DDT Blocks the Voltage-Gated Sodium Channel

 
Monday's Molecule #16 is 1,1,1-trichloro-2,2 bis(p-chlorophenyl) ethane, better known as DDT. DDT is a powerful insecticide. It binds to the voltage-gated sodium channel and locks it in the open state. Prolonged influx of sodium ions causes the nerves to fire repeatedly and this causes death of the insect.

The reason DDT is so powerful is due to its specificity. It binds to insect channel proteins but not to those of other animals (or plants, fungi, protists, and bacteria). Thus, it is an effective insecticide used to fight malaria and other insect borne diseases.

Unfortunately, even though DDT is not immediatly toxic to other animals it does have one disadvantage: it is extremely stable—its biological half-life is about eight years. Furthermore, DDT is stored in fatty tissues and its buildup in birds and fish resulted in considerable loss of these species. That, coupled with the evolution of DDT resistant insects, led to a ban of DDT in most countries by the 1970's.

Rachel Carson is largely credited with launching the environmental movement in 1962 with the publication of Silent Spring. The title refers to a world without birds. While I was writing this up I did a quick survey of the graduate students in the nearby labs and none of them had ever heard of Rachel Carson. Not only that, neither had several of my colleagues. I feel old.

One of the main targets of Silent Spring was DDT. By the time the book was published it was estimated that DDT had saved the lives of millions of people through prevention of malaria and thyphoid but it's effectiveness was much diminished. That's why the ban was not as controversial as it might have been.

Let's look at the biochemistry of DDT. We have already learned about the simple voltage-gated potassium channel. The Na⁺ (sodium) channel is closely related to the K⁺ channel protein. Recall that the K⁺ channel consists of four identical subunits surrounding a central hole through which K⁺ ions enter the cell.

The Na⁺ channel protein is much larger than the K⁺ channel subunit because it consists of four of the smaller subunits fused into a single polypeptide chain. The tolopology of the Na⁺ channel protein is shown below.

Each of the domains (I-IV) corresponds to a single subunit of the K⁺ channel. Like the K⁺ channel, the four domains of the Na⁺ channel protein are arranged around a central tunnel through which sodium ions enter the cell. The S5 and S6 helices line the tunnel.

The toplogy diagram above shows the locations of mutations conferring resistance to DDT and similar drugs. Each one represents mutants identified in resistant houseflies, fruit flies, mosquitos, or moths. The important mutations are substitutions at the 932 position normally occupied by leucine (L932) and at the 929 position normally occupied by threonine (T929) (blue dots). For example, the substition of isoleucine for threonine at 929 (T929I) confers almost complete resistance to DDT.

Incidently, the methionine at 918 (M918) is what confers sensitivity to DDT in the insect voltage-gated Na⁺ channel. Other animlas have a different amino acid at this position and they are not sensitive to DDT.

O'Reilly et al. (2006) modeled the structure of the Na⁺ channel using the known structures of the K⁺ channel proteins. This is necessary because the Na⁺ channel has not been crystallized. It's an excellent way to get a structure when you know that two proteins are homologous (descended from a common ancestor).

From the model, the authors were able to focus on the probable site of DDT binding based on the known mutations to resistance. In this case, they looked at the interface between helix S5 in Domain II and nearby helices S6 from Domain II (IIS6) and S6 from Domain III (IIIS6), which packs against IIS5 in the structure. They tried docking various insecticides in this region and came up with a good fit in all cases. The DDT binding site is shown below.

Note that the side chains of T929 and L932 interact directly with DDT. These are the sites of mutations to high levels of resistance. It looks like changes to these amino acids prevent binding of DDT and that's the basis of resistance.

The S4-S5 linker helix is shown in yellow in this figure. Recall that this is the helix that responds to membrane potential by reorienting to a more vertical position. This, in turn, shifts the S5 and S6 helices to more vertical postions and closes the channel. In the presence of DDT the S5 and S6 helices are effectively cross-linked and they cannot shift to a position where they move closer together. This prevents closing of the channel. DDT locks the channel in the open conformation leading to a continual influx of Na⁺, uncontrolled firing of the nerve, and eventual death.

O'Reilly, Andrias O., Khambay, Bhupinder P. S., Williamson, Martin S., Field, Linda M., Wallace, B. A., and Davies, T. G. Emyr (2006) Modelling insecticide-binding sites in the voltage-gated sodium channel. Biochem. J. (2006) 396:255–263.

Martin Rees Explains Accommodationism

 
Martin Rees is the President of the Royal Society in the UK. This is a position of enormous influence. When Rees speaks you can assume that he is representing the position of the Royal Society, or at least it's leaders.

Martin Rees was recently interviewed by The Independent [Martin Rees: 'We shouldn't attach any weight to what Hawking says about god'].

He is equally scathing about Hawking's more recent comments about there being no need for God in order to explain creation. "Stephen Hawking is a remarkable person whom I've know for 40 years and for that reason any oracular statement he makes gets exaggerated publicity. I know Stephen Hawking well enough to know that he has read very little philosophy and even less theology, so I don't think we should attach any weight to his views on this topic," he said.

Unlike many of the Fellows of the Royal Society he has presided over in the past five years, Lord Rees is not a militant atheist who goes out of his way to insult people of belief – Richard Dawkins once called him "a compliant quisling" for his tolerance of religion.

"I would support peaceful co-existence between religion and science because they concern different domains," Lord Rees said. "Anyone who takes theology seriously knows that it's not a matter of using it to explain things that scientists are mystified by."

His next popular science book is about these things that science still cannot explain, such as the origin of life on Earth and the scientific nature of human consciousness. This, he insisted, is what science is really about, and why it has the power to touch everyone of every culture.

I don't have time for a detailed explanation of this particular accommodationist position so I'll just note a few points.

• Hawking said there's no need for God but his views can be dismissed because (unlike Martin Rees?) he's not an expert on philosophy and theology.


• Rees does not go out of his way to insult people of belief. Good for him. Neither do lots of atheists, including Stephen Hawking. What's the point? Sounds to me like Dawkins might have been correct.


• Did Martin Rees just go out of his way to insult atheists like Stephen Hawking? I guess atheists don't deserve the same kid-gloves treatment that we owe to theists.


