Measuring Stacking Interactions

 
The two strands of double- stranded DNA are held together by a number of weak interactions such as hydrogen bonds, stacking interactions, and hydrophobic effects [The Three- Dimensional Structure of DNA].

Of these, the stacking interactions between base pairs are the most significant. The strength of base stacking interactions depends on the bases. It is strongest for stacks of G/C base pairs and weakest for stacks of A/T base pairs and that's why it's easier to melt A/T rich DNA at high temperature. (It is often incorrectly assumed that this is due to having only two hydrogen bonds between A/T base pairs and three between G/C base pairs.)

The figure below shows a melting curve of various DNAs. The curve shows the conversion of double-stranded DNA to denatured single strands by following the change in absorbance as the temperature is increased from left to right. When the double helix is unzipped the absorbance increases. Note that poly(AT) "melts" at a lower temperature (T_M = melting temperature) than poly(GC). This is because the average stacking interactions of G/C base pairs are two or three times stronger than A/T base pairs so more thermal energy is need to disrupt them.


The base stacking interactions have been measured in several different ways but most of these measurements are indirect and all of them have been with double-stranded DNA. Of the single-stranded polynucleotides, only polyA has a helical structure in solution and that's because of the stacking interactions between single adenylate resides in the polynucleotide. PolyT is somewhat unstructured and polyG and polyC have complex three-dimensional structures that are difficult to interpret.

Assuming that the stacking interactions of the adenylate residues is the only significant force maintaining the polyA helix, it's possible to measure the stacking interaction directly by pulling both ends to see how much pressure it takes to disrupt the helix. This can be done by fixing single-stranded polyA to a substrate and grabbing the other end with a molecular probe. The elasticity of the DNA can be measured by single-molecule atomic-force spectroscopy (Ke et al. 2007).

As the molecule is stretched, it resists up to the point were the bases become unstacked and the helix is disrupted. The force required can be used to directly calculate the stacking interactions between the adenylate residues. The value turns out to be 3.6 ± 0.2 kcal/mol per base (15 kJ/mol). This is very close to the stacking energies calculated for A/T base pairs in earlier experiments. (The stacking energies for G/C base pairs in DNA are about 61 kJ/mol.)

The experiment is independent, and direct, confirmation of the literature values for stacking interactions. The energies of these stacking interactions turn out to be significantly larger than the energies of the other weak interactions involved in holding double-stranded DNA together (hydrogen bonds, "normal" van der Waals interactions, and hydrophobic interactions).

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Changhong Ke, Michael Humeniuk, Hanna S-Gracz, and Piotr E. Marszalek (2007) Direct Measurements of Base Stacking Interactions in DNA by Single-Molecule Atomic-Force Spectroscopy. Phys. Rev. Lett. 99:018302

[The top figure is from Ke et al., 2007]

Junk DNA in New Scientist

I just got my copy of the July 14th issue of New Scientist so I can comment on the article Why 'junk DNA' may be useful after all by Aria Pearson. RPM at evolvgen thinks it's pretty good [Junk on Junk] and so does Ryan Gregory at Genomicron [New Scientist gets it right]. I agree. It's one of the best articles on the subject that I've seen in a long time.

First off, Aria Pearson does not make the common mistake of assuming that junk DNA is equivalent to non-coding DNA. The article makes this very clear by pointing out that we've known about regulatory sequences since the 1970's. The main point of the article is to discuss recent results that reveal new functions for some of the previously unidentified non-coding DNA that was classified as junk.

One such result is that reported Pennacchio et al. (2006) in Nature last year. They analyzed sequences in the human genome that showed a high degree of identity to sequences in the pufferfish genome. The idea is that these presumably conserved sequences must have a function. Pennacchio et al. (2006) tested them to see it they would help regulate gene expression and they found that 45% of the ones they tested functioned as enhancers. In other words, they stimulated the expression of adjacent genes in a tissue specific manner. The authors estimate that about half of the "conserved" elements play a role in regulating gene expression.

