IDiots have never heard of Gould and aren't aware of the debate in the New York Review of Books back in the mid-90's.

Denyse, here are some recent comments about evolutionary psychology [The Central Failure of Evolutionary Psychology]. I've listed some references below.

I don't know what proportion of evolutionary biologists are opposed to evolutionary psychology but I imagine it's significant. Opposition to evolutionary psychology comes from the same people who call themselves pluralists and who don't like to be called Darwinists. (You do recall, don't you, that many evolutionary biologists have moved well beyond Darwinism? You've been told more than once.)

Gould, S.J. (1997) Darwinian Fundamentalism The New York Review of Books Volume 44, Number 10 · June 12, 1997

Gould, S.J. (1997) Evolution: The Pleasures of Pluralism The New York Review of Books Volume 44, Number 11 · June 26, 1997

Rose, S., Lewontin, R.C. and Kamin, L.J. (1984) Not in Our Genes. Penguin Books, New York.

John Dennehy's Citation Classic for this Week

Check out The Evilutionary Biologist for This Week's Citation Classic. This paper is one of the most important papers in molecular biology. For most people it was the proof that DNA is the genetic material.

Hershey, A.D. and Chase, M. (1952) Independent functions of viral protein and nucleic acid in growth of bacteriophage. J. Gen. Physiol. 36:39-56.

Sandwalk Rating

What's My Blog Rated? From Mingle2 - Online Dating

Toronto City Council Approves Yellow Ribbons.

Yesterday's report of the deaths of three Canadian soldiers in Afghanistan stampeded Toronto City Council into approving the yellow ribbon decals on fire trucks and ambulances [What Does the "Support Our Troops" Ribbon mean to You?].

The press release from City Council says it all [Statement by the City of Toronto “Support Our Troops” ribbons to remain on Fire, EMS vehicles]. There's talk on the radio that the decals will also be placed on police cars. That's insane—I hope it's a false rumour.
Toronto City Council today voted unanimously to continue the ribbon campaign on Toronto Fire and EMS vehicles for an indefinite period of time as a show of support for the Canadian Forces Personnel Support Agency.

In recommending that City Council unanimously endorse the continuation of the ribbon campaign, Mayor David Miller said that all of Canada’s men and women serving in the military have the unwavering support of all Torontonians.

Toronto’s emergency services train and work closely with the Canadian Armed Forces. They share the common thread of personal sacrifice, dedication and professionalism with Canadian forces and the two services will continue to support and work with Canada’s military.

Circular University of Toronto

The main front campus of the University of Toronto is a popular place these days as parades of graduating students walk across it on their way to Convocation Hall.

This photograph was taken by Sam Javanrouh at daily dose of imagery [King's College Circle]. My building (Medical Sciences Building) is at 1 o'clock. The science library is at 10 o'clock and Convocation Hall is at 3 o'clock.

Evolution Ad Wins Award

No, unfortunately it's not that kind of evolution. This famous commercial produced by Ogilvy & Mather of Toronto, won the Grand Prix for viral marketing at Cannes as reported in today's Toronto Star [Dove's `Evolution' ad wins at Cannes]. It may not be biological evolution but the commercial sends a powerful message anyway.

Canadians Not So Sure About Evolution

A recent Angus Reid poll shows that only 59% of Canadians accept evolution [What does that Darwin know anyway?]. In Ontario that number drops to only 51%.

While the influence of Young Earth Creationism remains low (~25%), there are some surprising differences among the various regions in Canada. Only 9% of the people in Quebec are creationists but that number rises to 33% on the Prairies.

Evolution: Human
beings evolved from
less advanced life
forms over millions
of years

Creation: God
created human beings
in their present form
within the last
10,000 years

Not sure

British Columbia65%21%15%
Manitoba/Saskatchewan  56%33%11%
United States*53%66%3%

*USA Today/Gallup Poll (separate questions)

It's clear we have our work cut out for us if we are to convince Canadians that evolution is the correct view. We need to work on converting that 19% who still aren't sure.

[Hat Tip: a gloating John Pieret]

Thursday, June 21, 2007

Tangled Bank #82

The latest version of Tangled Bank has been posted on Greg Laden's blog.

This is a review by Derwin Darwin II with the title: Various Proofs of the Theory of Evolution presented in original form by my uncle, the honorable Charles Darwin in the year 1859 and in subsequent years.

Very entertaining. Thanks Greg.

Wednesday, June 20, 2007

Evolutionary Biologists Flunk Religion Poll

In a follow-up to previous studies, Gregory W. Graffin and William B. Provine surveyed prominent evolutionary biologists to find out what they thought about religion. The results are summarized in the latest issue of American Scientst [Evolution, Religion and Free Will]. (Click on the figure to see a larger version where you can read the fine print.)
Our study was the first poll to focus solely on eminent evolutionists and their views of religion. As a dissertation project, one of us (Graffin) prepared and sent a detailed questionnaire on evolution and religion to 271 professional evolutionary scientists elected to membership in 28 honorific national academies around the world, and 149 (55 percent) answered the questionnaire. All of them listed evolution (specifically organismic), phylogenetics, population biology/genetics, paleontology/paleoecology/paleobiology, systematics, organismal adaptation or fitness as at least one of their research interests. Graffin also interviewed 12 prestigious evolutionists from the sample group on the relation between modern evolutionary biology and religion.