• Religion and science concern different domains. So they do. Religion is firmly planted in the domain of mythology and superstition. What does that prove?


• Martin Rees is an astronomer. He's writing a book about the origin of life and human consciousness. I wonder if we should bother paying any attention to this book since he's not an expert in biology?

---

[Photo Credit: BBC]

The Three Domain Hypothesis (part 4)

[Part 1][Part 2][Part 3]
Ludwig and Schleifer question the reliability of the SSU tree. They begin by comparing trees constructed from the small ribosomal RNA subunit (SSU) and the large ribosomal RNA subunit (LSU). The example they use is 18 species of Enterococcus and they show that there are significant differences between the two trees. Surprisingly, they dismiss these differences as “minor local differences.” These authors are convinced that “SSU and LSU rRNA genes fulfill the requirements of ideal phylogenetic markers to an extent far greater than do protein coding genes.”

In spite of this bias, they compiled a database of protein trees from conserved genes that are found in all three of the proposed Domains. According to them, the Three Domain Hypothesis is supported by EF-Tu, the large subunits of RNA polymerase, Hsp60, and some aminoacyl-tRNA synthetases (aspartyl, leucyl, tryptophanyl, and tyrosyl).

The Three Domain Hypothesis is refuted by ATPase, DNA gyrase A, DNA gyrase B, Hsp70, RecA, and some aminoacyl-tRNA synthetases. Note the inclusion of ATPase in this list. The phylogeny of ATPase was one of the strongest bits of evidence for the Three Domain Hypothesis back in 1989 but further work has shown that these genes (proteins) now refute the hypothesis.

My own favorite is the HSP70 gene family, arguably the most highly conserved gene in all of biology and therefore an excellent candidate for studies of deep phylogeny. Hsp70 is the main chaperone in all species. It is responsible for the correct folding of proteins as they are synthesized. It forms a complex with DnaJ and GrpE in bacteria and similar proteins in eukaryotes. The complex associates with the translation machinery (ribomes etc.) during protein synthesis.

The conflict between trees constructed with HSP70 and the ribosomal RNA trees has been known for a long time. The actual pattern of the HSP70 tree can be interpreted in two different ways depending on where you place the root [see 1995] but neither one agrees with the Three Domain Hypothesis.

Here’s an example of an HSP70 tree that I just created using the latest sequences. It’s fairly typical of the trees that do not support the Three Domain Hypothesis. Eukaryotes cluster as a monophyletic group (lower left) and all prokaryotes form another distinct clade. The archaebacteria sequences (black dots) do not form a single clade, let alone a “domain.” Instead, they tend to be dispersed among the other bacterial groups.


Note that this tree, like many others, shows numerous short branches at the bottom of the bacteria tree suggesting that the diversity among bacteria is ancient. Phillippe and Forterre (1999) were among the first to document the serious differences between conserved protein trees and rRNA trees in “The Rooting of the Universal Tree of Life Is Not Reliable” (J. Mol. Evol. 49:509-523). It’s worth quoting their abstract in order to emphasize the controversy since Ludwig and Schleifer don’t do a very good job.

Several composite universal trees connected by an ancestral gene duplication have been used to root the universal tree of life. In all cases, this root turned out to be in the eubacterial branch. However, the validity of results obtained from comparative sequence analysis has recently been questioned, in particular, in the case of ancient phylogenies. For example, it has been shown that several eukaryotic groups are misplaced in ribosomal RNA or elongation factor trees because of unequal rates of evolution and mutational saturation. Furthermore, the addition of new sequences to data sets has often turned apparently reasonable phylogenies into confused ones. We have thus revisited all composite protein trees that have been used to root the universal tree of life up to now (elongation factors, ATPases, tRNA synthetases, carbamoyl phosphate synthetases, signal recognition particle proteins) with updated data sets. In general, the two prokaryotic domains were not monophyletic with several aberrant groupings at different levels of the tree. Furthermore, the respective phylogenies contradicted each others, so that various ad hoc scenarios (paralogy or lateral gene transfer) must be proposed in order to obtain the traditional Archaebacteria-Eukaryota sisterhood. More importantly, all of the markers are heavily saturated with respect to amino acid substitutions. As phylogenies inferred from saturated data sets are extremely sensitive to differences in evolutionary rates, present phylogenies used to root the universal tree of life could be biased by the phenomenon of long branch attraction. Since the eubacterial branch was always the longest one, the eubacterial rooting could be explained by an attraction between this branch and the long branch of the outgroup. Finally, we suggested that an eukaryotic rooting could be a more fruitful working hypothesis, as it provides, for example, a simple explanation to the high genetic similarity of Archaebacteria and Eubacteria inferred from complete genome analysis.

The problem is obvious. All trees, RNA and protein, have potential problems of saturation and long branch attraction. Although Ludwig and Schleifer argue in favor of the ribosomal RNA tree, there is still serious debate over which sequences are revealing the “true” phylogeny. Are there good reasons for rejecting those trees that refute the Three Domain Hypothesis as it's supporters maintain?

---

Microbobial Phylogeny and Evolution: Concepts and Controversies Jan Sapp, ed., Oxford University Press, Oxford UK (2005)

Jan Sapp The Bacterium’s Place in Nature

Norman Pace The Large-Scale Structure of the Tree of Life.

Woflgang Ludwig and Karl-Heinz Schleifer The Molecular Phylogeny of Bacteria Based on Conserved Genes.

Carl Woese Evolving Biological Organization.

W. Ford Doolittle If the Tree of Life Fell, Would it Make a Sound?.

William Martin Woe Is the Tree of Life.

Radhey Gupta Molecular Sequences and the Early History of Life.

C. G. Kurland Paradigm Lost.

---

What Should We Teach About the "Tree of Life"?

As most of you already know, I think the Three Domain Hypothesis is dead. The history of life is better explained as a net with rampant transfer of genes between species [The Web of Life]. This idea has been widely promoted by Ford Doolittle.

The debate over the tree of life has implications concerning the distinction between "prokaryote" and "eukaryote." I was checking some recent papers and came across one by Doolittle and Zhaxybayeva (2013) that seems particularly relevant. They discuss the evidence for and against the division of life into three domains and the attempt by Norm Pace to ban the word "prokaryote."

