Plasmodium falciparum Causes Malaria

 
Malaria is caused by a small protozoan parasite called Plasmodium falciparum (left). The Plasmodium is a single-celled organism with a complex life cycle. It is classified in the nebulous Protist kingdom within the phylum Apicomplexa [NCBI Taxonomy].

The life cycle is described in many places but one of the best comes from the Applied Biosystems website.

Human malaria is caused by infection with intracellular protozoan parasites of the genus Plasmodium that are transmitted by Anopheles mosquitoes. Four species of Plasmodium infect humans: P. falciparum, P. vivax, P. ovale, and P. malariae, with P. falciparum accounting for the majority of infections and being the most lethal. The causative agent of malaria was discovered in 1880 by Charles Alphonse Louis Laveran (Ref.1).

Plasmodium falciparum is exclusively transmitted by female Anopheles mosquitoes, mainly from members of the Anopheles gambiae complex. The parasites have a complicated life cycle that requires a vertebrate host for the asexual cycle and a female Anopheles mosquito for completion of the sexual cycle. Infection of humans by P. falciparum is initiated by injection of sporozoites into the bloodstream by an Anopheles mosquito (Ref.2). During a mosquito blood meal,infectious Sporozoites in the mosquito's saliva enter the host bloodstream and invade its hepatocytes. While some evidence indicates that Sporozoites are first trapped by Kupffer cells and then transported to hepatocytes,other findings suggest that Sporozoites home to hepatocytes directly. Sporozoite reaches liver via bloodstream in 30 minutes....

In the hepatocytes asexual multiplication (exoerythrocytic schizogony) occurs, leading to the production of several thousand merozoites. In 1 to 2 weeks, a single sporozoite can give rise to 30,000 merozoites. During this pre-erythrocytic stage,no illness is induced by malaria. In P. vivax infections, which are characterized by relapses,a dormant stage, called the hypnozoite, remains in the liver. From this stage relapsing infections may occur at a later stage. P. falciparum infection relapses do not occur. It is, therefore, assumed that the sporozoites of this species develop uniformly producing pre-erythocytic schizonts at the same time and these schizonts, once formed,discharge all the merozoites simultaneously; do not remain dormant as in P. vivax (Ref.3).


These Merozoites are released into the bloodstream and invade erythrocytes. The asexual erythrocytic cycle begins when a single merozoite invades a host red blood cell and is enclosed within a parasitophorous vacuole,separate from the host cell cytoplasm. Three morphologically distinct phases are then observed. The ring stage,lasting approximately 24 h in P. falciparum, accounts for about half of the intraerythrocytic cycle, but it is metabolically nondescript. It is followed by the trophozoite stage; a very active period during which most of the red blood cells cytoplasm is consumed. Finally,parasites undergo 4-5 rounds of binary divisions during the schizont stage, producing 8-36 new merozoites that burst from the host cell to invade new erythrocytes,beginning another round of infection. This phase of the infection (erythrocytic schizogony) is responsible for malaria pathogenesis. Much of the morbidity and mortality associated with malaria is caused by the rupture of iRBCs (Infected Red Blood Cells) during the asexual reproductive stages of the parasite. Intense fever, occurring in 24-72 hour intervals, is accompanied by nausea, headaches,and muscular pain among other symptoms. The characteristic fever spike has been correlated with incremental rises in serum levels of TNF-Alpha associated with the release of parasite proteins during erythrocytic rupture. Furthermore,a variety of potentially fatal symptoms,including liver failure, renal failure,and cerebral disease are associated with untreated P. falciparum. These symptoms are consequences of the unique ability of the parasite to bind to endothelial surfaces; this adherence inhibits circulation and causes localized oxygen-deprivation and sometimes hemorrhaging. It has been proposed that ICAM1 (Intercellular Adhesion Molecule-1), E-selectin,VCAM1 (Vascular Cell Adhesion Molecule-1), and CSA (Chondroitin Sulfate-A), and CD36 are some of the surface molecules responsible for parasite-endothelial adherence (Ref.4).