A primary complaint of scientists who answered the earlier polls was that the concept of God was limited to a "personal God." Leuba considered an impersonal God as equivalent to pure naturalism and classified advocates of deism as nonbelievers. We designed the current study to distinguish theism from deism—that is to day a "personal God" (theism) versus an "impersonal God" who created the universe, all forces and matter, but does not intervene in daily events (deism). An evolutionist can be considered religious, in our poll, if he calls himself a deist. ...

Perhaps the most revealing question in the poll asked the respondent to choose the letter that most closely represented where her views belonged on a ternary diagram. The great majority of the evolutionists polled (78 percent) chose A, billing themselves as pure naturalists. Only two out of 149 described themselves as full theists (F), two as more theist than naturalist (D) and three as theistic naturalists (B). Taken together, the advocacy of any degree of theism is the lowest percentage measured in any poll of biologists' beliefs so far (4.7 percent).

No evolutionary scientists in this study chose pure deism (I), but the deistic side of the diagram is heavy compared to the theistic side. Eleven respondents chose C, and 10 chose other regions on the right side of the diagram (E, H or J). Most evolutionary scientists who billed themselves as believers in God were deists (21) rather than theists (7).
When asked directly whether they believe in God, almost 80% said no. I wonder how many of them think of themselves as atheists as opposed to agnostics?

Here's the bad news. 79% of these eminent evolutionary biologists say they believe in free will (option A on the question). Even the authors of the study were surprised by that one.
We anticipated a much higher percentage for option B and a low percentage for A, but got just the opposite result. One of us (Provine) has been thinking about human free will for almost 40 years, has read most of the philosophical literature on the subject and polls his undergraduate evolution class (200-plus students) each year on belief in free will. Year after year, 90 percent or more favor the idea of human free will for a very specific reason: They think that if people make choices, they have free will. The professional debate about free will has moved far from this position, because what counts is whether the choice is free or determined, not whether human beings make choices. People and animals both certainly choose constantly. Comments from the evolutionists suggest that they were equating human choice and human free will. In other words, although eminent, our respondents had not thought about free will much beyond the students in introductory evolution classes. Evolutionary biology is increasingly applied to psychology. Belief in free will adds nothing to the science of human behavior.
There's one other surprise. 72% think that religion is part of evolution—it's an adaptation. One can only wonder what these evolutionary biologists think of themselves. Are they able to overcome their deterministic predisposition to God or are they mutants who lack the gene(s)? Maybe it explains why they believe in free will?

[Hat Tip: Denyse O'Leary]

Denyse O'Leary Has Advice for the Fans of Francis Collins

From Me?: Something against Francis Collins? No!.
On the other hand, I admit to deep disappointment in the intellectual substance of Collins’ arguments, which I unpack in the multipart review at Access Research Network.

Note to all, especially Collins fans: C. S. Lewis is not a security blanket, and the debate over the origin of free will, morality, altruism, and consciousness has moved on from his day. Today's atheist is not usually a genial, classical God-denier; he is a radical materialist who honestly believes that we are all just robots replicating our selfish genes. And he cannot wait to get his gospel onto the curriculum of publicly funded schools, as "evolutionary psychology," forcing everyone's nose into his nonsense.
Hmmm ... I don't know of very many radical materialist atheists who fall for evolutionary psychology. I wonder who she could be talking about? Maybe it's PZ?

Nobel Laureates: Richard Roberts and Phillip Sharp

The Nobel Prize in Physiology or Medicine 1993.

"for their discoveries of split genes"

Richard J. Roberts (1943 - ) and Phillip A. Sharp (1944 - ) received the Nobel Prize in Physiology or Medicine for their discovery of interrupted genes and splicing in eukaryotes [see RNA Splicing: Introns and Exons and Monday's Molecule #31].

Roberts and Sharp discovered that the genes in adenovirus were split into various segments that were combined during RNA processing. The results started to become widely known in 1975-76 and the key papers were published in 1977. Later this gene organization was found to be common in chromosomal eukaryotic genes. Unlike many Nobel Prize discoveries, this one really was revolutionary. Here's the presentation speech by Professor Bertil Daneholt of the Nobel Assembly of the Karolinska Institute.
Your Majesties, Your Royal Highnesses, Ladies and Gentlemen,

Why do children resemble their parents? This question has probably always fascinated humans, but not until the advent of natural science have we arrived at an increasingly satisfactory answer.

In the middle of the last century, the Austrian monk Gregor Mendel conducted his famous breeding experiments with the garden pea. He concluded that every trait of an individual plant is determined by a set of two genes, one obtained from each parental plant. To Mendel a gene was an abstract concept, which he used to interpret his breeding experiments. He had no idea of the physical properties of genes.

Only in the mid-1940s could it be established that in terms of chemistry, genetic material is composed of the nucleic acid DNA. About ten years later the double helical structure of DNA was revealed. Ever since then, progress within the field of molecular biology has been very rapid, and several Nobel prizes have been awarded in this area of research.

Initially, genetic material was studied mainly in simple organisms, particularly in bacteria and bacterial viruses. It was shown that a gene occurs in the form of a single continuous segment of the long, thread-like DNA, and it was generally assumed that the genes in all organisms looked this way. Therefore, it was a scientific sensation when this year's Nobel Laureates, Richard Roberts and Phillip Sharp, in 1977, independently of each other, observed that a gene in higher organisms could be present in the genetic material as several distinct and separate segments. Such a gene resembles a mosaic. Both Roberts and Sharp analyzed an upper respiratory virus, which is particularly suitable for studies of the genetic material in complex organisms. It soon became apparent that most genes in higher organisms, including ourselves, exhibited this mosaic structure.