The authors point out, once again, that eukaryotic genes are most closely related to genes from cyanobacteria, proteobacteria, and archaebacteria, in that order. The majority, by far, have their closest homologs in bacteria, not archaebacteria. The most likely explanation is that euakryotes are chimeras resulting from fusion of an archaebacterium and a eubacterium plus genes transferred from mitochondria and chloroplast to the nuclear genome.

The Three Domain Hypothesis (part 3)

The scientific dispute over The Three Domain Hypothesis is based on the validity of RNA trees, the importance of protein trees that disagree with the rRNA tree, the evidence for fusions, and the frequency of Lateral Gene Transfer (LGT). But, as usual, there’s more to it than just science. The side with the best advocates has a huge advantage in fights like this.

Let's set the stage by quoting from the article by William Martin.

Thus, it seems to me that there is a schisma abrew in cell evolution, with the rRNA tree and proponents of its infallibility on the one side and other forms of evidence, proponents of LGT, or proponents of a symbiotic origin of eukaryotes on the other. The former camp is well organized behind a unified view (be it right or wrong, still a view) and is arguing that we already have the answers to microbial evolution. The latter camp is not organized into castes of recognized leadership and followers, meaning that (if we are lucky) concepts and their merits, not position or power, will determine the outcome of the battle as to what ideas might or might not be worthwhile entertaining as a working hypothesis for the purpose of further scientific endeavour.

The article by Norman Pace represents the side that already has the answers. He is a strong proponent of the Three Domain Hypothesis. These days, the main thrust of his argument is that we should all jump on the bandwagon or risk being left behind. I heard him speak in San Francisco last April and he sounded more like a preacher than a scientist. His article in Nature, ”Time for a Change”, is an example of the way the Three Domain Hypothesis proponents have been arguing for 20 years.

One of the key problems in deep phylogeny is choosing the right gene. Pace argues in favor of ribosomal RNA—not a surprise since he has invested over 20 years in this molecule. Ideally, what kind of gene do we want to examine in order to determine the deepest branches in the tree of life? According to Pace there are three criteria ....

1. The gene must be universal.
2. The gene must have resisted lateral gene transfer.
3. The gene must be large enough to provide useful phylogenetic information.

Only ribosomal RNA meets all three criteria, says Pace.

There’s no question about #1. Ribosomal RNA genes are fond in all species. There are very few other genes that meet this criterion. Almost all other candidates are absent in at least a few species. Ribosomal RNA satisfies #3 as well. Even the small subunit is large enough.

What about #2? Which genes have “resisted” lateral gene transfer? You can’t just declare by fiat that ribosomal RNA genes haven’t been transferred. It’s a debatable question as we’ll see later on.

I would add three other criteria.

4. The gene must be unique, or if it isn’t, paralogues must be easily recognized.
5. The gene must encode a protein because it’s much more accurate to analyze amino acid sequences than nucleic acid sequences. (And easier to align.)
6. The gene must be highly conserved in order to retain significant sequence similarity at the deepest levels.

Ribosomal RNA doesn’t do so well when we add these criteria. Most bacterial genomes have multiple copies of ribosomal RNA genes. They are usually 99% similar but there are known examples of more divergent paralogues. This is not likely to be a serious problem for deep phylogeny, but it has caused problems at the species level.

Ribosomal RNA does not encode protein. That’s a serious problem that Pace never addresses.

Ribosomal RNA genes are well conserved but not as highly conserved as some others. This is why rRNA can be used to distinguish closely related species whereas the sequences of other genes are identical unless the species diverged more than 10-20 million years ago. Part of the problem with using rRNA sequences in deep phylogeny is that they are too divergent.

Having declared that ribosomal RNA genes are the best choice, Pace then goes on to show us the “true”universal tree of life. As you can see, it is divided into three distinct clusters separated by long branches. The clades represent Bacteria, Archaea, and Eukaryotes; the Three Domains. The prokarotes (Bacteria and Archaea) seem to associate and the eukaryotes seem to be more distantly related.

But first impressions can be misleading. Pace puts the root on the branch leading to bacteria and not on the long branch leading to Eukaryotes. This root is based entirely on two old 1989 papers, which he references. Both of these papers have been refuted, but that’s not something you would learn from reading Pace’s article. (There are other, more recent, experiments that root the tree on the bacterial branch and these should have been used. The fact that they weren’t reflects Pace’s degree of critical thinking on this problem. )

To many of us, the large scale structure of the tree of life just doesn’t look right. The long branches leading from the trifurcation point to Bacteria and Eukaryotes smack of artifact. The branching within each of the domains looks too simple. It’s part of the reason why there’s skepticism about the rRNA tree, as we’ll see.

The rest of the article is a passionate defense of the importance of bacteria. I agree with him, for the most part, and so do lots of evolutionary biologists. Bacteria are much more important than eukaryotes! :-)

Pace contributes very little to the debate since he is not willing to entertain any doubts about the Three Domain Hypothesis. For that we have to look at some other papers.

---

Microbobial Phylogeny and Evolution: Concepts and Controversies Jan Sapp, ed., Oxford University Press, Oxford UK (2005)

Jan Sapp The Bacterium’s Place in Nature

Norman Pace The Large-Scale Structure of the Tree of Life.

Woflgang Ludwig and Karl-Heinz Schleifer The Molecular Phylogeny of Bacteria Based on Conserved Genes.

Carl Woese Evolving Biological Organization.

W. Ford Doolittle If the Tree of Life Fell, Would it Make a Sound?.

William Martin Woe Is the Tree of Life.

Radhey Gupta Molecular Sequences and the Early History of Life.

C. G. Kurland Paradigm Lost.

---

John Mattick still claims that most lncRNAs are functional

Most of the human genome is transcribed at some time or another in some tissue or another. The phenomenon is now known as pervasive transcription. Scientists have known about it for almost half a century.

At first the phenomenon seemed really puzzling since it was known that coding regions accounted for less than 1% of the genome and genetic load arguments suggested that only a small percentage of the genome could be functional. It was also known that more than half the genome consists of repetitive sequences that we now know are bits and pieces of defective transposons. It seemed unlikely back then that transcripts of defective transposons could be functional.