Instead of producing new schizonts, some merozoites, after invasion of the erythrocyte, arrest their cell cycle and develop into male (micro) or female (macro) gametocytes, the forms that are required for transmission to the mosquito (asexual parasites do not survive ingestion by the insect). Inside the mid-gut of the mosquito, fertilization occurs, producing zygotes, which develop into ookinetes. The ookinetes form oocysts, which then grow and divide and rupture to give rise to sporozoites, which migrate to the salivary glands. Then the infectious cycle of malaria can repeat itself (Ref.5). While all four species of Plasmodium have a haemolytic component ie. when a new brood of parasites break out of the red blood cell this is usually of little consequence. The exception is falciparum malaria where the parasites multiply very rapidly and may occupy 30% or more of the red blood cells causing a very significant level of haemolysis. One reason for this is that P. falciparum invades red cells of all ages whereas P. vivax and P. ovale prefer younger red cells, while P. malariae seeks mature red cells. Malaria places an increasing burden on global public health resources. In the face of growing resistance of the malaria parasite to available antimalarial drugs, there is a need for new drugs and the identification of new chemotherapeutic targets (Ref.6).

---

Image Credits:

Plasmodium falciparum, the parasite that causes malaria in humans, needs a living host in …. [Photograph]. Retrieved July 25, 2007, from Encyclopædia Britannica Online: http://www.britannica.com/ebc/art-55545

Life Cycle diagram is from Don Forsdyke.

The red blood cell image is from The Scripps Research Institute.

Quinine and Malaria

 
Monday's Molecule #36 was quinine, an alkaloid isolated from the bark of Chichona, or quinine tree [Cinchona pubescens]. The tree originally grew only on the eastern slopes of the Andes in South America where the bark was widely used by the natives to prevent malaria and other diseases. Following the discovery of its amazing properties by Europeans, it was transported to other tropical parts of the world.

Quinine works by attacking the parasite that causes malaria. This protozoan parasite, Plasmodium falciparum, feeds on red blood cells. It can easily digest hemoglobin but can't handle the heme groups that are released when the protein is degraded. These heme groups are toxic to the parasite so they are stored in an inactive form inside a membrane-bound organelle called a digestive vacuole. Quinine interferes with this storage causing the hemes to remain free where they poison the cell. The exact mechanism is unknown but it is known that quinine has to enter the vacuole in order to be effective. The most likely mechanism is quinine binding to the heme molecule to prevent its conversion to the inactive form celled haemozoin.

Resistance to quinine and related compounds is usually due to mutations in transporter proteins that are found in the membranes of the digestive vacuole. The mutations prevent the accumulation of quinine in the vacuole.

Quinine is present in tonic water that was widely consumed in the last century to ward off malaria. The quinine imparts a bitter taste to tonic water so, as the story goes, British tourists used to dilute it with gin to hide the taste. The gin & tonic mixture became quire popular.

As a matter of fact, quinine is still present in modern bottles of tonic water. This can be easily demonstrated by shining ultraviolet light on a bottle of tonic water since quinine is fluorescent (left). To see how much quinine you get in a gin & tonic see [The Half-Decent Phamaceutical Chemistry Blog].

Quinine was synthesized after World War II but it isn't economical to make the drug and the only effective source is the bark of Chichona. However, a more effective drug called chloroquine (below) became widely available after World War II and it has mostly replaced quinine as the preferred drug against malaria.

Rosalind Franklin's Birthday

Today (July 25th) would have been Rosalind Franklin's 87th birthday if she had not died of cancer on April 16, 1958 [Rosalind Franklin: Wikipedia].

Rosalind Franklin's role in the elucidation of the structure of DNA was unknown and unappreciated, outside of a small group of friends, until the publication of Jim's Watson's book The Double Helix in 1968) [see The Story of DNA (Part 1) and The Story of DNA (Part 2)]. Watson revealed to the public the role that Franklin had played in the events leading up to April 1953. The picture he painted of "Rosy" (a name she never used) was not flattering and it was widely interpreted as misogynistic (probably unfairly, since Watson treats both men and women with an equal amounts of disrespect). The legend arose that Rosalind Franklin had been cheated out of the Nobel Prize.

As it turns out, Watson only met Franklin on a few brief occasions (three?) and got most of his information from Maurice Wilkins who was not on good terms with her.

The myth of Franklin as a persecuted woman scientist was reinforced by Anne Sayre in her 1975 book Rosalind Franklin & DNA. Today it is generally acknowledged that Sayre was a bit overzealous and that Franklin was not treated badly just because she was a women. This does not mean that she wasn't treated badly. Her problems with Maurice Wilkins are well-known and they stem from a personality conflict where there's enough blame on both sides to rule out a simple persecution story.