There are a total of 3,124 conserved elements and their average length is 1,270 bp. This accounts for 3.9 × 10⁶ bp out of a total genome size of 3.2 × 10⁹ bp or about 0.1% of the genome. The New Scientist article acknowledges, correctly, that more than 95% of the genome could still be junk.

Is this all junk DNA? Unlike most other science journalists, Pearson addresses this question with a certain amount of skepticism and she makes an effort to quote conflicting opinions. For example, Pearson mentions experiments claiming that ~90% of the genome is transcribed. Rather than just repeating the hype of the researchers making this claim, Pearson quotes skeptics who argue that this RNA might be just "noise."

Most articles on junk DNA eventually get around to mentioning John Mattick who has been very vocal about his claim that the Central Dogma has been overturned and most of the genome consists of genes that encode regulatory RNAs (Mattick, 2004; Mattick, 2007). This article quotes a skeptic to provide some sense of balance and demonstrate that the scientific community is not overly supportive of Mattick.

Others are less convinced. Ewan Birney of the European Bioinformatics Institute in Cambridge, UK, has bet Mattick that of the processed RNAs yet to be assigned a function - representing 14 per cent of the entire genome - less than 20 per cent will turn out to be useful. "I'll get a case of vintage champagne if I win," Birney says.

Under the subtitle "Mostly Useless," Pearson correctly summarizes the scientific consensus. (I wish she had used this as the title of the article. The actual title is somewhat misleading. Editors?)

Whatever the answer turns out to be, no one is saying that most of our genome is vital after all. "You could chuck three-quarters of it," Birney speculates. "If you put a gun to my head, I'd say 10 per cent has a function, maybe," says Lunter. "It's very unlikely to be higher than 50 per cent."

Most researchers agree that 50 per cent is the top limit because half of our genome consists of endless copies of parasitic DNA or "transposons", which do nothing except copy and paste themselves all over the genome until they are inactivated by random mutations. A handful are still active in our genome and can cause diseases such as breast cancer if they land in or near vital genes.

The ENCODE project made a big splash in the blogosphere last month (ENCODE Project Consortium, 2007). This study purported to show that much of the human genome was transcribed, leading to the suggestion that most of what we think is junk actually has some function. Aria Pearson interviewed Ewan Birney (see above) who is involved in the ENCODE project.

The real surprise is that ENCODE has identified many non-coding sequences in humans that seem to have a function, yet are not conserved in rats and mice. There seem to be just as many of these non-conserved functional sequences as there are conserved ones. One explanation is that these are the crucial sequences that make humans different from mice. However, Birney thinks this is likely to be true of only a tiny proportion of these non-conserved yet functional sequences. Instead, he thinks most are neutral. "They have appeared by chance and neither hinder nor help the organism."

Put another way, just because a certain piece of DNA can do something doesn't mean we really need it to do whatever it does. Such DNA may be very like computer bloatware: functional in one sense yet useless as far as users are concerned.

This is a perspective you don't often see in popular articles about junk DNA and Pearson is to be commended for taking the time and effort to find the right scientific perspective.

The article concludes by reporting the efforts to delete large amounts of mouse DNA in order to test whether they are junk or not. The results show that much of the conserved bits of DNA can be removed without any harmful effects. Some researchers urge caution by pointing out that very small effects may not be observed in laboratory mice but may be important for evolution in the long term.

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ENCODE Project Consortium (2007) Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447:799-816. [PubMed Abstract]

Mattick, J.S. (2004) The hidden genetic program of complex organisms. Sci. Am. 291:60-7.

Mattick, J.S. (2007) A new paradigm for developmental biology. J. Exp. Biol. 210:1526-47. [PubMed Abstract].

Pennacchio, L.A., Ahituv, N., Moses, A.M., Prabhakar, S., Nobrega, M.A., Shoukry, M., Minovitsky, S., Dubchak, I., Holt, A., Lewis, K.D., Plajzer-Frick, I., Akiyama, J., De Val, S., Afzal, V., Black, B.L., Couronne, O., Eisen, M.B., Visel, A., Rubin, E.M. (2006) In vivo enhancer analysis of human conserved non-coding sequences. Nature 444(7118):499-502.