Part of the problem was solved with the discovery of RNA processing, especially splicing. It soon became apparent (by the early 1980s) that a typical protein coding gene was stretched out over 37,000 bp of which only 1300 bp were coding region. The rest was introns and intron sequences appeared to be mostly junk.

How many genes do we have and what happened to the orphans?

How many genes in the human genome? There's only one correct answer to that question and that's "we don't know."

The main problem is counting the number of genes that produce functional RNA molecules. The latest Ensembl results are based on build CRch37 from February 2009 and the GENCODE annotation from last year (GENCODE 19) [see Human assembly and gene annotation and Harrow et al., 2014]

The most recent estimates are 20,807 protein-encoding genes, 9,096 genes for short RNAs, and 13,870 genes for long RNAs. This gives 43,773 genes. Nobody knows for sure how many of the putative genes for RNAs actually exist. They may only be a few thousand functional genes in this category.

It's a lot easier to figure out whether a gene really encodes a functional protein so most of the annotation effort is focused on those genes. I want to draw your attention to a recent paper by Ezkurdia et al. (2014) that discusses this issue. The authors begin with a bit of history ...

Science Doesn't Have All the Answers but Does It Have All the Questions?

Jerry Coyne has been following the debate between Steven Pinker and Leon Wieseltier on the topic of scientism [see The final round: Pinker vs. Wieseltier on scientism]. Jerry seems to agree with both Pinker and Wieseltier that there are "two magisteria" (science and humanities) ...

[Wieseltier] calls for a “two magisteria” solution, with science and humanities kept separate, but with “porous boundaries.” But that is exactly what Pinker called for, too! Wieseltier claims that Pinker and other advocates of scientism advocate “totalistic aspirations,” i.e., the complete takeover of humanities by the sciences (“unified field theories,” Wieseltier calls them), but Pinker explicitly said that he wasn’t calling for that.

...

As you can see above, Steve never argued that science is, or should be, supreme in all the contexts. Indeed, in his earlier piece he noted that art and literature, while they might be informed in some ways by science, nevertheless have benefits independent of science. To me, those benefits include affirming our common humanity, being moved by the plight of others, even if fictional, and luxuriating in the sheer beauty of music, words, or painting. (Note, though, that one day science might at least explain why we apprehend that beauty.)

I'm not sure how Pinker, Wieseltier, and Coyne are defining science but it's clear that they aren't using the same definition I use.

I think that science is a way of knowing based on evidence and logic and healthy skepticism. I think that all disciplines seeking knowledge use the scientific approach. This is the broad definition of science used by many philosophers and scientists.

Maarten Boudry discusses, and accepts, this definition in his chapter on "Loki's Wager and Lauden's Error" in Philosophy of Pseudescience: Reconsidering the Demarcation Problem. Boudry says that the distinction between the ways of knowing used by biologists, philosophers, and historians are meaningless and there's no easy way to distinguish them (territorial demarcation). On the other hand, there is a way to distinguish between good scientific reasoning and bad scientific reasoning like Holocaust denial.

I have expressed little confidence in the viability of the territorial demarcation problem, and even less interest in solving it. Not only is there no clear-cut way to disentangle epistemic domains like science and philosophy, but such a distinction carries little epistemic weight. The demarcation problem that deserves our attention is the one between science and pseudoscience (and the analogous ones between philosophy and pseudophilosophy and between history and pseudohistory).

Sven Ove Hanson is more specific because he actually defines "science in a broad sense" in a way that I have been using it for several decades. This is from his chapter on "Defining Pseudoscience and Science" in Philosophy of Pseudescience: Reconsidering the Demarcation Problem.

Unfortunately neither "science" nor any other established term in the English language covers all the disciplines that are parts of this community of knowledge disciplines. For lack of a better term, I will call them "science(s) in the broad sense." (The German word "Wissenschaft," the closest translation of "science" into that language, has this wider meaning; that is, it includes all the academic specialties, including the humanities. So does the Latin "scientia.") Science in a broad sense seeks knowledge about nature (natural science), about ourselves (psychology and medicine), about our societies (social science and history), about our physical constructions (technological science), and about our thought construction (linguistics, literary studies, mathematics, and philosophy). (Philosophy, of course, is a science in this broad sense of the word.)

If this is what we mean by science" then there's no difference between the ways we try to acquire knowledge in the humanities or the natural sciences and the debate between Pinker and Wieseltier takes on an entirely different meaning.

There aren't "two magisteria" but only one. Unless, of course, someone is willing to propose a successful non-scientific way of knowing. I have asked repeatedly for examples of knowledge ("truth") that have been successfully acquired by any other way of knowing. So far, nobody has come up with an answer so we can tentatively conclude that science (in the broad sense) is the only valid way of acquiring true knowledge.

Clearly we don't have all the answers to everything so it's clear that neither science nor anything else has all the answers. What about the questions? Are there any knowledge questions that science (in the broad sense) can't address? I don't think there are. I think "science" covers all the questions even though it doesn't (yet) have all the answers.

If this is "scientism" then I'm guilty. What is the alternative? Is it revelation (revealed truth)? Or is there some other way of knowing that I haven't heard about?

---

Is "Prokaryote" a Useful Term?

 
Coturnix (Bora Zivkovic) is the Online Community Manager at PLoS-ONE (Public Library of Science). Part of his job is to get people to post comments on the PLoS websites. [New in Science Publishing, etc.]

So when Bora suggested we get involved in a debate on "Is "prokaryotic" an outdated term?" I hopped on over to the PLoS website and read the comments. I discovered that you have to register on PLoS in order to comment so I went ahead and did that and posted a response to the question.

I don't like registering on websites, it's a painful process, especially in this case 'cause you have to answer a lot of questions. It took me about ten minutes to figure out what to do and to convince the program to let me register even though I didn't want to receive email spam from PLoS. I also had to make up a user ID—Larry_Moran, in this case—because, apparently your name isn't good enough. This is not a very open process.