The idea that Franklin deserves more credit for the discovery of DNA has been discussed at length in numerous books and articles since the publication of Sayre's polemical story in 1975. The most notable contributions are an appendix to Horace Judson's book The Eight Day of Creation when it was republished in 1996. In that appendix, titled In defense of Rosalind Franklin: The Myth of the wronged heroine, Judson attempts to sort out the myth from the reality. He concludes that Rosalind Franklin was unlucky and although she was close to figuring out the structure of DNA, she would not have got it on her own because she had abandoned the project entirely by the end of February 1953. Here's Judson's conclusion.

Franklin was poignantly unlucky. She had no collaborator. It's been said that Watson was her collaborator. She was stubborn—a virtue in science but with limitations, for she was too unwilling to speculate early on about the helical evidence, too set on analyzing the A form by classical mathematical means, and far too rigidly opposed to building models. She was doubly unlucky in Wilkins. Their preclusive scientific incompatibility stiffened her approach. He, shut out, had no understanding scientific auditors but Watson and Crick.

Could she have got it first? She had not perceived that the backbones ran in opposite directions. She had not started building the B form as a double helix and so had yet to even encounter the problem of fitting the bases inside. Furthermore she was moving. Randall, mean-spiritedly, no doubt set on by Wilkins, made her agree to wind up and publish what she had on DNA, then leave the problem behind. And yet, and still, she had been so close, two half-steps away, that she saw at once that the Watson and Crick structure was essentially correct. Watson was surprised at her gracious assent.

(But see Klug (2003) The Discovery of the DNA double helix for a slightly different opinion. Klug was a collaborator and good friend of Franklin's after she moved to Birbeck College.)

The definitive biography—as of today—is the one published by Brenda Maddox in 2002 (Rosalind Franklin: The Dark Lady of DNA). Maddox sorts out the various controversies and unweaves the myth of the persecuted woman from the fact of the unappreciated and competent scientist. With the publication of Maddox's book we begin to see that Franklin's contribution was important and should have been acknowledged more openly by Crick, Watson, and Wilkins. At the same time, we see that Watson, Crick and Franklin remained (became?) friends after the structure was solved. This is not the sort of thing you expect from someone who felt wronged by the evens leading up to February 1953.

Maddox has an article in Nature on the 50th anniversary of the publication of the Watson & Crick paper in 2003 [The double helix and the "wronged heroine"]. She concludes,

Belated credit

Watson and Crick seem never to have told Franklin directly what they subsequently have said from public platforms long after her death — that they could not have discovered the double helix of DNA in the early months of 1953 without her work. This is all the more surprising in view of the close friendship that developed among the three of them — Watson, Crick and Franklin — during the remaining years of her life. During this time, she was far happier at non-sectarian Birkbeck than she ever was at King's, and led a spirited team of researchers studying tobacco mosaic virus (TMV).

From 1954 until months before her death in April 1958, she, Watson and Crick corresponded, exchanged comments on each other's work on TMV, and had much friendly contact. At Wood's Hole, Massachusetts, in the summer of 1954 Watson offered Franklin a lift across the United States as he was driving to her destination, the California Institute of Technology. In the spring of 1956 she toured in Spain with Crick and his wife Odile and subsequently stayed with them in Cambridge when recuperating from her treatments for ovarian cancer. Characteristically, she was reticent about the nature of her illness. Crick told a friend who asked that he thought it was "something female".

In the years after leaving King's, Franklin published 17 papers, mainly on the structure of TMV (including four in Nature). She died proud of her world reputation in the research of coals, carbons and viruses. Given her determination to avoid fanciful speculation, she would never have imagined that she would be remembered as the unsung heroine of DNA. Nor could she have envisaged that King's College London, where she spent the unhappiest two years of her professional career, would dedicate a building — the Franklin–Wilkins building — in honour of her and the colleague with whom she had been barely on speaking terms.

Lynne Elkin wrote a brief review of the Rosalind Franklin controversy for Physics Today in 2003, after the publication of the Maddox book [Rosalind Franklin and the Double Helix]. The review emphasizes all of the complex twist and turns of this complicated story. She concludes with a sound piece of advice for all those who would exploit Rosalind Franklin to their own ends.

It is important to stop demeaning Franklin's reputation, but equally important to avoid obscuring her more difficult personality traits. She should not be put on a pedestal as a symbol of the unfair treatment accorded to many women in science. Her complicated relationship with Wilkins has been treated in overly simplistic ways. Distorted accounts, which inaccurately portray the three Nobel Prize winners as well as Franklin, are unfortunate and unnecessary: There was enough glory in the work of the four to be shared by them all.