DNA With Parallel Strands

 


A week ago I asked if any of you could identify a strange molecule that looked like a base pair [A Strange Molecule]. Steve LaBonne recognized that the bases were flipped and the strands were parallel.

Here's an image of the complete structure from the PDB Database [1R2L]. Unlike normal double-stranded DNA, in this structure the strands run in the same direction from top to bottom. The 5′ ends of each strand are at the bottom.

This is very unusual. So far, it's the first example of such a molecule. Nobody thinks that a parallel-stranded DNA can exist inside a cell but who knows?

Sandra Porter at Discovering Biology in a Digital World found it [It's still a DNA puzzle, but this is the answer]. Thanks Sandra.

The Story of DNA (Part 2)

We've discovered the secret of life.


.... Francis CrickWhere Jim and Francis Discover the Secret of Life

Following the disaster of their first attempt at a DNA structure, Francis Crick went back to studying proteins [The Story of DNA (Part 1)]. He and William Cochran worked out the theory of the X-ray diffraction pattern of helices. Crick became the leading expert on the interpretation of patterns due to helices and he was able to predict what kind of pattern a particular helical pattern would show. This study paid huge dividends later on. Crick also worked out the coiled coil arrangement of polypeptide chains.

Watson dabbled in a number of projects over the next year. Most importantly, he had Crick teach him diffraction theory and he applied it to the structure of tobacco mosaic virus showing that it was a helix. Watson too became extremely adept at recognizing helices from their X-ray diffraction pattern.

Franklin made one important discovery. She showed that there were two distinct forms of DNA and that the original Astbury pictures were composites of the two forms. She called them A and B and those are the same names that we give them today. The B form is the naturally occurring form and the DNA has to maintained at high humidity in order to persist in this form The A form is somewhat dehydrated, if the fibers dry out, the structure converts to the A form.

On April 10, 1952 Rosalind Franklin took a picture of the A form of DNA. This picture was complex but it had some significant new features. Franklin came to rely heavily on the images of dehydrated DNA (A-DNA). Over the spring and summer she convinced herself that DNA was not helical. In fact, on July 18th, 1952 she and Gosling announced “the death of the helix” by sending out small cards with black borders [Rosalind Franklin Anounces the Death of the Helix]..

Recall that Raymond Gosling was the former graduate student of Wilkins, now assigned to Franklin. Franklin was rapidly making herself a real pain in the you-know-what but everybody loved Raymond.

On May 2 and May 6, 1952, while immersed in the analysis of the A form of DNA, Franklin took two beautiful pictures of B-DNA (right). The photos screamed helix. A cursory glance by someone familiar with helical diffraction patterns showed that the bases were 0.34 nm apart; that there were ten nucleotides per turn; that each turn was 3.4 nm; that the phosphate groups were on the outside; that the diameter of the helix was 2 nm; and that there were most likely two polynucleotide strands. Rosalind Franklin did not recognize these features and she put the photos aside.

After announcing the death of the helix, Franklin set out to secure herself a new job at another institute. It is clear that she did not think that the structure of DNA was very important. She soon received an offer to move to Birkbeck College, London but delayed until the following spring. Meanwhile, she set herself the task of working out the structure directly from the X-ray diffraction patterns. She refused to engage in speculation or model building and preferred to try and let the data lead her directly to the correct structure. By January of 1953 she knew that she was not going to solve DNA and she prepared to abandon the problem and publish the data she had obtained.

With hindsight, it’s clear that Franklin needed a trusted collaborator in order to make progress on this difficult problem. While working in Wilkins group she found herself isolated because she and Wilkins did not get along. (Both were to blame.) Later on in her career she collaborated effectively with Aaron Klug and Francis Crick.

Franklin also proved that the unit cell of the DNA fibers was monoclinic, face centered. This was an absolutely crucial piece of information but one that Franklin failed to appreciate. As soon as Crick became aware of it, in January 1953, an important part of the structure became apparent (see below).