Roberts' and Sharp's discovery opened up a new perspective on evolution, that is, on how simple organisms develop into more complex ones. Earlier it was believed that genes evolve mainly through the accumulation of small discrete changes in the genetic material. But their mosaic gene structure also permits higher organisms to restructure genes in another, more efficient way. This is because during the course of evolution, gene segments - the individual pieces of the mosaic - are regrouped in the genetic material, which creates new mosaic patterns and hence new genes. This reshuffling process presumably explains the rapid evolution of higher organisms.

Roberts and Sharp also predicted that a specific genetic mechanism is required to enable split genes to direct the synthesis of proteins and thereby to determine the properties of the cell. Researchers had known for many years that a gene contains detailed instructions on how to build a protein. This instruction is first copied from DNA to another type of nucleic acid, known as messenger RNA. Subsequently, the RNA instruction is read, and the protein is synthesized. What Roberts and Sharp were now stating was that the messenger RNA in higher organisms has to be edited. The required process, called splicing, resembles the work that a film editor performs: the unedited film is scrutinized, the superfluous parts are cut out and the remaining ones are joined to form the completed film. Messenger RNA treated in this manner contains only those parts that match the gene segments. It later turned out that the same parts of the original messenger RNA are not always saved during the editing- there are choices. This implies that splicing can regulate the function of the genetic material in a previously unknown way.

Roberts' and Sharp's discovery also helps us understand how diseases arise. One example is a form of anemia called thalassemia, which is due to inherited defects in the genetic material. Several of these defects cause errors in the editing process during splicing; thus, an abnormal messenger RNA is formed and subsequently also a protein that functions poorly or not at all.

The discovery of split genes was revolutionary, triggering an explosion of new scientific contributions. Today this discovery is of fundamental importance for research in biology as well as in medicine.

Dr. Richard Roberts and Dr. Phillip Sharp,

Your discovery of split genes led to the prediction of a new genetic process, that of RNA splicing. The discovery also changed our view of how genes in higher organisms develop during evolution. On behalf of the Nobel Assembly of the Karolinska Institute I wish to convey to you our warmest congratulations, and I now ask you to step forward to receive the Nobel Prize from the hands of His Majesty the King.

What Does the "Support Our Troops" Ribbon mean to You?

Since last October emergency vehicles in Toronto have been displaying a decal in support of our troops in Afghanistan. The decals were placed on the vehicles at the request of firefighters and paramedics, whose unions are strong supporters of the soldiers. The original deal was that the decals would stay on for one year and then be removed when the vehicles came in for routine maintenance this Fall.

The issue has turned into a hot political fight that will be decided today at a City Council meeting [Time limit for 'Support Our Troops' ribbons is up].

As you might imagine, there are some city councilors who want the decals to stay on the ambulances and fire trucks.
Some councilors believe the decision to remove the decals is a black mark on the city.

"I was stunned this morning to hear on the radio that some official at the city had ordered emergency services, particularly ambulances, to take off the decal that supports our troops in Afghanistan," city councilor Brian Aston told CTV News on Tuesday.

"These decals are on there and it makes a very strong statement. To take them off, Toronto is the largest city, would just be an outrage. It would be a black eye on the reputation of our city," Ashton said.
It should also come as no surprise that some councilors want to stick to the original agreement and remove the decals in September.
Coun. Janet Davis said just as many councillors want to see the decals removed as those who support their presence on emergency vehicles.

Mayor David Miller said while emergency crews should continue to support Canadian troops, the one-year time limit for the decals was enough time.

"It's controversial on both sides. There are people who see it as support for the troops and there are people who see it as support for war," Miller said.
I'm one of those who believe that the "Support the Troops" ribbon is a political statement. I don't know very many people who are opposed to the war but have this sticker on their car. On the surface it seems like a no-brainer to offer support to our troops while opposing the mission. But, in fact, the term "no-brainer" is quite appropriate in this case. By blindly advertising support for the military you obscure the true difficulty in making rational decisions about how to deploy our army. It's no secret that most people who "support our troops" are also conservatives who are in favour of the war.

The idea that the "Support Our Troops" yellow ribbons are politically neutral is something that only a supporter of the war would say. It's ridiculous. It would be like putting peace symbols on the trucks on the grounds that surely everyone supports peace.

I am very supportive of individual soldiers who are posted to Afghanistan. It's not their fault that our government is insane. They have to follow orders. But that does not mean that I "support our troops" in the way that the decal signifies. As a matter of fact, I do not support our mission in Afghanistan and I would withdraw the troops tomorrow if I could. Every soldier who dies in Afghanistan will have died in vain. That's hardly a way to offer support to our troops.

Having those decals on city vehicles sends the wrong message. For those of us who oppose the war it signifies that the fire fighters and paramedics are on the other side of the issue. That makes me uncomfortable since these are people who deserve my respect and admiration but they're not going to get it if they push a political agenda through advertising on their vehicles.

Take the decals off. It's no place for politics.

Tuesday, June 19, 2007

RNA Splicing: Introns and Exons

Most eukaryotic protein-encoding genes are interrupted. The coding regions are divided into numerous blocks called "exons" and the exons are separated by "introns."