How Not to Get Elected in America

 
The chart below was published in The New York Times [God ’08: Whose, and How Much, Will Voters Accept?]. It's pretty scary when you think about it.

The numbers indicate the percentage of respondents who would said they would be less (or more) likely to vote for a candidate with the indicated traits. The data is from a Pew survey in February that was already blogged. It's worth a second look.

I can tell you one thing for certain—America is never going to have a President who is gay, atheist, 75 years old, and never before held public office. On the other hand, if you're a Christian and long time Washington politician with previous military service, then you're a shoo-in.


[Hat Tip: Alex Palazzo at The Daily Transcript]

There is no God

 
PZ found this first [Hide the guillotines, they're on to us!]. And you wonder why we call them IDiots?

The good part is that these people have finally realized that this is a fight between rationalism and superstition. At least I thought it would be a good thing before I saw this video. Who in their right mind would have associated rationalism with guillotines?

One School System Network [OSSN]

 
We learned today that the Progressive Conservative Party in Ontario is committed to expanding public funding of faith based schools [Faith-based school funding hailed by some]. According to the announcement by PC leader John Tory, the party would move rapidly to initiate funding of Christian, Muslim, and Hebrew schools.

John Tory, leader of the PCs, announced yesterday that former PC premier Bill Davis will look at ways to commit public money to faith-based schools if Tory becomes premier after the Oct. 10 vote.

The 53,000 students who attend schools outside the public and Catholic school systems deserve funding in the interest of fairness, Tory said. The funding for faith-based schools is part of his party platform.

Davis is to make recommendations and pilot programs could begin next fall. One of Davis's last acts as premier was to extend funding past Grade 10 for the province's Roman Catholic schools.

Ontario has two publicly funded school systems; a public school system and a separate school system. The "separate" schools are Roman Catholic schools. The "public" schools were originally Protestant but they evolved to become secular and open to students of all religions. The two school system was inherited from the time when Quebec and Ontario joined to form a single country (1867). The deal was that the Roman Catholic schools in Quebec would be funded and this was extended to cover students in Ontario.

Up until now, I have not been an advocate for change because the separate school system is not much different than the public school system in spite of the fact that it is Roman Catholic. However, it has always been difficult to resist funding other religious schools because we are already giving public money to the Roman Catholic schools.

Now that this is about to become a major election issue I've decided to take a more active position in advocating the abolition of the Roman Catholic School boards and the amalgamation of all students into a single school system. If you feel the same way then I urge you to support the One School System Network (OSSN). Many of the members of this groups also belong to the Center for Inquiry (CFI).

Here's part of the OSSN vision statement.

The organizations represented by the One School System Network [OSSN] are united in the conviction that:

Ontario's publicly funded school system brings students of all backgrounds together in an environment that fosters mutual respect and understanding while respecting their fundamental equality and helping them to realize their full potential as citizens.

To realize that vision, OSSN seeks the establishment of a single secular school system for each official language, namely English and French public school boards.

Furthermore, OSSN seeks the elimination of costly duplication in the Ontario school system in order to minimize infrastructure costs and to maximize the opportunities for student development.
Publicly funded schools in Ontario shall not discriminate on the basis of religion in any form including: school environment, enrollment of students, opportunities for all students, evaluation of students, employment and advancement of teachers and all other school board personnel, adherence to Ministry of Education curriculum guidelines including courses in World and Comparative Religion.

If the status quo isn't possible then in my opinion we have no choice but to terminate funding of Roman Catholic schools. The latest pools show that 58% of Ontario residents want a single school system. Let's give it to them.

Are You as Smart as a Third Year University Student? Q1

 
Over on the thread The Chemical Structure of Double-Stranded DNA we're having a little discussion about exam questions related to the structure of DNA and reading frames.

I thought it might be fun to post some multiple choice questions from old exams to see if the Sandwalk readers are as smart as my third year molecular biology students. Here's a question from 1999.

---

Examples of overlapping genes that are transcribed in opposite directions (i.e., opposite strands serve as templates) are very rare in biology. Part of the coding region from the middle of two such overlapping genes is shown below. In one of these genes a mutation results in the substitution of valine for methionine in the polypeptide (i.e., the normal protein has methionine). What effect would this have on the polypeptide sequence encoded by the other gene? (the sequence of the normal or wild-type gene is shown)

          a) no change
          b) substitution of methionine for arginine
          c) premature termination (shorter protein)
          d) substitution of threonine for isoleucine
          e) substitution of serine for phenylalanine

---

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).

---

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.

---

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.

---

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.