What were Watson and Crick up to in the summer of 1952 when Rosalind Franklin was announcing the death of the helix? Well, for one thing they were not ignoring DNA in spite of Bragg’s warning.

Crick had begun to consider the possibility that the bases might be on the inside of the helix. He asked his friend, John Griffith, to do some calculations to see whether the bases could interact with one another. Griffith replied that A and T are compatible and so are G and C. This has nothing to do with hydrogen bonds—that would come later—but it did confirm one idea in Crick’s mind. Both he and Watson were familiar with the idea of complementarity. Crick thought of it as a way of explaining how DNA was replicated since one part of DNA could give rise to its complement while the other could make the second part. Complementarity was often discussed among the phage group since Delbruck and Linus Pauling had published a paper on it just before the war.

They impressed me by their extreme ignorance … I never met two men who knew so little—and aspired to so much.

..... Erwin ChargaffErwin Chargaff visited Cambridge in May 1952. Chargaff met with Watson and Crick and explained his work on the base composition of DNA. The results were new to Crick but known to Watson. Crick immediately saw that A=T and G=C and that fitted in with his ideas about complementarity.

Chargaff was not impressed. He said later on that, "They impressed me by their extreme ignorance ... I never met two men who knew so little—and aspired to so much." Later on after the structure of DNA had been published Chargaff said, "That in our day such pygmies throw such giant shadows only shows how late in the day it has become."

After a round of conferences in the summer, life began to settle down again at the Cavendish labs in Cambridge. Watson and Crick were joined by two new members of the lab. Peter Pauling, the son of Linus Pauling, had become a graduate student and brought news from his father that Pauling senior was thinking about DNA. The other new member of the group was Jerry Donohue. He was a former graduate student of Pauling's who was joining Bragg's group as a post-doc.

On Wednesday, January 28th, 1953 a copy of the Pauling and Cory manuscript on the structure of DNA reached Watson and Crick. The structure was wrong. In fact, it was similar to the Watson and Crick model that Rosalind Franklin had destroyed fourteen months earlier. Watson and Crick were elated and they determined to try again in spite of the ban imposed by Bragg. (The ban was soon to be lifted.)

On Friday, January 30, 1953 Watson was in London and he stopped by to see Franklin in her lab (left). Watson showed her a copy of the Pauling and Cory manuscript and she too saw that it was wrong. Watson began lecturing Franklin about helices—remember that Franklin was, at this time, concentrating on the A form of DNA and had all but ruled out that it was a helix. However, she was beginning to have some doubts about her hasty announcement of the death of the helix [Rosalind Franklin Announces the Death of the Helix]. She resented Watson's lecture and advanced toward Watson with a view to dismissing him. Watson beat a hasty retreat. (Jim Watson is well over six feet tall and Rosalind Franklin is very much shorter.)

At that moment Wilkins came by and he and Watson walked off comforting one another in the knowledge that Franklin was impossible. Wilkins told Watson about the excellent pictures of B DNA that Franklin had taken eight months ago (May 1952). He showed Watson one of the pictures (see above). Watson left London with the knowledge that the B form of DNA was unmistakably helical, that the diameter was 2Å (2 nm), that there were 10 bases per turn, and that one turn was 34Å (3.4 nm). Some of this he got from the photo and some from measurements that Wilkins himself had made.

With this information, Watson started to build models. He began with the backbones inside but soon realized that it was impossible. Crick urged him to try to put the bases inside. Franklin had already concluded from her data that the phosphates were on the outside but it's not clear that Watson and Crick knew this.

Now comes a crucial bit of information. Rosalind Franklin had written a summary of her results for an institute report in December. Perutz gave Crick a copy. In that report Crick read for the first time that the crystalline form of DNA was based on a face-centered monoclinic unit cell. Why is this important?