An example is shown below. The triose phosphate isomerase (TPI) gene from maize is composed of 9 exons and 8 introns. (Triose phosphate isomerase is one of the enzymes in the glycolysis/gluconeogenesis pathway.)

The top line is a cartoon representation of the TPI gene with each exon in a different color. The thick gray lines between them represent the introns. The gene is transcribed from left (5′) to right (3′) beginning at the promoter (P). The long primary RNA transcript contains both intron and exon sequences. Subsequent processing of this primary transcript results in modification of the 5′ end by addition of an m7 GTP cap and modification of the 3′ end by addition of adenylate (A) residues to form the poly A tail. More importantly, the introns are spliced out and the exon sequences are fused to form the mature mRNA. This mRNA is then transported to the cytoplasm where it is translated into protein.

Note that all the coding regions in the exons (hatched) are contiguous in the mature mRNA. The relationship between the exons and the structure of the protein is shown on the right where the color of each segment of the protein corresponds to the color of the exons in the upper figure. There is no correlation between the exons and any protein domains or motifs. (It used to be thought that exons corresponded to domains in the protein.)

The splicing reaction is complicated. The cell must cleave the primary transcript at each end of the intron while holding on to the flanking exons so the chopped RNA transcript does not come apart. Then the two exons have to be joined together. For protein-encoding genes the splicing reactions are catalyzed by an RNA/protein complex called a spliceosome. In some cases, the introns can be thousands of nucleotides long—much longer than the exons.

Let's look at a simplified version of this reaction. The various components of the spliceosome have to assemble at the 5′ (left) end of an intron and at the 3′ end. There's a third site in the middle called the branch site. All three sites are identified by specific short sequences in the primary transcript as shown below.

These are the consensus sequences for vertebrates, including us. The splice site and branch site sequences in other species are similar but not identical.

In the first step of the splicing reaction, the various components of the spliceosome bind to the 5′ splice site, the 3′ splice site, and the branch site. Then the three complexes interact with each other to draw together the ends of the intron and position them near the branch site. This forms the spliceosome.

The first reaction involves an attack of the 2′ -OH group of the branch point adenylate residue on the 5′ splice site. This forms an intermediate where the branch site A residue is attached to three different ends of the primary transcript. The structure resembles a lariat or lasso. This is the structure depicted in Monday's Molecule #31.

Meanwhile, the 5′ end of the transcript is still bound to the spliceosome. This is important because it's about to be joined to the next exon and the reaction wouldn't work if the 5′ end were released following the first cleavage reaction.

In the next step, the spliceosome catalyzes the attack of the -OH group at the end of the 5′ exon on the 3′ splice site. This results in cleavage of the 3′ intron/exon junction and joining of the 5′ exon to the 3′ exon. The intron sequence (dark brown) is released as a lariat (looped) structure.

The two reactions are known as transesterification reactions because they require the breaking of one strand of RNA and formation of a new ester linkage. The details are not very important. What's important is to recognize that splicing depends on the correct interaction between the components of the spliceosome and the 5′ and 3′ splice site sequences (and the branch site).

These interactions are mediated by small RNAs that are bound to the spliceosome proteins. These RNAs are called small nuclear RNAs (snRNAs) and they're one example of a host of small RNAs produced by non-protein encoding genes. The snRNA/protein complexes are called small nuclear ribonuclear proteins or snRNPs (snurps).

The snRNAs are complimentary to the splice sites and branch sites and that's how the various snRNPs recognize them. This interaction is very weak since it depends on only three or four base pairs. It can be even less since there are many slice sites that are not perfect matches to the consensus sequences shown above. The relative lack of significant sequence similarity makes splicing a very error-prone reaction.

U1 snRNP recognizes 5′ splice sites, U2 snRNP binds to the branch site, and U5 snRNP binds to the 3′ splice site. A more detailed description of the formation of the splicesome is shown below.

What is a gene, post-ENCODE?

Back in January we had a discussion about the definition of a gene [What is a gene?]. At that time I presented my personal preference for the best definition of a gene.
A gene is a DNA sequence that is transcribed to produce a functional product.
This is a definition that's widely shared among biochemists and molecular biologists but there are competing definitions.

Now, there's a new kid on the block. The recent publication of a slew of papers from the ENCODE project has prompted many of the people involved to proclaim that a revolution is under way. Part of the revolution includes redefining a gene. I'd like to discuss the paper by Mark Gerstein et al. (2007) [What is a gene, post-ENCODE? History and updated definition] to see what this revolution is all about.

The ENCODE project is a large scale attempt to analyze and annotate the human genome. The first results focus on about 1% of the genome spread out over 44 segments. These results have been summarized in an extraordinarily complex Nature paper with massive amounts of supplementary material (The Encode Project Consortium, 2007). The Nature paper is supported by dozens of other papers in various journals. Ryan Gregory has a list of blog references to these papers at ENCODE links.

I haven't yet digested the published results. I suspect that like most bloggers there's just too much there to comment on without investing a great deal of time and effort. I'm going to give it a try but it will require a lot of introductory material, beginning with the concept of alternative splicing, which is this week's theme.

The most widely publicized result is that most of the human genome is transcribed. It might be more correct to say that the ENCODE Project detected RNA's that are either complimentary to much of the human genome or lead to the inference that much of it is transcribed.