Theme

The Three Domain Hypothesis
Anyway, the question is important. If you think the Three Domain Hypothesis is well established, then you believe there are two non-eukaryotic domains (Bacteria, Archaea). Furthermore, the eukaryotes cluster with the Archaea according to this hypothesis. Thus, the word "prokaryote" encompasses a paraphyletic group and becomes useless.

But we wouldn't be having this discussion if the Three Domain Hypothesis is incorrect. In that case, the root of the tree might well be a split between eukaryotes and prokaryotes. The point is that the discussion about usefulness of "prokaryote" is really a debate about the validity of the Three Domain Hypothesis and we shouldn't forget that. It's wrong to assume that your side has won that debate and then start to solidify your apparent victory by defining your opponent's point of view out of existence!

Theme: The Three Domain Hypothesis

 
This is a series of postings that describe the Three Domain Hypothesis. The Three Domain Hypothesis is the idea that life is divided into three domains—bacteria, archaebacteria, and eukaryotes—and that the archaebacteria and eukaryotes share a common ancestor. An example of this tree of life is shown on the Dept. of Energy (USA) Joint Genome Initiative website [JGI Microbial Genomes] (left).

The hypothesis was promoted by Carl Woese in the 1980's but the pure form has now been abandoned and replaced with a “net of life” concept of early evolution as shown in the figure below. This figure is taken from Ford Doolittle's Scientific American article "Uprooting the Tree of Life" (February 2000). © Scientific American




The Three Domain Hypothesis (part 1) (Nov. 17, 2006 )

The Three Domain Hypothesis (part 2) (Nov. 22, 2006)

The Three Domain Hypothesis (part 3) (Nov. 26, 2006)

The Three Domain Hypothesis (part 4) (Nov. 29, 2006)

The Three Domain Hypothesis (part 5) (Dec. 8, 2006)

The Three Domain Hypothesis (part 6) Carl Woese (Dec. 31, 2006)

Now the IDiots Don't Get Evolution (Feb. 14, 2007)

The Web of Life (March 15, 2007)

Is "Prokaryote" a Useful Term? (October 4, 2007)

Celebrating the Three Domain Hypothesis (October 18, 2007)

The Tree of Life (May 22, 2008)

Sequence Alignment (June 22, 2008)

On the Origin of Eukaryotes (December 27, 2008)

The Tree of Life (July 29, 2009)

Perspectives on the Tree of Life: Ford Doolittle (July 30, 2009)

Perspectives on the Tree of Life: Day One (July 31, 2009)

Perspectives on the Tree of Life: Day Two (August 1, 2009)

Perspectrives of the Tree of Life: Day Three (August 7, 2009)

Nobel Laureates: Gerald Edelman and Rodney Porter

 

The Nobel Prize in Physiology or Medicine 1972.

"for their discoveries concerning the chemical structure of antibodies"

Gerald M. Edelman (1929 - ) and Rodney R. Porter (1917 - 1985) received the Nobel Prize in Physiology or Medicine for elucidating the structure of immunoglobulins (antibodies). They determined that immunoglobulins were composed of two heavy chains and two light chains. There are three domains in the molecule. Two of them form binding sites for antigens and the third one links the two heavy chains together.

Edelman and Porter founded the field of molecular immunology, a field that today encompasses hundreds of labs. If you count all the clinical immunologists and cellular immunologists, there are as many immunology labs in the world as there are biochemistry labs. That was not true in the 1950's when Edelman and Porter began their work.

The presentation speech was in Swedish by Professor Sven Gard of the Karolinska Medico-Chirurgical Instit.

THEME:
Nobel Laureates

Your Royal Highnesses, Ladies and Gentlemen,

Immunebodies or antibodies is the designation of a group of proteins in the blood, that play an important part in the defense against infections and in the development of many different diseases. Their perhaps most characteristic property is the capacity to react and combine with substances, foreign to the organism, so-called antigens and to do so in a highly specific manner. There probably exist more than 50,000 different antibodies in the blood, each of them reactive against one particular antigen. Their main features are similar but they show individual characteristics and constitute, therefore, an extremely heterogeneous group. Since, in addition, they appear as very large molecules of a complex structure, it is understandable that the study of their chemistry for a long time offered great difficulties.

Up to 1959 the knowledge about their nature and mechanism of action was rather incomplete. That same year, however, Edelman and Porter separately and independently published their fundamental studies of the molecular structure of antibodies. Both of them had aimed at splitting the giant molecule into smaller, well defined fragments that might be more easily analysed than would the whole complex.

Porter's aim was to separate those parts of the antibody which are responsible for their specific reactivity. He hoped by this means to obtain a preparation lacking most of the biologic functions of the antibody but, on account of its capacity of combination, capable of competing with the antibody for the binding sites of the antigen. He succeeded in achieving this by means of treatment of the antibody, under strictly controlled conditions, with a protein-splitting enzyme called papain. By this treatment the antibody split into three parts. Two of these could combine specifically with the antigen and they were almost identical in other respects as well. The third fragment differed distinctly from the others, lacked binding capacity but possessed certain other biologic characteristics of the intact molecule.

Edelman for his part assumed the molecule, like those of many other proteins, to be composed of two or more separate chain structures held together by cross links of some kind, most probably so-called sulphide bonds. His assumption turned out to be correct. By means of a fairly rough treatment he was able to sever the cross bonds and release a number of separate chain molecules. Both he and Porter could later show that the antibody was in fact composed of four chains, one pair of identical, "light" chains and one pair of like- wise identical, "heavy" chains.

On the basis of the collected evidence Porter built a model of the molecule which has later, with overwhelming probability, been proven correct.

Accordingly the antibody molecule appears in the shape of the letter Y, with a stem and two angled branches. Each branch is composed of one light and one half of a heavy chain in side by side arrangement. The stem is made up of the remaining halves of the heavy chains. The specific combining capacity is accounted for by the structure of the free tips of the branches and in like measure by the light and the heavy chain; separately they are inactive. Porter's papain treatment attacks the molecule exactly at the point of branching and splits off the branches from the stem.