It's important because such a unit cell has a two-fold axis of symmetry. That means that the molecule looks the same whether it is right way up or upside down. This has important implications for the two strands of DNA. To see this, think about two pencils side-by-side with the points down and the erasers on top. If you turn the two pencils upside down they look very different. Now the tips are pointing upward. However, if you line up the two pencils side-by-side with the tip of one pointing up and the tip of the other pointing down, when you flip the pair upside down they look the same. It means the two strands of DNA must be anti-parallel [The Chemical Structure of Double-Stranded DNA].

The space group of Franklin's DNA just happened to be the same space group as that of hemoglobin, the molecule that Crick was working on as the subject of his Ph.D. thesis. Crick recognized immediately what this meant.

Watson worked out another argument that convinced them that there had to be two chains in the unit cell and not three. It had to do with the density and water content and we won't go into it here. Suffice to say that in the last days of February they knew that the backbones were on the outside, that there were two chains, and that the chains ran in opposite directions.

On Friday, February 20th Watson presented some ideas about base pairs to his colleagues. He had come up with a scheme involving like pairs (A/A, G/G etc.). Jerry Donohue instantly recognized a problem. Watson was using the standard textbook structures of the bases, the imino and lactim tautomers. Donohue knew that the predominant forms in living cells were the other tautomers, the amino and lactam conformations [Tautomers of Adenine, Cytosine, Guanine, and Thymine]. This was the final important clue. Like pairing with like was not an option; besides, it didn't conform to Chargaff's rules.

The next week Watson made some cardboard cutouts of the bases and began to try and fit them together into the middle of the backbones running in opposite directions. Crick urged him to think about complementarity—recall that the previous summer Crick had convinced himself that complementarity was the key to DNA replication. He had forgotten about the A=T and G=C data from Chargaff.

On Saturday, February 28, Watson was playing with his cardboard cutouts when he discovered that you could fit A/T and G/C base pairs into the model. Crick immediately confirmed that this was an elegant solution. They then realized that it explained the Chargaff ratios.

It took them about a week to build a detailed model. Many experts were called to give their opinion and all pronounced it sound. Wilkins, Gosling, and Franklin came up to Cambridge to see the model and agreed that it must be right. Raymond Gosling is an admirer of Wilkins and in reviewing Wilkins' autobiography in Nature (Gosling, 2003) Gosling writes,

Wilkins eloquently describes his feelings at seeing the double-helix structure for the first time: "It seemed that non-living atoms and chemical bonds had come together to form life itself. I was rather stunned by it all." This sums up beautifully how Franklin and I felt. It was so elegant an explanation of all of the complex properties required of DNA, and contained so many elements familiar from our own work using X-ray diffraction. At the time I did not know that Wilkins was offered co-authorship by Watson and Crick, but refused. It would certainly have been appropriate, and seems to be something that he later came to regret.

The paper was written up and sent off to Nature on April 2. It was published on April 25, 1953 along with papers by Franklin and Wilkins.

Before publication, Linus Pauling visited Cambridge and confirmed that the Watson/Crick model was correct and his model was wrong. The first announcement of the discovery was made by Bragg at a conference in Brussels in early April.

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Franklin, R. and Gosling, R.G. (1953) Molecular Configuration in Sodium Thymonucleate. Nature 171:740-741. [PDF]

Gosling, Raymond (2003) Completing the helix trilogy. Nature 425:901.

Watson, J.D. and Crick, F.H.C. (1953) Molecular structure of nucleic acids. Nature 171::737-738. [PDF]

Wilkins, M.H.F., Stokes, A.R., and Wilson, H.R. (1953) Molecular Structure of Deoxypentose Nucleic Acids. Nature 171:738-740. [PDF]

Bibliography

Clayton, J. and Denis, C. eds. (2003) 50 Years of DNA. Nature/Pallgrave/Macmillan

Judson, H.F. (1996} The Eighth Day of Creation: Makers of the Revolution in Biology. expanded ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. USA

Maddox, B. (2002) Rosalind Franklin: The Dark Lady of DNA. Perennial/HarperCollins

Watson, J.D. and Berry, A. (2003) DNA: The Secret of Life. Alfred A. Knope, New York, USA

Watson, J.D. (1168) The Double Helix. Atheneum, New York USA

Nobel Laureates 1962

 
The other day we were talking about good American writers and the name "John Steinbeck" came up (for unknown reasons). This led to the obligatory question about whether anyone had actually read The Grapes of Wrath (some had) or The Log from the Sea of Cortez (nobody had). Anyway, it reminded me of a famous photograph I had once seen so I tried to find it on the internet. Here it is.