This is not news. We've known about this kind of data for 15 years and it's one of the reasons why many scientists over-estimated the number of humans genes in the decade leading up to the publication of the human genome sequence. The importance of the ENCODE project is that a significant fraction of the human genome has been analyzed in detail (1%) and that the group made some serious attempts to find out whether the transcripts really represent functional RNAs.

My initial impression is that they have failed to demonstrate that the rare transcripts of junk DNA are anything other than artifacts or accidents. It's still an open question as far as I'm concerned.

It's not an open question as far as the members of the ENCODE Project are concerned and that brings us to the new definition of a gene. Here's how Gerstein et al. (2007) define the problem.
The ENCODE consortium recently completed its characterization of 1% of the human genome by various high-throughput experimental and computational techniques designed to characterize functional elements (The ENCODE Project Consortium 2007). This project represents a major milestone in the characterization of the human genome, and the current findings show a striking picture of complex molecular activity. While the landmark human genome sequencing surprised many with the small number (relative to simpler organisms) of protein-coding genes that sequence annotators could identify (~21,000, according to the latest estimate [see]), ENCODE highlighted the number and complexity of the RNA transcripts that the genome produces. In this regard, ENCODE has changed our view of "what is a gene" considerably more than the sequencing of the Haemophilus influenza and human genomes did (Fleischmann et al. 1995; Lander et al. 2001; Venter et al. 2001). The discrepancy between our previous protein-centric view of the gene and one that is revealed by the extensive transcriptional activity of the genome prompts us to reconsider now what a gene is.
Keep in mind that I personally reject the premise and I don't think I'm alone. As far as I'm concerned, the "extensive transcriptional activity" could be artifact and I haven't had a "protein-centric" view of a gene since I learned about tRNA and ribosomal RNA genes as an undergraduate in 1967. Even if the ENCODE results are correct my preferred definition of a gene is not threatened. So, what's the fuss all about?

Regulatory Sequences
Gerstein et al. are worried because many definitions of a gene include regulatory sequences. Their results suggest that many genes have multiple large regions that control transcription and these may be located at some distance from the transcription start site. This isn't a problem if regulatory sequences are not part of the gene, as in the definition quoted above (a gene is a transcribed region). As a mater of fact, the fuzziness of control regions is one reason why most modern definitions of a gene don't include them.
Overlapping Genes
According to Gerstein et al.
As genes, mRNAs, and eventually complete genomes were sequenced, the simple operon model turned out to be applicable only to genes of prokaryotes and their phages. Eukaryotes were different in many respects, including genetic organization and information flow. The model of genes as hereditary units that are nonoverlapping and continuous was shown to be incorrect by the precise mapping of the coding sequences of genes. In fact, some genes have been found to overlap one another, sharing the same DNA sequence in a different reading frame or on the opposite strand. The discontinuous structure of genes potentially allows one gene to be completely contained inside another one’s intron, or one gene to overlap with another on the same strand without sharing any exons or regulatory elements.
We've known about overlapping genes ever since the sequences of the first bacterial operons and the first phage genomes were published. We've known about all the other problems for 20 years. There's nothing new here. No definition of a gene is perfect—all of them have exceptions that are difficult to squeeze into a one-size-fits-all definition of a gene. The problem with the ENCODE data is not that they've just discovered overlapping genes, it's that their data suggests that overlapping genes in the human genome are more the rule than the exception. We need more information before accepting this conclusion and redefining the concept of a gene based on analysis of the human genome.
Splicing was discovered in 1977 (Berget et al. 1977; Chow et al. 1977; Gelinas and Roberts 1977). It soon became clear that the gene was not a simple unit of heredity or function, but rather a series of exons, coding for, in some cases, discrete protein domains, and separated by long noncoding stretches called introns. With alternative splicing, one genetic locus could code for multiple different mRNA transcripts. This discovery complicated the concept of the gene radically.
Perhaps back in 1978 the discovery of splicing prompted a re-evaluation of the concept of a gene. That was almost 30 years ago and we've moved on. Now, many of us think of a gene as a region of DNA that's transcribed and this includes exons and introns. In fact, the modern definition doesn't have anything to do with proteins.

Alternative splicing does present a problem if you want a rigorous definition with no fuzziness. But biology isn't like that. It's messy and you can't get rid of fuzziness. I think of a gene as the region of DNA that includes the longest transcript. Genes can produce multiple protein products by alternative splicing. (The fact that the definition above says "a" functional product shouldn't mislead anyone. That was not meant to exclude multiple products.)

The real problem here is that the ENCODE project predicts that alternative splicing is abundant and complex. They claim to have discovered many examples of splice variants that include exons from adjacent genes as shown in the figure from their paper. Each of the lines below the genome represents a different kind of transcript. You can see that there are many transcripts that include exons from "gene 1" and "gene 2" and another that include exons from "gene 1" and "gene 4." The combinations and permutations are extraordinarily complex.