These discoveries incited an intense activity in laboratories in the four corners of the world. Apparently there existed a latent need for immunochemical research that could not be satisfied until today's prize winners had opened the way and provided the means. During the two decades that have since past our knowledge about the processes of immunity has broadened and deepened to an extent that perhaps has not yet been fully appreciated, even by some specialists in closely related fields. Many novel and fascinating aspects on problems in the fields of molecular biology and genetics have grown out of the immunochemical studies. We have now a new and firmer grasp of the question of the role of immunity as defense against and as cause of disease. Our possibilities to make use of immune reactions for diagnostic and therapeutic purposes have improved. It is, thus, a very important pioneer contribution that has been rewarded with this year's prize in physiology or medicine.

Gerald Edelman, Rodney Porter,

By clarifying the principal chemical structure of immunoglobulins you achieved an extremely important break-through in the field of immunochemistry. You, so to speak, opened the sluice-gates and gave impetus to the flood of research that soon started gushing forth, irrigating previously arid land, making it fertile and producing rich harvests. By awarding you the prize in 'physiology or medicine the Karolinska Institute has recognized the great significance of your accomplishments for biology in general and medicine in particular. On behalf of the Institute I wish to express our admiration and extend to you our heart-felt felicitations.

Now I ask you to proceed to receive your prize from the hands of His Royal Highness the Crown Prince.

---

[Image Credit: The cartoon of an immunoglobulin molecule is from the Genetics Home Reference website of the National Institutes of Health (USA).]

The Three Domain Hypothesis (part 1)

 

The Three domain Hypothesis is dead. It passed away peaceably sometime in the past ten years. Most people didn't notice.

Last year a wake was held. Friends and enemies of the Three Domain Hypothesis were invited. Many gave eulogies and these were published in a book called Microbial Phylogeny and Evolution: Concepts and Controversies. This is a collection of papers by leading scientists in the field. It's edited by Jan Sapp, a Professor of Biology at York University here in Toronto. I've listed the most interesting articles at the bottom of the page and I'll stick to comments on these articles for now.

Surprisingly, some of the guests at the wake did not know the hypothesis had been falsified. They thought the corpse was still breathing!

The Three Domain Hypothesis refers to the proposal by Carl Woese that; (1) archaebacteria form a monophyletic group, (2) this clade is sufficiently different from all other prokaryotes to deserve elevation to a separate Domain called Archaea (the other two Domains are Bacteria and Eukarya), (3) eukaryotes are more closely related to archaebacteria than to other prokaryotes, and (4) the root of the universal tree of life lies in the branch leading to Bacteria.

The "standard" universal tree of life is based on the Three Domain Hypothesis. It is mostly derived from sequences of the small ribosomal RNA subunit (SSU).

In recent years, all four of the major claims of the Three Domain Hypothesis have been challenged. Some would say that two have been falsified. Furthermore, there is growing recognition that SSU-based trees are not as reliable as we once thought. Surprisingly, this skepticism among evolutionary biologists has not reached the ear of the average scientist who continues to act as though the Three Domain Hypothesis is a done deal.

The literature is large, varied, and controversial. I've been following it for twenty years and it's not possible to write a short note covering all the bases. Instead, I'll concentrate on reviewing a few of the papers in the book.

---

Norman Pace The Large-Scale Structure of the Tree of Life.

Woflgang Ludwig and Karl-Heinz Schleifer The Molecular Phylogeny of Bacteria Based on Conserved Genes.

Carl Woese Evolving Biological Organization.

W. Ford Doolittle If the Tree of Life Fell, Would it Make a Sound?.

William Martin Woe Is the Tree of Life.

Radhey Gupta Molecular Sequences and the Early History of Life.

C. G. Kurland Paradigm Lost.

Are most transcription factor binding sites functional?

The ongoing debate over junk DNA often revolves around data collected by ENCODE and others. The idea that most of our genome is transcribed (pervasive transcription) seems to indicate that genes occupy most of the genome. The opposing view is that most of these transcripts are accidental products of spurious transcription. We see the same opposing views when it comes to transcription factor binding sites. ENCODE and their supporters have mapped millions of binding sites throughout the genome and they believe this represent abundant and exquisite regulation. The opposing view is that most of these binding sites are spurious and non-functional.

The messy view is supported by many studies on the biophysical properties of transcription factor binding. These studies show that any DNA binding protein has a low affinity for random sequence DNA. They will also bind with much higher affinity to sequences that resemble, but do not precisely match, the specific binding site [How RNA Polymerase Binds to DNA; DNA Binding Proteins]. If you take a species with a large genome, like us, then a typical DNA protein binding site of 6 bp will be present, by chance alone, at 800,000 sites. Not all of those sites will be bound by the transcription factor in vivo because some of the DNA will be tightly wrapped up in dense chromatin domains. Nevertheless, an appreciable percentage of the genome will be available for binding so that typical ENCODE assays detect thousand of binding sites for each transcription factor.

This information appears in all the best textbooks and it used to be a standard part of undergraduate courses in molecular biology and biochemistry. As far as I can tell, the current generation of new biochemistry researchers wasn't taught this information.

John Mattick's seminar at the University of Toronto

I just learned that John Mattick gave a seminar this morning at the Department of Cell & Systems Biology at the University of Toronto. Unfortunately, I was unable to attend.

Most Sandwalk readers will recognize Mattick as one of the few remaining vocal opponents of junk DNA. He is probably best known for his dog-ass plot but this is only one of the ways he misrepresents science.

Good Science Writers: Jacques Monod

 
Jacques Monod (1910 - 1976) received the Nobel Prize in Physiology or Medicine (1965) for his work on the regulation of the lac operon (with François Jacob). While best known as a biochemist, Monod was also well respected for his many articles on politics and philosophy.

Dawkins didn't select anything from Monod for The Oxford Book of Modern Science Writing because his selections were limited to books written initially in English. Monod's most famous work is Le Hasard et la Nécessité first published in France in 1970. It is well known in the English version: Chance and Necessity: An Essay on the Natural Philosophy of Modern Biology (1971). The excepts below are from the translation by Austryn Wainhouse.

Various mutations have been identified as due to

1. The substitution of a single pair of nucleotides for another pair;
2. The deletion of addition of one or several pairs of nucleotides, and
3. Various kinds of "scrambling" of the genetic text by inversion, duplication, or fusion of more or less extended segments.