From left to right: Prof. Maurice Wilkins (Physiology & Medicine), Dr. Max Perutz (Chemistry), Francis Crick (Physiology & Medicine), John Steinbeck (Literature), James Watson (Physiology & Medicine), Dr. John Kendrew, (Chemistry). I hope Steinbeck was impressed.

Monday's Molecule #36

 
Today's molecule is complex. It has a strange-looking ring structure. The short common name of this molecule is well known but your task—should you choose to accept it— is to supply the correct IUPAC name. There's an indirect connection between this Monday's Molecule and Wednesday's Nobel Laureate(s). (The molecule also has a connection to Intelligent Design Creationism.)

The reward (free lunch) goes to the person who correctly identifies the molecule and predicts 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. The bonus is a free drink (alcoholic) with your lunch if you guess the connection to Intelligent Design Creationism.

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

THEME:
Malaria
UPDATE The molecule is quinine, a drug used in the treatment of malaria. The Noble Laureates are Charles Laveran (1907) and Ronald Ross (1902)

Are you an atheist, agnostic, or a believer?

 
Blogger has a new feature so I thought I'd try it out. There's a poll in the left-hand margin. Click on the appropriate response.

Hillary Clinton Is a Marxist According to Mitt Romney

 
American politics is like a reality TV show. It can be very entertaining at times but it's sort of like watching a train wreck. Here's Mitt Romney telling us how Europe is in decline and America is great. Why? Because America promotes individual freedom and initiative while Europe went for socialism. According to Romney, Hillary Clinton is a Marxist who couldn't even get elected in France these days because Europe is voting for conservatives.

The Frequency of Alternative Splicing

One of these days I'm going to get around to blogging about alternative splicing. As most of you know, the databases are full of information about alternatively spliced gene products in mammalian genomes. There are many scientists who believe that most mammalian genes have two or more different products as a result of alternative splicing of the primary transcript.


I think it's nonsense. When I look at my favorite genes, the HSP70 gene family, the predicted protein products make no sense whatsoever. (The image shows predicted splice variants of the HSPA5 (BiP) gene from the SpliceInfo database.) The alternatively spliced variant often removes a piece of the hydrophobic core of the protein or other parts that are known to be essential. Since these proteins are the most highly conserved proteins in all of biology, it makes no sense at all to predict that mammals have all of a sudden evolved variants that are missing large hunks of highly conserved amino acid sequence. I think that most predicted splice variants are artifacts of the EST databases.

The annotators of the human genome have pretty much rejected all of the splice variants of HSP70 genes (e.g., Entrez Gene HSPA5) and many other genes whose structures are known. They have not rejected the multiple splice variants of other genes that are less well studied.

Anyway, like I said, this discussion will have to wait for another time. Meanwhile, you can read my friend Deanne Taylor's views on alternative splicing (and her disagreement with me) on her blog [Alternative Culture]. She will be here in Toronto next summer to debate the issue so everyone should plan on attending a mini-Howlerfest. It will be at the same time as the Darwin Exhibit at the ROM [Charles Darwin Is Coming to Toronto].

Here's an Example of Pro-War Thinking

 
Steve Huntley has a column in Today's Chicago Sun-Times [Careful, Iraq may be key to al-Qaida]. It's a really good example of the irrational thinking behind the pro-war crowd. Huntley begins with,

The Iraq war critics seized upon a new intelligence report that al-Qaida has been rejuvenated by the Iraq war as proof that the invasion of Iraq was a distraction from the war on terror. OK, that should be good for a few minutes of bashing President Bush, but it doesn't change the reality that al-Qaida is in Iraq and is our enemy.