If this represents the true picture of gene expression in the human genome, then it would require a radical rethinking of what we know about molecular biology and evolution. On the other hand, if it's mostly artifact then there's no revolution under way. The issue has been fought out in the scientific literature over the past 20 years and it hasn't been resolved to anyone's satisfaction. As far as I'm concerned the data overwhelmingly suggests that very little of that complexity is real. Alternative splicing exists but not the kind of alternative splicing shown in the figure. In my opinion, that kind of complexity is mostly an artifact due to spurious transcription and splicing errors.
Trans-splicing refers to a phenomenon where the transcript from one part of the genome is attached to the transcript from another part of the genome. The phenomenon has been known for over 20 years—it's especially common in C. elegans. It's another exception to the rule. No simple definition of a gene can handle it.
Parasitic and mobile genes
This refers mostly to transposons. Gerstein et al say, "Transposons have altered our view of the gene by demonstrating that a gene is not fixed in its location." This isn't true. Nobody has claimed that the location of genes is fixed.
The large amount of "junk DNA" under selection
If a large amount of what we now think of as junk DNA turns out to be transcribed to produce functional RNA (or proteins) then that will be a genuine surprise to some of us. It won't change the definition of a gene as far as I can see.
The paper goes on for many more pages but the essential points are covered above. What's the bottom line? The new definition of an ENCODE gene is:
There are three aspects to the definition that we will list below, before providing the succinct definition:
  1. A gene is a genomic sequence (DNA or RNA) directly encoding functional product molecules, either RNA or protein.
  2. In the case that there are several functional products sharing overlapping regions, one takes the union of all overlapping genomic sequences coding for them.
  3. This union must be coherent—i.e., done separately for final protein and RNA products—but does not require that all products necessarily share a common subsequence.
This can be concisely summarized as:
The gene is a union of genomic sequences encoding a coherent set of potentially overlapping functional products.
On the surface this doesn't seem to be much different from the definition of a gene as a transcribed region but there are subtle differences. The authors describe how their new definition works using a hypothetical example.

How the proposed definition of the gene can be applied to a sample case. A genomic region produces three primary transcripts. After alternative splicing, products of two of these encode five protein products, while the third encodes for a noncoding RNA (ncRNA) product. The protein products are encoded by three clusters of DNA sequence segments (A, B, and C; D; and E). In the case of the three-segment cluster (A, B, C), each DNA sequence segment is shared by at least two of the products. Two primary transcripts share a 5' untranslated region, but their translated regions D and E do not overlap. There is also one noncoding RNA product, and because its sequence is of RNA, not protein, the fact that it shares its genomic sequences (X and Y) with the protein-coding genomic segments A and E does not make it a co-product of these protein-coding genes. In summary, there are four genes in this region, and they are the sets of sequences shown inside the orange dashed lines: Gene 1 consists of the sequence segments A, B, and C; gene 2 consists of D; gene 3 of E; and gene 4 of X and Y. In the diagram, for clarity, the exonic and protein sequences A and E have been lined up vertically, so the dashed lines for the spliced transcripts and functional products indicate connectivity between the proteins sequences (ovals) and RNA sequences (boxes). (Solid boxes on transcripts) Untranslated sequences, (open boxes) translated sequences.
This isn't much different from my preferred definition except that I would have called the region containing exons C and D a single gene with two different protein products. Gerstein et al (2007) split it into two different genes.

The bottom line is that in spite of all the rhetoric the "new" definition of a gene isn't much different from the old one that some of us have been using for a couple of decades. It's different from some old definitions that other scientists still prefer but this isn't revolutionary. That discussion has already been going on since 1980.

Let me close by making one further point. The "data" produced by the ENCODE consortium is intriguing but it would be a big mistake to conclude that everything they say is a proven fact. Skepticism about the relevance of those extra transcripts is quite justified as is skepticism about the frequency of alternative splicing.

Gerstein, M.B., Bruce, C., Rozowsky, J.S., Zheng, D., Du, J., Korbel, J.O., Emanuelsson, O., Zhang, Z.D., Weissman, S. and Snyder, M. (2007) What is a gene, post-ENCODE? History and updated definition. Genome Res. 17:669-681.

The ENCODE Project Consortium (2007) Nature 447:799-816. [PDF]

[Hat Tip: Michael White at Adaptive Complexity]

Monday, June 18, 2007

Gene Genie #9

The latest issue of the carnival Gene Genie has just been posted on DNAdirect talk.

Skepticism About "Out-of-Africa"

Alan R. Templeton has long been a critic of those who would over-interpret the genetic data on human origins. He's not alone. There are a surprisingly large number of biologists who refuse to jump on the "Out-of-Africa" bandwagon. This group does not get the same amount of publicity as the advocates of a recent (<100,000 years) wave of migration out of Africa. I think it's because skepticism of a new theory is seen as sour grapes. That, plus the fact that it's hard to publish criticisms of work that's already in the scientific literature.

An upcoming issue of the journal Evolution will contain a review by Templeton on human origins and the Out-of-Africa theory. Right now it's only available online [GENETICS AND RECENT HUMAN EVOLUTION]. Here's the abstract,
Starting with "mitochondrial Eve" in 1987, genetics has played an increasingly important role in studies of the last two million years of human evolution. It initially appeared that genetic data resolved the basic models of recent human evolution in favor of the "out-of-Africa replacement" hypothesis in which anatomically modern humans evolved in Africa about 150,000 years ago, started to spread throughout the world about 100,000 years ago, and subsequently drove to complete genetic extinction (replacement) all other human populations in Eurasia. Unfortunately, many of the genetic studies on recent human evolution have suffered from scientific flaws, including misrepresenting the models of recent human evolution, focusing upon hypothesis compatibility rather than hypothesis testing, committing the ecological fallacy, and failing to consider a broader array of alternative hypotheses. Once these flaws are corrected, there is actually little genetic support for the out-of-Africa replacement hypothesis. Indeed, when genetic data are used in a hypothesis-testing framework, the out-of-Africa replacement hypothesis is strongly rejected. The model of recent human evolution that emerges from a statistical hypothesis-testing framework does not correspond to any of the traditional models of human evolution, but it is compatible with fossil and archaeological data. These studies also reveal that any one gene or DNA region captures only a small part of human evolutionary history, so multilocus studies are essential. As more and more loci became available, genetics will undoubtedly offer additional insights and resolutions of human evolution.