We call these events accidental; we say that they are random occurrences. And since they constitute the only possible source of modification in the genetic text, itself the sole repository of the organism's hereditary structures, it necessarily follows that chance alone is at the source of every innovation, of all creation in the biosphere. Pure chance, absolutely free but blind, at the very root of the stupendous edifice of evolution: this central concept of modern biology is no longer one among other possible or even conceivable hypotheses. It is today the sole conceivable hypothesis, the only one that squares with observed and tested fact. And nothing warrants the supposition—or the hope—that on this score our position is ever likely to be revised.

I believe we can assert today that a universal theory, however completely successful in other domains, could never encompass the biosphere, its structure, and its evolution as phenomena deducible from first principles....

In a general manner the theory would anticipate the existence, the properties, the interrelations of certain classes of objects or events, but would obviously not be able to foresee the existence or the distinctive characteristics of any particular object or event.

The thesis that I shall present in this book is that the biosphere does not contain a predictable class of objects or of events but constitutes a particular occurrence, compatible indeed with first principles, but not deducible from those principles, and therefore essentially unpredictable.

Let there be no misunderstanding here. In saying that as a class living beings are not predictable upon the basis of first principles, I by no means intend to suggest that they are not explicable through these principles—that they transcend them in some way, and that other principles, applicable to living systems alone, must be invoked. In my view the biosphere is unpredictable for the very same reason—neither more nor less—that the particular configuration of atoms constituting this pebble I have in my hand is unpredictable. No one will find fault with a universal theory for not affirming and foreseeing the existence of this particular configuration of atoms; it is enough for us that this actual object, unique and real, be compatible with the theory. This object, according to the theory, is under no obligation to exist; but it has the right to.

That is enough for us as concerns the pebble, but not as concerns ourselves. We would like to think ourselves necessary, inevitable, ordained from all eternity. All religions, nearly all philosophies, and even a part of science testify to the unwearying, heroic effort of mankind desperately denying its own contingency.

---

Debating alternative splicing (Part III)

Proponents of massive alternative splicing argue that most human genes produce many different protein isoforms. According to these scientists, this means that humans can make about 100,000 different proteins from only ~20,000 protein-coding genes. They tend to believe humans are considerably more complex than other animals even though we have about the same number of genes. They think alternative splicing accounts for this complexity [see The Deflated Ego Problem].

Opponents (I am one) argue that most splice variants are due to splicing errors and most of those predicted protein isoforms don't exist. (We also argue that the differences between humans and other animals can be adequately explained by differential regulation of 20,000 protein-coding genes.) The controversy can only be resolved when proponents of massive alternative splicing provide evidence to support their claim that there are 100,000 functional proteins.

Religion may have evolved because of its ability to help people exercise self-control

 
Here's an example of evolutionary thinking by a psychologist at the University of Miami. Read the press release (below) and watch the video. It's only when you watch the video that you realize where Professor McCullough is coming from on this issue. He uses the word "evolution" to talk about cultural phenomena without necessarily including genetic changes. In other words, he is not talking about biological evolution.

This can be very confusing and I recommend that evolutionary psychologists change their practice. They should refer to "cultural evolution" and distinguish it from "biological evolution" whenever possible.

Religion may have evolved because of its ability to help people exercise self-control

A study by a University of Miami psychologist reveals that religion facilitates the exercise of self-control and attainment of long-term goals

CORAL GABLES, FL (December 30, 2008)—Self-control is critical for success in life, and a new study by University of Miami professor of Psychology Michael McCullough finds that religious people have more self-control than do their less religious counterparts. These findings imply that religious people may be better at pursuing and achieving long-term goals that are important to them and their religious groups. This, in turn, might help explain why religious people tend to have lower rates of substance abuse, better school achievement, less delinquency, better health behaviors, less depression, and longer lives.

In this research project, McCullough evaluated 8 decades worth of research on religion, which has been conducted in diverse samples of people from around the world. He found persuasive evidence from a variety of domains within the social sciences, including neuroscience, economics, psychology, and sociology, that religious beliefs and religious behaviors are capable of encouraging people to exercise self-control and to more effectively regulate their emotions and behaviors, so that they can pursue valued goals. The research paper, which summarizes the results of their review of the existing science, will be published in the January 2009 issue of Psychological Bulletin.

"The importance of self-control and self-regulation for understanding human behavior are well known to social scientists, but the possibility that the links of religiosity to self-control might explain the links of religiosity to health and behavior has not received much explicit attention," said McCullough. "We hope our paper will correct this oversight in the scientific literature." Among the most interesting conclusions that the research team drew were the following:

• Religious rituals such as prayer and meditation affect the parts of the human brain that are most important for self-regulation and self-control;
• When people view their goals as "sacred," they put more energy and effort into pursuing those goals, and therefore, are probably more effective at attaining them;
• Religious lifestyles may contribute to self-control by providing people with clear standards for their behavior, by causing people to monitor their own behavior more closely, and by giving people the sense that God is watching their behavior;
• The fact that religious people tend to be higher in self-control helps explain why religious people are less likely to misuse drugs and alcohol and experience problems with crime and delinquency.

McCullough's review of the research on religion and self-control contributes to a better understanding of "how the same social force that motivates acts of charity and generosity can also motivate people to strap bomb belts around their waists and then blow themselves up in crowded city buses," he explained. "By thinking of religion as a social force that provides people with resources for controlling their impulses (including the impulse for self-preservation, in some cases) in the service of higher goals, religion can motivate people to do just about anything."

Among the study's more practical implications is that religious people may have at their disposal a set of unique psychological resources for adhering to their New Year's Resolutions in the year to come.

I leave it up to you, dear readers, to decide whether non-religious people (atheists) tend to have higher rates of substance abuse, worse school achievement, more delinquency, worse health behaviors, more depression, and shorter lives. It would imply that countries like Sweden, where half the population is non-religious, are in much worse shape than America, where more than 80% is religious. It would imply that extremely religious countries like Saudi Arabia must be near-perfect societies full of very old people.

Incidentally, the idea of "self-control" is not well explained. If you behave in a certain way because you fear punishment from your god or your priests, then this isn't exactly what I think of when I use the term "self-control."