No, the reality is that al-Qaida is now in Iraq and it wasn't before the invasion. The reality is that al-Qaida has been rejuvenated by the invasion and occupation of Iraq but we were told that this wasn't going to happen. We were told that the invasion and occupation of Iraq was part of the war on terror, but it wasn't really. It is now, because terrorism has been unleashed in Iraq since the breakdown of civil order. The breakdown of civil order was caused by the invasion and by the presence of foreign occupying troops.

Here's another thought: What would be the reaction of the quit-Iraq advocates should al-Qaida in Iraq's fingerprints be found in a terrorist attack in America?

This is not an idle question. After all, the National Intelligence Estimate released last week also said Osama bin Laden's organization will "probably seek to leverage the contacts and capabilities of al-Qaida in Iraq, its most visible and capable affiliate and the only one known to have expressed a desire to attack the Homeland." Furthermore, the 9/11 Commission has said another attack on America by Islamist terrorists is inevitable, and a new threat assessment a week ago from the National Counterterrorism Center suggested al-Qaida is working to renew attacks on America. Now we're told al-Qaida in Iraq could be the agent for it.

My reaction would be "I told you so." The invasion of Iraq has caused a huge increase in the number of people who hate America. Some of these people are going to be easy recruits to al-Qaida and some of them are, quite possibly, going to attack America.

Most of the damage has been done but we may be able to prevent further damage by getting the heck out of the Middle East and letting the people there solve their own problems their own way.

No doubt, even as the bodies were being recovered, the wounded treated and survivors consoled, the implacable Bush haters would blame his policies for an attack by al-Qaida in Iraq. But what would be the view of the majority of Americans who have been telling pollsters that it's time for America to withdraw the troops from Iraq?

I'm hoping that the majority of Americans would see the truth. It's the Bush policy that led to more people hating American and an increased probability that the war would be brought to America. I'm hoping that the impeachment of Cheny would be swift and that it would be followed by the impeachment of George Bush.

But then, I tend to be overly optimistic about these things.

[The image is from the US Dept. of Defense and is in the public domain. See Wikipedia: Army.mil-2007-02-13-104034.jpg]

Shalini Doesn't Like Appeasers

 
Shalini, over at Scientia Natura: Evolution and Rationality has written a longish criticism of appeasers [ Appeasers: The spineless pushovers]. Appeasers are also called accommodationists, they are atheists who do not want to criticize religious beliefs out of a mistaken impression that it's wrong and counter-productive to question another person's faith. The accommodationists believe that religion deserves some kind of protective status that they do not grant to people who believe in astrology, bigfoot, and UFO's.

You have to read Shalini's entire article to appreciate what she has to say but here's an important paragraph that I agree with. It gives you the flavor of her argument.

Contrary to what appeasers think, this is not about one issue or another. It is not about young earth creationism, ID, evolution, climate change, stem cell research, marijuana or the latest hot-button issue. These are merely battles in the course of the real war -- the war between rationalism and superstition. In this war, only one side will be the winner. There is no room for appeasers, and the superstitious, at least, will have none of this cowardly garbage. They may be ignorant, deluded, liars or plain kooks, but they are certainly not cowards, and that is more than I can say for the appeasers. Remember, no change has ever been achieved by shutting up and bowing down to oppressive institutions. If we fail to make our voices heard, superstition has already won.

Rosalind Franklin Announces the Death of the Helix

 
If you've read The Story of DNA (Part 1) you'll know that we've reached the point in November 1951 where Watson and Crick have failed in their first attempt. Rosalind Franklin was more than happy to point out how little they knew about the chemistry of DNA.

In Part 2 we'll learn how Watson and Crick redeemed themselves and how they we able to turn the tables on Rosalind Franklin. This notice, which was sent out to everyone in London and Cambridge who were interested in DNA, was signed by Raymond Gosling and Rosalind Franklin. It announces that DNA is NOT a helix. The date is July 18, 1952.

[The image is from a Francis Crick website.]