[Hat Tip: Gene Expression]

Skepticism About Evo-Devo

The May issue of Evolution contains an article by Hoekstra and Coyne on Evolutionary-Developmental Biology or Evo-Devo. There are many evolutionary biologists who have serious doubts about the claims of evo-devo but these doubts don't often make it into the scientific literature because it's very hard to publish critiques. The Hoekstra and Coyne (2007) article is a welcome contribution to the debate.

Here's the abstract,
An important tenet of evolutionary developmental biology (“evo devo”) is that adaptive mutations affecting morphology are more likely to occur in the cis-regulatory regions than in the protein-coding regions of genes. This argument rests on two claims: (1) the modular nature of cis-regulatory elements largely frees them from deleterious pleiotropic effects, and (2) a growing body of empirical evidence appears to support the predominant role of gene regulatory change in adaptation, especially morphological adaptation. Here we discuss and critique these assertions. We first show that there is no theoretical or empirical basis for the evo devo contention that adaptations involving morphology evolve by genetic mechanisms different from those involving physiology and other traits. In addition, some forms of protein evolution can avoid the negative consequences of pleiotropy, most notably via gene duplication. In light of evo devo claims, we then examine the substantial data on the genetic basis of adaptation from both genome-wide surveys and single-locus studies. Genomic studies lend little support to the cis-regulatory theory: many of these have detected adaptation in protein-coding regions, including transcription factors, whereas few have examined regulatory regions. Turning to single-locus studies, we note that the most widely cited examples of adaptive cis-regulatory mutations focus on trait loss rather than gain, and none have yet pinpointed an evolved regulatory site. In contrast, there are many studies that have both identified structural mutations and functionally verified their contribution to adaptation and speciation. Neither the theoretical arguments nor the data from nature, then, support the claim for a predominance of cis-regulatory mutations in evolution. Although this claim may be true, it is at best premature. Adaptation and speciation probably proceed through a combination of cis-regulatory and structural mutations, with a substantial contribution of the latter.

Hoekstra, Hopi, E. and Coyne, Jerry, A. (2007) THE LOCUS OF EVOLUTION: EVO DEVO AND THE GENETICS OF ADAPTATION. Evolution 65:995–1016.

Monday's Molecule #31

Today's molecule is complicated but it makes a lot of sense if you know your basic biochemistry. We don't need a long complicated name this time. It's sufficient to simply describe what you're looking at and why it's significant. You have to identify the key residue to get credit for the answer.

As usual, there's a connection between Monday's molecule and this Wednesday's Nobel Laureate(s). This one is an obvious direct connection. Once you have identified the molecule you should be able to name the Nobel Laureate(s).

The reward (free lunch) goes to the person who correctly identifies the molecule and the reaction and the Nobel Laureate(s). Previous free lunch winners are ineligible for one month from the time they first collected the prize. There are no ineligible candidates for this Wednesday's reward since recent winners (including last week's winner, "Kyo") have declined the prize on the grounds that they live in another country and can't make it for lunch on Thursday (a feeble excuse, in my opinion, haven't you heard of airplanes?).

Comments will be blocked for 24 hours. Comments are now open.

UPDATE: The molecule is the lariat structure of the RNA splicing intermediate. The key residue is the adenylate residue that's joined through its 2′ hydroxyl group to the 5′ end of the intron [see RNA Splicing: Introns and Exons]. The Nobel Laureates are Rich Roberts and Phil Sharp. (See the comments for an interesting anecdote concerning the discovery of this molecule.)

Saturday, June 16, 2007

Cellular Respiration Ninja Enzymes

Here's a student video presentation of glycolysis and respiration. It's much better than most [e.g., An Example of High School Biochemistry]. However, there are two errors in the video. The first one is fairly serious. The second one is less serious but it's something we cover in my class and it helps illustrate a fundamental concept about how certain reactions work. The second error was very common in most biochemistry textbooks in the past but it's been eliminated from the majority of 21st century textbooks. Can you spot both errors?

[Hat Tip: Greg Laden]
[Hint: What are the products produced by glycolysis and by the Krebs cycle?]

Friday, June 15, 2007

Penicillin Resistance in Bacteria: After 1960

The widespread appearance of penicillin-resistant bacteria by 1960 prompted the introduction of new drugs that could not be degraded by newly evolved β-lactamases [see Penicillin Resistance in Bacteria: Before 1960].

The most important of these new drugs are the cephalosporins, modified β-lactams with bulky side chains at two different positions. These drugs still inhibit the transpeptidases and prevent cell wall formation but because of the bulky side chains they cannot be hydrolyzed by β-lactamases. Thus, they are effective against most of the penicillin-resistant strains that arose before 1960.

Other drugs, such as methicillin, were modified penicillins. They also had modified side chains that prevented degradation by the β-lactamases.