---

The Molecular Basis of Roundup® Resistance

Recall that glyphosate inhibits the enzyme EPSP synthase, an enzyme that catalyzes the following reaction in the chorsimate biosynthesis pathway [How Roundup® Works].

Funke et al. (2006) explored the molecular basis of this inhibition by looking at the structure of EPSP synthase from the C4 strain of Agrobacterium sp. This is the resistant form of the enzyme that has been genetically engineered into Roundup Ready® plants [Roundup Ready® Transgenic Plants].

Note that the structure of glyphosate resembles one of the substrates of the reaction; namely phosphoenolpyruvate (PEP). It was already known that glyphosate binds tightly to the active site of the enzyme and inhibits the reaction by preventing PEP binding. As it turns out, the site for glyphosate binding is exactly the same as the site for PEP binding and this explains the inhibition.

Funke et al. (2006) looked at the C4 EPSP enzyme with and without one of the other substrates: namely, shikimate-3-phosphate (sometimes called shikimate-5-phosphate). The results reveal the precise location of the active site of the enzyme at the base of a cleft between two domains. This form of the enzyme is called class II EPSP synthase because it is distantly related to the class I enzymes in other bacteria and eukaryotes (30% amino acid sequence identity). This is the first paper to examine the structure of a class II enzyme.

As an aside, notice that the enzyme closes up a little bit when the substrate binds—sort of like a Pacman icon. This mechanism of substrate binding is called induced fit and it's proving to be more common than most people realized.

The glyphosate resistant (Roundup Ready®) mutation in C4 EPSP synthase is a substitution of Alanine (A) for Glycine (G) at amino acid position 100. The glyphosate molecule fits nicely into the wild type G100 form of the enzyme (lower image) and it excludes PEP binding completely. Note that glyphosate (green) is in an extended configuration when it is bound. The dotted lines represent non-covalent interactions between the enzyme and the glyphosate molecule. The blue dots are "frozen" water molecules embedded in the active site.

In the mutant form of the enzyme the extra methyl group on alanine is just big enough to cause glyphosate to distort so it can no longer lie in the optimal extended configuration (top image). This means that glyphosate binds much more weakly and doesn't inhibit enzyme activity.

The important point is that the active site can still accommodate phosphoenolpyruvate because it is smaller than glyphosate. What this means is that the overall activity of the enzyme in the absence of glyphosate is unaffected. There are lots of EPSP synthase mutants that don't bind glyphosate but in almost all cases the rate of the reaction is drastically reduced because PEP binding is also weakened. For example, if you mutate the glycine to alanine at the equivalent position in other bacterial or plant enzymes you abolish PEP binding along with glyphosate binding.

What's special about the class II enzymes in general and the Agrobacterium sp. enzyme in particular, is that the amino acids surrounding the PEP binding pocket are positioned just right so that a slight shift can exclude glyphosate without affecting phosphoenolpyruvate. This is mostly due to the positions of the charged amino acid side chains that form weak interactions with the oxygen atoms and the nitrogen of glyphosate; for example, arginines (R) at 128, 357, and 405; lysine (K) at 28; and glutamate (E) at 354.

The results of this study not only shed light on the mechanism of glyphosate resistance but they also help explain the lack of Roundup® resistant plants. Apparently, the class I enzymes in plants have a binding pocket that is difficult to mutate in a way that excludes glyphosate while still allowing PEP binding. Nevertheless, some examples of Roundup® resistant plants are known. I'll describe them tomorrow.

(Funke et al. had to do a bit of sleuthing and reconstruction in order to solve the structure of the C4 EPSP synthase. The C4 strain of Agrobacterium sp. has, naturally enough, not been given out to scientists outside of Monsanto laboratories. So Funke et al. got the amino acid sequence from US Patent 5633435 and reverse engineered the nucleotide sequence of the gene. They synthesized the nucleotide sequence and amplified the fragments by PCR. They then tacked on a promoter and a transcription termination signal and cloned the articfial gene into an E. coli plasmid. The artificially reconstructed protein was then expressed in E. coli, isolated, purified, and crystallized.)

Funke, T., Han, H., Healy-Fried, M,L., Fischer, M., and Schonbrunn, E. (2006) Molecular basis for the herbicide resistance of Roundup Ready crops. Proc. Natl. Acad. Sci. (USA) 103:13010-13015. [PubMed]

Herbert Tabor/Journal of Biological Chemistry Lectureship

 
One of the big events for ASBMB is the Herbert Tabor JBC lecture. It was held Saturday night in one of the large ballrooms. There were about one thousand people attending.

The first lecture was by Tony Hunter from The Salk Institute in California (USA). He spoke about mammalian kinases and phosphorylases with an emphasis on tyrosine kinases, which he discovered back in 1979. Tyrosine kinases are enzymes that attach phosphate groups to tyrosine residues in proteins. They are important because the phosphorylation and dephosphorylation of enzymes regulates their activity. Many of the genes that cause cancer (oncogenes) encode tyrosine kinases.

Hunter is trying to find out how many different proteins kinases there are in humans. The latest count suggests about 900 different enzymes. This is a remarkable number when you think about it. It means that 3-4% of all genes in our genome are kinases.

The second award winner was Tony Pawson from the Samuel Lunenfeld Research Institute and the University of Toronto (Ontario, Canada). I've heard Tony speak many times so I wasn't quite as attentive during his lecture. Tony discovered a number of proteins domains, notably the SH2 domain, that interact with tyrosine kinases and their target proteins. The work of the two Tony's is complementary and that's why they received this joint award.

UPDATE: I forgot to mention that there was a reception after the talks. Lots of delicious munchies and an open bar. I had a beer (or two). Most biochemists drink wine or fruit juice. It was not a wild bunch.

The Last Universal Common Ancestor

 
Jeffrey Wong is a former member of our Department¹ and the author of the best theory on the origin of the genetic code [see: Amino Acids and the Racemization "Problem"]

LUCA, LECA and LBACA--Root of Life and Roots of the Biological Domains

Dr. Jeffrey Wong
Department of Biochemistry
Hong Kong University of Science and Technology

4:30 pm, September 10
Medical Sciences Building room 4171

---

His mother's photograph is on the wall of graduates on the first floor of our building. She graduated from medical school in 1929.