Ethidium Bromide Binds to DNA

 
Last Monday's Molecule was ethidium, better known by the name of its common salt, ethidium bromide [Monday's Molecule #35]. Ethidium is a large planer molecule that binds tightly to DNA. It is often used in biochemistry laboratories to visualize fragments of DNA that have been separated on gels. The ethidium molecule is fluorescent—when illuminated with ultraviolet light it shines in the visible range. Here's a picture (below right) of DNA fragments that are illuminated by ethidium binding. It's from an old paper of mine (Moran et al. 1979)—these days you usually can't publish simple experiments like this.

Ethidium binds by inserting itself bewteen the stacked bases in double-stranded DNA. Note that the ring structure of ethidium is hydrophobic and resembles the rings of the bases in DNA. Ethidium is capable of forming close van der Walls contacts with the base pairs and that's why it binds to the hydrophobic interior of the DNA molecule.

Molecules that bind in this manner are called intercalating agents because they intercalate into the compact array of stacked bases. In doing so, they distort the double helix and interfere with DNA replication, transcription, DNA repair, and recombination. This is why intercalating agents are often potent mutagens.

The cartoon below shows the distortion of the sugar-phosphate backbone when an intercalating agent bind and it also shows that the DNA is lengthened when intercalating agents bind. This changes the properties of DNA considerably. One of the tricks in separating closed circular molecules of DNA from linear fragments (such as genomic DNA) is to treat the DNA with ethidium bromide. The intercalating agent doesn't bind to closed circular molecules because they can't be lengthened enough to allow insertion of the chemical between the bases. The normal circular plasmid DNA can then be separated from linear DNA with bound ethidium because binding of ethidium changes the overall density of DNA.



The structure shown above (right) is from Reha et al. (2002). It shows a molecule of ethidium lying between two A/T base pairs.

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Moran,L., Mirault, M-E., Tissières, A., Lis, J., Schedl, P., Artavanis-Tsakonas, S., and Gehring, W. (1979) Physical Map of Two D. melanogaster DNA Segments Containing Sequences Coding for the 70,000 Dalton Heat Shock Protein. Cell 17:1-8.

Reha, D., Kabelác, M., Ryjácek, F., Sponer, J., Sponer, J.E., Elstner, M., Suhai, S., and Hobza, P. (2003) Intercalators. 1. Nature of stacking interactions between intercalators (ethidium, daunomycin, ellipticine, and 4',6-diaminide-2-phenylindole) and DNA base pairs. Ab initio quantum chemical, density functional theory, and empirical potential study. J. Am. Chem. Soc. 124:3366-76.

The Name of Buddy Holley's Airplane was "American Pie"

 
Friday's Urban Legend: FALSE

From americanpie.com.

Basic errors in American Pie interpretations have been carried forward and sometimes get reported as being fact. One of the most tedious theories of recent times is that the plane that crashed killing Buddy Holly, Ritchie Valens and the Big Bopper was called 'American Pie'. This is wholly untrue and Don McLean released a press statement in 1999 to confirm this:

"the growing urban legend that "American Pie" was the name of Buddy Holly’s plane the night it crashed, killing him, Ritchie Valens and the Big Boppper, is untrue. I created the term." - Don McLean, 1999

For those (one or two) of you who don't know what we're talking about, here's a video that interprets the song—one of the best pop songs of all time, especially for us old fogies who actually listened to Buddy Holly, Ritchie Valens and J. P. Richardson, Jr. (The Big Bopper).



[Hat Tip: Karmen at Chaotic Utopia]

200,000 Visits

 
According to Sitemeter I've had 200,000 visits to Sandwalk since I started last November. I have to admit that I'm fascinated by these numbers. It's exciting to see that some people are reading Sandwalk and it's fun to find out where they're coming from and which articles are the most popular.

When my six month trial period was up, I expressed some disappointment about the popularity of Sandwalk [My Six Months Are Up!]. Things look a little better now so I've decided to give it another six months and see how it works out. I don't know what I'll do when I run out of Nobel Laureates.