It wasn't long before cephalosporin- and methicillin-resistant strains began to appear in hospitals. As a general rule, these strains were not completely resistant to high doses of the new class of drugs but as time went on the resistant strains became more and more immune to the drugs.

The new version of drug resistance also involves the transpeptidase target but instead of developing into β-lactamases they evolve into enzymes that can no longer bind the cephalosporins. Usually the development of resistance takes place in several stages.

There are many different transpeptidases in most species of bacteria. The are usually referred to as penicillin-binding proteins or PBP's. Often the first sign of non-lactamase drug resistance is a mutant version of one PDP (e.g., PDP1a) and subsequent development of greater resistance requires the evolution of other PDB's that don't bind the drug. In the most resistant strains there will be one particular PDB (e.g., PDB2a) that is still active at high drug concentrations while the other transpeptidases will be inhibited.

Resistant enzymes have multiple mutations, which explains the slow, stepwise acquisition of drug resistance. An example is shown in the figure. This is PDP1a from Streptococcus pneumoniae (Contreras, et al. 2006) and the mutant amino acids are displayed as gold spheres. Most of the mutations do not affect the binding of the drug but those surrounding the entry to the active site are crucial. The necessary amino acid substitutions are numbered in the figure. You can see that they line the groove where the cephalosporin drug (purple) is bound. The effect of the mutations is to prevent the bulky β-lactam from inhibiting the enzyme. This is a very different form of drug resistance than the evolution of degradation enzymes that characterized the first stage of penicillin resistant bacteria.

Chambers, H.F. (2003) Solving staphylococcal resistance to beta-lactams. Trends Microbiol. 11:145-148.

Contreras-Martel, C., Job, V., Di Guilmi, A.M., Vernet, T., Dideberg, O. and Dessen, A. (2006) Crystal structure of penicillin-binding protein 1a (PBP1a) reveals a mutational hotspot implicated in beta-lactam resistance in Streptococcus pneumoniae. J. Mol. Biol. 355:684-696.

Livermore, D.M. (2000) Antibiotic resistance in staphylococci. Int. J. Antimicrob. Agents 16:s3-s10.

Penicillin Resistance in Bacteria: Before 1960

The Nobel Prize for the discovery and analysis of penicillin was awarded in 1945 [Nobel Laureates: Sir Alexander Fleming, Ernst Boris Chain, Sir Howard Walter Florey]. It was about this time that penicillin became widely available in Europe and North America.

By 1946 6% of Staphylococcus aureus strains were resistant to penicillin. Resistance in other species of bacteria was also detected in the 1940's. By 1960 up to 60% of Staphylococcus aureus strains were resistant with similar levels of resistance reported in other clinically relevant strains causing a wide variety of diseases (Livermore, 2000).

Penicillins are a class of antibiotics with a core structure called a β-lactam. The different types of penicillin have different R groups on one end of the core structure. A typical examples of a penicillin is penicillin G [Monday's Molecule #30]. Others common derivatives are ampicillin and amoxicillin.

The original resistance to this entire class of drugs was caused mostly by the evolution of bacterial enzymes that could degrade them before they could block cell wall synthesis. (Recall that bacteria have cell walls and penicillin blocks cell wall synthesis [How Penicillin Works to Kill Bacteria].)
It seems strange that the evolution of penicillin resistance would require a totally new enzyme for degrading the drug. Where did this enzyme come from? And how did it arise so quickly in so many different species?

The degrading enzyme is called penicillinase, β-lactamase, or oxacillinase. They all refer to the same class of enzyme that binds penicillins and then cleaves the β-lactam unit releasing fragments that are inactive. The enzymes are related to the cell wall transpeptidase that is the target of the drug. The inhibition of the transpeptidase is effective because penicillin resembles the natural substrate of the reaction: the dipeptide, D-alanine-D-alanine.

In the normal reaction, D-Ala-D-Ala binds to the enzyme and the peptide bond is cleaved causing release one of the D-Ala residues. The other one, which is part of the cell wall peptidoglycan, remains bound to the enzyme. In the second part of the reaction, the peptidoglycan product is transferred from the enzyme to a cell wall crosslinking molecule. This frees the enzyme for further reactions (see How Penicillin Works to Kill Bacteria for more information).

Penicillin binds to the peptidase as well and the β-lactam bond is cleaved resulting in the covalent attachment of the drug to the enzyme. However, unlike the normal substrate, the drug moiety cannot be released from the transpeptidase so the enzyme is permanently inactivated. This leads to disruption of cell wall synthesis and death.

Resistant strains have acquired mutations in the transpeptidase gene that allow the release of the cleaved drug. Thus, the mutant enzyme acts like a β-lactamase by binding penicillins, cleaving them, and releasing the products. Although the β-lactamases evolved from the transpeptidase target enzymes, the sequence similarity between them is often quite low in any given species. This is one of the cases where structural similarity reveals the common ancestry [see the SCOP Family beta-Lactamase/D-ala carboxypeptidase]. It's clear that several different β-lactamases have evolved independently but, in many cases, a particular species of bacteria seems to have licked picked up a β-lactamase gene by horizontal transfer from another species. The transfer can be mediated by bacteriophage or plasmids.

Livermore, D.M. (2000) Antibiotic resistance in staphylococci. Int. J. Antimicrob. Agents 16:s3-s10.